Estimating the Contribution of Spartina Anglica Biomass to Salt-Marsh Sediments Using Compound Speci®C Stable Carbon Isotope Measurements

Organic Geochemistry 30 (1999) 477±483

Estimating the contribution of Spartina anglica biomass to salt-marsh sediments using compound speci®c stable carbon isotope measurements

Ian D. Bull a, Pim F. van Bergen a, 1, Roland Bol b, Sue Brown c, Andrew R. Gledhill a, Alan J. Gray c, Douglas D. Harkness d, Simon E. Woodbury a, 2, Richard P. Evershed a,*

aOrganic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS, UK bInstitute of Grassland and Environmental Research, North Wyke, Okehampton EX20 2SB, UK cInstitute of Terrestrial Ecology, Furzebrook Research Station, Wareham, Dorset BH20 5AS, UK dNERC Radiocarbon Laboratory, Scottish Enterprise Technology Park, East Kilbride G75 0QF, UK Received 23 November 1998; accepted 11 February 1999 (Returned to author for revision 17 December 1998)

Abstract

13 Compound speci®c d C analyses were used to determine the relative input of a C4 temperate grass (Spartina anglica ) to primary biomass in a salt-marsh sediment. Lipid distributions revealed a C32 n-alkanol homologue as a characteristically dominant component of Spartina anglica whilst the cohabiting C3 species, Puccinellia maritima, exhibited a C26 maximum. The C32 n-alkanol component was used to create an isotopic mixing model, between organic matter derived from Spartina anglica and Puccinellia maritima, to estimate their relative contribution to the primary biomass input of salt-marsh sediments. The application of sedimentary lipid isotope data to the model gave values of Spartina anglica contributions ranging from 37 to 100%. This investigation represents the ®rst attempt to quantify inputs to sedimentary biomass based on compound speci®c stable carbon isotope techniques. # 1999 Elsevier Science Ltd. All rights reserved.

1. Introduction belonging to the family Poaceae and is a grass com- monly found in temperate salt-marshes (Vernberg, Spartina anglica is a monocotyledon angiosperm 1993). The origin of Spartina anglica can be traced to the accidental introduction of two species, Spartina maritima and Spartina alterni¯ora, to Britain in the early 19th century by shipping. Subsequent chromo- * Corresponding author. Tel.: +44-117-9287671; Fax: +44- some doubling of a sterile hybrid, Spartina townsendii, 117-9251295. produced the fertile amphidiploid hybrid, Spartina E-mail address: [email protected] (R.P. Evershed) anglica, which ``almost totally lacks in genetic vari- 1 Present address: Organic Geochemistry Group, Faculty of ation'' (Gray et al., 1991). As well as for its ability to Earth Sciences, Utrecht University, P.O. Box 80021, 3508 TA Utrecht, The Netherlands. stabilise/reclaim marsh-land, and the connected ben- 2 Present address: EKA Chemicals, 304 Worle Parkway, e®ts such as increased land for cattle grazing, Spartina Worle, Weston-super-Mare, Somerset BS22 6WA, UK. anglica has attracted additional attention. There has

0146-6380/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(99)00022-4 478 I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483 been speculation about using certain species of 2. Experimental Spartina as an `environmentally friendly' fuel source by taking advantage of the C4 metabolism, inherent to 2.1. Sampling the genus, which enables high eciencies of radiant energy conversion to be realised (Long, 1987; Long et Table 1 summarises the sediment and vegetation al., 1989; Carruthers, 1994). Indeed, studies concerning samples collected for this study. The majority of Spartina anglica have demonstrated that this attribute samples were collected from a salt-marsh at Wytch is retained even in cool temperate climates with night Farm which is part of the intertidal environment of and day temperatures of 7 and 108C respectively caus- Poole Harbour, Dorset, UK. Two algal samples, Ulva ing little change in the photosynthetic rate of the plant lactata and Enteromorpha intestinalis, were collected at (Matsuba et al., 1997). Furthermore, if relative quan- Cleaval Point, a lower energy environment, located ap- tities of Spartina anglica primary biomass in sediment proximately one mile east of the Wytch Farm site. can be ascertained these results could be correlated Sediment samples were exposed using a spade followed with the extensive recorded chronology of Spartina by careful sub-sampling of the disturbed sediment. All anglica to provide a data set from which inferences samples were air dried at 608C then crushed with a about carbon ¯ows in salt-marshes and the eects of pestle and mortar. Vegetation was frozen with N2(l) to climatic change on this species might be made. facilitate crushing. Sediment samples were Soxhlet Bulk stable carbon isotope analyses have already extracted using a dichloromethane:acetone (9:1 v/v) been utilised in determining the dietary composition of solvent system as per Lehtonen and Ketola (1993). macro-invertebrates indigenous to salt-marshes Dried algae and ¯ora were extracted ultrasonically also (Jackson et al., 1986). However, analogous sedimen- using a dichloromethane:acetone (9:1 v/v) solvent sys- tary studies are hampered by the incorporation of iso- tem. Total lipid extracts (TLEs) were fractionated topically heavy organic matter from marine algae, e.g. according to compound class and derivatized for Ulva lactata (16.0-). Such values fall between the analysis as per Bull et al. (1998). range bounded by Spartina anglica (12.1-) and Puccinellia maritima (26.9-), a marsh-grass currently 2.2. Gas chromatographic (GC) and gas invading the environmental niches occupied by chromatographic±mass spectrometric (GC±MS) Spartina anglica (Gray et al., 1991; Bull, 1997). By analyses using gas chromatography combustion±isotope ratio monitoring mass spectrometry (GCC±IRMS) it is GC analyses were performed using a Hewlett± possible to perform d13C measurements on speci®c Packard 5890 series II gas chromatograph ®tted with a sedimentary lipids derived from the primary biomass fused silica capillary column coated with a 100% inputs of higher plants. This route of study circum- dimethylpolysiloxane stationary phase (Chrompack vents the problem of algal contribution and reliable CPSil-5 CB, 50 m Â 0.32 mm Â 0.12 mm, H2 carrier measurements pertaining to the relative incorporation gas). Derivatized samples were injected (1.0 ml) via an of Spartina anglica biomass into sediments may be on-column injector as solutions in hexane. The tem- made. perature was programmed from 408C (1 min) to 2008C

Table 1 A summary of the samples collected

Sample Description

VegetationÐSpartina anglica Emergent aerial foliage obtained at the Wytch Farm site VegetationÐPuccinellia maritima Aerial foliage obtained at the Wytch Farm site AlgaeÐUlva lactata Whole algal sample obtained from a mud-¯at at Cleaval Point AlgaeÐEnteromorpha intestinalis Whole algal sample obtained from a mud-¯at at Cleaval Point AlgaeÐFilamentous (unidenti®ed) Thin mat present beneath and around Spartina anglica at Wytch Farm SedimentÐSpartina (oxic) Top (<7 cm) layer of brown sediment beneath Spartina anglica obtained at the Wytch Farm site SedimentÐSpartina (anoxic) Bottom (>7 cm) layer of grey sediment beneath Spartina anglica obtained at the Wytch Farm site SedimentÐPuccinellia Brown sediment beneath Puccinellia maritima obtained at the Wytch Farm site SedimentÐMud-¯at Grey sediment located 01 m away from the salt-marsh towards the water, obtained at the Wytch Farm site I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483 479 at a rate of 108C min1 then to 3008C at a rate of 38C ponents in each TLE are n-alkanols, dotriacontanol 1 min with an isothermal of 20 min (FID, 3008C). GC (C32: Spartina anglica ) and hexacosanol (C26: analyses of TLEs were made using an alternative col- Puccinellia maritima ), which have been established pre- umn capable of performing at higher temperatures viously as major lipid components in the epicuticular

(J&W Scienti®c DB1, 15 m Â 0.32 mm Â 0.1 mm, H2 leaf waxes of higher plants (Tulloch, 1976, 1981). carrier gas). The temperature was programmed from GCC±IRMS analysis of n-alkanols is relatively facile 508C (2 min) to 3508C at a rate of 108C min1 with an and is improved by a simple chemical separation of isothermal of 10 min (FID, 3508C). these components prior to analysis thereby making GC±MS analyses were performed on a Carlo Erba them ideal compounds to be used in this study. 5160 GC, using the same capillary columns and tem- Fig. 2 summarises the abundance of n-alkanol com- perature programs reported above but employing ponents observed to occur in the separated n-alkanol helium as a carrier gas and on-column injection fractions of Spartina anglica and Puccinellia maritima. coupled, via a heated transfer line, to a Finnigan MAT Quite clearly the C26 n-alkanol homologue in 4500 quadrupole mass spectrometer scanning in the Puccinellia maritima is excessively abundant compared range of m/z 50 to 850 with a cycle time of 1.5 s. The with that in Spartina anglica rendering any isotopic current was maintained at 300 mA with an ion source results obtained from its analysis prone to `swamping' temperature of 1908C. The mass spectrometer was eects biased towards the C3 grass. However, the operated with an electron voltage of 70 eV. abundance of the C32 homologue in the two species is similar making it a suitable component to monitor 2.3. Stable carbon isotope analyses relative contributions of Spartina anglica to the primary biomass of sediments. This may be done through Analyses were made on 1.0 ml sample aliquots using the construction of an isotopic mixing model. a Varian 3400 gas chromatograph on a fused silica capillary column coated with a 100% dimethylpolysi- 3.2. Construction of a mixing model loxane stationary phase (SGE BP-1, 50 m Â 0.32 mm Â 0.12 mm, He carrier gas), ®tted with a septum equipped Compound speci®c stable carbon isotope analyses temperature programmable injector (SPI) coupled to a have been used to great eect in the detection of veg-

Finnigan MAT Delta S stable isotope mass spec- etable oil adulteration where mixing models of C4 trometer (electron ionisation, 100 eV electron voltage, maize oil and a C3 adulterant vegetable oil, of lower 1mA electron current, 3 Faraday cup collectors for economic value, were constructed using d13C values m=z 44, 45 and 46, CuO/Pt combustion reactor main- obtained from C16:0,C18:1 and C18:2 n-alkanoic acids tained at a temperature of 8508C). d13C Values were (Woodbury et al., 1995). Binary oil mixture calibration obtained by correcting for the isotopic contribution of curves were constructed using an equation based on the derivatizing group as per Jones et al. (1991). six variables: the d13C value of a particular n-alkanoic acid and its relative abundance in each oil and the relative proportion of each oil in each mixture. The

3. Results and discussion mixing of Spartina anglica and C3 plant tissues can be considered analogous to the mixing of maize oil and

3.1. Lipid analysis C3 vegetable oil, however, it should be appreciated that the determination of relative inputs of a lipid to a Before undertaking any detailed analyses of sedi- sediment is several steps removed from determining ment samples it was necessary to establish a suitable that of biomass. Additional factors such as the water lipid component which could ultimately be used to content and growth rates of the plant species being assess relative contributions of Spartina anglica derived studied must be determined in order to ascertain the organic matter to sedimentary inputs of primary bio- contribution of a particular plant to a sedimentary mass. The main prerequisites for such a component `pool' of a lipid. The fraction of C32 n-alkanol derived were that it must be: (a) absent from the lipid pro®les from Spartina anglica in the primary biomass may be of indigenous algal species, (b) present in the lipid pro- represented thus: ®les of all ¯oral samples studied, (c) relatively abundant compared with other ¯ora derived lipid fs mslsrsps= mslsrsps mplprppp 1 components, and (d) present at roughly the same con- centration in the dierent species of ¯ora studied. where f is that fraction of the mixture derived from a Fig. 1 depicts partial gas chromatograms obtained particular plant, m is the mass of plant tissue, l is the from the TLEs of Spartina anglica and its primary relative abundance of C32 n-alkanol in the plant competitor, the C3 temperate grass Puccinellia mari- extract, r is the dry/wet weight tissue ratio of the plant tima. It may be observed that the most abundant com- and p is the primary production of the plant. A sub- 480 I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483

Fig. 1. Partial gas chromatograms of the total lipid extracts of: (a) Spartina anglica and (b) Puccinellia maritima. Bold text indicates the dominant component in a co-elution. (Key: ., n-alkane; q, n-alkanal; w, n-alkanol; Q, n-alkanoic acid; W, wax ester (integers in parentheses refer to the constituent n-alkanoic acid moieties as determined by MS); MAG, monoacylglycerol; DAG, diacylgly- cerol; TAG, triacylglycerol (notation before the glyceride designation refers to the identity of constituent n-alkanoic acid moieties as determined by MS); IS, internal standard). scripted `s' denotes values pertaining to Spartina diering periods of annual growth, the overall net pri- anglica whilst values related to Puccinellia maritima are mary production of Spartina anglica and Puccinellia identi®ed with a subscripted `p'. Since we are only maritima is about the same (Hussey and Long, 1982). dealing with a binary mixture the fraction of C32 n- Additionally, the dry/wet weight tissue ratios for the alkanol derived from Puccinellia maritima may be cal- two species are almost about equal (Bull, 1997). culated simply from the following equation: Hence, since rs 1 rp and ps 1 pp, fs may be calculated using a simpli®ed form of Eq. (1), i.e.: fp 1 fs 2 f m l = m l m l 4 The fractional values determined for the two plants s s s s s p p can then be used to predict the d13C value of the C 32 The model was tested by measuring the stable carbon n-alkanol component derived from an admixture of the isotope ratio of the n-alkanol component in arti®cial two plants thus: mixtures of Spartina anglica and Puccinellia maritima. Fig. 3 depicts the theoretical mixing curve generated dm fsds fpdp 3 by Eq. (3) superimposed by points relating to six arti®- where dm is the predicted isotope ratio of the C32 n- cial Spartina anglica:Puccinellia maritima mixes (0:1, alkanol with contributions from both Spartina anglica 3:7, 1:1, 7:3, 9:1 and 1:0). The C32 n-alkanol model and Puccinellia maritima. ds is the isotope ratio of the gives an acceptable correlation with the experimental 13 C32 n-alkanol component in Spartina anglica and dp is values. A similar model utilising d C values obtained the isotope ratio of the C32 n-alkanol component in from the C26 n-alkanoic acid component correlated Puccinellia maritima. It has been reported that despite very well with experimental values, however, the I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483 481

relative contribution between sediments with respect to Spartina anglica can be estimated from their physical

location. Estimates made using the C32 n-alkanol model give a wide range of values. The highest estimates are observed in the oxic (93%) and anoxic (100%) sediment sampled beneath a long standing sward of Spartina anglica indicating a dominant contribution from this plant to primary biomass. Sediment sampled beneath an invading patch of Puccinellia maritima gives a much lower value (46%) for Spartina anglica derived matter which is indicative of the rapid and substantial eect the new species has had on the primary biomass contribution to sedimentary organic matter. A correspondingly lower value (37%) is obtained from a mud-¯at sediment located next to the actual salt-marsh and currently supporting no vegetation. This indicates either a remnant signal from previous occupation by Spartina anglica or the result of physical movement of sediment and/or detritus from the salt-marsh true. It should be emphasised at this juncture that it is primary biomass which is being investigated this being distinct from total preserved sedimentary organic matter. Table 3 summarises the d13C values of primary biomass estimated, by substituting bulk d13C values for Spartina anglica and Puccinellia maritima into Eq. (3), after calculating the relative proportion contributed by each plant. It is interesting to note that the isotopic compositions of primary biomass in the Puccinellia maritima and mud-¯at sediments are close to that of the total sedimentary organic carbon. The d13C value for Puccinellia maritima sediment clearly re¯ects the

overlying C3 plant, whilst the somewhat similar value obtained for the mud-¯at sediment presumably re¯ects an admixture dominated by inputs from algal biomass

and terrestrial C3 biomass borne downstream. The Fig. 2. Histograms of the n-alkanol distributions in dried d13C values of sediments obtained from beneath samples of: (a) Spartina anglica and (b) Puccinellia maritima. Spartina anglica are not so similar to those estimated The black bars correspond to dry weight abundance whilst for primary biomass and exhibit dierences of 4- and the white bars correspond to wet weight abundance. 6- for the oxic and anoxic sediments respectively. Quanti®cations were made on n-alkanol fractions separated Such a dierence can be ascribed to an input from the from TLEs. unidenti®ed ®lamentous algae (18.9-) observed to grow in and around the swards of Spartina anglica. greater mobility of n-alkanoic acids in saline environ- This emphasises the need for a compound speci®c iso- ments has made their use, in this context, unfeasible tope method when determining primary biomass inputs (Meyers and Quinn, 1973; Bull, 1997). Having con- of Spartina anglica to salt-marsh sediments. Whilst the ®rmed the viability of the theoretical model the next choice of the species Spartina anglica and Puccinellia stage involved applying it to actual sedimentary data. maritima represents a signi®cant proportion of salt- marsh ¯ora it is by no means all encompassing. Future 3.3. Applying the model communications will address the incorporation of a greater number of ¯oral species into the model. Table 2 summarises the sediment samples used in this study and the associated values of Spartina anglica contributions determined using the model reported 4. Conclusions above. Whilst the exact quantity of Spartina anglica biomass in these sediments is currently unknown the This study has been concerned with the construction 482 I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483

Fig. 3. A plot of the theoretical Spartina anglica vs Puccinellia maritima stable carbon isotopic mixing curve obtained using the C32 n-alkanol component. The six experimental mixes and their associated errors are superimposed. of a lipid based stable carbon isotope mixing model to the methods used, we perceive this mixing model estimate the relative proportion of C4 Spartina anglica approach to be a potentially useful tool to be used in derived organic matter contributing to the primary the large scale mapping of Spartina anglica where: biomass in regions of a temperate salt-marsh. We (a) current vegetation or morphological remains might believe this method enables the ®rst determination of a not represent the actual composition of sedimentary quantity which cannot be obtained using more estab- organic matter or, (b) where there are no longer lished methods, i.e. lipid distributional analysis and any morphological remains from which to derive bulk d13C measurements. Although there is a recog- information concerning the growth of Spartina nised need for further re®nement and con®rmation of anglica.

Table 2 Estimates of the relative contribution of Spartina anglica to sedimentary biomass as determined by the proposed mixing model

Sample Estimated contribution (%) Maximum possible range due to errors (%)

SedimentÐSpartina (oxic) 93 80±100 SedimentÐSpartina (anoxic) 100 ± SedimentÐPuccinellia 46 35±58 SedimentÐMud-¯at 37 26±56

Table 3

A summary of stable carbon isotope values, obtained from the sedimentary C32 n-alkanol component [the fraction ( fs) derived from Spartina anglica is included] and bulk sediment, related to d13C values calculated for the primary biomass

13 13 13 Sample C32 n-alkanol d C/- fs Biomass d C/- Bulk d C/-

SedimentÐSpartina (oxic) 20.3 0.93 13.1 17.1 SedimentÐSpartina (anoxic) 19.7 1.00 12.1 18.1 SedimentÐPuccinellia 23.9 0.46 20.1 21.4 SedimentÐMud-¯at 25.0 0.37 21.4 20.4 I.D. Bull et al. / Organic Geochemistry 30 (1999) 477±483 483

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