Appendix H

PHYTOPLANKTON METHODS SUMMARY

I. Phytoplankton Sampling
Phytoplankton – defined as:
- minute, free-floating aquatic plants;
- photosynthetic or plant constituent of plankton; mainly unicellular algae.

Appendix A consists of the following:

1) FDEP protocols in Phytoplankton collection, including the equipment and
supplies required for sampling, method description, sample preservation and
handling, and laboratory quality control for sample identification.
2) Phytoplankton Methods Summary Table, and
3) Methods description

FDEP Protocols:
FS 7100 - Phytoplankton Sampling is outlined in DEP-SOP-001/01. For more
information on sample collection, see also FS 2100 (Surface water sampling). Refer also
to QASR – Appendix B, for discussion on equipment use and construction and sample
preservation, handling and holding times, if applicable.

1) Equipment and Supplies

● 1 L Nalgene wide-mouth dark sample bottle
● Van Dorn Bottle
● Lugol’s solution
● Buffered formalin
● Cooler with ice

2) Methods
1) Rinse the Nalgene sample bottle three times with ambient water. When sampling
from a boat, rinse the bottle on the side of the boat opposite from where samples are
collected in order to avoid disturbance of the surface algal community. When not
sampling from a boat, collect samples upstream from where you are standing.
2) When the algae are mostly near the surface, collect the grab sample by scooping
the bottle through the top 0.3 m of the surface water. Swirl the bottle to evenly mix
the contents. Collect samples from the top 0.3 meters to estimate algal populations
that would represent the “worst case” scenario. Algal populations are usually most
dense near the surface. When the algae are dispersed through the water column,
compositing samples from different depths is acceptable. When a fuel-powered boat
is used, collect samples from the bow to avoid contamination from the motor or from
sediment sampling activities at the rear of the boat.
3) You may collect samples directly with the sample bottles for surface collection or
with the additional equipment for various water depths.

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PHYTOPLANKTON METHODS SUMMARY

3) Sample Preservation and Handling

1) Place sample on ice.
2) Preserve the 1 L sample with 3 mL of Lugol’s solution (see FS 7001, section 2)
within 8 hours of collection time. For long-term storage, add buffered formalin (see
FS 7001, section 1) to achieve a minimum of 2.5% final concentration (approximately
25 mL). Depending on the study objectives and the organisms present, samples can
be initially preserved with buffered formalin.

4) Laboratory Quality Control for Algal Identification (LQ 7100) – For more
information on quality control procedures for Algal identification, refer to:
LQ7100, visit ftp://ftp.dep.state.fl.us/pub/labs/assessment/sopdoc/2004sops/lq1000.doc

1) General Requirements: Perform the following quality control activities for all
taxonomic identifications:
● Maintain copies of appropriate taxonomic identification keys
● Follow the quality control procedures to insure a new analyst has achieved a
minimum proficiency of Algal Identification.
● Achieve 60% correct at the genus level for soft algae or 60% correct at the species
level for Diatoms in the Consistency Identifications procedure (LQ 7130). A
recommended target level of 80% correct is suggested.
● The new analyst should re-identify a minimum of 10 samples previously
identified by a proficient analyst.

II. Phytoplankton Methods Summary

Some of the methodologies used for Phytoplankton sampling and monitoring, including
the parameters being measured, the required reporting units, references, and the detailed
method’s description are presented below:

II. Phytoplankton Methods Summary Table

III.
Method Parameter Method Reference Reporting
# Units
101 Phytoplankton Light and Dark Koch, M.S. and Madden, C. J., 2001. g C m -2 d -1
1o Productivity Bottle- Change in Patterns of primary production and (converted from
dissolved oxygen nutrient availability in a Bahamas lagoon mg l-1 O2 d-1
with fringing Mangroves. Mar. ecol.
Prog. Ser., 219:109-119.

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PHYTOPLANKTON METHODS SUMMARY

II. Phytoplankton Methods Summary Table

III.
Method Parameter Method Reference Reporting
# Units
102-a Phytoplankton Standard Downing, J.A. and Rigler, F.H., 1984. A a) cells ml-1
a) cell density and Procedures for Manual on Methods for the Assessment b) µm3 ml-1
b) biovolume Phytoplankton of Secondary Productivity in Freshwaters
Collection and (2nd ed.) Blackwell Scientific
Enumeration Publications, Oxford.

102-b Phytoplankton Inverted 1) Hasle, G. R., 1978. The inverted a) cells ml-1
a) cell density and Microscope and microscope method. Chapter 5.2.1 in b) µm3 ml-1
b) biovolume Modified Phytoplankton Manual. A. Sournia, ed.
Utermohl United Nations Educational, Scientific
Sedimentation and Cultural Organization. Paris. 337 pp.
Technique

102-c Phytoplankton Palmer-Maloney Palmer, C.M. and T. E. Maloney, 1954. A a) cells ml-1
a) cell density Counting Cells new counting slide for nanoplankton. b) µm3 ml-1
b) biovolume American Society of L&O Spec. Pub. 21.
6 pp.

103 Phytoplankton Gravimetric Hotzel, G. and R. Croome. 1999. A a) cells ml-1

a) cell density, sedimentation Phytoplankton Methods Manual for b) µm3 ml-1
b) biovolume and technique and Australian Freshwaters. LWRRDC a) c) Identification
c) taxonomy counting cells Occasional Paper 22/99. (ID) to lowest
taxonomic level
104-a a) Phytoplankton: Discreet Depth Hotzel, G. and R. Croome. 1999. A
Subsurface Grab Sampling Using Phytoplankton Methods Manual for NA
Sampling in Rivers Van Dorn, Australian Freshwaters. LWRRDC (collection only)
Ruttner, or Niskin Occasional Paper 22/99.
Bottle
104-b Phytoplankton: Discreet depth Hotzel, G. and R. Croome. 1999. A
Subsurface Grab sampling using Phytoplankton Methods Manual for NA
Sampling in Standing Van Dorn, Australian Freshwaters. LWRRDC (collection only)
Waters Ruttner, or Niskin Occasional Paper 22/99.
Bottle
104-c Phytoplankton: discreet depth Hotzel, G. and R. Croome. 1999. A
Subsurface Grab sampling using Phytoplankton Methods Manual for NA
Sampling in Estuaries Van Dorn, Australian Freshwaters. LWRRDC (collection only)
Ruttner, or Niskin Occasional Paper 22/99.
Bottle
105 Marine and Estuarine Acetone extraction Arar, E.J. and G.B. Collins, 1997. In Chl-a in µg/L
Chlorophyll-a and and fluorescence Vitro Determination of Chlorophyll a and Pheo-a in µg/L
Phaeophytin Phaeophytin a in Marine and Freshwater
Algae by Fluorescence. In Methods for
the Determination of Chemical
Substances in Marine and Estuarine
Environmental Samples, USEPA. Method

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PHYTOPLANKTON METHODS SUMMARY

II. Phytoplankton Methods Summary Table

III.
Method Parameter Method Reference Reporting
# Units
445.0-1.

106-a Phytoplankton: Algal Spectrophoto- Eaton, A.D., L.S. Clesceri and A.E. Chl a in µg l-1
Chlorophyll-a and metric Greenberg (Eds.) 1995. Standard with pheo
Phaeophytin determination of Methods for the Examination of Water correction (µg l-1)
chlorophyll and Wastewater. 19th ed. American Public
(phaeophytin Health Association, Washington, D.C.
corrected) Section 10200H.
106-b Phytoplankton: Algal Fluorometric Eaton, A.D., L.S. Clesceri and A.E. Chl-a in µg/L
Chlorophyll-a and determination of Greenberg (Eds.) 1995. Standard Pheo-a in µg/L
Phaeophytin chlorophyll Methods for the Examination of Water
(phaeophytin and Wastewater. 19th ed. American Public
corrected) Health Association, Washington, D.C.
Section 10200H.
106-c Phytoplankton Chlorophyll a- Hotzel, G. and R. Croome. 1999. A Chl-a in µg l-1
Chlorophyll a (used as a) spectrophoto- Phytoplankton Methods Manual for
biomass estimate) metric, Australian Freshwaters. LWRRDC
b) fluorometric Occasional Paper 22/99.
c) HPLC
107 Phytoplankton: a) Phytoplankton Havens, K.E., East, T.L., Beaver, J.R., a) cells ml-1
(a) density and Collection and 1996. Experimental studies of b) cm
(b) indirect density Enumeration zooplankton-phytoplankton interactions
estimate by light (Method 102) in a large subtropical lake (Lake
transmission b) Secchi depth Okeechobee, FL, USA) Freshwater
Biology 36:579-597.
108 Phytoplankton a) Light and dark Havens, K.E., East, T.L., Beaver, J.R., a) g C m-2d-1
a) productivity and bottle and 1996. Experimental studies of
b) biomass b) Filtration zooplankton-phytoplankton interactions b) mg l-1
in a large subtropical lake (Lake (dry weight)
Okeechobee, FL, USA) Freshwater
Biology 36:579-597.

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PHYTOPLANKTON METHODS SUMMARY

III. Phytoplankton Methods Description

Group Name Phytoplankton

Parameter Phytoplankton Primary (1º) Productivity
Method Light and Dark Bottles- Change in dissolved oxygen
Method # 101
Method Description

Phytoplankton and epiphyte metabolism is determined under in-situ conditions in 300 ml BOD bottles under
light and dark conditions. Epiphytes scraped from 10 cm sections of Thalassia testudium leaves and
phytoplankton were incubated for 4 h, and initial and final oxygen concentrations were measured.
Photosynthetic rates were determined by converting oxygen fluxes to carbon equivalents using a
photosynthetic quotient of 1.2 and a respiration quotient of 1.0. Daily respiration rates were calculated over 24
h, and photosynthesis over a 10 h light cycle based on PAR above 500 umol m-2 s-1.

Reference

Koch, M.S. and C.J. Madden, 2001. Patterns of Primary production and Nutrient Availability in a Bahamas
Lagoon with Fringing Mangroves. Mar. Eco. Prog. Ser., 219:109-119.

Quality Control
Reporting Name Community production/respiration
Units g C m-2 d-1 (converted from mg l-1 O2 d-1)
Database Elements NA

Quality Assurance Systems Requirements H-5 June 06

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PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: (a) cell density (b) biovolume
Method Standard Procedures for Phytoplankton Collection and Enumeration
Method # 102-a
Method Description

Collection: An integrated water column sampler is used to collect phytoplankton. A large plastic container
(20 L) is filled using the integrated water sampler. The composite sampler is mixed and a pre-labeled 125-ml
amber bottle is manually immersed into the container. The bottle should be pre-labeled with the date, station
id, sample type, replicate number, and preservative type. The sample is immediately preserved with 2-ml
Lugol’s solution per 100-ml of sample (Vollenweider 1974) and stored in the dark.

Enumeration: Phytoplankton are counted using the inverted microscope procedure of Utermohl as described
by Lund et al. (1958). Sub-samples are settled for at least 24 hours in a sedimentation chamber prior to
counting. Replicate areas are enumerated at a magnification of no less than 500X, until at least 400 animals
have been counted (Lund et al. 1958). For enumeration of rare, large taxa, the entire chamber is subsequently
scanned and counted at low magnification. Results are expressed as cells/ml and then converted to um3/ml
using appropriate geometric formulae (Downing and Rigler 1984) for all algal taxa.

References

Downing, J. A. and Rigler, F.H. 1984. A manual on Methods for the Assessment of Secondary Productivity in
fresh Waters (2nd ed.) Blackwell Scientific Publications, Oxford.

Additional References

Lund, J.W. Kipling, G.C., and Le Cren, E. E., 1958. The inverted microscope method of estimating algal
numbers and the statistical basis of estimation by counting.

Vollenweider, R. A. 1974. A Manual on Methods for Measuring Primary Production in Aquatic Environments
(2nd Ed.). Blackwell Scientific, Oxford.

Quality Control
Reporting Name a) cell density
b) biovolume
Units a) cells ml-1
b) um3 ml-1
Database Elements NA

Quality Assurance Systems Requirements H-6 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: (a) cell density and (b) biovolume
Method Inverted microscope and a modified Utermohl sedimentation technique
Method # 102-b
Method Description

The U.S. Geological Survey's (USGS) National Water-Quality Assessment Program (NAWQA) samples
phytoplanktonic algae by collecting whole-water samples (Porter et al. 1993, Moulton et al. 2002). This
protocol describes quantitative procedures for analyzing the soft-algal component of phytoplankton and
counting the total number of diatoms. This procedure is quantitative and designed to provide data on algal
densities (as cells per ml) and amount of algal biovolume (um3 per m]) at a sampling site.

Pre-Concentrate Subsamples. The initial concentration should be approximately 5-10 times the original
whole-water sample, leaving about 20 ml of concentrate for analysis. Samples are concentrated by settling and
decanting (settle for at least 2 days) or by centrifugation (1000 g for 20 min). It is important to measure and
record the original and final volumes, before and after concentration. This concentrated sample is then divided
into at least two subsamples one for soft-algae or phytoplankton analysis and one for diatom analysis.

Prepare Palmer-Maloney Counting Cell.

a. Spread a small drop of Rose Bengal solution on the base of the chamber of a clean Palmer-Maloney
counting cell and let dry.
b. Place a rectangular cover slip (#1 thickness, 22 x 50 mm) at 45° to the counting cell, covering about 1/3 of
the chamber, but not across the center of the cell.
c. Thoroughly mix the Palmer-Maloney fraction and draw it into an elongated Pasteur pipette (5.25 inch).
Quickly add the fraction drop-wise into the center of the chamber. When the surface tension starts to draw the
cover slip across the chamber, adjust the sides of the cover slip so that the ends of the chamber are covered and
the cover slip hangs over both sides of the ceramic portion of the counting cell. Then add glycerin to the area
where the cover slip hangs over the ceramic portion. This seals the cover slip to the counting cell temporarily;
without excess heat or vibration, the counting cell can be used for a week or more.

Choose to count random fields or along transects. Both methods (inverted microscope and Palmer-Maloney
counting cell) involve counting phytoplankton cells in a chamber, by counting either random fields or along
transects. Enumerate 300 natural algal units.

a) Enumerate algal forms using natural counting units. Natural counting units are defined as one for each
colony, filament, diatom cell (regardless if colonial or filamentous) or unicell. With the exception of diatoms,
identify algal forms to the lowest possible taxonomic level. Differentiate diatoms to the lowest practical
taxonomic level. Categorize diatoms as either "living" or "dead" at the time of collection and quantify them
separately.

b) Measure cell biovolumes. For each group of samples, measure the dimensions of the taxa hat contribute
most to sample biovolume. For each taxon requiring biovolume measurements, select a simple geometric
figure matching the shape of the taxon as best as possible, and determine the dimensions that must be
measured (see the "Shapes" table in the NADEDdat database).

Reference

Hasle, G.R. 1978. The inverted-microscope method. Chapter 5.2.1 in Phytoplankton Manual. A. Sournia, ed.
United Nations Educational, Scientific and Cultural Organization. Paris. 337 pp.
Additional References

Moulton, S.R., II, 1.G. Kennen, RM. Goldstein, lA. Hambrook. 2002. Revised protocols for sampling algal,

Quality Assurance Systems Requirements H-7 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

invertebrate, and fish communities in the National Water-Quality Assessment program, U.S. Geological
Survey Open-File Report 02-150. In Press.

Palmer, C.M. and T.E. Maloney. 1954. A new counting slide for nanoplankton. American Society of
Limnology and Oceanography Special Publication Number 21. 6 pp.

PCER, ANSP. 2002. Analysis of Diatoms in USGS NAWQA Program Quantitative Targeted-Habitat (RTH
and DTH) Samples. Protocol No. P-I3-39.

PCER, ANSP. 2002. Preparation of Algal Samples for Analysis Using Palmer-Maloney Cells. Protocol P-13-
50.

PCER, ANSP. 2002. Sub sampling Procedures for USGS NAWQA Program Periphyton Samples. Protocol P-
13-48.

Porter, S.D., T.F. Cuffney, M.E. Gurtz, M.R. Meador. ] 993. Methods for Collecting Algal Samples as Part of
the National Water Quality Assessment Program. U.S. Geological Survey Open-File Report 93-409, Raleigh,
NC [39 pp] http//water.usgs.gov/nawqalnawqa_home.html.

United States Geological Survey, National Water-Quality Assessment Program. 1997. Procedures for
Processing NAWQA Algal Samples. Draft Manuscript. February 1997.

Weber, C.I. 1973. Biological Field and Laboratory Methods for Measuring the Quality of Surface Waters and
Effluents. EPA-670/4- 73-001. National Environmental Research Center, Office of Research & Development,
U. S. Environmental Protection Agency. Cincinnati, OH.

Quality Control
Reporting Name a) cell density
b) biovolume
Units a) cells ml-1
b) µm3 ml-1
Database Elements
Enter data recorded on the bench sheets into the three tables of the PHYCLGY database.

Quality Assurance Systems Requirements H-8 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: (a) cell density and (b) biovolume
Method Palmer-Maloney Counting Cells
Method # 102-c
Method Description

The U.S. Geological Survey's (USGS) National Water-Quality Assessment Program (NAWQA) samples
phytoplanktonic algae by collecting whole-water samples (Porter et al. 1993, Moulton et al. 2002). This
protocol describes quantitative procedures for analyzing the soft-algal component of phytoplankton and
counting the total number of diatoms. This procedure is quantitative and designed to provide data on algal
densities (as cells per ml) and amount of algal biovolume (um3 per m]) at a sampling site.

Pre-Concentrate Subsamples. The initial concentration should be approximately 5-10 times the original
whole-water sample, leaving about 20 ml of concentrate for analysis. Samples are concentrated by settling and
decanting (settle for at least 2 days) or by centrifugation (1000 g for 20 min). It is important to measure and
record the original and final volumes, before and after concentration. This concentrated sample is then divided
into at least two subsamples one for soft-algae or phytoplankton analysis and one for diatom analysis.

Prepare Utermohl Sedimentation Chamber.

a. Attach a glass cover glass to the bottom of an Uterm6hl sedimentation chamber. For tubular varieties of
settling chambers, seal a cover glass to the threaded end of the tube and screw the tube into the base assembly.
Assemble the plate chamber type of settling chambers by sealing a cover glass on the bottom of the base,
locking it into place with the metal ring, and sealing the cylinder on top of the base. Use a light amount of
vacuum grease to seal the cover glasses and cylinders. It is critical that the cover glass be clean and grease-
free.
b. Homogenize the concentrated samples by repeatedly inverting the sample bottle. Place a 10-ml aliquot of
the sample into the assembled settling chamber. Let the sample settle for at least 8 hours. For the plate
chamber type ofUterm6hl chamber, drain the volumetric cylinder by sliding over the drainage hole. Slide the
cover plate over the chamber without allowing air bubbles to form. Analysis should proceed within a few
hours of removing the cylinder.

Choose to count random fields or along transects. Both methods (inverted microscope and Palmer-Maloney
counting cell) involve counting phytoplankton cells in a chamber, by counting either random fields or along
transects.

(a) Enumerate algal forms using natural counting units. Natural counting units are defined as one for each
colony, filament, diatom cell (regardless if colonial or filamentous) or unicell. With the exception of diatoms,
identify algal forms to the lowest possible taxonomic level. Differentiate diatoms to the lowest practical
taxonomic level. Categorize diatoms as either "living" or "dead" at the time of collection and quantify them
separately.

(b) Measure cell biovolumes. For each group of samples, measure the dimensions of the taxa hat contribute
most to sample biovolume. For each taxon requiring biovolume measurements, select a simple geometric
figure matching the shape of the taxon as best as possible, and determine the dimensions that must be
measured (see the "Shapes" table in the NADEDdat database).

Quality Assurance Systems Requirements H-9 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Reference

Palmer, C.M. and T.E. Maloney. 1954. A new counting slide for nanoplankton. American Society of
Limnology and Oceanography Special Publication Number 21. 6 pp.

Additional References

Hasle, G.R. 1978. The inverted-microscope method. Chapter 5.2.1 in Phytoplankton Manual. A. Sournia, ed.
United Nations Educational, Scientific and Cultural Organization. Paris. 337 pp.

Moulton, S.R., II, 1.G. Kennen, RM. Goldstein, lA. Hambrook. 2002. Revised protocols for sampling algal,
invertebrate, and fish communities in the National Water-Quality Assessment program, U.S. Geological
Survey Open-File Report 02-150. In Press.

PCER, ANSP. 2002. Analysis of Diatoms in USGS NAWQA Program Quantitative Targeted-Habitat (RTH
and DTH) Samples. Protocol No. P-I3-39.

PCER, ANSP. 2002. Preparation of Algal Samples for Analysis Using Palmer-Maloney Cells. Protocol P-13-
50.

PCER, ANSP. 2002. Subsampling Procedures for USGS NAWQA Program Periphyton Samples. Protocol P-
13-48.

Porter, S.D., T.F. Cuffney, M.E. Gurtz, M.R. Meador. ] 993. Methods for Collecting Algal Samples as Part of
the National Water Quality Assessment Program. U.S. Geological Survey Open-File Report 93-409, Raleigh,
NC [39 pp] http//water.usgs.gov/nawqalnawqa_home.html.

United States Geological Survey, National Water-Quality Assessment Program. 1997. Procedures for
Processing NAWQA Algal Samples. Draft Manuscript. February 1997.

Weber, C.I. 1973. Biological Field and Laboratory Methods for Measuring the Quality of Surface Waters and
Effluents. EPA-670/4- 73-001. National Environmental Research Center, Office of Research & Development,
U. S. Environmental Protection Agency. Cincinnati, OH.

Quality Control
Reporting Name a) cell density
b) biovolume
Units a) cells ml-1
b) µm3 ml-1
Database Elements
Enter data recorded on the bench sheets into the three tables of the PHYCLGY database.

Quality Assurance Systems Requirements H-10 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: (a) cell density (b) biovolume (c) taxonomy
Method Gravimetric sedimentation technique and counting cells
Method # 103
Method Description

While other methods are available, the concentration of algae by sedimentation is the preferred method.

After mixing, a subsample of between 5 mL and 1,000 mL (depending on cell density) is taken and
sedimented in a measuring cylinder of suitable size, allowing two hours for each 1 cm of water column at
20°e. If small diatoms are present, a settling time of six hours for every 1 cm of water column is recommended
(Furet and Benson-Evans, 1982). For example, if a 100 mL subsample is sedimented in a standard 100 mL
measuring cylinder (18.5 cm in height), sedimentation will take 2.3 days for average plankton and 4.6 days for
small centric diatoms. For most algal populations a sedimentation time of 48 hours for a standard 100 mL
measuring cylinder is recommended. Such a sedimentation time will also settle 'difficult' species such as
Cylindrospermopsis and Planktolyngbya. There are shorter 100 mL measuring cylinders available which will
reduce average sedimentation time to 24 hours. For occupational health and safety reasons, place
sedimentation cylinders under a fume hood or use stoppered cylinders. If these options are not available, at
least cover the top of the cylinders with a protective film or foil.

After sedimentation, the top 90% of volume is carefully siphoned off without disturbing the sedimented algae,
the remainder is shaken gently and a subsample of appropriate volume (see sections 4.1.7 to 4.1.9) is
transferred to the counting chamber and allowed to settle before counting. From time to time, examine the
supernatant for cells that have not sedimented. If the supernatant is siphoned off from a point well below the
meniscus, cells floating on the surface will be pulled down into the concentrate and will mix with the other
cells when the sample is shaken before taking the subs ample for counting.

Full details are available in the complete method text.

Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name a) cell density
b) biovolume
c) taxonomy
Units a) cells ml-1
b) µm3 ml-1
c) Identification to the lowest taxonomic level
Database Elements NA

Quality Assurance Systems Requirements H-11 June 06

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PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: Subsurface Grab Sample in Rivers
Method Discreet depth sampling using Van Dorn, Ruttner or Niskin Bottle
Method # 104-a
Method Description

Field Collection

Site selection will depend on the objectives of the individual phytoplankton program. Two aspects to be
considered: choosing the site along the stream or within the river basin and. choosing the exact location of
sampling at the sampling site.

The sampling method chosen should guarantee a representative sample and be easy to handle.
The method will vary according to in-stream conditions. The preferred method is a depth-integrated sample
taken from the main current. Water to be analyzed for all variables (e.g. algae and chemical and physical data)
should be collected in a single grab then sub sampled.
If the river appears well-mixed vertically and horizontally (presence of turbulence, lack of temperature
variability), a sample taken at mid-Stream 0.5 m below the surface (APHA, 1995) will suffice to characterize
the phytoplankton present. For specific purposes, for example sampling at a water supply off take for drinking
water, samples from a particular depth may be needed. Avoid sampling backwaters or back currents when
sampling for phytoplankton.

Full details are available in the complete method text.

Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name NA
Units Collection only
Database Elements NA

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PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: Subsurface Grab Sampling in Standing Waters
Method Discreet depth sampling using Van Dorn, Ruttner or Niskin Bottle
Method # 104-b
Method Description

Field Collection
Standing waters refer to lakes, reservoirs, lagoons and weir pools as well as rivers under unusual low-flow
conditions. Sampling sites may be chosen following a statistical approach or according to a set of criteria
related to the aim of the program.

Sampling methodology includes a vertical temperature profile should be taken at 1 m intervals to verify the
presence of stratification (Bartram and Ballance, 1996). The presence of stratification is best recognized if the
rate of temperature change with depth at the thermocline is significant (0.2C m-1, B. Sherman, pers. comm,).
In shallow waters, a temperature difference between the top and bottom layers of 1oC or greater under low
wind conditions may be taken as an indication of stratification.

The specific sampling method for phytoplankton depends on the depth at the water body. In shallow waters
with a depth of less than 2 m, a subsurface grab sample at 0.5 m is taken if the water body is well mixed.
However, if the water body is stratified or motile algae are present, a depth-integrated sample or discrete depth
samples are taken.

At each site, three samples are taken within an ecologically relevant sized area, mixed at equal volumes in a
clean container and the required volume subsampled. For work on large reservoirs, where usually only a
limited number of sites are sampled, at each site take five integrated samples within an area of 100 m by 100 m
and mix even volumes into a composite sample, In this way, the sampling regime takes into account the
horizontal patchiness of the phytoplankton without increasing the number of samples.

Full details are available in the complete method text.

Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name NA
Units Collection only
Database Elements NA

Quality Assurance Systems Requirements H-13 June 06

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PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: Subsurface Grab Sampling in Estuaries
Method Discreet depth sampling using Van Dorn, Ruttner or Niskin Bottle
Method # 104-c
Method Description

Field Collection
When deciding where and when to sample phytoplankton in an estuary, consideration needs to be given to the
physico-chemical condition of the system. Salinity stratification needs to be assessed in addition to
temperature stratification and integrated samples taken from layers of interest. When samples are integrated
across salinity layers, the total biomass sampled will include both fresh and marine species. The vertical and
horizontal salinity gradients move upstream or downstream within the estuary according to changes in
seasonal flow and weather conditions.

It is recommended that an experienced ecologist and/or statistician be consulted before commencing a

phytoplankton monitoring program in an estuary.

When sampling phytoplankton in an estuary, consideration should be given to both salinity stratification and
temperature stratification. It is therefore necessary to measure the temperature and electrical conductivity
profile at the sampling site and determine which depths of the water column are to be sampled in accordance
with project objectives. It may be that only the freshwater or saltwater layer is of interest, or it may be that
both are. Samples are then taken accordingly. To obtain an integrated sample, use a hosepipe sampler of
appropriate length. To obtain samples from different depths employ a discrete depth sampler. The discrete
depth samples may then be pooled into a composite sample if required.

Full details are available in the complete method text.

Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name NA
Units collection only
Database Elements NA

Quality Assurance Systems Requirements H-14 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Marine and Estuarine Chlorophyll-a and Phaeophytin
Method Acetone extraction and fluorescence
Method # 105
Method Description

This method provides a procedure for low level determination of chlorophyll a (chl a) and its magnesium free
derivative, phaeophytin a (pheo a), in marine and freshwater phytoplankton using fluorescence detection.

Chlorophyll-containing phytoplankton in a measured volume of sample water are concentrated by filtering at

low vacuum through a glass fiber filter. The pigments are extracted from the phytoplankton in 90% acetone
with the aid of a mechanical tissue grinder and allowed to steep for a minimum of 2 h, but not to exceed 24 h,
to ensure thorough extraction of the chlorophyll a. The filter slurry is centrifuged at 675 g for 15 min (or at
1000 g for 5 min) to clarify the solution. An aliquot of the supernatant is transferred to a glass cuvette and
fluorescence is measured before and after acidification. Sensitivity calibration factors, which have been
previously determined on solutions of pure chlorophyll a of known concentration, are used to calculate the
concentration of chlorophyll a and phaeophytin a in the sample extract. The concentration in the natural water
sample is reported in µg/L.

Full details are available in the complete method text.

Reference

Arar, E.J. and G.B. Collins, 1997. In Vitro Determination of Chlorophyll a and Phaeophytin a in Marine and
Freshwater Algae by Fluorescence. In Methods for the Determination of Chemical Substances in Marine and
Estuarine Environmental Samples. USEPA Method 445.0-1.
http://www.epa.gov/nerlcwww/m445_0.pdf

Quality Control
Reporting Name Chl-a
Pheo-a
Units µg l-1
µg l-1
Database Elements NA

Quality Assurance Systems Requirements H-15 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: Algal Chlorophyll-a and Phaeophytin
Method Spectrophotometric determination of chlorophyll (phaeophytin corrected)
Method # 106-a
Method Description

The concentration of photosynthetic pigments is used frequently used to estimate phytoplankton biomass. The
presence of absence of these pigments is used to distinguish major algal groups. One of the important
chlorophyll degradation products is phaeophytin. In the presence of pheopigments, significant error in
chlorophyll a values can occur. Chlorophyll and associated pheopigments are measured by spectrophotometry.

Samples are collected in opaque bottles and concentrated by filtration with a glass fiber filter (GF-F) as soon
as possible after collection. Held samples should be stored at 4ºC and protected from exposure to light.
Filtered samples can be frozen for up to three weeks prior to analysis.

Filtered samples are extracted with acetone and processed with a tissue grinder. Extracted samples are
analyzed with a spectrophotometer. The addition of acid, once the cholorphyll a value has been determined,
converts the chlorophyll a to phaeophytin a. An absorption peak ratio is used in correcting the chlorophyll a
concentration for phaeophytin a.

Full details are available in the complete method text.

Reference

Eaton, A.D., L.S. Clesceri and A.E. Greenberg (Eds.) 1995. Standard Methods for the Examination of Water
and Wastewater. 19th ed. American Public Health Association, Washington, D.C. Standard Methods for the
Examination of Water and Wastewater (20th ed.) Section 10200H.

Additional Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name Chl-a
Chl-a phaeophytin corrected
Units µg l-1
µg l-1
Database Elements NA

Quality Assurance Systems Requirements H-16 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Name Phytoplankton

Parameter Phytoplankton: Algal Chlorophyll-a and Phaeophytin
Method Fluorometric determination of chlorophyll (phaeophytin corrected)
(This method is normally not applicable to freshwater samples.)
Method # 106-b
Method Description

The concentration of photosynthetic pigments is used frequently used to estimate phytoplankton biomass. The
presence of absence of these pigments is used to distinguish major algal groups. One of the important
chlorophyll degradation products is phaeophytin. In the presence of pheopigments, significant error in
chlorophyll a values can occur. Chlorophyll and associated pheopigments are measured by flourometry. The
flourometric method for chlorophyll a is more sensitive than the spectrophotometric method and smaller
samples can be used.

Samples are collected in opaque bottles and concentrated by filtration with a glass fiber filter (GF-F) as soon
as possible after collection. Held samples should be stored at 4ºC and protected from exposure to light.
Filtered samples can be frozen for up to three weeks prior to analysis.

Filtered samples are extracted with acetone and processed with a tissue grinder. Chlorophyll Extracted samples
are first analyzed with a spectrophotometer (with and without the addition of acid) and then a calibrated
fluorometer. A series of calculations with formulas are used to determine the corrected chlorophyll a and
phaeophytin a concentrations. This method is normally not applicable to freshwater samples.

Full details are available in the complete method text.

Reference

Eaton, A.D., L.S. Clesceri and A.E. Greenberg (Eds.) 1995. Standard Methods for the Examination of Water
and Wastewater. 19th ed. American Public Health Association, Washington, D.C. Standard Methods for the
Examination of Water and Wastewater (20th ed.) Section 10200H.

Additional Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name Chl-a
Pheo-a
Units µg l-1
µg l-1
Database Elements NA

Quality Assurance Systems Requirements H-17 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Number Group I

Group Name Phytoplankton, Periphyton and Bacteria
Parameter Phytoplankton: Algal Chlorophyll-a
Method HPLC (high performance liquid chromatography) determination of chlorophylls and
their degradation products
Method # 106-c
Method Description

The concentration of photosynthetic pigments is frequently used to estimate phytoplankton biomass. The
presence of absence of these pigments is used to distinguish major algal groups. HPLC is a useful method for
quantifying photosynthetic pigments, such as chlorophyll a, accessory pigments and degradation products.
Pigment distribution is useful for quantitative assessment of phytoplankton community composition.

Samples are collected in opaque bottles and concentrated by filtration with a glass fiber filter (GF-F) as soon
as possible after collection. Held samples should be stored at 4ºC and protected from exposure to light.
Filtered samples can be frozen for up to three weeks prior to analysis.

Filtered samples are extracted with acetone and processed with a tissue grinder. HPLC system is set up,
equilibrated and calibrated with working standards prepared from primary standards. Add ion-pairing solution
to extracted samples and inject into instrument for analysis. Quantification is accomplished using the response
factor from a fluorescence detector and comparing known standards to sample results. See Eaton (1995) for
elution order and approximate retention times for chlorophyll pigments and their degradation products.

Full details are available in the complete method text.

Reference

Eaton, A.D., L.S. Clesceri and A.E. Greenberg (Eds.) 1995. Standard Methods for the Examination of Water
and Wastewater. 19th ed. American Public Health Association, Washington, D.C. Standard Methods for the
Examination of Water and Wastewater (20th ed.) Section 10200H.

Additional Reference

Hotzel, G. and R. Croome. 1999. A Phytoplankton Methods Manual for Australian Freshwaters. LWRRDC
Occasional Paper 22/99.

Quality Control
Reporting Name Chl-a
Units µg l-1
Database Elements NA

Quality Assurance Systems Requirements H-18 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Number Group I

Group Name Phytoplankton, Periphyton and Bacteria
Parameter Phytoplankton: (a) density and (b) indirect density estimate by light
transmission
Method a) Phytoplankton Collection and Enumeration (Method 102) and b) Secchi depth
Method # 107
Method Description

Over a 1-year period, twenty controlled experiments were performed using small mesocosms (20-l clear
plastic carboys) and plankton communities collected from four sites in shallow, subtropical Lake Okeechobee,
Florida. In replicated treatments, macro zooplankton grazers were excluded by size fractionation (115 mu m),
and/or nutrients (N and P) were added, and impacts on phytoplankton biomass and productivity were measured
after 3-day incubations.

Full details are available in the complete method text.

Reference

Havens, K. E., East, T.L., Beaver, J. R. 1996. Experimental studies of zooplankton-phytoplankton interactions
in a large subtropical lake (Lake Okeechobee, Florida, USA). Freshwater Biology 36: 579-597.

Quality Control
Reporting Name a) cell density (see Method 102)
b) Secchi depth
Units a) cells ml-1
b) cm
Database Elements

Quality Assurance Systems Requirements H-19 June 06

 Appendix H

PHYTOPLANKTON METHODS SUMMARY

Group Number Group I

Group Name Phytoplankton, Periphyton and Bacteria
Parameter Phytoplankton: (a) productivity and (b) biomass
Method a) Light and dark bottle and b) Filtration
Method # 108
Method Description

Over 1-yr period, twenty controlled experiments were performed using small mesocosms (20-l clear plastic
carboys) and plankton communities collected from four sites in Lake Okeechobee. In replicated treatments,
macrozooplankton grazers were excluded by size fractionation, and/or nutrients (N and P) were added, and
impacts on phytoplankton biomass and productivity were measured after 3-day incubations.

Full details are available in the complete method text.

Reference

Havens, K. E., East, T.L., Beaver, J. R. 1996. Experimental studies of zooplankton-phytoplankton interactions
in a large subtropical lake (Lake Okeechobee, Florida, USA). Freshwater Biology 36: 579-597.

Quality Control
Reporting Name a) productivity
b) biomass
Units a) cells ml-1
b) mg (dry weight) l-1
Database Elements

Quality Assurance Systems Requirements H-20 June 06