Method 446.0
In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in
Marine And Freshwater Algae by Visible Spectrophotometry
Adapted by
Elizabeth J. Arar
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 446.0
In Vitro Determination of Chlorophylls a, b, c1 + c2 and Pheopigments in
Marine and Freshwater Algae by Visible Spectrophotometry
1.0	Scope and Application
1.1	This method provides a procedure for
determination of chlorophylls a (chl a), b (chl b), c1 + c2
(chl c1 + c2) and pheopigments of chlorophyll a (pheo a)
found in marine and freshwater phytoplankton.
Chlorophyllide a is determined as chl a. Visible
wavelength spectrophotometry is used to measure the
pigments in sub-parts per million (ppm) concentrations.
The trichromatic equations of Jeffrey and Humphrey'1' are
used to calculate the concentrations of chl a, chl b, and
chl c^+c2. Modified monochromatic equations of
Lorenzen(2) are used to calculate pheopigment-corrected
chl a and pheo a.
1.2	This method differs from previous descriptions of
the spectrophotometric technique in several important
aspects. Quality assurance/quality control measures are
described in Sect. 9.0. Detailed sample collection and
extraction procedures are described in Sect. 8.0, and
most importantly, interference data, heretofore only
presented in research journals, is included so the analyst
may know the potential limitations of the method.
Multilaboratory data is included in Section 13.
Chemical Abstracts Service
Analyte Registry Number (CASRN)
Chlorophyll a
Chlorophyll b
Chlorophyll c1
Chlorophyll c2
479-61-8
519-62-0
18901-56-9
27736-03-4
1.3 Instrumental detection limits (IDLs) of 0.08 mg
chl a/L, 0.093 mg chl Jb/L and 0.085 mg pheo a/L in pure
solutions of 90% acetone were determined by this
laboratory using a 1-cm glass cell. Lower detection limits
can be obtained using 2, 5 or 10-cm cells. An IDL for
chlorophylls c^+c2 was not determined due to
commercial unavailability of the pure pigments.
Estimated detection limit (EDL) determinations were
made by analyzing seven replicate filtered phytoplankton
samples containing the pigments of interest. Single-
laboratory EDLs (S-EDL) were as follows: chl a - 0.037
mg/L, chl b - 0.07 mg/L, chl c1 + p - 0.087 mg/L,
pheopigment-corrected chl a - 0.053 mg/L, and pheo a -
0.076 mg/L. The trichromatic equations lead to
inaccuracy in the measurement of chlorophylls b and
c^+c2 at chl a concentrations greater than ~5X the
concentration of the accessory pigment or in the
presence of pheo a. The upper limit of the linear dynamic
range (LDR) for the instrumentation used in this method
evaluation was approximately 2.0 absorbance units (AU)
which corresponded to pigment concentrations of 27 mg
chl a/L, 30 mg chl Jb/L and approximately 45 mg pheo a/L.
No LDR for chl c1 + q was determined. It is highly
unlikely that samples containing chl c^+c2 at
concentrations approaching the upper limit of the LDR will
be encountered in nature.
1.4	Chl c^+c2 is not commercially available,
therefore, the minimum indicator of laboratory
performance for this pigment is precision of chl c1 + c,
determinations in natural samples known to contain the
pigments.
1.5	This method uses 90% acetone as the extraction
solvent because of its efficiency for extracting chl a from
most types of algae. (NOTE: There is evidence that
certain chlorophylls and carotenoids are more thoroughly
extracted with methanol'3 5' or dimethyl sulfoxide.'6' Using
high performance liquid chromatography (HPLC),
Mantoura and Llewellyn'7' found that methanol led to the
formation of chl a derivative products, whereas 90%
acetone did not. Bowles, et al.'5' found that for chl a 90%
acetone was an effective solvent when the steeping
period was optimized for the predominant species
present.)
1.6	One of the limitations of absorbance
spectrophotometry is low sensitivity. It may be preferable
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to use a fluorometric'8"10' or HPLC(1115) method if high
volumes of water (>4 L) must be filtered to obtain
detectable quantities of chl a. The user should be aware
of the inaccuracies of flu oro metric methods when chl b is
also present in the sample.
1.7 This method is for use by analysts experienced in
handling photosynthetic pigments and in the operation of
visible wavelength spectrophotometers 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
filtration 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 exceeding
24 h, to ensure thorough extraction of the pigments. 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 cell and absorbance
is measured at four wavelengths (750, 664, 647 and 630
nm) to determine turbidity, chlorophylls a, b, and c1 + c2,
respectively. If pheopigment-corrected chl a is desired,
the sample's absorbance is measured at 750 and 664 nm
before acidification and at 750 and 665 nm after
acidification with 0.1 N HCI. Absorbance values are
entered into a set of equations that utilize the extinction
coefficients of the pure pigments in 90% acetone to
simultaneously calculate the concentrations of the
pigments in a mixed pigment solution. No calibration of
the instrument with standard solutions is required.
Concentrations are reported in mg/L (ppm).
3.0	Definitions
3.1	Field Replicates — Separate samples collected
at the same time and place under identical circumstances
and treated exactly the same throughout field and
laboratory procedures. Analyses of field replicates give
a measure of the precision associated with sample
collection, preservation and storage, as well as with
laboratory procedures.
3.2	Instrument Detection Limit (IDL) - The
minimum quantity of analyte or the concentration
equivalent that gives an analyte signal equal to three
times the standard deviation of a background signal at the
selected wavelength, mass, retention time, absorbance
line, etc. In this method the instrument is zeroed on a
background of 90% acetone resulting in no signal at the
measured wavelengths. The IDL is determined instead
by serially diluting a solution of known pigment
concentration until the signal at the selected wavelength
is between .005 and .008 AU.
3.3	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. For this method the LRB is a
blank filter that has been extracted as a sample.
3.4	Linear Dynamic Range (LDR) — The absolute
quantity or concentration range over which the instrument
response to an analyte is linear.
3.5	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.
3.6	Estimated Detection Limit (EDL) - The EDL
is determined in a manner similar to an EPA MDL. It is
not called an MDL in this method because there are
known spectral interferences inherent to this method that
make 99% confidence that the chlorophyll concentration
is greater than zero impossible.
3.7	Quality Control Sample (QCS) — A solution of
method analytes of known concentrations that 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. The USEPA no longer provides
QCSs for this method.
4.0	Interferences
4.1	Any compound extracted from the filter or
acquired from laboratory contamination that absorbs light
between 630 and 665 nm may interfere in the accurate
measurement of the method analytes. An absorbance
measurement is made at 750 nm to assess turbidity in the
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sample. This value is subtracted from the sample's
absorbance at 665, 664, 647 and 630 nm. A 750 nm
absorbance value that is > .005 AU indicates a poorly
clarified solution. This is usually remedied by further
centrifugation or filtration of the sample prior to analysis.
4.2 The relative amounts of chlorophyll a, b and c1 +
c2 vary with the taxonomic composition of the
phytoplankton. Due to the spectral overlap of the
chlorophylls and pheo a, over- or underestimation of the
pigments is inevitable in solutions containing all of these
pigments.
Chi a is overestimated by the trichromatic equation of
Jeffrey and Humphrey when pheo a is present (Figure 1).
Lorenzen's modified monochromatic equation only
slightly overestimates chl a in the presence of chl b
(Figure 2). The degree of error in the measurement of
any pigment is directly related to the concentration of the
interfering pigment. Knowledge of the taxonomic
composition of the sample, proper storage and good
sample handling technique (to prevent degradation of chl
a) can aid in determining whether to report trichromatic or
pheopigment-corrected chl a. If no such knowledge
exists, it is advisable to obtain values for all of the
pigments and to compare the chl a results in light of the
apparent concentrations of the other pigments.
Obviously, if the chl a values vary widely, sound
judgement must be used in deciding which pigments, chl
b and chl c1 + q, or pheo a, are in greatest abundance
relative to each other and to chl a. The method of
standard additions, explained in most analytical chemistry
textbooks, is recommended when greater accuracy is
required.
Accuracy of chl b measurements is highly dependent
upon the concentration of chl a and pheo a.(16) In pure
solutions of chl a and b, underestimation of chl b is
observed with increasing concentrations of chl a (Figure
3). Using the method of standard additions, the same
phenomenon was confirmed to occur in natural samples.
The underestimation of chl b is due in part to the spectral
component of chl a that is subtracted from chl b as chl c1
+ c2 in the trichromatic equation. Chl a concentrations
that range from 4 to 10 times the concentration of chl b
lead to 13% to 38% underestimation of chl b. The highest
chl Jb:chl a ratio likely to occur in nature is 1:1.
Pheo a:chl a ratios rarely exceed 1:1. Pheo a is
overestimated in the presence of certain carotenoids(16)
and when chl b is converted to pheo b in the acidification
step required to determine pheopigment-corrected chl a
and pheo a. The rate of conversion of chl b to pheo b,
however, is slower than that of chl a to pheo a. It is
important, therefore, to allow the minimum time required
for conversion of chl a to pheo a before measuring
absorbance at 665 nm. Ninety seconds is recommended
by this method.
When a phytoplankton sample's composition is known
(i.e., green algae, diatoms, dinoflagellates) Jeffrey and
Humphrey's dichromatic equations for chl a, b, and c1 +
c2 are more accurate than the trichromatic equations
used here.(1)
4.3	Precision and recovery for any of the pigments is
related to efficient maceration of the filtered sample and
to the steeping period of the macerated filter in the
extraction solvent (Table 1). Precision improves with
increasing steeping periods. A drawback to prolonged
steeping periods, however, is the extraction of interfering
pigments. For example, if the primary pigment of interest
is chl a, extended steeping periods may extract more of
the other pigments but not necessarily more chl a.
Statistical analysis revealed steeping period to be a
significant factor in the recovery of chl b and pheo a from
a mixed assemblage containing these pigments in
detectable quantities, but not a significant factor in the
recovery of chl a. Chl b and pheo a are mutual
interferents so that an actual increase in the recovery of
chl b leads to a slight apparent increase in pheo a.
4.4	Sample extracts must be clarified by
centrifugation prior to analysis.
4.5	All photosynthetic pigments are light and
temperature sensitive. Work must be performed in
subdued light and all standards, QC materials, and
filtered samples must be stored in the dark at -20 or
-70°C to prevent rapid degradation.
5.0	Safety
5.1	Each chemical used in this method 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 also should be made
available to all personnel involved in the chemical
analysis.
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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	Spectrophotometer — Visible, multiwavelength,
with a bandpass (resolution) not to exceed 2 nm.
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 round-bottomed, 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 (20 KPa).
6.10	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.
6.10.1	Assorted Class A calibrated pipets.
6.10.2	Graduated cylinders, 500-mL and 1-L.
6.10.3	Volumetric flasks, Class A calibrated, 25-mL, 50-
mL, 100-mL and 1-L capacity.
6.10.4	Glass rods.
6.10.5	Disposable Pasteur type pipets or medicine
droppers.
6.10.6	Glass cells for the spectrophotometer, 1, 2, 5 or
10 cms in length. If using multiple cells, they must be
matched.
6.10.7	Filtration apparatus consisting of 1 or 2-L filtration
flask, 47-mm fritted glass disk base and a glass filter
tower.
6.10.8	Centrifuge tubes, polypropylene or glass, 15-mL
capacity with nonpigmented screw-caps.
6.10.9	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	Chi a free of chl b and chl b substantially free of
chl a may be obtained from a commercial supplier such
as Sigma Chemical (St. Louis, MO).
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%
ASTM Type I water. Carefully measure 100 mL of the
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) -
Chl a (MW = 893.5) and chl b (MW = 907.5) from a
commercial supplier is shipped in amber glass ampules
that have been flame sealed. The dry standards must be
stored at -20°C in the dark. Tap the ampule until all the
dried pigment is in the bottom of the ampule. In subdued
light, carefully break the tip off the ampule. Transfer the
entire contents of the ampule into a 25-mL volumetric
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flask. Dilute to volume with 90% acetone, label the flask
and wrap with aluminum foil to protect from light. Pheo a
may be prepared by the mild acidification of chl a (to .003
N HCI) followed by a 1:1 molar neutralization with a base
such as dilute sodium hydroxide solution. When stored in
a light- and air-tight container at -20°C, the SSS is stable
for at least six months. All dilutions of the SSS must be
determined spectrophotometrically using the equations in
Sect. 12.
7.8	Laboratory Reagent Blank (LRB) - A blank
filter that 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	Quality Control Sample (QCS) — Since there
are no commercially available QCSs, dilutions of a stock
standard 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 and frequency'21' 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.
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 populations,
thus chlorophyll a concentration, can change in a
relatively short period 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
filtration pressures or excessively long filtration times (>10
min) may damage cells and result in loss of chlorophyll.
Care must be taken not to overload the filters. Do not
increase the vacuum during filtration.
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). Typically, 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,
wrap the container with aluminum foil to protect the
phytoplankton from light and store the filter at -20°C or
-70°C. Short term storage (2 to 4 h) on ice is acceptable,
but samples should be stored at -20°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°C for as long as 31/2 weeks without significant loss of
chl a.(22)
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 replicates and
QC samples as a continuing check on performance. The
laboratory is required to maintain performance records
that define the quality of the data generated.
9.2	Initial Demonstration of Performance
(Mandatory)
9.2.1	The initial demonstration of performance is used
to characterize instrument performance (IDLs and LDRs)
and laboratory performance (MDLs and analyses of
QCSs) prior to sample analyses.
9.2.2	Standard Reference Material (SRM) 930e
(National Institute of Standards and Technology,
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Gaithersburg, MD) or other suitable spectrophotometric
filter standards that test wavelength accuracy must be
analyzed yearly and the results compared to the
instrument manufacturer's specifications. If wavelength
accuracy is not within manufacturer's specifications,
identify and repair the problem.
9.2.3	Linear Dynamic Range (LDR) — The LDR should
be determined by analyzing a minimum of 5 standard
solutions ranging in concentration from 1 to 15 mg/L.
Perform the linear regression of absorbance response (at
pigment's wavelength maximum) 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 absorbance
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 and absorbance response defines the
upper limit of the LDR for your instrument. Absorbance
responses for samples should be well below the upper
limit of the LDR, ideally between .1 and 1.0 AU.
9.2.4	Instrumental Detection Limit (IDL) — Zero the
spectrophotometer with a solution of 90% acetone. Pure
pigment in 90% acetone should be serially diluted until it
yields a response at the selected wavelength between
.005 and .008 AU.
9.2.5	Estimated Detection Limit (EDL) — At least seven
natural phytoplankton samples known to contain the
pigments of interest should be collected, extracted and
analyzed according to the procedures in Sects. 8 and 11,
using clean glassware and apparatus. The concentration
of the pigment of interest should be between 2 and 5
times the IDL. Dilution or spiking of the sample extract
solution to the appropriate concentration may be
necessary. Inaccuracies occur in the measurement of
chlorophylls b and c1 + c2 when the chl a concentration is
greater than ~5X the concentration of the accessory
pigment. Perform all calculations to obtain concentration
values in mg/L in the extract solution. Calculate the EDL
as fo I lows'23':
EDL = (3) X (S)
S = Standard deviation of the replicate analyses.
9.2.6 Quality Control Sample (QCS) - When beginning
to use this method, on a quarterly basis or as required to
meet data quality needs, verify instrument performance
with the analysis of a QCS (Sect. 7.9). 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.
9.2.7	Extraction Proficiency - Personnel performing
this method for the first time should demonstrate
proficiency in the extraction of sampled filters (Sect. 11.1).
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 trichromatic chl a should not exceed
15% for samples that are at least 10X the IDL.
9.2.8	Corrected Chl a - Multilaboratory testing of this
method revealed that many analysts do not adequately
mix the acidified sample when determining the corrected
chl a. The problem manifests itself by highly erratic
pheo a results, high %RSDs for correctetd 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 2.0 ppm chl a solution in 90%
acetone. The new analyst should analyze 5-10 separate
aliquots, using carefully rinsed 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
acceptable results are obtained.
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 in a sample, fresh
samples or field replicates must be analyzed after the
contamination has been corrected and acceptable LRB
values have been obtained.
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10.0	Calibration and Standardization
10.1	Daily calibration of the spectrophotometer is not
required when using the equations discussed in this
method. It is extremely important, therefore, to perform
regular checks on instrument performance. By analyzing
a standard reference material such as SRM 930e
(National Institute of Standards and Technology,
Gaithersburg, MD) at least quarterly, wavelength
accuracy can be compared to instrument manufacturer's
specifications. Filter kits that allow stray light, bandpass
and linearity to be evaluated are also commercially
available. Although highly recommended, such kits are
not required for this method if the LDR is determined for
the pigment of interest and QCSs are routinely analyzed.
10.2	Allow the instrument to warm up for at least 30
min. Use a 90% acetone solution to zero the instrument
at all of the selected wavelengths. 750 nm, 664 nm, 647
nm and 630 nm are used for the determination of chl a,
chl b and chl c1 + c2. 750 nm, 665 nm and 664 nm are
used for the determination of pheopigment-corrected chl
a and pheo a. The instrument is now ready to analyze
samples.
11.0	Procedure
11.1	Extraction of Filter Samples
11.1.1	For convenience, a 10-mL final extraction volume
is described in the following procedure. A larger
extraction volume may be necessary if using a low-
volume 10-cm cell. On the other hand, a smaller
extraction volume can be used to obtain a concentration
factor. The filter residue retains 2-3 mL of solution after
centrifugation and a 1-cm cell requires approximately 3
mL of solution so that a recommended minimum
extraction volume is 6 mL.
11.1.2	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 laboratory 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. After the
filter has been converted to a slurry, grind the filter for
approximately 1 min at 500 rpm. (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 the
aqueous 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.
To reduce the volume of reagent grade solvents used for
rinsing between extractions, thoroughly rinse the grinding
tube and glass rod with tap water prior to a final rinse with
ASTM Type I water and acetone. 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 last filter extracted should be a blank. The
entire extraction with transferring and rinsing takes
approximately 5 min. Approximately 500 mL of acetone
and water waste are generated per 20 samples from the
rinsing of glassware and apparatus.
11.1.3	Shake each tube vigorously again 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. Tubes should be shaken at least once, preferably
two to three times, during the steeping period to allow the
extraction solution to have maximum contact with the filter
slurry.
11.1.4	After steeping is complete, centrifuge samples for
15 min at 675 g or for 5 min at 1000 g.
11.2 Sample Analysis
11.2.1 The instrument must be zeroed on a 90%
acetone solution as described in Sect. 10.2. In subdued
lighting, pour or pipet the supernatant of the extracted
sample into the glass spectrophotometer cell. If the
absorbance at 750 nm exceeds .005 AU, the sample
must be recentrifuged or filtered through a glass fiber
filter (syringe filter is recommended). The volume of
sample required in the instrument's cell must be known if
the pheopigment-corrected chl a and pheo a will be
determined so that acidification to the correct acid
concentration can be performed. For example, a cell that
Revision 1.2 September 1997
446.0-8

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holds 3 mL of extraction solution requires .09 mL of the .1
N HCI solution to obtain an acid concentration of .003 N.
Measure the sample's absorbance at the selected
wavelengths for chl a, chl b and chl c1 + q. Dilute and
reanalyze the sample if the signal at the selected
wavelength is >90% of the signal previously determined
as the upper limit of the LDR. If pheopigment-corrected
chl a and pheo a will be determined, acidify the sample in
the cell to .003 N HCI using the .1 N HCI solution. Use a
disposable 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 and
measure the sample's absorbance at 750 and 665 nm.
NOTE: Proper mixing of the acidified sample is critical for
accurate and precise results.
12.0	Data Analysis and Calculations
12.1	Jeffrey and Humphrey's Trichromatic
Equations — Subtract the absorbance value at 750 nm
from the absorbance values at 664, 647 and 630 nm.
Calculate the concentrations (mg/L) of chl a, b, and c1 +
c2 in the extract solution by inserting the 750 nm-corrected
absorbance values into the following equations:
CEa = 11.85 (Abs 664) -1.54 (Abs 647) - .08 (Abs 630)
CEb = 21.03 (Abs 647) - 5.43 (Abs 664) - 2.66 (Abs 630)
CEc = 24.52 (Abs 630) - 7.60 (Abs 647) -1.67 (Abs 664)
where:
CEa = concentration (mg/L) of chlorophyll a in the
extraction solution analyzed,
CEb = concentration (mg/L) of chlorophyll b in the extract
solution.
CEc = concentration (mg/L) of chlorophyll c, + c2 in the
extract solution analyzed.
12.2	Lorenzen's Pheopigment-corrected Chl a and
Pheo a - Subtract the absorbance values at 750 nm from
the absorbance values at 664 and 665 nm. Calculate the
concentrations (mg/L) in the extract solution, CE, by
inserting the 750 nm corrected absorbance values into
the following equations:
CEa = 26.7(Abs 664b - Abs 665a)
PEa = 26.7 [1.7 X (Abs 665a) - (Abs 664b)]
where,
CEa = concentration (mg/L) of chlorophyll a in the extract
solution measurted,
PEa = concentration (mg/L) of pheophytin a in the
extraction measured.
Abs 664b = sample absorbance at 664 nm (minus
absorbance at 750 nm) measured before acidification,
and
Abs 665a = sample absorbance at 665 nm (minus
absorbance at 750 nm) measured after acidification.
12.3 Calculate the conentration of pigment in the
whole water sample using the following generalized
equation:
Cs = Cr (a.b. or cl X extract volume (LI X DF
sample volume (L) X cell length (cm)
where:
Cs = concentration (mg/L) of pigment in the whole water
sample.
CE(a,b,or c> = concentration (mg/l) of pigment in extract
measured in the cuvette.
extract volume = volume (L) of extract (before any
dilutions), typically 0.0104).
DF = any dilution factors.
sample volume = volume (L) of whole water sample that
was filtered, and
cell length = optical path length (cm) of cuvette used
(typically 1 cm).
For example, calculate the conentration of chlorophyll a
in the whole water sample as:
446.0-9
Revision 1.2 September 1997

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CEa X extract volume (L) DF
s b sample volume (L) X cell length (cm)
12.4 LRB and QCS data should be reported with each
sample data set.
13.0	Method Performance
13.1	Single Laboratory Performance
13.1.1	Replicate analyses were performed on low level
dilutions of the pure pigments in 90% acetone. The
results, contained in Table 2, give an indication of the
variability not attributable to sampling and extraction or
pigment interferences.
13.1.2	The IDLs and S-EDLs for the method analytes
are reported in Table 3.
13.1.3	Precision (%RSD) for replicate analyses of two
distinct mixed assemblages are contained in Table 4.
13.1.4	Three QCS ampules were obtained from the
USEPA, analyzed and compared to the reference values
in Table 5. (NOTE: The USEPA no longer provides
pigment QCSs.)
13.2	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 24
volunteer participants in the spectrophotometric methods
component that returned data. The primary goals of the
study were to determine detection limits and to assess
precision and bias (as percent recovery) for select
unialgal species, and natural seawater.
13.2.1 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.24 method that does not assume 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 6. It is referred to as the 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 pooled
EDL (p-EDL) for each method. Results (in ppm) are
given in Table 6. The standard fluorometric and HPLC
methods gave the lowest p-EDLs while the
spectrophotometric (monochromatic equations) gave the
highest p-EDLs.
13.2.2 To address precision and bias in chlorophyll a
determination for different algal species three pure
uniagal cultures (amphidinium, dunnnaliella 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 respectiave methods. The "true"
concentration was assigned by taking the average of the
HPLC results for the highest concentration algae sample
since chlorophyll a is separatead from other interfereing
pigments prior to determination. Pooled precision data
(%RSD) are presented in Tables 7-9 and accuracy data
(as percent recovery) are presented in Table 10. No
significant differences in precision were observed across
conentrations 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 10) and
in this case the median is a better measurement 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
Revision 1.2 September 1997
446.0-10

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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
chlorophylide 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 tahe 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 11. Only one filtration volume (100 mL) was
provided in duplicate to partaicpant 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
USEPA has established a preferred hierarchy of
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 U.S. 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.	Jeffrey, S.W. and G.F. Humphrey, "New
Spectrophotometric Equations for Determining
Chlorophylls a, b, c1 + c2 in Higher Plants, Algae
and Natural Phytoplankton," Biochem. Physiol.
Pflanzen. Bd, 167, (1975), S. pp. 191-4.
2.	Lorenzen, C.J., "Determination of Chlorophyll
and Pheo-Pigments: Spectrophotometric
Equations," Limnol. Oceanogr., 12 (1967), pp.
343-6.
3.	Holm-Hansen, O., "Chlorophyll a determination:
improvements in methodology," OIKOS, 30
(1978), pp. 438-447.
4.	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.
5.	Bowles, N.D., H.W. Paerl, and J. Tucker,
"Effective solvents and extraction periods
employed in phytoplankton carotenoid and
chlorophyll determination," Can. J. Fish. Aquat.
Sci., 42 (1985) pp. 1127-1131.
6.	Shoaf, W.T. and B.W. Lium, "Improved extraction
of chlorophyll a and b from algae using dimethyl
sulfoxide," Limnol. and Oceanogr., 21(6) (1976)
pp. 926-928.
446.0-11
Revision 1.2 September 1997

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7.	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.
8.	Yentsch, C.S. and D.W. Menzel, "A method for
the determination of phytoplankton chlorophyll
and phaeophytin by fluorescence," Deep Sea
Res., 10 (1963), pp. 221-231.
9.	Strickland, J.D.H. and T.R. Parsons, A Practical
Handbook of Seawater Analysis. Bull. Fish. Res.
Board Can., 1972, No.167, p. 201.
10.	USEPA Method 445.0, "In vitro determination of
chlorophyll a and pheophytin a in marine and
freshwater phytoplankton by fluorescence,"
Methods for the Determination of Chemical
Substances in Marine and Estuarine
Environmental Samples. EPA/600/R-92/121.
11.	Wright, S.W., S.W. Jeffrey, R.F.C. Mantoura,
C.A.	Llewellyn, T. Bjornland, D. Repeta, and N.
Welschmeyer, "Improved HPLC method for the
analysis of chlorophylls and carotenoids from
marine phytoplankton," Mar. Ecol. Prog. Ser.,
77:183.
12.	Brown, L.M., B.T. Hargrave, and M.D.
MacKinnon, "Analysis of chlorophyll a in
sediments by high-pressure liquid
chromatography," Can. J. Fish. Aquat. Sci., 38
(1981) pp. 205-214.
13.	Bidigare, R.R., M.C. Kennicutt, II, and J.M.
Brooks, "Rapid determination of chlorophylls and
their degradation products by HPLC," Limnol.
Oceanogr., 30(2) (1985) pp. 432-435.
14.	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.
15.	Neveux.J., D. Delmas, J.C. Romano, P. Algarra,
L. Ignatiades, A. Herbland, P. Morand, A. Neori,
D.	Bonin, J. Barbe, A. Sukenik and T. Berman,
"Comparison of chlorophyll and pheopigment
determinations by spectrophotometric,
fluorometric, spectrofluorometric and HPLC
methods," Marine Microbial Food Webs, 4(2),
(1990) pp. 217-238.
16.	Sartory, D.P., "The determination of algal
chlorophyllous pigments by high performance
liquid chromatography and spectrophotometry,"
Water Research, 19(5), (1985), pp. 605-10.
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.	Marshall, C.T., A. Morin and R.H. Peters,
"Estimates of Mean Chlorophyll-a concentration:
Precision, Accuracy and Sampling design," Wat.
Res. Bull., 24(5), (1988), pp. 1027-1034.
22.	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.
23.	Code of Federal Regulations 40. Ch.1.
Pt.136, Appendix B.
24.	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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446.0-12

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17.0 Tables, Diagrams, Flowcharts, and Validation Data
Dilutions of a 1:1 Mixture of Chla
and Pheo.a
Concentration pigment Ippm)
2
5
1
5
0
0
Concentration pheo.a and chla lppm|
	 True Chla Value	+ Trichromatic chla
HE Corrected chla	~ Pheophytin a
FIGURE 1 - The effect of pheo a on calculated pigment
concentrations.
446.0-13
Revision 1.2 September 1997

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Corrected Chi a vs, Chi b
Closeness of Fit
Corrected chla Concentration Ippm]
5
3
5
2
5
1
5
0
0
chl b Concentration Ippml
	 True chl a Value ~ a:b~3:1 0 a:b-l:l
FIGURE 2 - The effect of Chl b on pheopigment - corrected Chl a.
Revision 1.2 September 1997
446.0-14

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Increasing Ratios of chl a:ch 1 b
The Underestimation of chl b
Calculated chl b (ppm|
Concentration chl b (ppm|
True chl b Value
FIGURE 3 - The underestimation of Chl b with increasing concentrations of Chl a.
446.0-15
Revision 1.2 September 1997

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TABLE 1. COMPARISON OF PRECISION AND RECOVERY OF PIGMENTS FOR 4 h AND 24 h STEEPING PERIODS
chl a	chl b	chl c^+c2	pheo a	corr a
4h	24h	4h	24h	4h	24h	4h	24h	4h 24h
N	6	6	6	6	66	6666
SD	1.22	0.88	0.42	0.21	0.44 0.37	1.08 1.23 1.46 1.04
Mean	26.14	25.73	0.49	1.72	5.87 5.26	1.38 2.88 24.47 23.29
%RSD	24.67	3.40	6.35	12.00	7.43 7.04	78.35 42.62 5.97 4.47
N - Number of samples
SD - Standard deviation
Mean - Concentration in natural water, mg/L
%RSD - Percent relative standard deviation
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TABLE 2. REPLICATE ANALYSES OF PURE PIGMENTS AT LOW CONCENTRATIONS
Trichromatic Equations
Modified
Monochromatic Equations
N
SD
Mean
%RSD
chl a
7
.000612
.102 mg/L
.60
chl b
7
.009792
.109 mg/L
8.9
N
SD
Mean
%RSD
chl a
1
.010091
.103 mg/L
9.8
chl b
6
.011990
.171 mg/L
7.0
TABLE 3. INSTRUMENTAL AND METHOD DETECTION LIMITS
INSTRUMENTAL DETECTION LIMITS1
(Concentrations in mg/L)
Modified
Trichromatic Equations	Monochromatic Equation
chl a	.080	pheo a .085
chl b	.093
S-ESTIMATED DETECTION LIMITS1
(Concentrations in mg/L)
Modified
Trichromatic Equations	Monochromatic Equation
chl a	.0372	chl a .0532
chl b	.0702	pheo a .0762
chl c, + c, .0873
1	Determinations made using a 1-cm path length cell.
2	Mixed assemblage samples from San Francisco Bay.
3	Predominantly diatoms from Raritan Bay.
446.0-17
Revision 1.2 September 1997

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TABLE 4. ANALYSES OF NATURAL SAMPLES
SAN FRANCISCO BAY
Modified
Trichromatic Equations	Monochromatic Equations
chl a	chl b	chl c^+c2	pheo a	corr a
N
7
7
7
7
7
SD
0.0118
0.0062
0.0096
0.0244
0.0168
Mean
0.2097
0.04271
0.03561
0.0806
0.1582
%RSD
5.62
14.50
26.82
30.21
0.64



RARITAN BAY






Modified


Trichromatic Equations

Monochromatic Equations

chl a
chl b
chl c^+c2
pheo a
corr a
N
1
7
1
1
1
SD
0.0732
0.0223
0.0277
0.0697
0.0521
Mean
1.4484
0.0914
0.2867
0.1720
1.3045
%RSD
5.06
24.43
9.65
40.53
3.99
Mean concentrations (mg/L) reported in final extraction volume of 10 mL. Samples were macerated and allowed to steep for
approximately 24 h.
N - Number of samples
SD - Standard deviation
Mean - Concentration in natural water
%RSD - Percent relative standard deviation
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TABLE 5. ANALYSES OF USEPA QC SAMPLES
Ampule 1 (3 separate ampules, chl a only)
Modified
Monochromatic Equations
chl a
pheo a
Mean
2.56 mg/L
ND
Reference
2.70
%RSD
Trichromatic Equations
Mean Reference %RSD
chl a 2.54 mg/L 2.59	.61
ND - None detected
Ampule 2 (3 separate ampules, all method pigments)
Trichromatic Equations
Mean Reference %RSD
Modified
Monochromatic Equations
Mean
Reference %RSD
chl a	4.87 mg/L 4.86
chlb	1.12 mg/L 1.02
chl q + c2 .29 mg/L	.37
.1
1.3
4.9
chl a
pheo a
3.70 mg/L
1.79 mg/L
3.76
1.70
2.3
4.4
446.0-19
Revision 1.2 September 1997

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TABLE 6. 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
9
0.082
HPLC
4
0.081
SP-M
15
0.229
SP-T
15
0.104
(1)	See Section 13.2.1 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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446.0-20

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TABLE 7. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES
mLs of
culture
Method'1'	filtered	N(2)
SP-M	5	17
10	19
50	19
100	19
Mean (ma chla/D	Std. Dev.	%RSD
0.068	0.026	37.8
0.139	0.037	26.6
0.679	0.150	22.1
1.366	0.205	15
SP-T
5
10
50
100
16
18
18
18
0.059
0.130
0.720
1.408
0.021
0.027
0.102
0.175
35.1
20.8
14.2
12.4
(1)	SP-M = Pheophytin a - corrected chlorophyll a method using monochromatic equations.
SP-T = Trichromatic equations method.
(2)	N = Number of volunteer labs whose data was used.
446.0-21
Revision 1.2 September 1997

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TABLE 8. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES
mLs of
culture
Method'1' filtered	N(2) Mean fma chla/L)	Std. Dev.	%RSD
SP-M 5	19	0.166	0.043	26.0
10	19	0.344	0.083	24.0
50	19	1.709	0.213	12.5
100	19	3.268	0.631	19.3
SP-T
5
10
50
100
0.161
0.339
1.809
3.500
0.030
0.058
0.190
0.524
18.4
17.1
10.5
15.0
(1)	SP-M = Pheophytin a corrected chlorophyll a method using monochromatic equations.
SP-T = Trichromatic equationss method.
(2)	N = number of volunteer labs whose data was used.
Revision 1.2 September 1997
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TABLE 9. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES
Method'1'
mLs of
culture
filtered
N(2)
Mean fma chla/L)
Std. Dev.
%RSD
SP-M
5
10
50
100
19
19
19
19
0.223
0.456
2.042
4.083
0.054
0.091
0.454
0.694
24.1
19.9
22.2
17.0
SP-T
5
10
50
100
0.224
0.465
2.223
4.422
0.031
0.077
0.217
0.317
14.0
16.5
9.7
7.2
(1) SP-M = Pheophytin a corrected chorophyll a method using monochromatic equations.
(2) N = number of volunteer labs whose data was used.
446.0-23
Revision 1.2 September 1997

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TABLE 10. 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


SP-M
240
247
247
243
Revision 1.2 September 1997	446.0-24

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Table 10 cont'd
Species
Statistic
Method
Percent Recovery
Cone.
Level 1
Cone.
Level 2
Cone.
Level 3
Cone.
Level 4


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
446.0-25	Revision 1.2 September 1997

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TABLE 11. CHLOROPHYLL A CONCENTRATIONS IN mg/L DETERMINED IN FILTERED SEA WATER
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.
Revision 1.2 September 1997
446.0-26

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