Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytin a
in Marine and Freshwater Algae by Fluorescence
Elizabeth J. Arar
and
Gary B. Collins
Revision 1.2
September 1997
National Exposure Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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Method 445.0
In Vitro Determination of Chlorophyll a and Pheophytin a
in Marine and Freshwater Algae by Fluorescence
1.0	Scope and Application
1.1	This method provides a procedure for low level
determination of chlorophyll a (chl a) and its magnesium-
free derivative, pheophytin a (pheo a), in marine and
freshwater phytoplankton using fluorescence detection.'1,2'
Phaeophorbides present in the sample are determined
collectively as pheophytin a. For users primarily
interested in chl a there is currently available a set of very
narrow bandpass excitation and emission filters (Turner
Designs, Sunnyvale, CA) that nearly eliminates the
spectral interference caused by the presence of pheo a
and chlorophyll b. The difference between the modified
method and the conventional fluorometric method is that
the equations used for the determination of chlor a
without pheo a correction (uncorrected chlor a), are used
instead of the equations for "corrected chlor a". This EPA
laboratory has evaluated the modified filters and found
the technique to be an acceptable alternative to the
conventional fluorometric method using pheo a
correction.(3)
Chemical Abstracts Service
Analyte Registry Number (CASRN)
Chlorophyll a 479-61-8
1.2 Instrumental detection limits (IDL) of 0.05 |jg chl
a/L and 0.06 |jg pheo a/L in a solution of 90% acetone
were determined by this laboratory. Method detection
limits (MDL) using mixed assemblages of algae provide
little information because the fluorescence of other
pigments interferes in the fluorescence of chlorophyll a
and pheophytin a.(4) A single lab estimated detection limit
for chlorophyll a was determined to be 0.11 |jg/L in 10 mL
of final extraction solution. The upper limit of the linear
dynamic range for the instrumentation used in this
method evaluation was 250 |jg chl a/L.
1.3	This method was multilaboratory validated in
1996.(5) Results from that study may be found in Section
13. Additional QC procedures also have been added as
a result of that study.
1.4	This method uses 90% acetone as the extraction
solvent because of its efficiency for most types of algae.
There is evidence that certain chlorophylls and
carotenoids are more thoroughly extracted with
methanol'5"8' or dimethyl sulfoxide.'9' Bowles, et al.'8'
found that for chlorophyll a, however, 90% acetone was
an effective extractant when the extraction period was
optimized for the dominant species present in the sample.
1.5	Depending on the type of algae under
investigation, this method can have uncorrectable
interferences (Sect. 4.0). In cases where taxomonic
classification is unavailable, a spectrophotometric or high
performance liquid chromatographic (HPLC) method may
provide more accurate data for chlorophyll a and
pheophytin a.
1.6	This method is for use by analysts experienced in
the handling of photosynthetic pigments and in the
operation of fluorescence detectors or by analysts under
the close supervision of such qualified persons.
2.0	Summary of Method
2.1	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 to
0.003 N HCIwith 0.1 N HCI. Sensitivity calibration factors,
which have been previously determined on solutions of
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pure chlorophyll a of known concentration, are used to
calculate the concentration of chlorophyll a and
pheophytin a in the sample extract. The concentration in
the natural water sample is reported in |jg/L.
3.0	Definitions
3.1	Estimated Detection Limit (EDL) — The
minimum concentration of an analyte that yields a
fluorescence 3X the fluorescence of blank filters which
have been extracted according to this method.
3.2	Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.3	Instrument Detection Limit (IDL) — The
minimum quantity of analyte or the concentration
equivalent which gives an analyte signal equal to three
times the standard deviation of the background signal at
the selected wavelength, mass, retention time,
absorbance line, etc. For this method the background is
a solution of 90% acetone.
3.4	Stock Standard Solution (SSS) - A
concentrated solution containing one or more method
analytes prepared in the laboratory using assayed
reference materials or purchased from a reputable
commercial source.
3.5	Primary Dilution Standard Solution (PDS) — A
solution of the analytes prepared in the laboratory from
stock standard solutions and diluted as needed to
prepare calibration solutions and other needed analyte
solutions.
3.6	Calibration Standard (CAL) — A solution
prepared from the primary dilution standard solution or
stock standard solutions containing the internal standards
and surrogate analytes. The CAL solutions are used to
calibrate the instrument response with respect to analyte
concentration.
3.7	Response Factor (RF) - The ratio of the
response of the instrument to a known amount of analyte.
3.8	Laboratory Reagent Blank (LRB) - An aliquot
of reagent water or other blank matrices that are treated
exactly as a sample including exposure to all glassware,
equipment, solvents, reagents, internal standards, and
surrogates that are used with other samples. The LRB is
used to determine if method analytes or other
interferences are present in the laboratory environment,
reagents, or apparatus.
3.9	Field Duplicates (FD1 and FD2) - Two separate
samples collected at the same time and place under
identical circumstances and treated exactly the same
throughout field and laboratory procedures. Analyses of
FD1 and FD2 give a measure of the precision associated
with sample collection, preservation and storage, as well
as with laboratory procedures.
3.10	Quality Control Sample (QCS) — A solution of
method analytes of known concentrations which is used
to fortify an aliquot of LRB or sample matrix. Ideally, the
QCS is obtained from a source external to the laboratory
and different from the source of calibration standards. It
is used to check laboratory performance with externally
prepared test materials.
3.11	Material Safety Data Sheet (MSDS) - Written
information provided by vendors concerning a chemical's
toxicity, health hazards, physical properties, fire, and
reactivity data including storage, spill, and handling
precautions.
4.0	Interferences
4.1	Any substance extracted from the filter or
acquired from laboratory contamination that fluoresces in
the red region of the spectrum may interfere in the
accurate measurement of both chlorophyll a and
pheophytin a.
4.2	The relative amounts of chlorophyll a, b and c
vary with the taxonomic composition of the phytoplankton.
Chlorophylls b and c may significantly interfere with
chlorophyll a measurements depending on the amount
present. Due to the spectral overlap of chlorophyll b with
pheophytin a and chlorophyll a, underestimation of
chlorophyll a occurs accompanied by overestimation of
pheophytin a when chlorophyll b is present in the sample.
The degree of interference depends upon the ratio of a:b.
This laboratory found that at a ratio of 5:1, using the
acidification procedure to correct for pheophytin a,
chlorophyll a was underestimated by approximately 5%.
Loftis and Carpenter'10' reported an underestimation of
16% when the a.b ratio was 2.5:1. A ratio of 1:1 is the
highest ratio likely to occur in nature. They also reported
overestimation of chlorophyll a in the presence of
chlorophyll c of as much as 10% when the a.c ratio was
1:1 (the theoretical maximum likely to occur in nature).
The presence of chlorophyll c also causes the under-
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estimation of pheophytin a. The effect of chlorophyll c is
not as severe as the effect of chlorophyll b on the
measurement of chlorophyll a and pheophytin a.
Knowledge of the taxonomy of the algae under
consideration will aid in determining if the
spectrophotometric method using trichromatic equations
to determine chlorophyll a, b, and c or an HPLC method
would be more appropriate.(1116) In the presence of
chlorophyll b or pheopigments, the modified fluorometric
method described here is also appropriate.'5'
4.3	Quenching effects are observed in highly
concentrated solutions or in the presence of high
concentrations of other chlorophylls or carotenoids.
Minimum sensitivity settings on the fluorometer should be
avoided; samples should be diluted instead.
4.4	Fluorescence is temperature dependent with
higher sensitivity occurring at lower temperatures.
Samples, standards, LRBs and QCSs must be at the
same temperature to prevent errors and/or low precision.
Analyses of samples at ambient temperature is
recommended in this method. Ambient temperature
should not fluctuate more than ± 3°C between
calibrations or recalibration of the fluorometer will be
necessary.
4.5	Samples must be clarified by centrifugation prior
to analysis.
4.6	All photosynthetic pigments are light and
temperature sensitive. Work must be performed in
subdued light and all standards, QC materials and filter
samples must be stored in the dark at -20°C or -70°C to
prevent degradation.
5.0	Safety
5.1	The toxicity or carcinogenicity of the chemicals
used in this method have not been fully established.
Each chemical should be regarded as a potential health
hazard and handled with caution and respect. Each
laboratory is responsible for maintaining a current
awareness file of Occupational Safety and Health
Administration (OSHA) regulations regarding the safe
handling of the chemicals specified in this method.(17 20) A
file of MSDS should also be made available to all
personnel involved in the chemical analysis.
5.2	The grinding of filters during the extraction step of
this method should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.
6.0	Apparatus and Equipment
6.1	Fluorometer - Equipped with a high intensity
F4T.5 blue lamp, red-sensitive photomultiplier, and filters
for excitation (CS-5-60) and emission (CS-2-64). A
Turner Designs Model 10 Series fluorometer was used in
the evaluation of this method. The modified method
requires excitation filter (436FS10) and emission filter
(680FS10).
6.2	Centrifuge, capable of 675 g.
6.3	Tissue grinder, Teflon pestle (50 mm X 20 mm)
with grooves in the tip with 1/4" stainless steel rod long
enough to chuck onto a suitable drive motor and 30-mL
capacity glass grinding tube.
6.4	Filters, glass fiber, 47-mm or 25-mm, nominal
pore size of 0.7 |jm unless otherwise justified by data
quality objectives. Whatman GF/F filters were used in this
work.
6.5	Petri dishes, plastic, 50 X 9-mm, or some other
solid container for transporting and storing sampled
filters.
6.6	Aluminum foil.
6.7	Laboratory tissues.
6.8	Tweezers or flat-tipped forceps.
6.9	Vacuum pump or source capable of maintaining
a vacuum up to 6 in. Hg.
6.10	Room thermometer.
6.11	Labware - All reusable labware (glass,
polyethylene, Teflon, etc.) that comes in contact with
chlorophyll solutions should be clean and acid free. An
acceptable cleaning procedure is soaking for 4 h in
laboratory grade detergent and water, rinsing with tap
water, distilled deionized water and acetone.
6.11.1	Assorted Class A calibrated pipets.
6.11.2	Graduated cylinders, 500-mL and 1-L.
6.11.3	Volumetric flasks, Class A calibrated, 25-mL, 50-
mL, 100-mL and 1-L capacity.
6.11.4	Glass rods.
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6.11.5	Pasteur type pipets or medicine droppers.
6.11.6	Disposable glass cuvettes for the fluorometer.
6.11.7	Filtration apparatus consisting of 1 or 2-L filtration
flask, 47-mm fritted glass disk base and a glass filter
tower.
6.11.8	Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.11.9	Polyethylene squirt bottles.
7.0	Reagents and Standards
7.1	Acetone, HPLC grade, (CASRN 67-64-1).
7.2	Hydrochloric acid (HCI), concentrated (sp. gr.
1.19), (CASRN 7647-01-0).
7.3	Chlorophyll a free of chlorophyll b. May be
obtained from a commercial supplier such as Sigma
Chemical (St. Louis, MO). Turner Designs (Sunnyvale,
CA) supplies ready-made standards.
7.4	Water - ASTM Type I water (ASTM D1193) is
required. Suitable water may be obtained by passing
distilled water through a mixed bed of anion and cation
exchange resins.
7.5	0.1 N HCI Solution - Add 8.5 mL of
concentrated HCI to approximately 500 mL water and
dilute to 1 L.
7.6	Aqueous Acetone Solution - 90% acetone
/10% water. Carefully measure 100 mL of water into the
1-L graduated cylinder. Transfer to a 1-L flask or storage
bottle. Measure 900 mL of acetone into the graduated
cylinder and transfer to the flask or bottle containing the
water. Mix, label and store.
7.7	Chlorophyll Stock Standard Solution (SSS) -
Chlorophyll a from a commercial supplier will be shipped
in an amber glass ampoule which has been flame sealed.
This dry standard should be stored at -20 or -70°C in the
dark and the SSS prepared just prior to use. Tap the
ampoule until all the dried chlorophyll is in the bottom of
the ampoule. In subdued light, carefully break the tip off
the ampoule. Transfer the entire contents of the
ampoule into a 50-mL volumetric flask. Dilute to volume
with 90% acetone, label the flask and wrap with
aluminum foil to protect from light. The concentration of
the solution must be determined spectrophotometrically
using a multiwavelength spectrophotometer.'10' When
stored in a light and airtight container at freezer
temperatures, the SSS is stable for at least six months.
The concentration of all dilutions of the SSS must be
determined spectrophotometrically each time they are
made.
7.8	Laboratory Reagent Blank (LRB) - A blank
filter which is extracted and analyzed just as a sample
filter. The LRB should be the last filter extracted of a
sample set. It is used to assess possible contamination
of the reagents or apparatus.
7.9	Chlorophyll a Primary Dilution Standard
Solution (PDS) - Add 1 mL of the SSS (Sect. 7.8) to a
clean 100-mL flask and dilute to volume with the aqueous
acetone solution (Sect. 7.7). If exactly 1 mg of pure
chlorophyll a was used to prepare the SSS, the
concentration of the PDS is 200 |jg/L. Prepare fresh just
prior to use.
7.10	Quality Control Sample (QCS) - Since there
are no commercially available QCSs, dilutions of a stock
standard of a different lot number from that used to
prepare calibration solutions may be used.
8.0	Sample Collection, Preservation and
Storage
8.1	Water Sample Collection - Water may be
obtained by a pump or grab sampler. Data quality
objectives will determine the depth at which samples are
taken. Healthy phytoplankton, however, are generally
obtained from the photic zone (depth at which the
illumination level is 1% of surface illumination). Enough
water should be collected to concentrate phytoplankton
on at least three filters so that precision can be assessed.
Filtration volume size will depend on the particulate load
of the water. Four liters may be required for open ocean
water where phytoplankton density is usually low,
whereas 1 L or less is generally sufficient for lake, bay or
estuary water. All apparatus should be clean and acid-
free. Filtering should be performed in subdued light as
soon as possible after sampling since algal poulations,
thus chlorophyll a concentration, can change in relatively
short periods of time. Aboard ship filtration is highly
recommended.
Assemble the filtration apparatus and attach the vacuum
source with vacuum gauge and regulator. Vacuum
filtration should not exceed 6 in. Hg (20 kPa). Higher
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filtration pressures and excessively long filtration times (>
10 min) may damage cells and result in loss of
chorophyll.
Prior to drawing a subsample from the water sample
container, thoroughly but gently agitate the container to
suspend the particulates (stir or invert several times).
Pour the subsample into a graduated cylinder and
accurately measure the volume. Pour the subsample into
the filter tower of the filtration apparatus and apply a
vacuum (not to exceed 20 kPa). A sufficient volume has
been filtered when a visible green or brown color is
apparent on the filter. Do not suck the filter dry with the
vacuum; instead slowly release the vacuum as the final
volume approaches the level of the filter and completely
release the vacuum as the last bit of water is pulled
through the filter. Remove the filter from the fritted base
with tweezers, fold once with the particulate matter inside,
lightly blot the filter with a tissue to remove excess
moisture and place it in the petri dish or other suitable
container. If the filter will not be immediately extracted,
then wrap the container with aluminum foil to protect the
phytoplankton from light and store the filter at -20 or
-70°C. Short term storage (2 to 4 h) on ice is acceptable,
but samples should be stored at -20 or -70°C as soon as
possible.
8.2	Preservation — Sampled filters should be stored
frozen (-20°C or -70°C) in the dark until extraction.
8.3	Holding Time — Filters can be stored frozen at
-20 or -70°C for as long as 31/2 weeks without significant
loss of chlorophyll a.(21)
9.0	Quality Control
9.1	Each Laboratory using this method is required to
operate a formal quality control (QC) program. The
minimum requirements of this program consist of an initial
demonstration of laboratory capability and the continued
analysis of laboratory reagent blanks, field duplicates and
quality control samples as a continuing check on
performance. The laboratory is required to maintain
performance records that define the quality of the data
thus generated.
9.2	Initial Demonstration of Performance
(Mandatory)
9.2.1 The initial demonstration of performance is used
to characterize instrument performance (instrumental
detection limits, linear dynamic range and MDLs) and
laboratory performance (analyses of QCSs) prior to
sample analyses.
9.2.2	Linear Dynamic Range (LDR) - The LDR should
be determined by analyzing a minimum of 5 calibration
standards ranging in concentration from 0.2 |jg/L to 200
|jg chl a/L across all sensitivity settings of the fluorometer.
If using an analog fluorometer or a digital fluorometer
requiring manual changes in sensitivity settings, normalize
responses by dividing the response by the sensitivity
setting multiplier. Perform the linear regression of
normalized response vs. concentration and obtain the
constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher
concentration until the measured fluorescence response,
R, of a standard no longer yields a calculated
concentration, Cc, that is ± 10% of the known
concentration, C, where Cc = (R - b)/m. That
concentration defines the upper limit of the LDR for your
instrument. Should samples be encountered that have a
concentration which is 90% of the upper limit of the LDR,
these samples must be diluted and reanalyzed.
9.2.3	Instrumental Detection Limit (IDL) — Zero the
fluorometer with a solution of 90% acetone on the
maximum sensitivity setting. Pure chlorophyll a in 90%
acetone should be serially diluted until it is no longer
detected by the fluorometer on a maximum sensitivity
setting.
9.2.4	Estimated Detection Limit (EDL) — Several blank
filters should be extracted according to the procedure in
Sect. 11, using clean glassware and apparatus, and the
fluorescence measured. A solution of pure chlorophyll a
in 90% acetone should be serially diluted until it yields a
response which is 3X the average response of the blank
filters.
9.2.5	Quality Control Sample (QCS) - When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify the calibration standards
and acceptable instrument performance with the analysis
of a QCS (Sect. 7.10). If the determined value is not
within the confidence limits established by project data
quality objectives, then the determinative step of this
method is unacceptable. The source of the problem
must be identified and corrected before continuing
analyses.
9.2.6	Extraction Proficiency - Personnel performing
this method for the first time should demonstrate
proficiency in the extraction of sampled filters (Sect. 11.1).
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Twenty to thirty natural samples should be obtained using
the procedure outlined in Sect. 8.1 of this method. Sets
of 10 or more samples should be extracted and analyzed
according to Sect. 11.2. The percent relative standard
deviation (%RSD) of uncorrected values of chlorophyll a
should not exceed 15% for samples that are
approximately 10X the IDL. RSD for pheophytin a might
typically range from 10 to 50%.
9.2.7 Corrected Chi a — Multilaboratory testing of this
method revealed that many analysts do not adequately
mix the acidified sample when determining corrected chl
a. The problem manifests itself by highly erratic pheo-a
results, high %RSDs for corrected chl a and poor
agreement between corrected and uncorrected chl a. To
determine if a new analyst is performing the acidification
step properly, perform the following QC procedure:
Prepare 100 mL of a 50 ppb chl a solution in 90%
acetone. The new analyst should analyze 5-10 separate
aliquots, using separate cuvettes, according to
instructions in Section 11.2. Process the results
according to Section 12 and calculate separate means
and %RSDs for corrected and uncorrected chl a. If the
means differ by more than 10%, then the stock chl a has
probably degraded and fresh stock should be prepared.
The %RSD for corrected chl a should not exceed 5%. If
the %RSD exceeds 5%, repeat the procedure until the
%RSD 5%.
9.3 Assessing Laboratory Performance
(Mandatory)
9.3.1 Laboratory Reagent Blank (LRB) — The
laboratory must analyze at least one blank filter with each
sample batch. The LRB should be the last filter
extracted. LRB data are used to assess contamination
from the laboratory environment. LRB values that exceed
the IDL indicate contamination from the laboratory
environment. When LRB values constitute 10% or more
of the analyte level determined for a sample, fresh
samples or field duplicates must be analyzed after the
contamination has been corrected and acceptable LRB
values have been obtained.
10.0	Calibration and Standardization
10.1	Calibration — Calibration should be performed
bimonthly or when there has been an adjustment made
to the instrument, such as replacement of lamp, filters or
photomultiplier. Prepare 0.2, 2, 5, 20 and 200 |jg chl a/L
calibration standards from the PDS (Sect. 7.11). Allow
the instrument to warm up for at least 15 min. Measure
the fluorescence of each standard at sensitivity settings
that provide midscale readings. Obtain response factors
for chlorophyll a for each sensitivity setting as follows:
Fs = Ca/Rs
where:
Fs = response factor for sensitivity setting, S.
Rs = fluorometer reading for sensitivity
setting, S.
Ca = concentration of chlorophyll a.
NOTE: If you are using special narrow bandpass filters
for chl a determination, DO NOT acidify. Use the
"uncorrected" chl a calculation described in Section 12.1.
If pheophytin a determinations will be made, it will be
necessary to obtain before-to-after acidification response
ratios of the chlorophyll a calibration standards as follows:
(1) measure the fluorescence of the standard, (2) remove
the cuvette from the fluorometer, (3) acidify the solution
to .003 N HCI(6) with the 0.1 N HCI solution, (4) use a
pasteur type pipet to thoroughly mix the sample by
aspirating and dispensing the sample into the cuvette,
keeping the pipet tip below the surface of the liquid to
avoid aerating the sample, (5) wait 90 sec and measure
the fluorescence of the standard solution again. Addition
of the acid may be made using a medicine dropper. It will
be necessary to know how many drops are equal to 1 mL
of acid. For a cuvette that holds 5 mL of extraction
solution, it will be necessary to add 0.15 mL of 0.1 N HCI
to reach a final acid concentration of 0.003N in the 5 mL.
Calculate the ratio, r, as follows:
r = Rb/Ra
where:
Rb = fluorescence of pure chlorophyll a
standard solution before acidification.
Ra = fluorescence of pure chlorophyll a
standard solution after acidification.
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11.0 Procedure
11.2 SAMPLE ANALYSIS
11.1 Extraction of Filter Samples
11.1.1	If sampled filters have been frozen, remove them
from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand tissues and squirt bottles
containing water and acetone. Workspace lighting should
be the minimum that is necessary to read instructions and
operate instrumentation. Remove a filter from its
container and place it in the glass grinding tube. The filter
may be torn into smaller pieces to facilitate extraction.
Push it to the bottom of the tube with a glass rod. With a
volumetric pipet, add 4 mL of the aqueous acetone
solution (Sect. 7.6) to the grinding tube. Grind the filter
until it has been converted to a slurry. (NOTE: Although
grinding is required, care must be taken not to overheat
the sample. Good judgement and common sense will
help you in deciding when the sample has been
sufficiently macerated.) Pour the slurry into a 15-mL
screw-cap centrifuge tube and, using a 6-mL volumetric
pipet, rinse the pestle and the grinding tube with 90%
acetone. Add the rinse to the centrifuge tube containing
the filter slurry. Cap the tube and shake it vigorously.
Place it in the dark before proceeding to the next filter
extraction. Before placing another filter in the grinding
tube, use the acetone and water squirt bottles to
thoroughly rinse the pestle, grinding tube and glass rod.
The last rinse should be with acetone. Use a clean tissue
to remove any filter residue that adheres to the pestle or
to the steel rod of the pestle. Proceed to the next filter
and repeat the steps above. The entire extraction with
transferring and rinsing steps takes 5 min. Approximately
500 mL of acetone and water waste are generated per 20
samples from the rinsing of glassware and apparatus.
11.1.2	Shake each tube vigorously before placing them
to steep in the dark at 4°C. Samples should be allowed
to steep for a minimum of 2 h but not to exceed 24 h.
The tubes should be shaken at least once during the
steeping period.
11.1.3	After steeping is complete, shake the tubes
vigorously and centrifuge samples for 15 min at 675 g or
for 5 min at 1000 g. Samples should be allowed to come
to ambient temperature before analysis. This can be
done by placing the tubes in a constant temperature
water bath or by letting them stand at room temperature
for 30 min. Recalibrate the fluorometer if the room
temperature fluctuated ± 3°C from the last calibration
date.
11.2.1	After the fluorometer has warmed up for at least
15 min, use the 90% acetone solution to zero the
instrument on the sensitivity setting that will be used for
sample analysis.
11.2.2	Pour or pipet the supernatant of the extracted
sample into a sample cuvette. The volume of sample
required in your instrument's cuvette should be known so
that the correct amount of acid can be added in the
pheophytin a determinative step. For a cuvette that holds
5 mL of extraction solution, 0.15 mL of the 0.1 N HCI
solution should be used. Choose a sensitivity setting that
yields a midscale reading when possible and avoid the
minimum sensitivity setting. If the concentration of
chlorophyll a in the sample is 90% of the upper limit of
the LDR, then dilute the sample with the 90% acetone
solution and reanalyze. Record the fluorescence
measurement and sensitivity setting used for the sample.
Remove the cuvette from the fluorometer and acidify the
extract to a final concentration of 0.003 N HCI using the
0.1 N HCI solution. Use a pasteur type pipet to
thoroughly mix the sample by aspirating and dispensing
the sample into the cuvette, keeping the pipet tip below
the surface of the liquid to avoid aerating the sample.
Wait 90 sec before measuring fluorescence again.
NOTE: Proper mixing is critical for precise and accurate
results. Twenty-five to thirty-five samples can be
extracted and analyzed in one 8 hr day.
NOTE: If you are using special narrow bandpass filters
for chl a determination, DO NOT acidify samples. Use
the "uncorrected" chl a calculations described in Section
12.1.
12.0	Data Analysis and Calculations
12.1	For "uncorrected chlorophyll a," calculate the
chlorophyll a concentration in the extract as:
Ce,u = Rf> x Fs
where CE„ = uncorrected chlorophyll a concentration
(|jg/L) in the extract solution analyzed,
R^ = fluoresence response of sample extract
before acidification, and
Fs = fluoresence respnse factor for sensitivity
setting S.
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Calculate the "uncorrected" concentration of chlorophyll
a in the whole water sample as follows:
CEu x extract volume (L) X DF
Co II ~
sample volume (L)
where Cs„ = uncorrected chlorophyll a concentration
(|jg/L) in the whole water sample,
extract volume = volume (L) of extraction
prepared before any dilutions,
DF = dilution factor,
sample volume = volume (L) of whole water
sample.
12.2 For "corrected chlorophyll a", calculate the
chlorophyll a concentration in the extract as :
CE,c= Fs (r/r-1) (Rb - Ra)
where:
CEc= corrected chlorophyll a concentration (|jg/L) in the
extract solution analyzed,
Fs = response factor for the sensitivity setting S,
r = the before-to-after acidification ratio of a
pure chlorophyll a solution (Sect. 10.1),
Rb = fluorescence of sample extract before
acidification, and
Ra = fluorescence of sample extract after
acidification.
Calculate the "corrected" concentration of chlorophyll a
in the whole water sample as follows:
CEu x extract volume (L) X DF
C = 	:	
sample volume (L)
where CSc = corrected chlorophyll a concetration (|jg/L)
in the whole water sample,
extract volume = volume (L) of extract prepared
before dilution,
12.3	Calculate the pheophytin a concentration as
follows:
PE = Fs (r/r-1) (rRa-R6)
PF X extract volume (L) X DF
Ps = —	—	
sample volume (L)
where PE = pheophytin a concentration (|jg/L) in the
sample extract; and
Ps = pheophytin a concentration (|jg/L) in the
whole water sample.
12.4	LRB and QCS data should be reported with each
sample data set.
13.0	Method Performance
13.1	The single lab EDL forthe instrument used in the
evaluation of this method was 0.05 |jg/L for chlorophyll a
and 0.06 |jg/L pheophytin a.
13.2	The precision (%RSD) for chlorophyll a in mostly
blue-green and green phytoplankton natural samples
which were steeped for 2 h vs 24 h is reported in Table 1.
Although the means were the same, precision was better
for samples which were allowed to steep for 24 h prior to
analysis. Since pheophytin a was found in the samples,
the chlorophyll a values are "corrected" (Sect. 12.2).
Table 2 contains precision data for pheophytin a. A
statistical analysis of the pheophytin a data indicated a
significant difference in the mean values at the 0.05
significance level. The cause of the lower pheophytin a
values in samples extracted for 24 h is not known.
13.3	Three QCS ampoules obtained from the USEPA
were analyzed and compared to the reported confidence
limits in Table 3. NOTE: The USEPA no longer provides
these QCSs.
13.4	Multilaboratory Testing - A multilaboratory
validation and comparison study of EPA Methods 445.0,
446.0 and 447.0 for chlorophyll a was conducted in 1996
by Research Triangle Institute, Research Triangle Park,
N.C. (EPA Contract No. 68-C5-0011). There were 21
volunteer participants in the fluorometric methods
445.0-9
Revision 1.2 September 1997

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component that returned data; 10 that used the modified
fluorometric method and 11 that used the conventional
method. The primary goals of the study were to
determine estimated detection limits and to assess
precision (%RSD) and bias (as percent recovery) for
select unialgal species, and natural seawater.
13.4.1	The term, pooled estimated detection limit (p-
EDL), is used in this method to distinguish it from the EPA
defined method detection limit (MDL). An EPA MDL
determination is not possible nor practical for a natural
water or pure species sample due to known spectral
interferences and to the fact that it is impossible to
prepare solutions of known concentrations that
incorporate all sources of error (sample collection,
filtration, processing). The statistical approach used to
determine the p-EDL was an adaptation of the Clayton,
et.al.(22) method that does not assume constant error
variances across concentration and controls for Type II
error. The statistical approach used involved calculating
an estimated DL for each lab that had the desired Type I
and Type II error rates (0.01 and 0.05, respectively). The
median DLs over labs was then determined and is
reported in Table 4. It is referred to as pooled-EDL (p-
EDL).
Solutions of pure chlorophyll a in 90% acetone were
prepared at three concentrations (0.11, 0.2 and 1.6 ppm)
and shipped with blank glass fiber filters to participating
laboratories. Analysts were instructed to spike the filters
in duplicate with a given volume of solution and to
process the spiked filters according to the method. The
results from these data were used to determine a p-EDL
for each method. Results (in ppm) are given in Table 4.
The standard fluorometric and HPLC methods gave the
lowest p-EDLs while the spectrophotometric
(monochromatic equations) gave the highest p-EDLs.
Due to the large dilutions required to analyze these
solutions, the fluorometric p-EDLs are unrealistically high
compared to what is achievable by a single lab. Typical
single lab EDLs can easily be 1000 fold lower than the p-
EDL reported in Table 4.
13.4.2	To address precision and bias in chlorophyll a
determination for different algal species, three pure
unialgal cultures (Amphidinium, Dunaliella and
Phaeodactylum) were cultured and grown in the
laboratory. Four different "concentrations" of each
species were prepared by filtering varying volumes of the
algae. The filters were frozen and shipped to participant
labs. Analysts were instructed to extract and analyze the
filters according to the respective methods. The "true"
concentration was assigned by taking the average of the
HPLC results for the highest concentration algae sample
since chlorophyll a is separated from other interfering
pigments prior to determination. Pooled precision (as
determined by %RSD) data are presented in Tables 5-7
and accuracy data (as percent recovery) are presented in
Table 8. No significant differences in precision were
observed across concentrations for any of the species. It
should be noted that there was considerable lab-to-lab
variation (as exhibited by the min and max recoveries in
Table 8) and in this case the median is a better measure
of central tendency than the mean.
In summary, the mean and median concentrations
determined for Amphidinium carterae (class
dinophyceae) are similar for all methods. No method
consistently exhibited high or low values relative to the
other methods. The only concentration trend observed
was that the spectrophotometric method-trichromatic
equations (SP-T) showed a slight percent increase in
recovery with increasing algae filtration volume.
For Dunaliella tertiolecti (class chlorophyceae) and
Phaeodactylum tricornutum (class bacillariophyceae)
there was generally good agreement between the
fluorometric and the spectrophotometric methods,
however, the HPLC method yielded lower recoveries with
increasing algae filtration volume for both species. No
definitive explanation can be offered at this time for this
phenomenon. A possible explanation for the
Phaeodactylum is that it contained significant amounts of
chlorophyllide a which is determined as chlorophyll a in
the fluorometric and spectrophotometric methods. The
conventional fluorometric method (FL-STD) showed a
slight decrease in chlorophyll a recovery with increasing
Dunaliella filtration volume. The spectrophotometric-
trichromatic equations (SP-T) showed a slight increase in
chlorophyll a recovery with increasing Dunaliella filtration
volume. The fluorometric and the spectrophotometric
methods both showed a slight decrease in chlorophyll a
recovery with increasing Phaeodactylum filtration volume.
Results for the natural seawater sample are presented in
Table 9. Only one filtration volume (100 mL) was
provided in duplicate to participant labs.
14.0	Pollution Prevention
14.1	Pollution prevention encompasses any technique
that reduces or eliminates the quantity or toxicity of waste
at the point of generation. Numerous opportunities for
pollution prevention exist in laboratory operation. The
EPA has established a preferred hierarchy of
Revision 1.2 September 1997
445.0-10

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environmental management techniques that places
pollution prevention as the management option of first
choice. Whenever feasible, laboratory personnel should
use pollution prevention techniques to address their waste
generation (e.g., Sect. 11.1.1). When wastes cannot be
feasibly reduced at the source, the Agency recommends
recycling as the next best option.
14.2 For information about pollution prevention that
may be applicable to laboratories and research
institutions, consult Less is Better: Laboratory Chemical
Management for Waste Reduction, available from the
American Chemical Society's Department of Government
Relations and Science Policy, 1155 16th Street N.W.,
Washington D.C. 20036, (202)872-4477.
15.0	Waste Management
15.1	The Environmental Protection Agency requires
that laboratory waste management practices be
conducted consistent with all applicable rules and
regulations. The Agency urges laboratories to protect the
air, water, and land by minimizing and controlling all
releases from hoods and bench operations, complying
with the letter and spirit of any sewer discharge permits
and regulations, and by complying with all solid and
hazardous waste regulations, particularly the hazardous
waste identification rules and land disposal restrictions.
For further information on waste management consult
The Waste Management Manual for Laboratory
Personnel, available from the American Chemical Society
at the address listed in the Sect. 14.2.
16.0 References
1.	Yentsch, C.S. and D.W. Menzel, "A method for
the determination of phytoplankton chlorophyll
and pheophytin by fluorescence", Deep Sea
Res.. 10 (1963), pp. 221-231.
2.	Strickland, J.D.H. and T.R. Parsons, A Practical
Handbook of Seawater Analysis, Bull. Fish. Res.
Board Can., 1972, No.167, p. 201.
3.	Arar, E., "Evaluation of a new technique that uses
highly selective interference filters for measuring
chlorophyll a in the presence of chlorophyll b and
pheopigments," USEPA Summary Report, 1994,
NTIS No. PB94-210622.
4.	Trees, C.C., M.C. Kennicutt, and J.M. Brooks,
"Errors associated with the standard fluorometric
determination of chlorophylls and
phaeopigments", Mar. Chem.. 17 (1985) pp. 1-
12.
5.	Method 445, "Multi-Laboratory Comparison and
Validation of Chlorophyll Methods," Final Report,
USEPA Contract 68-C5-0011, WA1-03, August
1997.
6.	Holm-Hansen, O., "Chlorophyll a determination:
improvements in methodology", OKI OS. 30
(1978), pp. 438-447.
7.	Wright, S.W. and J.D. Shearer, "Rapid extraction
and HPLC of chlorophylls and carotenoids from
marine phytoplankton", J. Chrom.. 294 (1984),
pp. 281-295.
8.	Bowles, N.D., H.W. Paerl, and J. Tucker,
"Effective solvents and extraction periods
employed in phytoplankton carotenoid and
chlorophyll determination", Can. J. Fish. Aauat.
Sci.. 42 (1985) pp. 1127-1131.
9.	Shoaf, W.T. and B.W. Lium, "Improved extraction
of chlorophyll a and b from algae using dimethyl
sulfoxide", Limnol. and Oceanoar.. 21(6) (1976)
pp. 926-928.
10.	Loftis, M.E. and J.H. Carpenter, "A fluorometric
method for determining chlorophylls a, b, and c,1"
J. Mar. Res.. 29 (1971) pp.319-338.
11.	Standard Methods for the Analysis of Water and
Wastes. 17th Ed., 1989, 10200H, Chlorophyll.
12.	Wright, S.W., S.W. Jeffrey, R.F.C. Manntoura,
C.A. Llewellyn, T. Bjornland, D. Repeta, and N.
Welschmeyer, "Improved HPLC method for the
analysis of chlorophylls and carotenoids from
marine phytoplankton", paper submitted for
publication in 1991.
13.	Mantoura, R.F.C. and C.A. Llewellyn, "The rapid
determination of algal chlorophyll and carotenoid
pigments and their breakdown products in
natural waters by reverse-phase high
performance liquid chromatography", Anal.
Chim. Acta.. 151 (1983) pp. 297-314.
445.0-11
Revision 1.2 September 1997

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14.	Brown, L.M., B.T. Hargrave, and M.D.
MacKinnon, "Analysis of chlorophyll a in
sediments by high-pressure liquid
chromatography", Can. J. Fish. Aauat. Sci.. 38
(1981) pp. 205-214.
15.	Bidigare, R.R., M.C. Kennicutt, II, and J.M.
Brooks, "Rapid determination of chlorophylls and
their degradation products by HPLC", Limnol.
Oceanoar.. 30(2) (1985) pp. 432-435.
16.	Minguez-Mosquera, M.I., B. Gandul-Rojas, A.
Montano-Asquerino, and J. Garrido-Fernandez,
"Determination of chlorophylls and carotenoids
by HPLC during olive lactic fermentation", J,
Chrom.. 585 (1991) pp. 259-266.
17.	Carcinogens - Working With Carcinogens,
Department of Health, Education and Welfare,
Public Health Service, Center for Disease
Control, National Institute for Occupational Safety
and Health, Publication No. 77-206, 1977.
18.	"OSHA Safety and Health Standards, General
Industry", (29 CFR 1910), Occupational Safety
and Health Administration, OSHA 2206, revised
January 1976.
19.	Safety in Academic Chemistry Laboratories,
American Chemical Society publication,
Committee on Chemical Safety, 3rd Edition,
1979.
20.	"Proposed OSHA Safety and Health Standards,
Laboratories", Occupational Safety and Health
Administration, Federal Register. July 24, 1986.
21.	Weber, C.I., L.A. Fay, G.B. Collins, D.E. Rathke,
and J. Tobin, "A Review of Methods for the
Analysis of Chlorophyll in Periphyton and
Plankton of Marine and Freshwater Systems",
work funded by the Ohio Sea Grant Program,
Ohio State University. Grant No.NA84AA-D-
00079, 1986, 54 pp.
22.	Clayton, C.A., J.W. Hines and P.D. Elkins,
"Detection limits within specified assurance
probabilities," Analytical Chemistry. 59 (1987),
pp. 2506-2514.
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17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA
TABLE 1. COMPARISON OF PRECISION OF TWO EXTRACTION PERIODS
CORRECTED CHLOROPHYLL a

SamDle A(1)
SamDle B(2)

2 h(3)
24 h(3)
2 h(3)
24 h(3)
Mean Concentration (|jg/L)
49.6
52.9
78.6
78.8
Standard Deviation (|jg/L)
4.89
2.64
6.21
2.77
Relative Standard Deviation (%)
9.9
5.0
7.9
3.5
Values reported are the mean measured concentrations (n=6) of chlorophyll a in the natural water based
on a 100-mL filtration volume.
Values reported are the mean measured concentrations (n=9) of the extraction solution. Sample filtration
volume was 300 mL.
The length of time that the filters steeped after they were macerated.
445.0-13
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TABLE 2. COMPARISON OF PRECISION OF TWO EXTRACTIONS PERIODS FOR Pheophytin a
Pheophytin a

SamDle A(1)
SamDle B(2)

2 h(3)
24 h(3)
2 h(3)
24 h(3)
Mean Concentration (|jg/L)
9.22
8.19
13.1
10.61
Standard Deviation (|jg/L)
2.36
3.55
3.86
2.29
Relative Standard Deviation (%)
25.6
43.2
29.5
21.6
Values reported are the mean measured concentrations (n=6) of pheophytin a in the natural water based
on a 100-mL filtration volume.
Values reported are the mean measured concentrations (n=9) of pheophytin a the extraction solution.
Sample filtration volume was 300 mL.
The length of time that the filters steeped after they were macerated.
Revision 1.2 September 1997
445.0-14

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TABLE 3. ANALYSES OF USEPA QC SAMPLES
ANALYTE	REFERENCE VALUE	CONFIDENCE LIMITS
Chlorophyll a	2.1 |jg/L	0.5 to 3.7 |jg/L
Pheophytin a	0.3 |jg/L	-0.2 to 0.8 |jg/L
MEAN	% Relative Standard1
ANALYTE	MEASURED VALUE	Deviation
Chlorophyll a	2.8 |jg/L	1.5
Pheophytin a	0.3 |jg/L	33
N = 3
445.0-15
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TABLE 4. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS'1'
Method'2'
N'3'
p-EDL'4' (mg/L)
FL -Mod'5'
8
0.096
FL - Std'5'
9
0.082
HPLC
4
0.081
SP-M
15
0.229
SP-T
15
0.104
(1)	See Section 13.4.1 for a description of the statistical approach used to determine p-EDLs.
(2)	FL-Mod = fluorometric method using special interference filters.
FL-Std = conventional fluorometric method with pheophytin a correction.
HPLC = EPA method 447.0
SP-M = EPA method 446.0, monochromatic equation.
SP-T = EPA method 446.0, trichromatic equations.
(3)	N = number of labs whose data was used.
(4)	The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.
(5)	Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are
unrealistically high.
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445.0-16

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TABLE 5. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES
mLs of
culture
Method'1'	filtered	N(2)
Fl-Mod	5	7
10	7
50	7
100	7
Mean (ma chla/D	Std. Dev.	%RSD
0.163	0.037	22.8
0.298	0.080	26.7
1.684	0.385	22.9
3.311	0.656	19.8
Fl-Std
5
10
50
100
8
8
8
8
0.185
0.341
1.560
3.171
0.056
0.083
0.311
0.662
30.4
24.4
19.9
20.9
(1)	Fl-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2)	N = number of volunteer labs whose data was used.
445.0-17
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TABLE 6. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES
Method'1'
Fl-Mod
mLs of
culture
filtered
5
10
50
100
N(2)
7
7
7
7
Mean (ma chla/D
0.066
0.142
0.757
1.381
Std. Dev.
0.010
0.045
0.208
0.347
%RSD
14.6
31.5
27.5
25.1
Fl-Std
5
10
50
100
8
8
8
8
0.076
0.165
0.796
1.508
0.018
0.040
0.140
0.324
23.2
24.3
17.5
21.5
(1)	Fl-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2)	N = number of volunteer labs whose data was used.
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TABLE 7. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES
Method'1'
mLs of
culture
filtered
N(2)
Mean (ma chla/D
Std. Dev
%RSD
Fl-Mod
5
10
50
100
7
7
7
7
0.221
0.462
2.108
3.568
0.040
0.094
0.491
1.186
18.0
20.3
23.3
33.2
Fl-Std
5
10
50
100
8
8
8
8
0.214
0.493
2.251
4.173
0.053
0.091
0.635
0.929
24.8
18.4
28.2
22.3
(1)	Fl-Mod = fluorometric method using special interference filters.
Fl-Std = conventional fluorometric method with pheophytin a correction.
(2)	N = number of volunteer labs whose data was used.
NOTE: The phaeodactylum extract contained significant amounts of chlorophyll c and chlorophyllide a which
interferes in chlorophyll a measurement in the fluorometric method, therefore, the concentration of chlorophyll a is
overestimated compared to the HPLC method which separates the three pigments. The FL-Mod interference filters
minimize this interference more so than the conventional filters.
445.0-19
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TABLE 8. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND
CONCENTRATION LEVEL
Species
Statistic
Method
Percent Recovery
Cone.
Level 1
Cone.
Level 2
Cone.
Level 3
Cone.
Level 4
Amphidinium
Minimum
FL-MOD
70
73
75
76


FL-STD
66
91
91
90


HPLC
82
85
87
88


SP-M
36
48
68
64


SP-T
21
63
71
70

Median
FL-MOD
105
112
105
104


FL-STD
109
107
111
109


HPLC
102
106
112
105


SP-M
99
101
101
101


SP-T
95
96
106
107

Maximum
FL-MOD
121
126
143
146


FL-STD
156
154
148
148


HPLC
284
210
131
116


SP-M
141
133
126
125


SP-T
115
116
119
117







Dunaliella
Minimum
FL-MOD
162
159
157
156


FL-STD
179
171
165
164


HPLC
165
109
64
41


SP-M
120
188
167
164


SP-T
167
169
166
165

Median
FL-MOD
206
246
227
223


FL-STD
250
228
224
210


HPLC
252
177
89
80
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Table 8 cont'd
Species
Statistic
Method
Percent Recovery
Cone.
Level 1
Cone.
Level 2
Cone.
Level 3
Cone.
Level 4


SP-M
240
247
247
243


SP-T
225
244
256
256
Dunaliella
Maximum
FL-MOD
295
277
287
288


FL-STD
439
385
276
261


HPLC
392
273
172
154


SP-M
342
316
296
293


SP-T
291
283
283
283
Phaeodactylum
Minimum
FL-MOD
216
183
157
154


FL-STD
189
220
223
219


HPLC
150
119
84
75


SP-M
161
138
156
160


SP-T
203
195
216
244

Median
FL-MOD
292
285
250
245


FL-STD
296
263
254
254


HPLC
225
203
114
90


SP-M
287
274
254
253


SP-T
286
281
277
274

Maximum
FL-MOD
357
337
320
318


FL-STD
371
415
415
334


HPLC
394
289
182
139


SP-M
446
344
330
328


SP-T
357
316
318
299
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TABLE 9. CHLOROPHYLL A CONCENTRATIONS IN MG/L DETERMINED IN FILTERED SEAWATER
SAMPLES
Method
Con.(1)
No. Obs.
No. Labs
Mean
Std. Dev.
RSD(%)
Minimum
Median
Maxium
FL-MOD
100
14
7
1.418
0.425
30.0
0.675
1.455
2.060
FL-STD
100
15
8
1.576
0.237
15.0
1.151
1.541
1.977
HPLC
100
10
5
1.384
0.213
15.4
1.080
1.410
1.680
SP-M
100
38
19
1.499
0.219
14.6
0.945
1.533
1.922
SP-T
100
36
18
1.636
0.160
9.8
1.250
1.650
1.948
All Methods
100
113
57
1.533
0.251
16.4
0.657
1.579
2.060
(1) Con = mLs of seawater filtered.
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