Journal of Oceanography, Vol. 60, pp. 259 to 261, 2004
Short Contribution
Comparison of Chlorophyll a Concentrations Obtained
with 90% Acetone and N,N-dimethylformamide
Extraction in Coastal Seawater
K UNINAO TADA1*, HITOMI Y AMAGUCHI2 and SHIGERU MONTANI2
1
2
Department of Life Sciences, Kagawa University, Miki, Kagawa 761-0795, Japan
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan
(Received 27 December 2002; in revised form 30 May 2003; accepted 9 June 2003)
Determinations of Chlorophyll a (Chl a) in eutrophic coastal marine waters were compared using N,N-dimethylformamide (DMF) and 90% acetone techniques. Measured
Chl a concentrations ranged from 0.89 to 10.65 µg l –1 for 90% acetone extracts and
from 0.97 to 12.92 µg l –1 for DMF extracts, respectively, for 24 surface water samples
from the Harima-nada, Seto Inland Sea, Japan. Chl a concentrations using DMF as a
solvent were consistently higher than those found using 90% acetone (p < 0.001, n =
24). Chl a is thus likely to be underestimated (by 13%) when 90% acetone is used for
eutrophic waters.
Keywords:
⋅ Chlorophyll a,
⋅ N,Ndimethylformamide,
⋅ 90% acetone,
⋅ coastal water.
of solvents can be compared without using a correction
factor. Comparability of results is particularly important
when investigating long-term variations of Chl a concentration.
This paper shows that the fluorometric determination of phytoplankton chlorophyll in coastal waters using DMF extraction gives consistently higher values than
those obtained by using 90% acetone.
1. Introduction
Chlorophyll a (Chl a) is routinely measured to quantify phytoplankton abundance in the aquatic environment.
The most commonly used solvent for measuring Chl a is
90% acetone (e.g. Strickland and Parsons, 1972), but N,Ndimethylformamide (DMF) is also very useful and is
widely used, especially in Japan, since the technique gives
high extraction efficiency with little vaporization at room
temperature (Speziale et al., 1984; Suzuki and Ishimaru,
1990). Additionally, DMF has recently been reported to
be an efficient solvent for HPLC analysis of
phytoplankton pigments due to its efficient extraction,
which expedites the extraction process (Suzuki et al.,
1993; Furuya et al., 1998).
Suzuki and Ishimaru (1990) compared the extraction
efficiency of DMF to that of 90% acetone for cultured
phytoplankton; however, few comparisons have been reported of these Chl a determination solvents for natural
seawater samples. The unavailability of Chl a
determinations using both solvents for natural seawaters
is a potentially serious omission. In particular, it is not
clear whether DMF extraction gives a higher Chl a value
in natural seawater samples because of its higher extraction efficiency. Furthermore, it is very important to know
whether Chl a field data determined by using the two kinds
2. Materials and Methods
Chl a was determined in field samples collected at
24 stations throughout Harima-Nada, Seto Inland Sea,
Japan. In general, diatoms predominated all year round
in the study area. Surface seawater samples were collected
with a clean plastic bucket on September 18, 2002. Water
samples were immediately filtered through a Whatman
GF/F filter (47 mm) and preserved in DMF or 90% acetone. Extracts using DMF and 90% acetone were stored
for one night in the dark at 4°C until analysis. Supersonic
treatment was performed after solvent extraction. A 200
ml seawater sample was filtered, the particulate matter
collected on the filter, and Chl a was extracted with 10
ml of both solvents. Moreover, the filter extracted with
90% acetone was placed in another tube and Chl a was
again measured after the filter was treated with DMF.
Fluorometric measurement of Chl a was performed
by the method of Holm-Hansen et al. (1965) using a
Turner fluorometer (10-AU), as described by Parsons et
al. (1984). Standard Chl a calibration for fluorometric
* Corresponding author. E-mail: [email protected]
Copyright © The Oceanographic Society of Japan.
259
determination was performed by the method described
by UNESCO (1994): a Chl a reagent (Wako Pure Co. Ltd.,
super grade) was dissolved in DMF and 90% acetone and
the concentrations of Chl a solutions were determined
spectrophotometrically using the equation:
respectively (Jeffly and Humphrey, 1975; Porra et al.,
1989). Using these precise Chl a concentrations, a factor
(ti) of the equation for fluorometric Chl a determination
was calculated for DMF and 90% acetone, respectively:
Chl a concentration (µg l–1) = ti·(Fo – Fa)·v
–1
Chl a concentration (µg ml )
= Achl/specific absorption coefficient
where Achl is the difference between absorbance at 663.8
nm and 750 nm. The specific absorption coefficient is
88.74 and 87.67 in the case of DMF and 90% acetone,
Fig. 1. Comparison of Chl a concentrations extracted with DMF
or 90% acetone for coastal waters collected at Harima-Nada,
the Seto Inland sea, Japan. - - - line shows y = x.
where Fo is the original fluorescence, Fa is the fluorescence after acidification, and v is the dilution factor from
the volume of filtered and extracted seawater solution. In
this study, all Chl a determinations were performed in
duplicate. The average mean deviations of the duplicate
analyses for DMF and 90% acetone were 2.2 ± 1.7% (n =
23) and 3.3 ± 2.7% (n = 24) respectively.
3. Results and Discussion
Chl a concentrations in coastal waters ranged from
0.89 to 10.65 µg l–1 for 90% acetone extracts and from
0.97 to 12.92 µg l–1 for DMF extracts, respectively. A
good correlation was obtained between Chl a concentrations determined by using the two kinds of solvents (Fig.
1). The equation for all Chl a values was [Chl a]DMF =
1.13 × [Chl a]90% acetone + 0.23 (r2 = 0.987, p < 0.0001,
n = 24), where [Chl a]DMF and [Chl a]90% acetone are Chl a
concentrations in DMF and 90% acetone, respectively.
This means that Chl a concentrations obtained using DMF
were higher than those using 90% acetone. This agrees
with previous results, which showed that DMF extracts
Chl a more effectively than other solvents for various
taxonomic groups (e.g. Moran and Porath, 1980; Speziale
et al., 1984; Suzuki and Ishimaru, 1990). Suzuki and
Ishimaru (1990) also reported that DMF extraction gives
a higher Chl a value than 90% acetone extraction in natural algal samples collected from Harumi Pier in Tokyo
Fig. 2. Chl a extracted with 90% acetone and additional Chl a extracted with DMF after 90% acetone extraction.
260
K. Tada et al.
Bay, although Speziale et al. (1984) reported no significant differences in Chl a concentrations determined for
field samples collected from some lakes and ponds.
In our measurements, Chl a was further extracted by
DMF from the residue of 90% acetone extraction (Fig.
2), which means that Chl a extraction using 90% acetone
is less efficient than DMF. However, Chl a extracted with
90% acetone, plus the additional amount extracted with
DMF after 90% acetone extraction, accounted for 88.2 ±
12.4% of Chl a extracted with DMF alone. Unfortunately,
11.8% of Chl a extracted with DMF remains unaccounted
for. The reason for this inconsistency is probably due to
the loss of some particles on the filter during the first
90% acetone extraction. Nonetheless, we believe that the
difference in Chl a concentration between DMF and 90%
acetone extracts was due to the high extraction efficiency
of DMF for coastal water samples. Our results indicate
that Chl a concentration determined on field samples using the 90% acetone extract may be underestimated for
coastal water samples in Seto Inland Sea. While the degree of Chl a underestimation using the 90% acetone extract method may depend on the in-situ phytoplankton
assemblage, our results show that Chl a concentrations
determined using the 90% acetone extract may underestimate true values by 13%.
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Comparison of Chlorophyll a Obtained with 90% Acetone and DMF Extraction
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