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FLUORESCENCE EXCITATION AND EMISSION SPECTRA
OF DISSOLVED ORGANIC MATTER IN SEA WATER
EUGENE D. TRAGANZA
U. S. Naval Academy, Annapolis, Maryland 21402
ABSTRACT
Fluorescence spectra of waters from the Sargasso Sea, the Atlantic
Shelf, a Trichodesmium "bloom," and a cultured concentrate of suspended
matter were obtained on board R/V CHAIN with the Baird-Atomic tluorescence spectrophotometer. Excitation (peaks at 282 m,u and 365 m,u) and
emission (peaks at 350 m,u and 450 m,u) spectra of water from the
Trichodesmium bloom were well defined, suggesting that specific sources
of fluorescence may be identifiable after recent biological events. Spectra
of several samples from the surface showed recurring peaks at 340 m,u,
suggesting that fluorophors may concentrate at this interface. Within the
limits of sensitivity of this instrument, no spectra were seen for water from
the Sargasso Sea. Spectra of water from the Atlantic Shelf were very broad,
with an emission peak ranging from 410 m,u to 430 m,u. A remarkable
similarity was seen in the emission (peaks at 340 mj.L and 450 m,u) of
dissolved materials in a laboratory culture of Skeletonema costatum and
an incubated concentrate of suspended matter in surface water from the
shelf.
INTRODUCTION
Sea water fluoresces in the blue end of the spectrum when it is irradiated
with ultraviolet light. This response is thought to be due to the presence
of nanogram quantities of largely unidentified organic compounds (Jeffrey
& Hood, 1958; Fogg, 1966; Strickland, 1965; Kalle, 1966; Duursma,
1965). Historically, interest in the fluorescence of sea water began with
Kane's studies of the famous "Gelbstoff." During this work Kalle discovered that, in addition to the "Gelbstoff" dissolved in sea water, there
was another organic material which emitted a blue fluorescence. According
to Kalle, the blue flourescence may be associated with organic compounds
present during the formation of "melanoidines." The "melanoidines" are
soluble, yellow compounds that are a part of the "Gelbstoff." The remainder
of the "Gelbstoff" consists of the so-called phenol humic acids. Duursma
( 1965) suggested that theoretical examination of fluorescence spectra may
yield useful information about the compounds emitting the blue fluorescence.
Fluorometry can be useful in the study of the trace quantities of dissolved
organic matter in sea water. The more sensitive filter fluorometers can
detect as little as 1 mp.g/ml (Udenfriend, 1962). These instruments can
be used for continuous detection in situ by towing or pumping techniques
(Turner, 1968; Lorenzen, 1966; Karabashev, 1965). Fluorescence spectrophotometers may serve as an additional tool for identification of compounds
and selection of excitation wavelengths. The potential of both techniques
898
Bulletin of Marine Science
[19(4)
offers some hope to the oceanographer in the difficult task of finding and
identifying dissolved organic compounds in sea water. The new accessibility
of the fluorescence spectrophotometer should encourage its use and contribute to the study of the ocean as a biochemical system. This paper
describes some preliminary studies using a Baird-Atomic fluorescence
spectrophotometer.
METHODS
A Baird-Atomic fluorescence spectrophotometer was used on board
the Woods Hole Oceanographic Institution's R/V CHAIN to determine
emission and excitation characteristics of compounds dissolved in sea water.
Samples were collected in August 1967, along a transect between Woods
Hole, Massachusetts, and the Sargasso Sea. In the operation of this instrument, sea water was irradiated at selected wavelengths between 200 mp'
and 700 mp' by manual adjustment of the excitation monochromator. The
remainder of the wave band was scanned each time for emission with the
emission monochromator. Once an emission peak was detected, the emission monochromator was set at the peak wavelength. The search with the
excitation monochromator was continued until the maximum excitation
wavelength was determined. Finally, with the maximum setting on the
excitation monochromator, the remainder of the wave band was scanned
automatically. A spectrograph was recorded of relative emission intensity
vs. wavelength. The procedure was reversed to obtain an excitation
spectrograph. A base line was established and checked repeatedly during
the course of all measurements. For this purpose, water was distilled and
irradiated with ultraviolet light. This treatment is presumed to reduce the
content of organic matter below detectable levels (Hamilton & Carlucci,
1965).
Some samples were obtained as the ship passed through a surface
"bloom" of Trichodesmium. To avoid contamination from the ship, a
plastic bucket was cast forward of the ship's bow. Shelf water was collected for a suspended-matter culture in a 10-1 glass sampler known as
the Dazzler. Water from the Dazzler was emptied into a 5-gal glass carboy.
The suspended matter in 20 1 of sample was concentrated to 500 m!. A
gentle inverted filtration technique was employed to avoid rupture of
organisms (Dodson & Thomas, 1964). Filters used to concentrate the
suspended matter were prewashed with HCl and distilled water. All equipment and glassware were washed with Calgon, which leaves no trace of
fluorescence after rinsing with distilled water from freshly opened bottles.
(Opened bottles of distilled water develop fluorescence on aging.) All
other samples were collected in Nansen bottles as a part of standard
hydrographic casts.
A shipboard "culture" was prepared by adding a nutrient mixture of
1969]
Traganza: Fluorescence of Dissolved Organic Matter
899
phosphate, silicate, nitrate, and iron to the suspended-matter concentrate
described above. Fluorescence was checked at the beginning of the experiment. The "culture" was allowed to incubate for 7 days at 15°C with
alternating 12-hour periods of fluorescent light and darkness. Suspended
materials were centrifuged off, and the supernatant was tested for fluorophoric compounds. No attempt was made to determine the effect of light
or nutrients on the growth rate of the "culture" because of the preliminary
nature of the experiment. A filtered sea-water control was not used because
of the qualitative nature of the test. The nutrient mixture had no initial
effect on sea water alone.
Prior to the cruise, a 3-1 culture of Skeletonema costatum was prepared
in a glass vessel by Dr. Yentsch, at the Woods Hole Oceanographic Institution on July 19, 1967. Phosphate, silicates, nitrate, and trace elements were
used to enrich the sea-water base. A constant pH of 8.1 was maintained
by metering CO2 gas into the culture on demand, as sensed by a pH meter.
The culture was incubated in a coldroom at 22°C before a fluorescent light
bank. Constant stirring was provided by a magnetic motor and stirring bar.
Samples were withdrawn through a siphon at daily intervals.
All analyses were conducted at approximately 28°C.
RESULTS
Distinctive spectra were obtained from surface water collected in a
"bloom" of Trichodesmium sp. encountered in the Sargasso Sea (Fig. 1)
on August 27, 1967, at R/V CHAINstation 805 (36°25.8' N, 67°19' W).
There were two excitation peaks, 282 mp' and 365 mp', and two emission
peaks, 350 mp' and 450 mI" Irradiation at the 282-mp. excitation peak
produced maximum fluorescence. Rupture of a concentration of cells
by freezing produced a red pigment with the same spectra. Ten months
later, the same peaks were obtained from a "bloom" sample stored in a
reagent bottle in the dark at ambient laboratory temperature. The later
results
indicate
either
a long stability
for the fluorophor,
or that
the
organisms remained viable. Microscopic examination of one sample revealed what appeared to be intact cells of some type.
Figure 2 shows the optimum fluorescence emissions of Sargasso Sea
water and water from the Continental Shelf at R/V CHAINstations 805A
(August 27) and 810 (August 31), respectively. Water samples were
collected at standard depths for both stations. No fluorescence was detectable in Sargasso Sea water at any excitation wavelength or at any depth;
traces for all samples were recorded on the same graph. For shelf water
(Fig. 2), irradiation at 305 mp' gave maximum fluorescence; traces were
recorded on separate graphs. The broad indistinct "peak" for the
sample from 30 m typifies this water, which apparently contained a dilute
mixture of fluorophors.
Bulletin of Marine Science
900
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1. Left, excitation spectrum of sea water collected in a surface concentration of Trichodesmium sp., near 35°25.S'N, 67°19'W; right, fluorescence
spectrum of same. (The irregular traces were caused by the roll of the ship.)
FIGURE
The emission spectrum in Figure 3 (left) was produced by the shipboard-incubated concentrate of suspended matter from surface shelf water
taken at R/V CHAIN station 796 on August 22, 1967. The emission trace
on the right of Figure 3 was produced by the laboratory culture of Skeletonema costatum. The similarity of the emission spectra of the two samples
is apparent; both show peaks at 340 mp' and 450 mp.. In the Skeletonema
experiment, maximum excitation for the 340-mp. emission peak occurred
at 292 mp' and 305 mI,/,. The 292-mp. wavelength of maximum excitation
for the 340-mp. emission peak was common to both cultures. The excitation maximum for the 450-mp. peak was 365 mp' in the culture of Skeletonema, but excitation at 305 mp' and 365 mp' was not checked for the
concentrate culture. Skeletonema may be a source of fluorophors in the sea.
At several stations, surface samples showed peaks at 340 mp' while nothing was detected at other depths. It is difficult to assess the importance of
this finding, since contamination may have come from the ship. However,
the possibility that it is indigenous should not be overlooked.
Finally, in addition to the above, Dr. Guillard of the Woods Hole Oceanographic Institution supplied cultures of 12 different plankters for this study.
All of these indicated the presence of marine fluorophors in solution. Emission peaks fell within 335 m,u-345 mp' and 435 mp.-450 mp', and excitation
peaks within 282 mp.-292 m,u and 365 m,u-375 mp..
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FIGURE 3. Left, fluorescence spectrum of a 7-day culture of concentrated
suspended matter, incubated at sea. The sample consisted of surface water
from the Continental Shelf near 40 43'N, 69°11'W. Right, fluorescence spectrum
of a 6-day culture of Skeletonema costatum at the Woods Hole Oceanographic
Institution.
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DISCUSSION
The origin and nature of fluorescent substances in sea water is not known,
but it is supposed that they are derived from the metabolism and decomposition of organisms. The fluorescent biochemicals include phenols, like those
related to tyrosine; aromatic amines, such as anthranilic acid; polycyclic
compounds (e.g., the homocyclic vitamin K, and the heterocyclic compounds
including the indole precursors and metabolites of tryptophan, the purines,
riboflavin, quinolines and porphyrins); and conjugated polyenes like vitamin
A (Udenfriend, 1962).
Any of the substances could have an important, direct or indirect, influence on the ocean as a biochemical system. It is known, for example, that
damage to plants and animals resulting from light can be markedly increased
in the presence of fluorescent substances (Friedrich, 1961). This suggests
an important photodynamic role in ecological relations. It would be interesting to compare water from the euphotic, disphotic, and aphotic zones,
as well as from the equator, the poles, and areas of upwelling.
Greater use of fluorescence excitation and emission spectra is warranted
in the study of organic matter in sea water. As for direct or indirect
(Stryer, 1968) characterization of specific compounds in mixtures, it will
1969]
Traganza: Fluorescence of Dissolved Organic Matter
903
probably give trouble because many compounds emit in the same region
of the spectrum. Nevertheless, fluorometry shows much promise for
detecting and studying areas of recent biological events.
ACKNOWLEDGMENTS
This work was supported by the U. S. Naval Academy Research Council.
The Woods Hole Oceanographic Institution, Woods Hole, Massachusetts,
provided a summer appointment with full use of its facilities, and the BairdAtomic Company, Cambridge, Massachusetts, provided the use of a fluorescence spectrophotometer. The author thanks these organizations and A. W.
Hornig of Baird-Atomic. I also thank the following people at Woods Hole
Oceanographic Institution: Paul Fye, Director; John Ryther, Head of the
Department of Biology; the department personnel; and especially Charles
Yentsch, who shared his laboratory and equipment.
SUMARIO
ESPECTROSDE EXCITACI6NDE LA FLUORESCENCIA
Y DE EMISI6N
EN MATERIAORGANICADISUELTAEN AGUA DE MAR
A bordo del barco de investigaciones CHAINse obtuvieron espectros de
la f1uorescencia de aguas procedentes del Mar de los Sargazos, de la Plataforma Atlantica, de un afloramiento de Trichodesmium y de un cultivo de
un concentrado de materia en suspension, utilizando el espectofotometro
de fluorescencia atomico de Baird. Los espectros de excitacion (maximos
a 282 m,u y 365 m,u) y de emision (maximos a 350 m,u y 450 m,u) del agua
de un afloramiento de Trichodesmium esuvieron bien definidos, sugiriendo
que fuentes especificas de fluorescencia pueden identificarse despues de
eventos biologicos recientes. Los espectros de varias muestras procedentes
de la superficie presentaron maximas recurrentes a 340 mp', sugiriendo que
pueden concentrarse fluoroforos en esta interfacie. Dentro de los limites
de ]a sensibilidad de este instrumento, no se vieron espectros en e] agua
del Mar de los Sargazos. Los espectros del agua de la Plataforma Atlantica
fueron muy amplios, con un maximo en la emision que iba de 410 m,u a
430 m,u. Una similaridad notable fue vista en la emision (maximos a
340 m,u y 450 m,u) de materiales disueltos en un cultivo de laboratorio de
Skeletonema costatum y un concentrado incubado de materia en suspension
en agua de la superficie procedente de la plataforma.
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