Solid-phase ¯uorescence determination of chlorins
in marine sediments
R.F. Chen
a,*, Y. Jiang
a, M. Zhao
baEnvironmental, Coastal and Ocean Sciences, UMassBoston, 100 Morrissey Boulevard, Boston, MA 02125, USA bDepartment of Earth Science, Dartmouth College, Hanover, NH 03755, USA
Abstract
Chlorophyll degradation products are preserved in marine sediments over timescales of thousands of years. The production of chlorophyll in the water column is related to biological productivity, so chlorophyll degradation pro-ducts (chlorins) preserved in marine sediments can be used as indicators of paleoproductivity. A new, rapid, non-destructive method of determining chlorin concentrations in marine sediments is presented. Potential interferences associated with the solid-phase ¯uorescence (SPF) method are explored using reference materials, yet this method compares favorably with spectroscopic and high performance liquid chromatographic (HPLC) methods of analysis using marine sediments from Boston Harbor and the continental shelf o northwest Africa.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Chlorins; Paleoproductivity; Fluorescence; Analysis; Non-destructive
1. Introduction
Biological productivity in the ocean plays an impor-tant role in the global cycling of carbon and, therefore, has a major eect on the global climate (Berger et al., 1989; Mix, 1989; Falkowski and Wilson, 1992; Falk-owski, 1994). Phytoplankton biomass can be estimated from the concentration of chlorophyllain seawater, and attempts have been made to relate chlorophyll con-centration determined using satellite data to productiv-ity (Longhurst et al., 1995; Behrenfeld and Falkowski, 1997). Chlorophyllais transformed rapidly in the water column and surface marine sediments (e.g. Eckardt et al., 1992; Harradine et al., 1996), but the macrocyclic ring, the tetrapyrrole backbone of the chlorophyll a
molecule, is quite stable over long periods of time (e.g. Treibs, 1936; Eckardt et al., 1991). The green transfor-mation products of chlorophylls, the major pigments
required by all phytoplankton for photosynthesis, have been termed ``chlorins'' and have been used as an indi-cator of present day and paleoproductivity (Harris et al., 1996).
Freshly produced organic matter in the ocean under-goes a number of degradation processes including graz-ing, microbial degradation, sinkgraz-ing, and sediment diagenesis before being buried in marine sediments (e.g. Welschmeyer and Lorenzen, 1985; Hedges and Keil, 1995). Yet, biogenic debris buried in deep-sea sediments has often been used to explore oceanic productivity in the past. Several chemical parameters and biomarkers have been suggested as proxies for paleoproductivity (e.g. Martinez et al., 1996; Hinrichs et al., 1999), each with its own strengths and weaknesses. Total organic carbon in sediments has been used to indicate total organic carbon rain rate and thus surface production (Reichart et al., 1997), although inputs from terrestrial sources might alter this signal (Westerhausen et al., 1993). Biogenic opal (Stein et al., 1989; Harris et al., 1996) yields information on the silica-producing phyto-plankton community. Barium (McManus et al., 1999), aluminum and titanium (Dymond et al., 1997), and
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* Corresponding author. Tel.: 617-287-7491; fax: +1-617-287-7474.
231Pa/230Th ratios (Kumar et al., 1993; Walter et al.,
1997) have been examined as paleoproductivity indica-tors. Total organic d13C values (Bouloubassi et al.,
1997) and d13C of individual biomarkers such as C 37
alkenones and C22±C33alkanes have also been studied
(Villaneuva et al., 1997). Common to all these indicators is the extraction of various components and the destructive analysis of the sediment sample.
Chlorins have been found to be ubiquitous and abundant in immature marine and lacustrine sediments (e.g. Keely and Maxwell, 1991; Prowse and Maxwell, 1991; Keely et al., 1994; Harris et al., 1995; Soma et al., 1996). They are directly related to photosynthesis and so if conserved should act as a measure of total primary productivity (Harris et al., 1996). In order to maximize our understanding of paleoproductivity, an analysis of chlorins in marine sediments should have a resolution at least similar to concurrent measurements of oxygen-18 (d18O), total organic carbon (TOC), and biogenic
opal.
Spectroscopic and chromatographic methods have both been used to measure chlorin concentrations in marine sediments (Trees et al., 1985; Meyns et al., 1994; Harris and Maxwell, 1995). Chromatographic methods involve the extraction of a sediment sample and quanti-®cation and identiquanti-®cation of major chlorophyll trans-formation products by comparing the chromatographic peaks with those of authentic standards. This procedure is destructive and requires long analysis times, making high resolution (n>50) stratigraphic measurements practically impossible. Spectroscopic methods rely on extraction and a comparison of the UV±vis absorbance or ¯uorescence spectrum of the extract with that of chlorophyll a or authentic chlorin standard. An auto-mated spectroscopic method using on-line extraction and ¯uorescence determination has allowed high reso-lution chlorin determinations that may serve as mea-surements of total primary productivity variations (Harris and Maxwell, 1995; Harris et al., 1996).
The stable macrocyclic ring structure is aromatic with highly delocalized electron density, so it can absorb near the sunlight maximum at sea level, around 420±450 nm. This unique property has been used to determine chlor-ophyll concentrations in living plants and as well as porphyrins in source rocks by measuring their re¯ec-tance spectra (Holden and Gaey, 1990; Holden et al., 1991; Yamada and Fujimura, 1991; Gitelson and Mer-zlak, 1994, 1996). Chlorophylls are also highly ¯uor-escent so ¯uorescence spectroscopy of marine sediments may oer increased sensitivity by reducing the inherent scattering interference associated with re¯ectance spec-tra. We present here a new solid-phase ¯uorescence method for the non-destructive determination of chlor-ins in marine sediments and compare this new rapid method with two established methods for chlorin deter-mination.
2. Methods
2.1. Samples
Boston Harbor surface sediments collected by grab and box core and sediments from the continental shelf o NW Africa (Ocean Drilling Program Leg 108, Site 658C; Ruddiman et al., 1988) were frozen until analysis. Samples were analyzed wet or after drying at 60C for
12 h. Chlorophyllastandards (extracted fromAnacystis nidulans) were obtained from Sigma Chemical and were free from chlorophyll b interferences. Sediment stan-dards were obtained from Wards Natural Science Establishment (Rochester, New York, USA). ``Clean'' sediments were prepared by sonicating sediment stan-dards in acetone repetitively until no chlorin was present in the solvent phase as determined by ¯uorescence with 420 nm excitation.
2.2. Fluorescence spectrophotometry
A Photon Technologies International (PTI) Quantum Master-1 spectro¯uorometer equipped with a powder sample holder was used for solid-phase ¯uorescence analysis. This spectro¯uorometer utilizes a 150 W xenon lamp excitation source, 2 x 0.25 m excitation mono-chromators, a single emission monochromator and a cooled photomultiplier tube detector. A variable angle solid sample holder allows minimization of scattered light interference. In addition, a 420 nm narrow band pass interference ®lter on the excitation side and a 530 nm long wavelength pass ®lter on the emission side were used to decrease scattering and stray light (see Section 3). As scattering was dependent on the sample matrix, no common ``clean'' or ``blank'' sediment could be pre-pared. Instead, ¯uorescence emission from 570 to 700 nm was smoothed with a second order polynomial ®t, and the peak above the curve between 620 and 700 nm was integrated and compared with ``clean'' sediments spiked with chlorophyllato provide quanti®cation.
2.3. High performance liquid chromatography
In addition, a high performance liquid chromato-graphy (HPLC) method and a standard spectroscopic method (Trees et al., 1985) were utilized to provide comparison with the new method. Sediments were extracted with acetone by sonication, volume-reduced by rotoevaporation, and analyzed with a Dynamax HPLC system equipped with a UV absorbance detector. An Econosphere C18 5 mm reversed phase column
components as chlorins was con®rmed by analyzing collected peaks with an HP 8453 UV±vis spectro-photometer and obtaining the characteristic spectrum (including absorption at 650 nm) of chlorins rather than carotenoids. Peaks were quanti®ed with respect to chlorophylla standards which provides only a relative quanti®cation as the ¯uorescence and absorbance char-acteristics of individual chlorins and chlorophyllavary somewhat. While absolute identi®cation of the HPLC-produced chlorin peaks was not provided, UV-vis spec-tra provide secondary con®rmation of our interpreta-tion.
2.4. Sediment extract ¯uorescence
Sediment acetone extracts were also analyzed in the solution phase with the PTI QM-1 spectro¯uorometer operating in the 90con®guration. Samples were placed
in a 1 cm2quartz cell, and the integrated emission from
620 to 700 nm was compared to chlorophyllastandards
for quanti®cation. A 530 nm long wavelength pass ®lter was used to decrease scattering.
3. Results
Fluorescence excitation scans show a maximum of 410±420 nm for chlorophyllaand chlorins in sediments (Fig. 1a). Second order scattering below 350 nm was apparent but does not interfere in normal measurements at 420 nm excitation where a 420 nm narrow band interference ®lter was used. Emission scans from 500 to 700 nm were recorded as the chlorophyll/chlorin peak was broad and shifted slightly (640±670 nm) for dier-ent samples (Fig. 1b). The appardier-ent peak at 540 nm and the long wavelength tail extending to 700 nm is a pro-duct of light scattering and the long wavelength passes ®lters used (Fig. 1b).
In order to optimize the solid-phase ¯uorescence method, various experimental parameters were explored.
First, the incident angle of the excitation radiation was altered aording optimal signal intensity at 25, similar
to the 22optimal angle observed in ¯uorescence
inves-tigations of fuels in soils (Fig. 2; Apitz et al., 1992a). This maximum is a result of reducing scattering while maintaining signi®cant signal and may be somewhat dependent on the optical con®guration used.
Next, water content also showed an eect on ¯uores-cence eciency. Fig. 3 shows dried standard sediments (sand and kaolinite, not organic-free) spiked with chlorophyll a (added in acetone and then dried) with consecutive amounts of deionized water added. Fluor-escence response decreases with increasing water con-tent. Possible explanations for this behavior are: (1)
Fig. 2. The eect of incident angle of the excitation beam to the window of the powder sample holder in the PTI QM-1 spectro-¯uorometer on the ¯uorescence response of chlorophylla.
absorption of light by the organic compounds asso-ciated with the standard mineral grains and which dis-solve into the water, (2) increased re¯ection of the excitation light by water, or (3) a hydration of the pigment on the sediment surface, thus altering its geo-metry and reducing its quantum yield. Water extrac-tions of the sediments did not show an appreciable absorption of light at 420 nm or 680 nm compared to the absorption due to chlorins at relevant concentra-tions. It is known that the ¯uorescence of chlorophyll can be either increased or decreased by the association of the chromophore with various surfaces and by its interaction with various solvents (Mamleeva et al., 1988; Komleva et al., 1989). Most probably, water interacts with the surface of the mineral grain, ``releasing'' the chlorin from the surface, with a concomitant reduction in the surface-enhanced ¯uorescence of the chlorin. Another possibility is that water reduces the re¯ectivity of the sediment (wet sediments are darker) thus reducing the excitation and emission light throughput (Loh-mannsroben and Schober, 1999).
While chlorophyll aspiked on to all sediment stan-dards yielded good linearity (r2>0.85) over various
concentrations ranges varying from 0±1.5mg/g for kao-linite to 0±60mg/g for silt, ¯uorescence intensity actually decreases with increasing concentration above some threshold concentration, probably due to chromophore stacking and quenching. In any case, detection limits were around 1mg/g for all standard sediments.
Sediment composition also has a dramatic eect on ¯uorescence response (Apitz et al., 1992b). Response
factors for the various matrices are shown in Fig. 4. Kaolinite and opal yield the highest sensitivity of detec-tion and bentonite yields the lowest for the standard minerals measured. This variability in ¯uorescence response is probably due to complex surface interactions between the chromophore and the mineral surfaces (Mamleeva et al., 1988). Formation of complexes would generally reduce ¯uorescence while certain surfaces could enhance the pigment ¯uorescence. Thus the solid-phase ¯uorescence method may be in¯uenced by varia-tions (especially in the opal and kaolinite fraction) in sediment composition. A full description of the lithol-ogy of the analyzed sediments would aid in the inter-pretation of the relative abundances of chlorins in those sediments.
Finally, the grain size of the same minerals shows an eect on the ¯uorescence response factors (Fig. 5). Increasing grain size generally decreases ¯uorescence response. There are two possible explanations. First, the more re¯ecting ®ne grained sediments, especially for opal with respect to quartz, may lead to an increase in the eective path length of the excitation radiation and the resulting ¯uorescence emission. The ¯uorescence response is thus enhanced because light enters and exits the small grains more eciently. The dramatic decrease in ¯uorescence response with grain size for opal but not for sand supports this explanation. It has been shown that ¯uorescence responses in sediments can be cor-rected by taking into account sediment re¯ectance (Lohmannsroben and Schoder, 1999). Second, as grain size increases, more chlorin may be forced out of the
sediment grain interstices and would, therefore, be more prone to stack together, self-quenching the ¯uorescence emission of the chlorin. This is the opposite eect that was seen with smaller chromophores, the polynuclear aromatics, in marine diesel fuel as observed by Apitz (1992a). In that study, the smaller grain sizes oered more surface areas for PAH to adsorb and be hidden from excitation radiation, thus the ¯uorescence response increased with increasing grain size. The large planar geometry of the chlorin probably dominates the adsorption factors in our case. Again, knowledge of grain size would aid in the calibration of solid-phase ¯uorescence for determining the relative abundances of chlorins.
Humic substances, major constituents of marine sedi-ments, are known to ¯uoresce (Hayase and Tsubota, 1985; Chen and Bada, 1995), but do not show appreci-able interference in the 650±700 nm range. A ¯uores-cence emission scan, given the optical con®guration (including a 530 nm long wavelength pass ®lter), of humics obtained from Aldrich Chemical yielded an apparent maximum at550 nm. The tail of this
¯uor-escent peak in the 650±700 nm range was simply back-ground-subtracted along with scattered light and random noise and did not interfere with quanti®cation of the chlorins.
To correct for all these possible interferences, a cor-rection factor for the ¯uorescence response of chlorins could be calculated given information on sediment water content, mineral composition and grain size. However, using real marine sediments from a wide
range of environments [Boston Harbor surface sedi-ments and sedisedi-ments from the continental shelf o NW Africa (Ocean Drilling Program Leg 108, Site 658C; Ruddiman et al., 1988)] and wide range of chlorin con-centrations (0±50 mg/g), the solid phase ¯uorescence technique does agree quite well with both the spectro-scopic method (Fig. 6a; r2=0.82) and the HPLC
method (Fig. 6b; r2=0.77) for 40 samples. A closer
examination of the HPLC method shows a signi®cant intercept using this method while the solid phase method shows no ¯uorescence (Fig. 6b). This may be due to the HPLC quanti®cation (based on absorbance) of peaks that are not chlorins or chlorins that are not as ¯uorescent as chlorophyll a. Because the solid and liquid phase ¯uorescence methods correlate with a nearly one-to-one relationship (slope=0.84), it would appear that the HPLC method quanti®es only a fraction of the ¯uorescent chlorins (slope=0.30), but also responds to a few peaks that are not ¯uorescent. Initial investigations into the identity of the HPLC peaks used for quanti®cation showed that these peaks were not carotenoids but had absorption spectra similar to chlorins. However, further investigation of some of these peaks by comparison with puri®ed standards was not carried out because detailed characterization of the individual chlorins in marine sediments is not the focus of this paper; rather, this new, non-destructive method for the approximate determination of chlorin con-centrations in marine sediments shows promise as a rapid screening tool for estimating paleoproductivity changes.
4. Discussion
Solid-phase ¯uorescence determination of chlorins in marine sediments appears to be a rapid, non-destructive method of estimating paleoproductivity changes. The
method has been shown to give similar results to extraction methods using ¯uorescence spectroscopy (Harris and Maxwell, 1995; Fig. 6a) and HPLC (Trees et al., 1985; Fig. 6b). In the concentration range of 1±50
mg/g chlorin, the SPF method shows some (<25%)
deviation from the other methods; this observation may be due to the possible interferences of water content, grain size, mineral composition, or self-quenching that are associated with the solid-phase ¯uorescence method. While the method might not be as accurate as the solvent extraction methods, solid-phase ¯uorescence analysis of chlorins has some distinct advantages: it is fast and non-destructive, and can, therefore, potentially be applied as a routine analysis for new cores or for archived sediment cores. By using a dierent optical geometry, such as a laser-®ber optic excitation source (Rudnick and Chen, 1997) and simple ®lters to dis-criminate the 650±700 nm emission lines, solid-phase ¯uorescence could be applied directly to sediment sur-faces much in the same manner as color analysis. This would give an estimation of chlorin concentration and therefore paleoproductivity (Harris et al., 1996) in sedi-ment cores at high resolution (the spot size of the exci-tation light can be focused to <1 mm) without disturbance to the core. Sediment could subsequently be sampled and analyzed for other chemical, geological, or biological properties, or archived intact. Archived sedi-ment cores such as the Ocean Drilling Program archives, could all be scanned relatively rapidly and inexpensively using this new technique, and the additional parameter related to paleoproductivity would greatly enhance subsequent detailed studies on those cores.
Acknowledgements
We thank John Warner for use of his HPLC, Dan Repeta for supplying authentic chlorophyll standards, Drs. Keely and Maxwell for helpful reviews and the UMassBoston internal grants program for support.
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