445.0-1

Method 445.0

In Vitro Determination of Chlorophyll a and Pheophytin a

in Marine and Freshwater Algae by Fluorescence

Elizabeth J. Arar

and

Gary B. Collins

Revision 1.2

September 1997

National Exposure Research Laboratory

Office of Research and Development

U.S. Environmental Protection Agency

Cincinnati, Ohio 45268
Revision 1.2 September 1997 445.0-2

Method 445.0

In Vitro Determination of Chlorophyll a and Pheophytin a

in Marine and Freshwater Algae by Fluorescence

1.0 Scope and Application

1.1 This method provides a procedure for low level

determination of chlorophyll a (chl a) and its magnesiumfree derivative, pheophytin a (pheo a), in marine and

freshwater phytoplankton using fluorescence detection.(1,2)

Phaeophorbides present in the sample are determined

collectively as pheophytin a. For users primarily

interested in chl a there is currently available a set of very

narrow bandpass excitation and emission filters (Turner

Designs, Sunnyvale, CA) that nearly eliminates the

spectral interference caused by the presence of pheo a

and chlorophyll b. The difference between the modified

method and the conventional fluorometric method is that

the equations used for the determination of chlor a

without pheo a correction (uncorrected chlor a), are used

instead of the equations for "corrected chlor a". This EPA

laboratory has evaluated the modified filters and found

the technique to be an acceptable alternative to the

conventional fluorometric method using pheo a

correction.(3)

 

Chemical Abstracts Service

Analyte Registry Number (CASRN)

 

Chlorophyll a 479-61-8

 

1.2 Instrumental detection limits (IDL) of 0.05 μg chl

a/L and 0.06 μg pheo a/L in a solution of 90% acetone

were determined by this laboratory. Method detection

limits (MDL) using mixed assemblages of algae provide

little information because the fluorescence of other

pigments interferes in the fluorescence of chlorophyll a

and pheophytin a. A single lab estimated detection limit (4)

for chlorophyll a was determined to be 0.11 μg/L in 10 mL

of final extraction solution. The upper limit of the linear

dynamic range for the instrumentation used in this

method evaluation was 250 μg chl a/L.

1.3 This method was multilaboratory validated in

1996. Results from that study may be found in Section (5)

13. Additional QC procedures also have been added as

a result of that study.

1.4 This method uses 90% acetone as the extraction

solvent because of its efficiency for most types of algae.

There is evidence that certain chlorophylls and

carotenoids are more thoroughly extracted with

methanol or dimethyl sulfoxide. Bowles, et al. (5-8) (9) (8)

found that for chlorophyll a, however, 90% acetone was

an effective extractant when the extraction period was

optimized for the dominant species present in the sample.

1.5 Depending on the type of algae under

investigation, this method can have uncorrectable

interferences (Sect. 4.0). In cases where taxomonic

classification is unavailable, a spectrophotometric or high

performance liquid chromatographic (HPLC) method may

provide more accurate data for chlorophyll a and

pheophytin a.

1.6 This method is for use by analysts experienced in

the handling of photosynthetic pigments and in the

operation of fluorescence detectors or by analysts under

the close supervision of such qualified persons.

2.0 Summary of Method

2.1 Chlorophyll-containing phytoplankton in a

measured volume of sample water are concentrated by

filtering at low vacuum through a glass fiber filter. The

pigments are extracted from the phytoplankton in 90%

acetone with the aid of a mechanical tissue grinder and

allowed to steep for a minimum of 2 h, but not to exceed

24 h, to ensure thorough extraction of the chlorophyll a.

The filter slurry is centrifuged at 675 g for 15 min (or at

1000 g for 5 min) to clarify the solution. An aliquot of the

supernatant is transferred to a glass cuvette and

fluorescence is measured before and after acidification to

0.003 N HCl with 0.1 N HCl. Sensitivity calibration factors,

which have been previously determined on solutions of
 445.0-3 Revision 1.2 September 1997 

pure chlorophyll a of known concentration, are used to interferences are present in the laboratory environment,

calculate the concentration of chlorophyll a and

pheophytin a in the sample extract. The concentration in

the natural water sample is reported in μg/L.

3.0 Definitions

3.1 Estimated Detection Limit (EDL) -- The

minimum concentration of an analyte that yields a

fluorescence 3X the fluorescence of blank filters which

have been extracted according to this method.

3.2 Linear Dynamic Range (LDR) -- The absolute

quantity or concentration range over which the instrument

response to an analyte is linear.

3.3 Instrument Detection Limit (IDL) -- The

minimum quantity of analyte or the concentration

equivalent which gives an analyte signal equal to three

times the standard deviation of the background signal at

the selected wavelength, mass, retention time,

absorbance line, etc. For this method the background is

a solution of 90% acetone.

3.4 Stock Standard Solution (SSS) -- A

concentrated solution containing one or more method

analytes prepared in the laboratory using assayed

reference materials or purchased from a reputable

commercial source.

3.5 Primary Dilution Standard Solution (PDS) -- A

solution of the analytes prepared in the laboratory from

stock standard solutions and diluted as needed to

prepare calibration solutions and other needed analyte

solutions. vary with the taxonomic composition of the phytoplankton.

3.6 Calibration Standard (CAL) -- A solution

prepared from the primary dilution standard solution or

stock standard solutions containing the internal standards

and surrogate analytes. The CAL solutions are used to

calibrate the instrument response with respect to analyte

concentration.

3.7 Response Factor (RF) -- The ratio of the

response of the instrument to a known amount of analyte.

3.8 Laboratory Reagent Blank (LRB) -- An aliquot

of reagent water or other blank matrices that are treated

exactly as a sample including exposure to all glassware,

equipment, solvents, reagents, internal standards, and

surrogates that are used with other samples. The LRB is

used to determine if method analytes or other

reagents, or apparatus.

3.9 Field Duplicates (FD1 and FD2) -- Two separate

samples collected at the same time and place under

identical circumstances and treated exactly the same

throughout field and laboratory procedures. Analyses of

FD1 and FD2 give a measure of the precision associated

with sample collection, preservation and storage, as well

as with laboratory procedures.

3.10 Quality Control Sample (QCS) -- A solution of

method analytes of known concentrations which is used

to fortify an aliquot of LRB or sample matrix. Ideally, the

QCS is obtained from a source external to the laboratory

and different from the source of calibration standards. It

is used to check laboratory performance with externally

prepared test materials.

3.11 Material Safety Data Sheet (MSDS) -- Written

information provided by vendors concerning a chemical's

toxicity, health hazards, physical properties, fire, and

reactivity data including storage, spill, and handling

precautions.

4.0 Interferences

4.1 Any substance extracted from the filter or

acquired from laboratory contamination that fluoresces in

the red region of the spectrum may interfere in the

accurate measurement of both chlorophyll a and

pheophytin a.

4.2 The relative amounts of chlorophyll a, b and c

Chlorophylls b and c may significantly interfere with

chlorophyll a measurements depending on the amount

present. Due to the spectral overlap of chlorophyll b with

pheophytin a and chlorophyll a, underestimation of

chlorophyll a occurs accompanied by overestimation of

pheophytin a when chlorophyll b is present in the sample.

The degree of interference depends upon the ratio of a:b.

This laboratory found that at a ratio of 5:1, using the

acidification procedure to correct for pheophytin a,

chlorophyll a was underestimated by approximately 5%.

Loftis and Carpenter reported an underestimation of

(10)

16% when the a:b ratio was 2.5:1. A ratio of 1:1 is the

highest ratio likely to occur in nature. They also reported

overestimation of chlorophyll a in the presence of

chlorophyll c of as much as 10% when the a:c ratio was

1:1 (the theoretical maximum likely to occur in nature).

The presence of chlorophyll c also causes the under-
Revision 1.2 September 1997 445.0-4

estimation of pheophytin a. The effect of chlorophyll c is

not as severe as the effect of chlorophyll b on the

measurement of chlorophyll a and pheophytin a.

Knowledge of the taxonomy of the algae under

consideration will aid in determining if the

spectrophotometric method using trichromatic equations

to determine chlorophyll a, b, and c or an HPLC method

would be more appropriate. In the presence of

(11-16) 

chlorophyll b or pheopigments, the modified fluorometric

method described here is also appropriate.(5)

4.3 Quenching effects are observed in highly

concentrated solutions or in the presence of high

concentrations of other chlorophylls or carotenoids.

Minimum sensitivity settings on the fluorometer should be

avoided; samples should be diluted instead.

4.4 Fluorescence is temperature dependent with

higher sensitivity occurring at lower temperatures.

Samples, standards, LRBs and QCSs must be at the

same temperature to prevent errors and/or low precision.

Analyses of samples at ambient temperature is

recommended in this method. Ambient temperature

should not fluctuate more than ± 3EC between

calibrations or recalibration of the fluorometer will be

necessary.

4.5 Samples must be clarified by centrifugation prior

to analysis.

4.6 All photosynthetic pigments are light and

temperature sensitive. Work must be performed in

subdued light and all standards, QC materials and filter

samples must be stored in the dark at -20 C or -70 C to o o

prevent degradation.

5.0 Safety

5.1 The toxicity or carcinogenicity of the chemicals

used in this method have not been fully established.

Each chemical should be regarded as a potential health

hazard and handled with caution and respect. Each

laboratory is responsible for maintaining a current

awareness file of Occupational Safety and Health

Administration (OSHA) regulations regarding the safe

handling of the chemicals specified in this method. A (17-20)

file of MSDS should also be made available to all

personnel involved in the chemical analysis.

5.2 The grinding of filters during the extraction step of

this method should be conducted in a fume hood due to

the volatilization of acetone by the tissue grinder.

6.0 Apparatus and Equipment

6.1 Fluorometer -- Equipped with a high intensity

F4T.5 blue lamp, red-sensitive photomultiplier, and filters

for excitation (CS-5-60) and emission (CS-2-64). A

Turner Designs Model 10 Series fluorometer was used in

the evaluation of this method. The modified method

requires excitation filter (436FS10) and emission filter

(680FS10).

6.2 Centrifuge, capable of 675 g.

6.3 Tissue grinder, Teflon pestle (50 mm X 20 mm)

with grooves in the tip with 1/4" stainless steel rod long

enough to chuck onto a suitable drive motor and 30-mL

capacity glass grinding tube.

6.4 Filters, glass fiber, 47-mm or 25-mm, nominal

pore size of 0.7 μm unless otherwise justified by data

quality objectives. Whatman GF/F filters were used in this

work.

6.5 Petri dishes, plastic, 50 X 9-mm, or some other

solid container for transporting and storing sampled

filters.

6.6 Aluminum foil.

6.7 Laboratory tissues.

6.8 Tweezers or flat-tipped forceps.

6.9 Vacuum pump or source capable of maintaining

a vacuum up to 6 in. Hg.

6.10 Room thermometer.

6.11 Labware -- All reusable labware (glass,

polyethylene, Teflon, etc.) that comes in contact with

chlorophyll solutions should be clean and acid free. An

acceptable cleaning procedure is soaking for 4 h in

laboratory grade detergent and water, rinsing with tap

water, distilled deionized water and acetone.

6.11.1 Assorted Class A calibrated pipets.

6.11.2 Graduated cylinders, 500-mL and 1-L.

6.11.3 Volumetric flasks, Class A calibrated, 25-mL, 50-

mL, 100-mL and 1-L capacity.

6.11.4 Glass rods.
 445.0-5 Revision 1.2 September 1997 

6.11.5 Pasteur type pipets or medicine droppers. the solution must be determined spectrophotometrically

6.11.6 Disposable glass cuvettes for the fluorometer.

6.11.7 Filtration apparatus consisting of 1 or 2-L filtration

flask, 47-mm fritted glass disk base and a glass filter

tower.

6.11.8 Centrifuge tubes, polypropylene or glass, 15-mL

capacity with nonpigmented screw-caps.

6.11.9 Polyethylene squirt bottles.

7.0 Reagents and Standards

7.1 Acetone, HPLC grade, (CASRN 67-64-1).

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

1.19), (CASRN 7647-01-0).

7.3 Chlorophyll a free of chlorophyll b. May be

obtained from a commercial supplier such as Sigma

Chemical (St. Louis, MO). Turner Designs (Sunnyvale,

CA) supplies ready-made standards.

7.4 Water -- ASTM Type I water (ASTM D1193) is

required. Suitable water may be obtained by passing

distilled water through a mixed bed of anion and cation

exchange resins.

7.5 0.1 N 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% water. Carefully measure 100 mL of water into the

1-L graduated cylinder. Transfer to a 1-L flask or storage

bottle. Measure 900 mL of acetone into the graduated

cylinder and transfer to the flask or bottle containing the

water. Mix, label and store.

7.7 Chlorophyll Stock Standard Solution (SSS) --

Chlorophyll a from a commercial supplier will be shipped

in an amber glass ampoule which has been flame sealed.

This dry standard should be stored at -20 or -70EC in the

dark and the SSS prepared just prior to use. Tap the

ampoule until all the dried chlorophyll is in the bottom of

the ampoule. In subdued light, carefully break the tip off

the ampoule. Transfer the entire contents of the

ampoule into a 50-mL volumetric flask. Dilute to volume

with 90% acetone, label the flask and wrap with

aluminum foil to protect from light. The concentration of

using a multiwavelength spectrophotometer. When

(10)

stored in a light and airtight container at freezer

temperatures, the SSS is stable for at least six months.

The concentration of all dilutions of the SSS must be

determined spectrophotometrically each time they are

made. 

7.8 Laboratory Reagent Blank (LRB) -- A blank

filter which is extracted and analyzed just as a sample

filter. The LRB should be the last filter extracted of a

sample set. It is used to assess possible contamination

of the reagents or apparatus.

7.9 Chlorophyll a Primary Dilution Standard

Solution (PDS) -- Add 1 mL of the SSS (Sect. 7.8) to a

clean 100-mL flask and dilute to volume with the aqueous

acetone solution (Sect. 7.7). If exactly 1 mg of pure

chlorophyll a was used to prepare the SSS, the

concentration of the PDS is 200 μg/L. Prepare fresh just

prior to use.

7.10 Quality Control Sample (QCS) -- Since there

are no commercially available QCSs, dilutions of a stock

standard of a different lot number from that used to

prepare calibration solutions may be used.

8.0 Sample Collection, Preservation and

Storage

8.1 Water Sample Collection -- Water may be

obtained by a pump or grab sampler. Data quality

objectives will determine the depth at which samples are

taken. Healthy phytoplankton, however, are generally

obtained from the photic zone (depth at which the

illumination level is 1% of surface illumination). Enough

water should be collected to concentrate phytoplankton

on at least three filters so that precision can be assessed.

Filtration volume size will depend on the particulate load

of the water. Four liters may be required for open ocean

water where phytoplankton density is usually low,

whereas 1 L or less is generally sufficient for lake, bay or

estuary water. All apparatus should be clean and acidfree. Filtering should be performed in subdued light as

soon as possible after sampling since algal poulations,

thus chlorophyll a concentration, can change in relatively

short periods of time. Aboard ship filtration is highly

recommended.

Assemble the filtration apparatus and attach the vacuum

source with vacuum gauge and regulator. Vacuum

filtration should not exceed 6 in. Hg (20 kPa). Higher
Revision 1.2 September 1997 445.0-6

filtration pressures and excessively long filtration times (> laboratory performance (analyses of QCSs) prior to

10 min) may damage cells and result in loss of sample analyses.

chorophyll. 

Prior to drawing a subsample from the water sample

container, thoroughly but gently agitate the container to

suspend the particulates (stir or invert several times).

Pour the subsample into a graduated cylinder and

accurately measure the volume. Pour the subsample into

the filter tower of the filtration apparatus and apply a

vacuum (not to exceed 20 kPa). A sufficient volume has

been filtered when a visible green or brown color is

apparent on the filter. Do not suck the filter dry with the

vacuum; instead slowly release the vacuum as the final

volume approaches the level of the filter and completely

release the vacuum as the last bit of water is pulled

through the filter. Remove the filter from the fritted base

with tweezers, fold once with the particulate matter inside,

lightly blot the filter with a tissue to remove excess

moisture and place it in the petri dish or other suitable

container. If the filter will not be immediately extracted,

then wrap the container with aluminum foil to protect the

phytoplankton from light and store the filter at -20 or

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

but samples should be stored at -20 or -70EC 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 or -70 C for as long as 31⁄2 weeks without significant o

loss of chlorophyll a.(21)

9.0 Quality Control

9.1 Each Laboratory using this method is required to

operate a formal quality control (QC) program. The

minimum requirements of this program consist of an initial

demonstration of laboratory capability and the continued

analysis of laboratory reagent blanks, field duplicates and

quality control samples as a continuing check on

performance. The laboratory is required to maintain

performance records that define the quality of the data

thus generated.

9.2 Initial Demonstration of Performance

(Mandatory)

9.2.1 The initial demonstration of performance is used

to characterize instrument performance (instrumental

detection limits, linear dynamic range and MDLs) and

9.2.2 Linear Dynamic Range (LDR) -- The LDR should

be determined by analyzing a minimum of 5 calibration

standards ranging in concentration from 0.2 μg/L to 200

μg chl a/L across all sensitivity settings of the fluorometer.

If using an analog fluorometer or a digital fluorometer

requiring manual changes in sensitivity settings, normalize

responses by dividing the response by the sensitivity

setting multiplier. Perform the linear regression of

normalized response vs. concentration and obtain the

constants m and b, where m is the slope and b is the yintercept. Incrementally analyze standards of higher

concentration until the measured fluorescence response,

R, of a standard no longer yields a calculated

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

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

concentration defines the upper limit of the LDR for your

instrument. Should samples be encountered that have a

concentration which is 90% of the upper limit of the LDR,

these samples must be diluted and reanalyzed. 

9.2.3 Instrumental Detection Limit (IDL) -- Zero the

fluorometer with a solution of 90% acetone on the

maximum sensitivity setting. Pure chlorophyll a in 90%

acetone should be serially diluted until it is no longer

detected by the fluorometer on a maximum sensitivity

setting.

9.2.4 Estimated Detection Limit (EDL) -- Several blank

filters should be extracted according to the procedure in

Sect. 11, using clean glassware and apparatus, and the

fluorescence measured. A solution of pure chlorophyll a

in 90% acetone should be serially diluted until it yields a

response which is 3X the average response of the blank

filters. 

9.2.5 Quality Control Sample (QCS) -- When beginning

to use this method, on a quarterly basis or as required to

meet data quality needs, verify the calibration standards

and acceptable instrument performance with the analysis

of a QCS (Sect. 7.10). If the determined value is not

within the confidence limits established by project data

quality objectives, then the determinative step of this

method is unacceptable. The source of the problem

must be identified and corrected before continuing

analyses.

9.2.6 Extraction Proficiency -- Personnel performing

this method for the first time should demonstrate

proficiency in the extraction of sampled filters (Sect. 11.1).
 445.0-7 Revision 1.2 September 1997 

Twenty to thirty natural samples should be obtained using the instrument to warm up for at least 15 min. Measure

the procedure outlined in Sect. 8.1 of this method. Sets the fluorescence of each standard at sensitivity settings

of 10 or more samples should be extracted and analyzed that provide midscale readings. Obtain response factors

according to Sect. 11.2. The percent relative standard for chlorophyll a for each sensitivity setting as follows:

deviation (%RSD) of uncorrected values of chlorophyll a

should not exceed 15% for samples that are F = C /R

approximately 10X the IDL. RSD for pheophytin a might

typically range from 10 to 50%.

 

9.2.7 Corrected Chl a -- Multilaboratory testing of this

method revealed that many analysts do not adequately

mix the acidified sample when determining corrected chl

a. The problem manifests itself by highly erratic pheo-a

results, high %RSDs for corrected chl a and poor

agreement between corrected and uncorrected chl a. To

determine if a new analyst is performing the acidification

step properly, perform the following QC procedure:

Prepare 100 mL of a 50 ppb chl a solution in 90%

acetone. The new analyst should analyze 5-10 separate

aliquots, using separate cuvettes, according to

instructions in Section 11.2. Process the results

according to Section 12 and calculate separate means

and %RSDs for corrected and uncorrected chl a. If the

means differ by more than 10%, then the stock chl a has

probably degraded and fresh stock should be prepared.

The %RSD for corrected chl a should not exceed 5%. If

the %RSD exceeds 5%, repeat the procedure until the

%RSD 5%.

9.3 Assessing Laboratory Performance

(Mandatory)

9.3.1 Laboratory Reagent Blank (LRB) -- The

laboratory must analyze at least one blank filter with each

sample batch. The LRB should be the last filter

extracted. LRB data are used to assess contamination

from the laboratory environment. LRB values that exceed

the IDL indicate contamination from the laboratory

environment. When LRB values constitute 10% or more

of the analyte level determined for a sample, fresh

samples or field duplicates must be analyzed after the

contamination has been corrected and acceptable LRB

values have been obtained.

10.0 Calibration and Standardization

10.1 Calibration -- Calibration should be performed

bimonthly or when there has been an adjustment made

to the instrument, such as replacement of lamp, filters or

photomultiplier. Prepare 0.2, 2, 5, 20 and 200 μg chl a/L

calibration standards from the PDS (Sect. 7.11). Allow

s a s

where:

F = response factor for sensitivity setting, S. s

Rs = fluorometer reading for sensitivity

 setting, S.

C = concentration of chlorophyll a. a

NOTE: If you are using special narrow bandpass filters

for chl a determination, DO NOT acidify. Use the

“uncorrected” chl a calculation described in Section 12.1.

If pheophytin a determinations will be made, it will be

necessary to obtain before-to-after acidification response

ratios of the chlorophyll a calibration standards as follows:

(1) measure the fluorescence of the standard, (2) remove

the cuvette from the fluorometer, (3) acidify the solution

to .003 N HCl with the 0.1 N HCl solution, (4) use a

(6)

pasteur type pipet to thoroughly mix the sample by

aspirating and dispensing the sample into the cuvette,

keeping the pipet tip below the surface of the liquid to

avoid aerating the sample, (5) wait 90 sec and measure

the fluorescence of the standard solution again. Addition

of the acid may be made using a medicine dropper. It will

be necessary to know how many drops are equal to 1 mL

of acid. For a cuvette that holds 5 mL of extraction

solution, it will be necessary to add 0.15 mL of 0.1 N HCl

to reach a final acid concentration of 0.003N in the 5 mL.

Calculate the ratio, r, as follows:

 r = Rb/Ra

where:

Rb = fluorescence of pure chlorophyll a

 standard solution before acidification. 

R = fluorescence of pure chlorophyll a a

 standard solution after acidification.
Revision 1.2 September 1997 445.0-8

11.0 Procedure

11.1 Extraction of Filter Samples

11.1.1 If sampled filters have been frozen, remove them

from the freezer but keep them in the dark. Set up the

tissue grinder and have on hand tissues and squirt bottles

containing water and acetone. Workspace lighting should

be the minimum that is necessary to read instructions and

operate instrumentation. Remove a filter from its

container and place it in the glass grinding tube. The filter

may be torn into smaller pieces to facilitate extraction.

Push it to the bottom of the tube with a glass rod. With a

volumetric pipet, add 4 mL of the aqueous acetone

solution (Sect. 7.6) to the grinding tube. Grind the filter

until it has been converted to a slurry. (NOTE: Although

grinding is required, care must be taken not to overheat

the sample. Good judgement and common sense will

help you in deciding when the sample has been

sufficiently macerated.) Pour the slurry into a 15-mL

screw-cap centrifuge tube and, using a 6-mL volumetric

pipet, rinse the pestle and the grinding tube with 90%

acetone. Add the rinse to the centrifuge tube containing

the filter slurry. Cap the tube and shake it vigorously.

Place it in the dark before proceeding to the next filter

extraction. Before placing another filter in the grinding

tube, use the acetone and water squirt bottles to

thoroughly rinse the pestle, grinding tube and glass rod.

The last rinse should be with acetone. Use a clean tissue

to remove any filter residue that adheres to the pestle or

to the steel rod of the pestle. Proceed to the next filter

and repeat the steps above. The entire extraction with

transferring and rinsing steps takes 5 min. Approximately

500 mL of acetone and water waste are generated per 20

samples from the rinsing of glassware and apparatus.

11.1.2 Shake each tube vigorously before placing them

to steep in the dark at 4 C. Samples should be allowed o

to steep for a minimum of 2 h but not to exceed 24 h.

The tubes should be shaken at least once during the

steeping period.

11.1.3 After steeping is complete, shake the tubes

vigorously and centrifuge samples for 15 min at 675 g or

for 5 min at 1000 g. Samples should be allowed to come

to ambient temperature before analysis. This can be

done by placing the tubes in a constant temperature

water bath or by letting them stand at room temperature

for 30 min. Recalibrate the fluorometer if the room

temperature fluctuated ± 3EC from the last calibration

date. 

11.2 SAMPLE ANALYSIS

11.2.1 After the fluorometer has warmed up for at least

15 min, use the 90% acetone solution to zero the

instrument on the sensitivity setting that will be used for

sample analysis. 

11.2.2 Pour or pipet the supernatant of the extracted

sample into a sample cuvette. The volume of sample

required in your instrument's cuvette should be known so

that the correct amount of acid can be added in the

pheophytin a determinative step. For a cuvette that holds

5 mL of extraction solution, 0.15 mL of the 0.1 N HCl

solution should be used. Choose a sensitivity setting that

yields a midscale reading when possible and avoid the

minimum sensitivity setting. If the concentration of

chlorophyll a in the sample is 90% of the upper limit of

the LDR, then dilute the sample with the 90% acetone

solution and reanalyze. Record the fluorescence

measurement and sensitivity setting used for the sample.

Remove the cuvette from the fluorometer and acidify the

extract to a final concentration of 0.003 N HCl using the

0.1 N HCl solution. Use a pasteur type pipet to

thoroughly mix the sample by aspirating and dispensing

the sample into the cuvette, keeping the pipet tip below

the surface of the liquid to avoid aerating the sample.

Wait 90 sec before measuring fluorescence again.

NOTE: Proper mixing is critical for precise and accurate

results. Twenty-five to thirty-five samples can be

extracted and analyzed in one 8 hr day.

NOTE: If you are using special narrow bandpass filters

for chl a determination, DO NOT acidify samples. Use

the “uncorrected” chl a calculations described in Section

12.1.

12.0 Data Analysis and Calculations

12.1 For “uncorrected chlorophyll a,” calculate the

chlorophyll a concentration in the extract as:

CE,u = Rb x Fs

where CE,u = uncorrected chlorophyll a concentration

 (μg/L) in the extract solution analyzed,

 R = fluoresence response of sample extract b

 before acidification, and 

 Fs = fluoresence respnse factor for sensitivity

 setting S.
CS,u

'

CE,ux extract volume (L) X DF

sample volume (L)

Cs,c '

CE,ux extract volume (L) X DF

sample volume (L)

Ps'

PE X extract volume (L) X DF

sample volume (L)

 445.0-9 Revision 1.2 September 1997 

Calculate the “uncorrected” concentration of chlorophyll extract volume = volume (L) of extract prepared

a in the whole water sample as follows: before dilution,

where CS,u = uncorrected chlorophyll a concentration

 (μg/L) in the whole water sample,

 extract volume = volume (L) of extraction

 prepared before any dilutions,

 DF = dilution factor, sample extract; and 

 sample volume = volume (L) of whole water P = pheophytin a concentration (μg/L) in the

 sample.

12.2 For “corrected chlorophyll a”, calculate the

chlorophyll a concentration in the extract as :

CE,c = F (r/r-1) (R - R ) s b a

where:

CE,c = corrected chlorophyll a concentration (μg/L) in the

extract solution analyzed,

F = response factor for the sensitivity setting S, s

r = the before-to-after acidification ratio of a

 pure chlorophyll a solution (Sect. 10.1),

Rb = fluorescence of sample extract before

 acidification, and

R = fluorescence of sample extract after a

 acidification.

Calculate the “corrected” concentration of chlorophyll a

in the whole water sample as follows:

where CS,c = corrected chlorophyll a concetration (μg/L)

 in the whole water sample,

12.3 Calculate the pheophytin a concentration as

follows:

 PE = F (r/r-1) (rR - R ) s a b

where PE = pheophytin a concentration (μg/L) in the

s 

 whole water sample.

12.4 LRB and QCS data should be reported with each

sample data set.

13.0 Method Performance

13.1 The single lab EDL for the instrument used in the

evaluation of this method was 0.05 μg/L for chlorophyll a

and 0.06 μg/L pheophytin a.

13.2 The precision (%RSD) for chlorophyll a in mostly

blue-green and green phytoplankton natural samples

which were steeped for 2 h vs 24 h is reported in Table 1.

Although the means were the same, precision was better

for samples which were allowed to steep for 24 h prior to

analysis. Since pheophytin a was found in the samples,

the chlorophyll a values are "corrected" (Sect. 12.2).

Table 2 contains precision data for pheophytin a. A

statistical analysis of the pheophytin a data indicated a

significant difference in the mean values at the 0.05

significance level. The cause of the lower pheophytin a

values in samples extracted for 24 h is not known.

13.3 Three QCS ampoules obtained from the USEPA

were analyzed and compared to the reported confidence

limits in Table 3. NOTE: The USEPA no longer provides

these QCSs.

13.4 Multilaboratory Testing - A multilaboratory

validation and comparison study of EPA Methods 445.0,

446.0 and 447.0 for chlorophyll a was conducted in 1996

by Research Triangle Institute, Research Triangle Park,

N.C. (EPA Contract No. 68-C5-0011). There were 21

volunteer participants in the fluorometric methods
Revision 1.2 September 1997 445.0-10

component that returned data; 10 that used the modified HPLC results for the highest concentration algae sample

fluorometric method and 11 that used the conventional since chlorophyll a is separated from other interfering

method. The primary goals of the study were to

determine estimated detection limits and to assess

precision (%RSD) and bias (as percent recovery) for

select unialgal species, and natural seawater.

13.4.1 The term, pooled estimated detection limit (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 constant error

(22)

variances across concentration and controls for Type II

error. The statistical approach used involved calculating

an estimated DL for each lab that had the desired Type I

and Type II error rates (0.01 and 0.05, respectively). The

median DLs over labs was then determined and is

reported in Table 4. It is referred to as pooled-EDL (pEDL).

Solutions of pure chlorophyll a in 90% acetone were

prepared at three concentrations (0.11, 0.2 and 1.6 ppm) definitive explanation can be offered at this time for this

and shipped with blank glass fiber filters to participating phenomenon. A possible explanation for the

laboratories. Analysts were instructed to spike the filters Phaeodactylum is that it contained significant amounts of

in duplicate with a given volume of solution and to chlorophyllide a which is determined as chlorophyll a in

process the spiked filters according to the method. The

results from these data were used to determine a p-EDL

for each method. Results (in ppm) are given in Table 4.

The standard fluorometric and HPLC methods gave the

lowest p-EDLs while the spectrophotometric

(monochromatic equations) gave the highest p-EDLs.

Due to the large dilutions required to analyze these

solutions, the fluorometric p-EDLs are unrealistically high

compared to what is achievable by a single lab. Typical

single lab EDLs can easily be 1000 fold lower than the pEDL reported in Table 4.

13.4.2 To address precision and bias in chlorophyll a

determination for different algal species, three pure

unialgal cultures (Amphidinium, Dunaliella and

Phaeodactylum) were cultured and grown in the

laboratory. Four different “concentrations” of each

species were prepared by filtering varying volumes of the

algae. The filters were frozen and shipped to participant

labs. Analysts were instructed to extract and analyze the

filters according to the respective methods. The “true”

concentration was assigned by taking the average of the

pigments prior to determination. Pooled precision (as

determined by %RSD) data are presented in Tables 5-7

and accuracy data (as percent recovery) are presented in

Table 8. No significant differences in precision were

observed across concentrations for any of the species. It

should be noted that there was considerable lab-to-lab

variation (as exhibited by the min and max recoveries in

Table 8) and in this case the median is a better measure

of central tendency than the mean. 

In summary, the mean and median concentrations

determined for Amphidinium carterae (class

dinophyceae) are similar for all methods. No method

consistently exhibited high or low values relative to the

other methods. The only concentration trend observed

was that the spectrophotometric method-trichromatic

equations (SP-T) showed a slight percent increase in

recovery with increasing algae filtration volume.

For Dunaliella tertiolecti (class chlorophyceae) and

Phaeodactylum tricornutum (class bacillariophyceae)

there was generally good agreement between the

fluorometric and the spectrophotometric methods,

however, the HPLC method yielded lower recoveries with

increasing algae filtration volume for both species. No

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 the spectrophotometric

methods both showed a slight decrease in chlorophyll a

recovery with increasing Phaeodactylum filtration volume.

Results for the natural seawater sample are presented in

Table 9. Only one filtration volume (100 mL) was

provided in duplicate to participant labs.

14.0 Pollution Prevention

14.1 Pollution prevention encompasses any technique

that reduces or eliminates the quantity or toxicity of waste

at the point of generation. Numerous opportunities for

pollution prevention exist in laboratory operation. The

EPA has established a preferred hierarchy of
 445.0-11 Revision 1.2 September 1997 

environmental management techniques that places 4. Trees, C.C., M.C. Kennicutt, and J.M. Brooks,

pollution prevention as the management option of first "Errors associated with the standard fluorometric

choice. Whenever feasible, laboratory personnel should determination of chlorophylls and

use pollution prevention techniques to address their waste phaeopigments", Mar. Chem., 17 (1985) pp. 1-

generation (e.g., Sect. 11.1.1). When wastes cannot be 12.

feasibly reduced at the source, the Agency recommends

recycling as the next best option. 5. Method 445, “Multi-Laboratory Comparison and

14.2 For information about pollution prevention that USEPA Contract 68-C5-0011, WA1-03, August

may be applicable to laboratories and research 1997.

institutions, consult Less is Better: Laboratory Chemical

Management for Waste Reduction, available from the

American Chemical Society's Department of Government

Relations and Science Policy, 1155 16th Street N.W.,

Washington D.C. 20036, (202)872-4477.

15.0 Waste Management

15.1 The Environmental Protection Agency requires

that laboratory waste management practices be

conducted consistent with all applicable rules and

regulations. The Agency urges laboratories to protect the

air, water, and land by minimizing and controlling all

releases from hoods and bench operations, complying

with the letter and spirit of any sewer discharge permits

and regulations, and by complying with all solid and

hazardous waste regulations, particularly the hazardous

waste identification rules and land disposal restrictions.

For further information on waste management consult

The Waste Management Manual for Laboratory

Personnel, available from the American Chemical Society

at the address listed in the Sect. 14.2.

16.0 References

1. Yentsch, C.S. and D.W. Menzel, "A method for

the determination of phytoplankton chlorophyll

and pheophytin by fluorescence", Deep Sea

Res., 10 (1963), pp. 221-231.

2. Strickland, J.D.H. and T.R. Parsons, A Practical

Handbook of Seawater Analysis, Bull. Fish. Res.

Board Can., 1972, No.167, p. 201.

3. Arar, E., "Evaluation of a new technique that uses

highly selective interference filters for measuring

chlorophyll a in the presence of chlorophyll b and

pheopigments," USEPA Summary Report, 1994,

NTIS No. PB94-210622.

Validation of Chlorophyll Methods,” Final Report,

6. Holm-Hansen, O., “Chlorophyll a determination:

improvements in methodology”, OKIOS, 30

(1978), pp. 438-447.

7. Wright, S.W. and J.D. Shearer, "Rapid extraction

and HPLC of chlorophylls and carotenoids from

marine phytoplankton", J. Chrom., 294 (1984),

pp. 281-295.

8. Bowles, N.D., H.W. Paerl, and J. Tucker,

"Effective solvents and extraction periods

employed in phytoplankton carotenoid and

chlorophyll determination", Can. J. Fish. Aquat.

Sci., 42 (1985) pp. 1127-1131.

9. Shoaf, W.T. and B.W. Lium, "Improved extraction

of chlorophyll a and b from algae using dimethyl

sulfoxide", Limnol. and Oceanogr., 21(6) (1976)

pp. 926-928.

10. Loftis, M.E. and J.H. Carpenter, "A fluorometric

method for determining chlorophylls a, b, and c, "

1

J. Mar. Res., 29 (1971) pp.319-338.

11. Standard Methods for the Analysis of Water and

Wastes, 17th Ed., 1989, 10200H, Chlorophyll.

12. Wright, S.W., S.W. Jeffrey, R.F.C. Manntoura,

C.A. Llewellyn, T. Bjornland, D. Repeta, and N.

Welschmeyer, "Improved HPLC method for the

analysis of chlorophylls and carotenoids from

marine phytoplankton", paper submitted for

publication in 1991.

13. Mantoura, R.F.C. and C.A. Llewellyn, "The rapid

determination of algal chlorophyll and carotenoid

pigments and their breakdown products in

natural waters by reverse-phase high

performance liquid chromatography", Anal.

Chim. Acta., 151 (1983) pp. 297-314.
Revision 1.2 September 1997 445.0-12

14. Brown, L.M., B.T. Hargrave, and M.D. 20. "Proposed OSHA Safety and Health Standards,

MacKinnon, "Analysis of chlorophyll a in Laboratories", Occupational Safety and Health

sediments by high-pressure liquid

chromatography", Can. J. Fish. Aquat. Sci., 38

(1981) pp. 205-214.

15. Bidigare, R.R., M.C. Kennicutt, II, and J.M.

Brooks, "Rapid determination of chlorophylls and

their degradation products by HPLC", Limnol.

Oceanogr., 30(2) (1985) pp. 432-435.

16. Minguez-Mosquera, M.I., B. Gandul-Rojas, A.

Montano-Asquerino, and J. Garrido-Fernandez,

"Determination of chlorophylls and carotenoids

by HPLC during olive lactic fermentation", J.

Chrom., 585 (1991) pp. 259-266.

17. Carcinogens - Working With Carcinogens,

Department of Health, Education and Welfare,

Public Health Service, Center for Disease

Control, National Institute for Occupational Safety

and Health, Publication No. 77-206, 1977.

18. "OSHA Safety and Health Standards, General

Industry", (29 CFR 1910), Occupational Safety

and Health Administration, OSHA 2206, revised

January 1976.

19. Safety in Academic Chemistry Laboratories,

American Chemical Society publication,

Committee on Chemical Safety, 3rd Edition,

1979.

Administration, Federal Register, July 24, 1986.

21. Weber, C.I., L.A. Fay, G.B. Collins, D.E. Rathke,

and J. Tobin, "A Review of Methods for the

Analysis of Chlorophyll in Periphyton and

Plankton of Marine and Freshwater Systems",

work funded by the Ohio Sea Grant Program,

Ohio State University. Grant No.NA84AA-D00079, 1986, 54 pp.

22. Clayton, C.A., J.W. Hines and P.D. Elkins,

“Detection limits within specified assurance

probabilities,” Analytical Chemistry, 59 (1987),

pp. 2506-2514. 
 445.0-13 Revision 1.2 September 1997 

17.0 TABLES, DIAGRAMS, FLOWCHARTS, AND VALIDATION DATA

TABLE 1. COMPARISON OF PRECISION OF TWO EXTRACTION PERIODS

CORRECTED CHLOROPHYLL a

 Sample A Sample B (1) (2)

2 h 24 h 2 h 24 h (3) (3) (3) (3)

Mean Concentration (μg/L) 49.6 52.9 78.6 78.8

Standard Deviation (μg/L) 4.89 2.64 6.21 2.77 

Relative Standard Deviation (%) 9.9 5.0 7.9 3.5

Values reported are the mean measured concentrations (n=6) of chlorophyll a in the natural water based 1

on a 100-mL filtration volume. 

Values reported are the mean measured concentrations (n=9) of the extraction solution. Sample filtration 2

volume was 300 mL. 

The length of time that the filters steeped after they were macerated. 3
Revision 1.2 September 1997 445.0-14

TABLE 2. COMPARISON OF PRECISION OF TWO EXTRACTIONS PERIODS FOR Pheophytin a

Pheophytin a

 Sample A Sample B (1) (2)

2 h 24 h 2 h 24 h (3) (3) (3) (3)

Mean Concentration (μg/L) 9.22 8.19 13.1 10.61

Standard Deviation (μg/L) 2.36 3.55 3.86 2.29

Relative Standard Deviation (%) 25.6 43.2 29.5 21.6

Values reported are the mean measured concentrations (n=6) of pheophytin a in the natural water based 1

on a 100-mL filtration volume. 

Values reported are the mean measured concentrations (n=9) of pheophytin a the extraction solution.

2

Sample filtration volume was 300 mL. 

The length of time that the filters steeped after they were macerated. 3
 445.0-15 Revision 1.2 September 1997 

TABLE 3. ANALYSES OF USEPA QC SAMPLES

ANALYTE REFERENCE VALUE CONFIDENCE LIMITS

Chlorophyll a 2.1 μg/L 0.5 to 3.7 μg/L

Pheophytin a 0.3 μg/L -0.2 to 0.8 μg/L

 

MEAN % Relative Standard 

1

ANALYTE MEASURED VALUE Deviation

Chlorophyll a 2.8 μg/L 1.5

Pheophytin a 0.3 μg/L 33

 

 N = 3 1
Revision 1.2 September 1997 445.0-16

TABLE 4. 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 (5)

HPLC 4 0.081

SP-M 15 0.229

SP-T 15 0.104

_____________________________________________________________

(1) See Section 13.4.1 for a description of the statistical approach used to determine p-EDLs. 

(2) FL-Mod = fluorometric method using special interference filters.

 FL-Std = conventional fluorometric method with pheophytin a correction.

 HPLC = EPA method 447.0

 SP-M = EPA method 446.0, monochromatic equation.

 SP-T = EPA method 446.0, trichromatic equations.

(3) N = number of labs whose data was used.

(4) The p-EDL was determined with p = 0.01 and q (type II error rate) = 0.05.

(5) Due to the large dilutions required to analyze the solutions by fluorometry, the fluormetric p-EDLs are

 unrealistically high.
 445.0-17 Revision 1.2 September 1997 

TABLE 5. POOLED PRECISION FOR DUNALIELLA TERTIOLECTI SAMPLES

 mLs of 

 culture

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

Fl-Mod 5 7 0.163 0.037 22.8

 10 7 0.298 0.080 26.7

 50 7 1.684 0.385 22.9

100 7 3.311 0.656 19.8

Fl-Std 5 8 0.185 0.056 30.4

 10 8 0.341 0.083 24.4

 50 8 1.560 0.311 19.9

100 8 3.171 0.662 20.9

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.
Revision 1.2 September 1997 445.0-18

TABLE 6. POOLED PRECISION FOR AMPHIDINIUM CARTERAE SAMPLES

 mLs of 

 culture

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

Fl-Mod 5 7 0.066 0.010 14.6

 10 7 0.142 0.045 31.5

 50 7 0.757 0.208 27.5

100 7 1.381 0.347 25.1

Fl-Std 5 8 0.076 0.018 23.2

 10 8 0.165 0.040 24.3

 50 8 0.796 0.140 17.5

100 8 1.508 0.324 21.5

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.
 445.0-19 Revision 1.2 September 1997 

TABLE 7. POOLED PRECISION FOR PHAEODACTYLUM TRICORNUTUM SAMPLES

 mLs of 

 culture

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

Fl-Mod 5 7 0.221 0.040 18.0

 10 7 0.462 0.094 20.3

 50 7 2.108 0.491 23.3

100 7 3.568 1.186 33.2

Fl-Std 5 8 0.214 0.053 24.8

 10 8 0.493 0.091 18.4

 50 8 2.251 0.635 28.2

100 8 4.173 0.929 22.3

___________________________________________________________________________________

(1) Fl-Mod = fluorometric method using special interference filters.

 Fl-Std = conventional fluorometric method with pheophytin a correction.

(2) N = number of volunteer labs whose data was used.

NOTE: The phaeodactylum extract contained significant amounts of chlorophyll c and chlorophyllide a which

interferes in chlorophyll a measurement in the fluorometric method, therefore, the concentration of chlorophyll a is

overestimated compared to the HPLC method which separates the three pigments. The FL-Mod interference filters

minimize this interference more so than the conventional filters.
Revision 1.2 September 1997 445.0-20

TABLE 8. 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
Table 8 cont’d

 445.0-21 Revision 1.2 September 1997 

Species Statistic Method

Percent Recovery

Conc. Conc. Conc. Conc.

Level 1 Level 2 Level 3 Level 4

SP-M 240 247 247 243

SP-T 225 244 256 256

Dunaliella Maximum FL-MOD 295 277 287 288

FL-STD 439 385 276 261

HPLC 392 273 172 154

SP-M 342 316 296 293

SP-T 291 283 283 283

Phaeodactylum Minimum FL-MOD 216 183 157 154

FL-STD 189 220 223 219

HPLC 150 119 84 75

SP-M 161 138 156 160

SP-T 203 195 216 244

Median FL-MOD 292 285 250 245

FL-STD 296 263 254 254

HPLC 225 203 114 90

SP-M 287 274 254 253

SP-T 286 281 277 274

Maximum FL-MOD 357 337 320 318

FL-STD 371 415 415 334

HPLC 394 289 182 139

SP-M 446 344 330 328

SP-T 357 316 318 299
Revision 1.2 September 1997 445.0-22

TABLE 9. CHLOROPHYLL A CONCENTRATIONS IN MG/L DETERMINED IN FILTERED SEAWATER 

 SAMPLES

Method Con. No. Obs. No. Labs Mean Std. Dev. RSD(%) Minimum Median Maxium (1)

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.