446.0-1

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
Revision 1.2 September 1997 446.0-2

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 c + c ) and pheopigments of chlorophyll a (pheo a) 1 2

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 are (1)

used to calculate the concentrations of chl a, chl b, and

chl c +c . Modified monochromatic equations of 1 2

Lorenzen are used to calculate pheopigment-corrected (2)

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 479-61-8

Chlorophyll b 519-62-0

Chlorophyll c1 18901-56-9

Chlorophyll c2 27736-03-4

 

1.3 Instrumental detection limits (IDLs) of 0.08 mg

chl a/L, 0.093 mg chl b/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 c1+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. Singlelaboratory EDLs (S-EDL) were as follows: chl a - 0.037

mg/L, chl b - 0.07 mg/L, chl c + c - 0.087 mg/L, 1 2

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

c1+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 b/L and approximately 45 mg pheo a/L.

No LDR for chl c1 + c2 was determined. It is highly

unlikely that samples containing chl c +c at 1 2

concentrations approaching the upper limit of the LDR will

be encountered in nature.

1.4 Chl c +c is not commercially available, 1 2 

therefore, the minimum indicator of laboratory

performance for this pigment is precision of chl c1 + c2

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 or dimethyl sulfoxide. Using (3-5) (6)

high performance liquid chromatography (HPLC),

Mantoura and Llewellyn found that methanol led to the (7)

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
 446.0-3 Revision 1.2 September 1997

to use a fluorometric or HPLC method if high selected wavelength, mass, retention time, absorbance

(8-10) (11-15)

volumes of water (>4 L) must be filtered to obtain line, etc. In this method the instrument is zeroed on a

detectable quantities of chl a. The user should be aware background of 90% acetone resulting in no signal at the

of the inaccuracies of fluorometric 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 c + c , 1 2

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 HCl. 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

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
Revision 1.2 September 1997 446.0-4

sample. This value is subtracted from the sample's step required to determine pheopigment-corrected chl a

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.

Chl 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 extraction solvent (Table 1). Precision improves with

any pigment is directly related to the concentration of the increasing steeping periods. A drawback to prolonged

interfering pigment. Knowledge of the taxonomic steeping periods, however, is the extraction of interfering

composition of the sample, proper storage and good pigments. For example, if the primary pigment of interest

sample handling technique (to prevent degradation of chl is chl a, extended steeping periods may extract more of

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 significant factor in the recovery of chl b and pheo a from

pigments and to compare the chl a results in light of the

apparent concentrations of the other pigments. detectable quantities, but not a significant factor in the

Obviously, if the chl a values vary widely, sound recovery of chl a. Chl b and pheo a are mutual

judgement must be used in deciding which pigments, chl interferents so that an actual increase in the recovery of

b and chl c + c , or pheo a, are in greatest abundance chl b leads to a slight apparent increase in pheo a. 1 2

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. In pure

(16)

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

+ c2in 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 b: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

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

the other pigments but not necessarily more chl a.

Statistical analysis revealed steeping period to be a

a mixed assemblage containing these pigments in

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. o

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. A file of MSDS also should be made

(17-20)

available to all personnel involved in the chemical

analysis.
 446.0-5 Revision 1.2 September 1997

5.2 The grinding of filters during the extraction step of 6.10.5 Disposable Pasteur type pipets or medicine

this method should be conducted in a fume hood due to droppers.

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 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in 7.1 Acetone, HPLC grade, (CASRN 67-64-1).

this work.

6.5 Petri dishes, plastic, 50 X 9-mm, or some other 1.19), (CASRN 7647-01-0).

solid container for transporting and storing sampled

filters. 7.3 Chl a free of chl b and chl b substantially free of

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.

6.10.6 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

7.2 Hydrochloric acid (HCl), concentrated (sp. gr.

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 HCl Solution -- Add 8.5 mL of

concentrated HCl 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 -20EC 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
Revision 1.2 September 1997 446.0-6

flask. Dilute to volume with 90% acetone, label the flask Prior to drawing a subsample from the water sample

and wrap with aluminum foil to protect from light. Pheo a container, thoroughly but gently agitate the container to

may be prepared by the mild acidification of chl a (to .003

N HCl) 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 o

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 at

(21)

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 acidfree. 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.

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 o

-70 C. Short term storage (2 to 4 h) on ice is acceptable, o

but samples should be stored at -20EC as soon as

possible.

8.2 Preservation -- Sampled filters should be stored

frozen (-20 C or -70 C) in the dark until extraction. o o

8.3 Holding Time -- Filters can be stored frozen at

-20 C for as long as 31⁄2 weeks without significant loss of o

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,
 446.0-7 Revision 1.2 September 1997

Gaithersburg, MD) or other suitable spectrophotometric value is not within the confidence limits established by

filter standards that test wavelength accuracy must be project data quality objectives, then the determinative step

analyzed yearly and the results compared to the of this method is unacceptable. The source of the

instrument manufacturer's specifications. If wavelength problem must be identified and corrected before

accuracy is not within manufacturer's specifications, continuing analyses.

identify and repair the problem.

9.2.3 Linear Dynamic Range (LDR) -- The LDR should this method for the first time should demonstrate

be determined by analyzing a minimum of 5 standard proficiency in the extraction of sampled filters (Sect. 11.1).

solutions ranging in concentration from 1 to 15 mg/L. Twenty to thirty natural samples should be obtained using

Perform the linear regression of absorbance response (at the procedure outlined in Sect. 8.1 of this method. Sets

pigment's wavelength maximum) vs. concentration and of 10 or more samples should be extracted and analyzed

obtain the constants m and b, where m is the slope and b according to Sect. 11.2. The percent relative standard

is the y-intercept. Incrementally analyze standards of

higher concentration until the measured absorbance 15% for samples that are at least 10X the IDL. 

response, R, of a standard no longer yields a calculated

concentration, Cc, that is ± 10% of the known

concentration, C, where C = (R - b)/m. That

c

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 step properly, perform the following QC procedure:

spectrophotometer with a solution of 90% acetone. Pure

pigment in 90% acetone should be serially diluted until it Prepare 100 mL of a 2.0 ppm chl a solution in 90%

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 follows :

(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

9.2.7 Extraction Proficiency -- Personnel performing

deviation (%RSD) of trichromatic chl a should not exceed

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

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.
Revision 1.2 September 1997 446.0-8

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 lowvolume 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

o

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
 446.0-9 Revision 1.2 September 1997

holds 3 mL of extraction solution requires .09 mL of the .1 C = 26.7(Abs 664 - Abs 665 ) 

N HCl 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 + c2. 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 HCl using the .1 N HCl 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:

C = 11.85 (Abs 664) - 1.54 (Abs 647) - .08 (Abs 630) E,a 

C = 21.03 (Abs 647) - 5.43 (Abs 664) - 2.66 (Abs 630) E,b

C = 24.52 (Abs 630) - 7.60 (Abs 647) - 1.67 (Abs 664) E,c 

where:

CE,a = concentration (mg/L) of chlorophyll a in the

extraction solution analyzed,

C = concentration (mg/L) of chlorophyll b in the extract E,b

solution.

CE,c = concentration (mg/L) of chlorophyll c 1 + c 2 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:

E,a b a

P = 26.7 [1.7 X (Abs 665 ) - (Abs 664 )] E,a a b

where,

C = concentration (mg/L) of chlorophyll a in the extract E,a 

solution measurted,

PE,a = 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 = CE (a,b, or c) X extract volume (L) X DF

sample volume (L) X cell length (cm)

where:

C = concentration (mg/L) of pigment in the whole water s

sample.

C = concentration (mg/l) of pigment in extract E(a,b,or c)

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:
Cs,b

'

CE,a X extract volume (L) DF

sample volume (L) X cell length (cm)

Revision 1.2 September 1997 446.0-10

12.4 LRB and QCS data should be reported with each median DLs over labs was then determined and is

sample data set. reported in Table 6. It is referred to as the pooled-EDL

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 (pEDL), 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. method that does not assume error variances

24

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

(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
 446.0-11 Revision 1.2 September 1997

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 spectrophotometrictrichromatic 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.
Revision 1.2 September 1997 446.0-12

7. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid determinations by spectrophotometric,

determination of algal chlorophyll and carotenoid fluorometric, spectrofluorometric and HPLC

pigments and their breakdown products in methods," Marine Microbial Food Webs, 4(2),

natural waters by reverse-phase high

performance liquid chromatography," Anal.

Chim. Acta., 151 (1983) pp. 297-314. 16. Sartory, D.P., "The determination of algal

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

(1990) pp. 217-238. 

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-D00079, 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. 
 446.0-13 Revision 1.2 September 1997

17.0 Tables, Diagrams, Flowcharts, and Validation Data

FIGURE 1 - The effect of pheo a on calculated pigment

 concentrations.
Revision 1.2 September 1997 446.0-14

FIGURE 2 - The effect of Chl b on pheopigment - corrected Chl a.
 446.0-15 Revision 1.2 September 1997

FIGURE 3 - The underestimation of Chl b with increasing concentrations of Chl a.
Revision 1.2 September 1997 446.0-16

TABLE 1. COMPARISON OF PRECISION AND RECOVERY OF PIGMENTS FOR 4 h AND 24 h STEEPING PERIODS

 chl a chl b chl c1+c2 pheo a corr a

 4h 24h 4h 24h 4h 24h 4h 24h 4h 24h

 

N 6 6 6 6 6 6 6 6 6 6

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
 446.0-17 Revision 1.2 September 1997

TABLE 2. REPLICATE ANALYSES OF PURE PIGMENTS AT LOW CONCENTRATIONS

 Modified

 Trichromatic Equations Monochromatic Equations 

chl a chl b chl a chl b

 N 7 7 N 7 6

SD .000612 .009792 SD .010091 .011990

Mean .102 mg/L .109 mg/L Mean .103 mg/L .171 mg/L

%RSD .60 8.9 %RSD 9.8 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 .037 chl a .053 

2 2

chl b .070 pheo a .076 2 2

chl c1 + c2.0873

 

 Determinations made using a 1-cm path length cell. 1

 Mixed assemblage samples from San Francisco Bay. 2

 Predominantly diatoms from Raritan Bay. 3
Revision 1.2 September 1997 446.0-18

TABLE 4. ANALYSES OF NATURAL SAMPLES

 

SAN FRANCISCO BAY 

 Modified

 Trichromatic Equations Monochromatic Equations

 chl a chl b chl c1+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 c1+c2 pheo a corr a

 

N 7 7 7 7 7

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
 446.0-19 Revision 1.2 September 1997

TABLE 5. ANALYSES OF USEPA QC SAMPLES

Ampule 1 (3 separate ampules, chl a only)

 Modified

 Trichromatic Equations Monochromatic Equations

 Mean Reference %RSD Mean Reference %RSD

chl a 2.54 mg/L 2.59 .61 chl a 2.56 mg/L 2.70 .8

pheo a ND

 

 ND - None detected

Ampule 2 (3 separate ampules, all method pigments)

 Modified

 Trichromatic Equations Monochromatic Equations

 Mean Reference %RSD Mean Reference %RSD

chl a 4.87 mg/L 4.86 .1 chl a 3.70 mg/L 3.76 2.3

chl b 1.12 mg/L 1.02 1.3 pheo a 1.79 mg/L 1.70 4.4

chl c1 + c2.29 mg/L .37 4.9

 
Revision 1.2 September 1997 446.0-20

TABLE 6. POOLED ESTIMATED DETECTION LIMITS FOR CHLOROPHYLL A METHODS(1)

_____________________________________________________________

Method N p-EDL (mg/L) (2) (3) (4)

FL -Mod 8 0.096 (5)

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.
 446.0-21 Revision 1.2 September 1997

TABLE 7. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

SP-M 5 17 0.068 0.026 37.8

 10 19 0.139 0.037 26.6

 50 19 0.679 0.150 22.1

100 19 1.366 0.205 15

SP-T 5 16 0.059 0.021 35.1

 10 18 0.130 0.027 20.8

 50 18 0.720 0.102 14.2

100 18 1.408 0.175 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.
Revision 1.2 September 1997 446.0-22

TABLE 8. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

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 18 0.161 0.030 18.4

 10 18 0.339 0.058 17.1

 50 18 1.809 0.190 10.5

100 18 3.500 0.524 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.
 446.0-23 Revision 1.2 September 1997

TABLE 9. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES

 mLs of 

 culture

Method filtered N Mean (mg chla/L) Std. Dev. %RSD (1) (2)

SP-M 5 19 0.223 0.054 24.1

 10 19 0.456 0.091 19.9

 50 19 2.042 0.454 22.2

100 19 4.083 0.694 17.0

SP-T 5 18 0.224 0.031 14.0

 10 18 0.465 0.077 16.5

 50 18 2.223 0.217 9.7

100 18 4.422 0.317 7.2

___________________________________________________________________________________

(1) SP-M = Pheophytin a corrected chorophyll a method using monochromatic equations.

(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 446.0-24

TABLE 10. MINIMUM, MEDIAN, AND MAXIMUM PERCENT RECOVERIES BY GENERA, METHOD, AND

 CONCENTRATION LEVEL

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 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
Table 10 cont’d

 446.0-25 Revision 1.2 September 1997

Species

Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 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
Revision 1.2 September 1997 446.0-26

TABLE 11. 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.