Chemosphere xxx (2009) xxx–xxx

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Chemosphere

journal homepage: www.elsevier .com/locate /chemosphere

Compost may affect volatile and semi-volatile plant emissions throughnitrogen supply and chlorophyll fluorescence

Elena Ormeño a,1, Romain Olivier b,1, Jean Philippe Mévy b, Virginie Baldy b, Catherine Fernandez b,*

a Division of Ecosystem Sciences, Department of Environmental Science, Policy, and Management (ESPM), Berkeley University of California, 251A Mulford Hall Berkeley, CA 94720, USAb Aix-Marseille Université – Institut Méditerranéen d’Ecologie et de Paléoécologie (IMEP, UMR CNRS 6116), Equipe Diversité Fonctionnelle des CommunautésVégétales – Centre de St Charles, Case 4, 13331 Marseille Cedex 03, France

a r t i c l e i n f o a b s t r a c t

Article history:Received 5 February 2009Received in revised form 11 May 2009Accepted 14 May 2009Available online xxxx

Keywords:TerpenesSesquiterpenesBiogenic emissionsChlorophyll fluorescenceNutrientsEnvironmental stress

0045-6535/$ - see front matter � 2009 Published bydoi:10.1016/j.chemosphere.2009.05.014

* Corresponding author. Tel.: +33 (0) 4 88 57 69 94E-mail address: catherine.fernandez@univ-provenc

1 Equal contribution to the work.

Please cite this article in press as: Ormeño, E., etfluorescence. Chemosphere (2009), doi:10.1016

The use of composted biosolids as an amendment for forest regeneration in degraded ecosystems isgrowing since sewage–sludge dumping has been banned in the European Community. Its consequenceson plant terpenes are however unknown. Terpene emissions of both Rosmarinus officinalis (a terpene-storing species) and Quercus coccifera (a non-storing species) and terpene content of the former, werestudied after a middle-term exposure to compost at intermediate (50 t ha�1: D50) and high (100 t ha�1:D100) compost rates, in a seven-year-old post-fire shrubland ecosystem. Some chlorophyll fluorescenceparameters (Fv/Fm, ETR, UPSII), soil and plant enrichment in phosphorus (P) and nitrogen (N) were mon-itored simultaneously in amended and non-amended plots in order to establish what factors wereresponsible for possible compost effect on terpenes. Compost affected all studied parameters with theexception of Fv/Fm and terpene content. For both species, mono- and sesquiterpene basal emissions wereintensified solely under D50 plots. On the contrary leaf P, leaf N levels reached in D50 were partly respon-sible of terpene changes, suggesting that optimal N conditions occurred therein. N also affected ETR andUPSII which were, in turn, robustly correlated to terpene emissions. These results imply that emissions ofterpene-storing and non-storing species were under nitrogen and chlorophyll fluorescence control, andthat a correct management of compost rates applied on soil may modify terpene emission rate of plants,which in turn has consequences in air quality and plant defense mechanisms.

� 2009 Published by Elsevier Ltd.

1. Introduction

Since sewage–sludge dumping has been banned in the Euro-pean Community and its disposal has low cost, there is renewedinterest in studying novel ways for recycling it. This interest isadditionally reinforced since the annual amount of sewage–sludgeproduced in Europe is expected to increase in the future, mainlydue to larger amounts of high quality drinking water needed byan increasing population and to increasing demands for cleanersewage water (Laturnus et al., 2007). Due to these circumstances,fresh and composted sludge from sewage treatment plants has reg-ularly been amended in cultivated areas as a way to valorize com-post in agriculture, forestry, and landscaping. In France, the totalsewage–sludge production accounts for 11.3% of the total sew-age–sludge production in the European Union (Laturnus et al.,2007). Particularly, the annual sludge production of urban originis 85 000 t (dried matter); 60% of these residues are recycled in

Elsevier Ltd.

; fax: +33 (0) 4 91 10 70 91.e.fr (C. Fernandez).

al. Compost may affect volatile/j.chemosphere.2009.05.014

agriculture, 20–25% are stored in controlled landfills, and 10–15%are incinerated (Courtois, 2000).

The possible impact of soil enrichment with compost on Bio-genic Volatile Organic Compound (BVOC) emissions has hithertonever been tested, whereas BVOCs, such as mono- and sesquiter-penes, affect the plant, the ecosystem and the atmosphere. Terp-enes account for plant defenses against pests, herbivores,diseases and environmental pressures (Gouinguene and Turlings,2002; Boege and Marquis, 2005). They have a role in protectingleaves against oxidative and thermal stresses (Loreto et al., 2004;Grote and Niinemets, 2008) through such processes as enhancingmembrane stability and scavenging reactive oxygen species owingto the presence of double bonds in their molecular structure. Inaddition, BVOC are involved in the ecosystem functioning and per-turbation partly by affecting plant flammability and fire risk (Or-meño et al., 2009). BVOC also participate to the photochemicalbalance of the troposphere as they are readily oxidized by O3,OH, and NO3. They also contribute to ozone formation, via photo-chemical reactions with anthropogenic Nitrogen oxides (NOx),and contribute to OH formation even under low NOx environments(Lelieveld et al., 2008). Finally, deposition of BVOC-reaction

and semi-volatile plant emissions through nitrogen supply and chlorophyll

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products may favor aerosol formation which in turn affects theclimate (Lelieveld et al., 2008).

The study of the possible impact of compost on BVOCs of Med-iterranean species is of special interest, since soil amendment withsewage–sludge compost is especially frequent in the Mediterra-nean area as a mean for improving soils that are naturally rela-tively poor in organic matter, phosphorus (P) and nitrogen (N).This practice also aims to prevent or reduce soil erosion andimpoverishment caused by fire recurrence and violent precipita-tions. Compost could affect plant terpene emissions since N andP, which are supplied via compost amendment (Larchevêqueet al., 2009), may induce long-term changes in soil chemical com-position and hence, positive or negative fluctuations of mono- andsesquiterpene emissions (Ormeño et al., 2007a). N is expected to beinvolved in terpene emissions since it increases leaf photosyntheticactivity (Lerdau et al., 1995), which generally influences light-dependent terpene emissions (in species without specific terpenestorage pools) but also light-independent terpene emissions (inspecies with storage pools), since the carbon for any terpene syn-thesis ultimately comes from photosynthesis (Niinemets et al.,2002a). In addition, terpene precursors contain high-energy phos-phate bonds and P is a key component of ATP and NADPH whichare required for terpene synthesis. Niinemets et al. (2002b) esti-mated that Quercus coccifera requires 28 moles of NADPH and40 moles of ATP to synthesize monoterpenes. P could hence be akey limiting nutrient involved in terpene emission.

Some studies have been carried out on the effect of fertilizationon BVOC emissions. Most of them are focused on isoprene, a non-stored compound (e.g. Fares et al., 2008), rather than monoter-penes and sesquiterpenes (Blanch et al., 2007). However, onlyfew studies have provided information on the consequences ofcompost on terpene content in plants (Tanu et al., 2004; Husseinet al., 2006). The observed effects of fertilizers vary according tothe terpene compound (Powell and Raffa, 1999), the leaf develop-mental status (mature or young leaves) (Kainulainen et al., 1996),the plant phenology (Lerdau et al., 1995), and the species (Blanchet al., 2007). Although one could presumably claim that storedand emitted terpenes have the same pattern against a given envi-ronmental factor, their variation is not necessarily the same, par-tially because the terpene emission rate is usually not properlypredicted from their concentration in leaves (Peñuelas and Llusià,1997; Ormeño et al., 2007b). An explanation to this lack of rela-tionship can be found in the study of Steinbrecher et al. (1999)which suggested that emissions from both specific leaf reservoirsand de novo synthesis occur in storing species.

The main purpose of this work was to study whether a middle-term exposure to compost influences terpene emission and contentof a storing species, and terpene emission of a non-storing species,through changes in (i) nitrogen (N) and phosphorus (P) contents, or(ii) possible changes in chlorophyll fluorescence, as terpene emis-sions may be highly sensitive to light reactions of photosynthesis(Niinemets et al., 2002a). Secondarily, we also tested the relation-ship between (i) stored and emitted terpenes, (ii) mono- and ses-quiterpenes, to evaluate whether the former could be used tocalculate the later.

2. Materials and methods

2.1. Experimental set-up, species studied and site conditions

The experiment was carried out in a post-fire shrubland, burnedin 1995, in the vicinity of Marseille (Provence, Southeastern France;5�180600E–43�2901000N), 240 m above sea level and under Mediterra-nean climatic conditions. The silty–clayey chalky soil had a highpercentage of stones (70%) and a low average depth of about

Please cite this article in press as: Ormeño, E., et al. Compost may affect volatilefluorescence. Chemosphere (2009), doi:10.1016/j.chemosphere.2009.05.014

24 cm. On this site, compost was surface-applied in January 2002.The experiment was arranged in randomized blocks with threecompost treatments and three repetitions (blocks) per composttreatment. Plot surface was 500 m2, making a total surface of4500 m2. Three plots did not receive any compost (D0 = controlplots), three received 50 t ha�1 (D50) and the three others100 t ha�1 of fresh compost (D100).

The compost, produced by a local company (Biotechna Ensuès,Southeastern France), was made with greenwastes (1/3 volume),pine bark (1/3 volume) and local municipal sewage sludge (1/3 vol-ume). The mixture was composted for 30 days at 75 �C to kill path-ogenic microorganisms and decompose phytotoxic substances, andthen sieved (<20 mm mesh) to remove large bark pieces and storedin swathes. The swathes were mixed several times over the nextsix months to promote organic matter humification. The finalsix-month maturity compost met the French legal standards (NFU 44-095) for pathogenic microorganisms, organic trace elementsand trace metals, on composts made from materials of water treat-ment origin (Larchevêque et al., 2006).

Two typical Mediterranean sclerophyl evergreen shrub species:Rosmarinus officinalis L. and Q. coccifera L. were selected to investi-gate the impact of compost on BVOC storage and emission. Thefirst species presents leaf specialized structures (i.e. gland tric-homes) where terpenes are stored, while the latter species doesnot show such specialized structures, i.e. it only exhibits non-spe-cific terpene storage in intercellular spaces or small newly synthe-sized pools in chloroplasts, temporarily. Hence, whereas for R.officinalis both BVOC emissions and storage were considered, onlyBVOC emissions were analyzed for Q. coccifera.

2.2. BVOC emission sampling and analyses

BVOC emission measurements were carried out from 10th to20th July 2007. Emissions were sampled on three plants per plot.For each plant, a little healthy and sun exposed twig was carefullyenclosed with a dynamic Teflon-made bag. On average the total fo-liar mass enclosed was 1.3 ± 0.1 g of Dry Matter (DM). The system,fully described in Ormeño et al. (2007a,b,c) consisted of 16 bagenclosure systems. Each bag enclosure (3 l) was designed with in-let and outlet air streams. Before any sampling was undertaken, airin the bag was renewed with filtered air (Alphagaz Type 1, 99.99%purity) at 695.5 ± 40.5 mL min�1 during 30 min. Air flow was mea-sured with a digital mass flow controller (Aalborg� CFC17. 0–500 mL). Terpene sampling took place with continued inflow ofclean air. The outgoing terpenes from each bag enclosure were col-lected on glass tubes, filled with preconditioned Tenax TA (Var-ian�), using pumping system, placed downstream of theadsorbent tubes. Outflowing air (Qout: 53.9 ± 7.1 mL min�1) at eachbag enclosure was precisely measured with a bubble flowmeter(0–280 mL min�1 GPE, ‘MeTeRate’ 314–140/084 tube; PrecisionEngineering Ltd., Hemel Hempstead, UK), placed immediately aftereach Tenax. Terpene sampling took place during 10 min. After sam-pling, all Tenax TA were immediately placed in a portable refriger-ator at +4 �C until being stored at �20 �C in the laboratory within aperiod not longer than 2 h.

Photosynthetically Active Radiation (PAR) was measured (Porta-ble photo system plant and canopy transmission meter, EMS-7Model; Surechem� Marketing SDN, BHD, Kuala Lumpur, Malaysia)outside bag enclosures. PAR values were automatically recordedevery minute (Organizer II, Psion�, Digitron instrumentation,LZ64 Model, Psion PLC, UK). Air temperatures inside the bag enclo-sure were measured (38.9 ± 2.2 �C). Emissions were sampled from12:00 to 3:00 pm (local hour), during which environmental tem-perature and PAR values ranged from 34 to 38 �C and 1157 to1364 lmol s�1 m�2, respectively. The order of sampled plants wasrandomized to minimize any eventual effect of circadian rhythm.

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After emission trapping, leaves of each twig were cut off andstored in liquid nitrogen, until being stored at �20 �C in the labo-ratory. They were then lyophilized (Liovac-GT-2E Steris� Hürth,Germany) to calculate their foliage DM (dry mass).

2.3. Terpene emission analyses (GC–FID) and basal emissioncalculation

Tenax TA cartridges with adsorbed terpenes were analyzed byGas Chromatography (GC) fitted with a Flame Ionization Detector(FID) (HP�5890 series II, Agilent, Palo Alto, CA, USA) and equippedwith a non-polar chromatographic column (Ultra 2,50 m � 0.2 mm � 0.25 lm). Further details on the chromatographysystem can be found in Ormeño et al. (2007a,b,c). The identity ofpeaks was checked by comparing their retention times with thoseof the commercial standards (purity higher than 90%) (Sigma–Al-drich� and Firmenich).

For the storing species R. officinalis, monoterpene and sesquiter-pene basal emissions were calculated using the relationship be-tween temperature and monoterpene emissions shown inGuenther et al. (1995), since temperature is considered as the mainenvironmental parameter controlling monoterpene emissions ofstoring species (Hansen et al., 1997). Through this algorithm, emis-sions are standardized at 30 �C temperature. Emissions for the non-storing Q. coccifera were normalized to the basal conditions understandard conditions of 30 �C temperature and 1000 lmol m�2 s�1

PAR after the algorithm shown in Guenther et al. (1995), sinceemissions of this Mediterranean oak are temperature and lightdependent (Niinemets et al., 2002a). Throughout the paper, emis-sions shown are basal emission unless field emission is specified.

2.4. BVOC content extraction and analyses

Terpene extracts were obtained on the same leaves enclosed forterpene emission sampling. Prior to their terpene extraction, leaveswere lyophilized in order to express results on a leaf DM basis. Thisdrying process allows water removal from plant tissues withoutlosses of terpene content (Ormeño et al., 2007b). The method usedfor terpene extraction is based in that shown in Ormeño et al.(2008). Briefly, it consisted in dissolving 1 gDM of ground leavesin cyclohexane for 60 min, under constant shaking at room tem-perature. A non-terpenoid volatile internal standard (dodecane)was added to the cyclohexane extraction.

Analyses were performed using GC (Hewlett Packard GC6890�,Agilent, Palo Alto, CA, USA) coupled to a Mass Selective Detector(MSD, HP 5973 N). The HP-5MS capillary column (0.25 mm,30 m, 0.25 lm – JW Scientific), in constant flow mode, was con-nected directly to the MSD. All further details on injection andanalysis parameters are given in Ormeño et al. (2007b), with theexception of the electron multiplayer energy of the MSD whichwas 1950 V in this study. Identity of stored terpenes was estab-lished by comparison of the retention time and the mass spectrumof detected compounds with those of the authentic reference sam-ples (Aldrich–Firmenich).

2.5. Chlorophyll fluorescence measurements

Fluorescence measurements, performed to assess their impactin terpene emissions, were carried out simultaneously to BVOCsampling, but on a different branch. For each branch, these mea-surements were carried out on three full expanded and sun ex-posed leaves per plant. Measurements were performed using aportable Fluorescence Monitoring System (FMS 2, Hansatech�,Kings Lynn, Norfolk, UK), a leaf clip holder for adapting leaves todarkness conditions, and a PAR/temperature leaf-clip for ambientlight environment.

Please cite this article in press as: Ormeño, E., et al. Compost may affect volatilefluorescence. Chemosphere (2009), doi:10.1016/j.chemosphere.2009.05.014

The variable to maximum fluorescence ratio (Fv/Fm), the actualphotochemical quantum yield (UPSII) and the Electron TransportRate (ETR) were studied. Fv/Fm, measured in the non-energizedstate after darkness adaptation for about 30 min, is a reliable mea-sure of the maximal (potential) efficiency of excitation capture byopen PSII in dark-adapted conditions. Fv/Fm is used as an estimateof the functional state of the photosynthetic apparatus at a givenenvironmental situation (Krause and Weis, 1991). A decrease inFv/Fm may indicate photo-inhibition of PSII. UPSII and ETR weremeasured from light-adapted leaves. UPSII measures the efficiencyof PSII electron transport and was calculated according to theequation:

UPSII ¼ ðF 0m � FsÞ=F 0m

where Fs is the fluorescence under ambient light pulses and F 0m un-der saturating light pulses. ETR represents the apparent photosyn-thetic electron transport rate leading to carbon fixation and isproportional to the photosynthetic activity during the course ofthe day (Flexas et al., 1999). This parameter was calculated by mul-tiplying the quantum yield of PSII by 0.84, which is the averageabsorbance of leaves, and by half PAR (Photosynthetically ActiveRadiation), since it is assumed that radiant energy is equally ab-sorbed by photosystems I and II:

ETR ¼ PAR � 0:5�UPSII � 0:84

2.6. Soil and leaf properties analyses

In soils, total N (N), extractable P (PE), total organic carbon andpH were analyzed in the laboratory. The Kjeldahl and Anne meth-ods were used for N and total organic carbon analyses, respec-tively. PE was determined in a sodium bicarbonate solutionusing spectrophotometry, following the Olsen method. pHH2O

was measured in distilled water (PHM 240, Radiometer SAS, Neu-illy Plaisance, France). Soils were collected from (i) the humusfraction of the organic horizon, and (ii) the overall accessible min-eral horizon down to bedrock, since mineral horizons could not bedistinguished as usually occurs in other Mediterranean areas. Foreach soil horizon, a sample was collected within each plot. Each ofthese samples was a composite of three samples randomly col-lected on each 500 m2 plot. Before analyses, soil samples weresieved (2 mm sieve) and oven-dried (70 �C) till constant weight.The mass of dry soil used for analyses ranged between 2 and2.5 g.

In leaves, total N was analyzed in the laboratory through theKjeldahl method. Leaf P content was analyzed in an external labo-ratory (SAS laboratory, France) by extracting P with nitric and chlo-ridric acid, and quantifying it by ICP (Inductively Coupled Plasma).Leaves were harvested from three plants per plot. These plantswere the same as those used for sampling BVOCs, but leaves camefrom a close branch with the same sun exposure since leaf DM con-tained in the BVOC sampled branch was not enough to carry outboth nutrient and terpene content analyses. Before analyses, leafsamples were dried by lyophilization. The mass of dry leaves usedfor N and P analyses was 2 and 0.5 gDM, respectively.

2.7. Statistical analyses

Linear and non-linear regression analyses were applied to testthe relationship between (i) leaf N content and terpene emissions,(ii) leaf P content and terpene emissions (iii) chlorophyll fluores-cence and emitted terpenes, (iv) stored and emitted terpenes,excluding oxygenated stored sesquiterpenes since oxygenated ses-quiterpenes were not detected in emission analysis, and (v) mono-terpenes and sesquiterpenes whether emitted or stored. Only forpoints (iv) and (v) field emissions instead of basal emission were

and semi-volatile plant emissions through nitrogen supply and chlorophyll

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used. Then, the effect of compost on leaf terpene storage, terpenebasal emissions, N, P, chlorophyll fluorescence and soil propertieswas tested through a one-way ANOVA test. For basal emissions,data were previously log-transformed in order to conform to thenormal data distribution and the homocedasticity (not significantdifferences between variances) required for the ANOVA test. Then,post-hoc Tukey test was applied to compare pairs of means be-tween the different compost treatments. Statistical analyses werecarried out with Statistical Graphics Plus�, version 4.1 (StatpointOnc, North Virginia, USA).

3. Results

3.1. Compost effect on soil properties and plant nutrition

While in the humic organic fraction both N and PE were signif-icantly higher in D50 and D100 plots in comparison to control plots(ANOVA and Tukey test, P < 0.05, Table 1), in the mineral fraction ofthe soil, only concentration of PE was significantly higher in com-post-amended plots (ANOVA, and Tukey test, P < 0.05, Table 1).As total organic carbon did not vary significantly with compostin humic fractions, C/N ratio was lower in amended plots (ANOVAand Tukey tests, P < 0.05, Table 1).

N content in leaves of R. officinalis and Q. coccifera varied signif-icantly among compost rates (ANOVA, P < 0.05, Table 1). As com-post rate increased, leaf N content for both species increased(Tukey test), indicating a greater leaf N uptake. P content in leavesof R. officinalis varied significantly according to the compost rate(ANOVA, P < 0.05, Table 1), with plants in D50 and D100 presentingapproximately twice P concentration of non-amended plants (Tu-key test, Table 1). Contrary to R. officinalis, P content in Q. cocciferafoliage was not sensitive to amendment (ANOVA, P > 0.05, Table 1),since plants growing on D0, D50 and D100 plots showed the sameconcentrations (0.06%DM, Table 1).

Table 1Mineral and organic soil properties in D0, D50 and D100 plots (plots without compost, withcontents in leaves for the different compost treatments are also shown. Values shown are mbetween plots with different compost rates are tested through ANOVA followed by Tukdifference between plots with different compost rates (a < b < c, and thus similar letters indnot occur. TOC: total organic carbon. F: ANOVA value. ns: not significant differences.

pH (H2O) TOC (%) Total N (K

Soil horizMineral horizon

D0 8.05 ± 0.05 3.86 ± 0.58 2.09 ± 0.5D50 7.82 ± 0.08 6.69 ± 1.28 3.69 ± 1.0D100 7.95 ± 0.06 4.64 ± 0.91 1.80 ± 0.2F 3.20ns 2.22ns 2.37ns

Organic horizonD0 7.22 ± 0.22 16.29 ± 2.32 6.88 ± 1.1D50 7.37 ± 0.05 17.22 ± 1.02 12.95 ± 0D100 7.24 ± 0.09 19.65 ± 1.60 13.24 ± 0F 0.35ns 1.00ns 18.46**

LeaveRosmarinus officinalisD0 0.82 ± 0.0D50 0.95 ± 0.0D100 1.05 ± 0.0F 28.42***

Quercus cocciferaD0 0.86 ± 0.0D50 0.98 ± 0.0D100 1.08 ± 0.0F 18.42***

* P < 0.05.** P < 0.01.*** P < 0.001.

Please cite this article in press as: Ormeño, E., et al. Compost may affect volatilefluorescence. Chemosphere (2009), doi:10.1016/j.chemosphere.2009.05.014

Regarding pH, compost pH was initially very close to soil pH(less than 0.4 unit difference; Larchevêque et al., 2006) and no sig-nificant differences between treatments were found in the frac-tions analyzed (ANOVA, P > 0.05, Table 1).

3.2. Effect of compost and nutrients in chlorophyll fluorescence

Values obtained for the maximal efficiency of PSII (Fv/Fm) werecomprised between 0.74 and 0.76 for the two species, indepen-dently of the compost rate (Fig. 1A and B, ANOVA, P > 0.05). ForQ. coccifera, a significant deficiency in the photochemical quantumyield (UPSII) (Fig. 1D) and the Electron Transport (ETR) (Fig. 1F)were observed in D0 and D100 plots, in comparison with plantsin D50 (ANOVA, P < 0.05). R. officinalis showed a similar trend,although changes in UPSII (Fig. 1C) and ETR (Fig. 1E) were not signif-icantly different according to the compost rate (ANOVA, P = 0.42).

When data from D0 and D50 were considered in the regressionanalysis, leaf N was positively correlated with ETR and UPSII of bothQ. coccifera and R. officinalis, (P < 0.05, Fig. 2A and C). Contrastingly,when data from D50 and D100 plants were taken into account,high leaf N concentration resulted in a significant diminution ofETR (P < 0.05, Fig. 2B and D). Because Q. coccifera showed the sameleaf P concentrations in amended and non-amended plots (cf. 3.1)the relationship between P and fluorescence parameters was onlyperformed for R. officinalis (Fig. 2E–H). When leaf P ranged from0.07 (D0) to 0.13%DM (D50) high P concentrations favored ETRand UPSII of R. officinalis (P < 0.001, Fig. 2E–G), while no relationshipwas detected when PE concentrations ranged between 0.13%DM

(D50) and 0.15%DM (D100) (P > 0.10, Fig. 2F and H).

3.3. Leaf terpene content and emissions

R. officinalis stored 15.22 ± 3.85 mg gDM�1 on average. Monoter-

penes were the main stored compounds with a-pinene, 1,8-cineole

50 t ha�1 and 100 t ha�1 of fresh compost rate, respectively). Nitrogen and phosphorusean ± standard error (SE) for three replicates per compost rate. Significant differences

ey post-hoc test. Different letters obtained through Tukey test denote a significanticate no significant differences). When letters are not shown, significant differences do

jeldhal) (%DM) Extractable P (%DM) C/N

ons

3 10.05 ± 2.40 (a) 21.67 ± 7.720 31.27 ± 6.30 (b) 20.20 ± 6.293 28.66 ± 5.17 (b) 27.58 ± 7.44

5.55* 0.30ns

4 (a) 24.67 ± 4.91 (a) 23.90 ± 0.74 (b).62 (b) 252.08 ± 4.23 (b) 13.28 ± 0.16 (a).66 (b) 258.25 ± 15.58 (b) 14.80 ± 0.68 (a)

186.46** 94.75**

Total P (%DM)s

1 (a) 0.07 ± 0.003 (a)2 (b) 0.13 ± 0.008 (b)3 (c) 0.15 ± 0.009 (c)

31.75***

5 (a) 0.06 ± 0.0023 (b) 0.06 ± 0.0013 (c) 0.06 ± 0.002

0.68ns

and semi-volatile plant emissions through nitrogen supply and chlorophyll

0.0

0.1

0.2

0.3

0.4

0.5P

SII

F = 0.82nsCa

b

a

F = 3.58*D

0

100

200

300

400

ETR

(µ

mol

ele

ctro

ns m

-2s-1

)

F = 0.97nsE

D0 D50 D100 D0 D50 D100

ab

a

F = 3.78* F

0.0

0.2

0.4

0.6

0.8

1.0

Fv/F

m

F = 0.75nsA F = 0.19nsB

Rosmarinus officinalis Quercus coccifera

Fig. 1. Fv/Fm (maximal quantum efficiency of PSII in dark-adapted state), UPSII (actual quantum yield of PSII in the light-adapted state) and electron transport rate (ETR), ofRosmarinus officinalis and Quercus coccifera in D0, D50 and D100 plot (control pots: without fresh compost, plots with 50 t ha�1 and 100 t ha�1, respectively). ANOVA followedby Tukey post hoc test was applied to test significant differences in chlorophyll fluorescence between plots with different compost rates. Different letters, obtained throughTukey test, denote significant statistical differences (a < b < c). Bars indicate mean for n = 9 ± SE. The absence of letters denotes that differences were not statisticallysignificant (ns) at 95% of confidence.

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and camphor as major compounds (Fig 3A and B). Sesquiterpenes,mainly represented by a- and b-caryophyllene, accounted only for13.4% of the total content. There was, however, a great diversity ofboth monoterpenes (22 different compounds) and sesquiterpenes(25 different compounds). Overall monoterpene and sesquiterpenecontents of R. officinalis were highly correlated (P < 0.01, Fig. 4A).

Mean basal terpene emission rates of R. officinalis and Q. coccif-era were 4.79 and 4.35 lg gDM

�1 h�1, respectively and were mostlyrepresented by monoterpenes (Fig. 3C and E). Sesquiterpenes, witha-humulene, a-copaene and b-caryophyllene as the most repre-sentative, accounted only for 6.1% and 8.8% of total emissions,respectively. Major compounds (i.e. those accounting for at least10% of total emissions) released by both species were a-pinene,myrcene and b-pinene (Fig. 3D and F). For R. officinalis, limonenealso accounted for an important fraction of the total emitted terp-enes (9.3%, Fig 3D), closely followed by 1.8-cineole (8.4 ± 0.1%, Sup-plementary material). As for terpene content, a positive linearrelationship between overall sesquiterpenes and monoterpenes(field emissions) was found both for Q. coccifera and R. officinalis(P < 0.001, Fig. 4B). Moreover, monoterpene emissions (field emis-sions instead of basal emissions) of R. officinalis could not be signif-icantly estimated from monoterpene content (linear and non-linear regression analyses, P > 0.05). Only leaf sesquiterpene emis-sions were significantly and positively correlated with leaf sesqui-terpene content (P < 0.01, best fitting model: y = 2.19 � 10�6 x1.55,R2 = 0.26, figure not shown).

3.4. Effect of compost, nutrients and chlorophyll fluorescence onterpenes

For R. officinalis, neither the concentration of the overall content(monoterpenes and sesquiterpenes) (Fig. 3A), nor that of majorcompounds (Fig. 3B) was significantly modified by compost rate(ANOVA, P > 0.05). In addition, foliar N and P were not significantly

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correlated with leaf terpene content (0.2 < R2 < 0.10, data notshown).

Nevertheless, overall and major terpene emissions of both R.officinalis and Q. coccifera were significantly modified by compost(Fig. 3C and F, ANOVA, P < 0.05). For both species, mono- and ses-quiterpene emissions were significantly elicited by intermediatecompost rates (D50), while under high compost rates (D100), emis-sions were as low as in control plots (Tukey test). Terpene emis-sions of plants from non- and highly amended plots representedonly 35% and 19%, respectively of emissions from plants growingunder intermediate compost rates.

The study of the relationship between foliar N content and ter-pene emissions only clarified, in part, results reported above(Fig. 5A–D). When data from control and D50 plots were taken intoaccount, corresponding to a N range from 0.735%DM to 1.051%DM, apositive relationship (Fig. 5A and C, P < 0.05) appeared between fo-liar N content and either mono- or sesquiterpenes. When data fromD50 and D100 were considered, corresponding to a N range from0.870%DM to 1.217%DM, monoterpene emissions of R. officinalisand Q. coccifera were negatively correlated with foliar N content(Fig. 5B, P < 0.05). For sesquiterpene emissions, the same tendencywas observed (Fig. 5D, 0.05 < P < 0.10).

Unlike N, leaf P content of Q. coccifera could not be used to pro-vide information about changes in terpene emission rates, since itsfoliage was not enriched in P after compost amendment (Table 1).Additionally, leaf P content of R. officinalis tended to be positivelycorrelated with basal terpene emissions only when data from D0and D50 were considered in the linear and non-linear regressionanalyses (Fig. 5E and G, 0.05 < P < 0.10).

ETR and UPSII were positively correlated with both mono- andsesquiterpene basal emissions of Q. coccifera and R. officinalis(P < 0.05, Fig. 6A–D). The relationship was especially significant be-tween the photosynthetic ETR and monoterpene emissions of Q.coccifera (P < 0.001, Fig. 6A).

and semi-volatile plant emissions through nitrogen supply and chlorophyll

210

240

270

300

330

360

390

ETR

(µm

ol e

lect

rons

m -2 s

-1)

A( ) y =150.3x + 139.9 R2 =0.48**

(+) y =102.8x + 183.5 R2 = 0.31*

B( ) y = - 237.6x + 476.8 R2 = 0.23*

(+) y = - 176.0x + 453.8 R2 =0.31*

210

240

270

300

330

360

390

ETR

(µm

ol e

lect

rons

m -2 s

-1)

E( ) y = 238.4x + 355.1 R2 = 0.66*** F

P > 0.10

0.20

0.25

0.30

0.35

0.40

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

C

( ) y = 0.5x + 0.1R2 = 0.51***

(+) y = 0.3x + 0.1R2 = 0.30*

ΦPS

II

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

D

P > 0.10

(+) y = -0.2x + 0.5R2 =0.28*

Total leaf nitrogen content (%DM)

0.20

0.25

0.30

0.35

0.40

0.05 0.10 0.15 0.20

G

ΦPS

II

( ) y =0.2x + 1.1 R2 = 0.49***

0.05 0.10 0.15 0.20

HP > 0.10

Total leaf phosphorus content (%DM)

INTERMEDIATE AND HIGHLYAMENDED PLOTS

CONTROL AND INTERMEDIATE AMENDED PLOTS

Fig. 2. Relationship between foliar N (A–D) or P (E–H) contents of Rosmarinus officinalis (dotted line and s) and Quercus coccifera (continued line and +) and chlorophyllfluorescence, measured through ETR and UPSII. Data from control and intermediate amended plots (left graphs) and data from intermediate and highly amended plots (rightgraphs) are analyzed separately. For leaf P, only results for Rosmarinus officinalis are shown since P concentration in leaves of Quercus coccifera was the same in D0, D50 andD100 plots. P: relationship significance. (n = 18). *0.01 < P < 0.05, and **0.001 < P < 0.01, ***P < 0.001.

6 E. Ormeño et al. / Chemosphere xxx (2009) xxx–xxx

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4. Discussion

4.1. Terpene content and emissions

Quantitative and qualitative results reported here for terpenecontent of R. officinalis are mostly in accordance with results re-ported in Ormeño et al. (2007b). This study corroborates that bothR. officinalis and Q. coccifera are essentially monoterpene emitters.For R. officinalis, Hansen et al. (1997) showed a basal emission rate(at 30 �C) of 1.84 lg gDM

�1 h�1 in the Mediterranean area duringspring, which is lower than the emission rate here reported. Thesedifferences are likely to be attributable to seasonality, since BVOCemissions of R. officinalis fluctuate over the seasonal cycle (Peñu-elas and Llusià, 1997). For Q. coccifera, summer basal emissions

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(at 30 �C and 1000 lmol s�1 m�2), measured by Ormeño et al.(2007c), were very close to the emission rates shown in the presentstudy. Furthermore, major compounds found for the studied spe-cies (a-pinene, myrcene and ß-pinene) are consistent with previ-ous literature (R. officinalis: Ormeño et al., 2007a; Q. coccifera:Ormeño et al., 2007c).

4.2. Relationship between emitted and stored terpenes

Consistently with Peñuelas and Llusià (1997), and Ormeño et al.(2007b), this research study highlights the lack of relationship be-tween emitted and stored monoterpenes for R. officinalis, suggest-ing that a fraction of the overall monoterpenes of R. officinalis couldbe emitted to the atmosphere directly after synthesis, instead of

and semi-volatile plant emissions through nitrogen supply and chlorophyll

0

4

8

12

16A

Rosmarinus officinalis

(mg

gDM

-1)

Ove

ral t

erpe

ne c

onte

nt

0

2

4

6

8B

Maj

or te

rpen

e co

nten

t(m

g gD

M-1)

Rosmarinus officinalis

Maj

or b

asal

em

issi

ons

(µg

g D

M-1

h -1)

0.0

0.5

1.0

1.5

2.0

ab aab

b

aab

b

a

ab b

a

b

DRosmarinus officinalis

Quercus coccifera

0.0

0.5

1.0

1.5

2.0

a

b

ba

b

aa

aa

F

D0 D50 D100

0

1

2

3

4

5

a

b

ab

a

Rosmarinus officinalis

a

CO

vera

ll b

asal

em

issi

ons

(µg

g D

M-1h-1

)

D0 D50 D1000

1

2

3

4

5

a

b

aaba

Quercus cocciferaE

Compost dose Compost dose

1.8 cineolecamphor

- pinenemonoterpenes sesquiterpenes

myrcene

- pinene

limonene

Fig. 3. Emission and concentration of overall monoterpenes, sesquiterpenes, and major compounds of leaves of Quercus coccifera and/or Rosmarinus officinalis in D0, D50 andD100 plots (plots without compost, plots with 50 t ha�1 and 100 t ha�1, respectively). Different letters, obtained through Tukey post hoc test, denote significant statisticaldifferences (P < 0.05) between emissions in plots with different compost rates (a < b, i.e. a = ab). Bars indicate mean for nine replicates ±SE.

0

1

2

3

4

5

0 10 20 30

(mg

g D

M-1

)O

vera

ll se

squi

terp

ene

cont

ent

Overall monoterpene content(mg gDM-1)

( ) R2= 0.34**y = 459.68x + 0.12

A

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 10 20 30

(µg

g D

M-1

h-1)

Overall monoterpene emissions(µg gDM-1 h -1)

( ) R2 = 0.51**y = 0.06x + 0.16

Ove

rall

sesq

uite

rpen

e em

issi

ons

B(+) R2 = 0.86***y = 0.09x + 0.13

Fig. 4. Relationship between overall monoterpene and sesquiterpene content in leaves of R. officinalis (A) and between overall leaf monoterpene and sesquiterpene emissions(field emissions used instead of basal emissions) of R. officinalis (s) and Q. coccifera (+) (B) (n = 27). P: relationship significance, **0.001 < P < 0.01, and ***P < 0.001.

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ARTICLE IN PRESS

being stored in expensive storage pools. This hypothesis of connec-tion between photosynthesis and monoterpene metabolism in ter-pene-storing species was proposed by Schürmann et al. (1993)who demonstrated, by means of 13C labeling, that assimilatedCO2 in Norway Spruce was used much more quickly in the synthe-sis of directly emitted compounds than in that of stored com-pounds, and that this synthesis was apparently not restricted tothe secretory cells of the resin ducts. Likewise, Staudt et al.(1997) noted that some emissions of Pinus pinea were stopped orstrongly reduced in darkened conditions. Noe et al. (2006) also

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demonstrated that linalool and trans-b-ocimene needle emissionsof the same species mostly relied on a recently synthesized poolof monoterpenes rather than the emission from resin ducts. Fur-thermore, Steinbrecher et al. (1999) reported that monoterpeneemissions of conifers was not only the result of volatilization fromresin vessels but also originated from de novo synthesis in the pho-tosynthetic tissue of the leaves. Therefore, the authors proposedthat the total twig emission from conifers may be better parame-terized by an algorithm that takes into account the emission frompools and the emission related to actual synthesis, rather than only

and semi-volatile plant emissions through nitrogen supply and chlorophyll

0.0

0.2

0.4

0.6

0.8

1.0

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

(+) y = exp(5.56x -7.16) P = 0.020, R2 = 0.33

( ) y = 1.28x - 0.95 P = 0.036, R2 = 0.26

C

0

2

4

6

8

10

12(+) y = exp (5.36x - 4.58) P = 0.019, R2 = 0.33

( ) y = 13.78x - 9.84 P = 0.041, R2 = 0.25

A

Mon

oter

pene

bas

al e

mss

ions

(µ

g h

-1 gD

M-1

)

0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3

( ) y = 1/(-67.67/x + 84.47) P = 0.088, R2 = 0.17

(+) y = 1/ (-23.64/x + 31.83) P = 0.056, R2 = 0.22

D

Sesq

uite

rpen

e ba

sal e

mss

ions

(µ

g h

-1 gD

M-1

)

0

3

6

9

12E ( ) y = 18.03(X0.99)

P= 0.07, R2 =0.18

0.0

0.2

0.4

0.6

0.8

1.0

0 0.05 0.1 0.15 0.2

G ( ) y = 0.24lnx + 0.75 P = 0.09, R2 = 0.18

F P > 0.10

0 0.05 0.1 0.15 0.2

H P > 0.10

Leaf nitrogen content (%DM)

Leaf phosphorus content (%DM)

B (+) y =exp (6.345x + 6.60) P = 0.030, R2 = 0.27

( ) y = exp (-0.63x + 6.60) P = 0.030, R2 = 27.6

Mon

oter

pene

bas

al e

mss

ions

(µ

g h

-1 gD

M-1

)Se

squi

terp

ene

basa

l em

ssio

ns

(µg

h -1

gDM

-1)

INTERMEDIATE AND HIGHLYAMENDED PLOTS

CONTROL AND INTERMEDIATE AMENDED PLOTS

Fig. 5. Relationship between foliar N (A–D) or P (E–H) content and basal emissions of monoterpenes and sesquiterpenes of Rosmarinus officinalis (dotted line and s) andQuercus coccifera (continued line and +) (n = 18). The relationship is shown separately for low (D0) and intermediate (D50) leaf N concentrations (left graphs), and forintermediate (D50) and high (D100) leaf N concentrations (right graphs). P: relationship significance.

8 E. Ormeño et al. / Chemosphere xxx (2009) xxx–xxx

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considering emissions from pools. As underlined by Niinemetset al. (2004), both emission processes are not regulated in the sameway: direct emissions after synthesis are controlled by physiolog-ical parameters related to metabolic activity and precursor avail-ability, while emissions from storage pools are mainly underphysicochemical controls such as volatility and diffusion rates ofspecific compounds.

Although we suggest a dual emission origin for species thatshow specialized structures, it is likely that de novo synthesized

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compound emission, i.e. compounds whose synthesis relies onlight rate instead of preexisting stored terpenes, is even more com-plex, as shown by Loreto et al. (1996). In fact, the authors demon-strated, by labeling with 13C, three different classes of terpenes forQuercus ilex, a species without such structures: (i) pinene class,rapidly light-induced, whose carbon skeleton is entirely dependenton photosynthetic intermediates (ii) ocimene class, more slowlylight-induced, that is made from one or more terpenes of thepinene class and (iii) the most slowly light-induced class, including

and semi-volatile plant emissions through nitrogen supply and chlorophyll

0

2

4

6

8

10

12

Mon

oter

pene

bas

al e

mis

sion

s (µ

g g

DM -1

h-1)

( ) y = 0.05x -11.6 p = 0.0046R2 = 0.28

(+) y = 0.04x -9.6 p = 0.0005R2 = 41.6

A

0.0

0.2

0.4

0.6

0.8

1.0

210 260 310 360

ETR (µmol electrons m-2 s-1)

Ses

quite

rpen

e ba

sal e

mis

sion

s (µ

g g

DM -1

h-1)

( ) y = 0.004x -1.0 p = 0.0076R2 = 0.25

(+) y = 0.003x - 0.6p = 0.0012R2 = 0.37

C

B( ) y = 22.9x -4.9 p = 0.017R2 = 0.23

(+) y = -3.3 + 17.3x p = 0.0143R2 = 0.201

0.20 0.25 0.30 0.35 0.40

PSII

D( ) y = 1.9x - 0.4 p = 0.0033R2 = 0.30

(+) y = 1.7x - 0.3p = 0.015R2 = 0.23

Fig. 6. Relationship between basal emissions of monoterpenes (upper graphs) and sesquiterpenes (bottom graphs), and ETR or UPSII of Rosmarinus officinalis (dotted line ands) and Quercus coccifera (continued line and +) (n = 18). P: relationship significance. *0.01 < P < 0.05, **0.001 < P < 0.01, and ***P < 0.001. N = 27.

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3-methyl-3-buten-1-ol and linalool, likely formed in non-photo-synthetic plastids.

The relationship observed between monoterpenes and sesqui-terpenes, both in content and emissions, could be a result of thecross-talk between their biosynthesis pathways whose existenceis now being admitted (Hemmerlin et al., 2003). Although mono-terpenes and sesquiterpenes biosynthesis classically consists oftwo spatially separated pathways – the classical cytosolic MVAfor most sesquiterpenes, and the plastidial MEP for monoterpenesand some sesquiterpenes – the presence of a bidirectional trans-port of MVA intermediates from the cytosol to the plastid has beendemonstrated, as well as the transport of MEP intermediates fromthe plastid to the cytosol, across de chloroplast envelope (Hem-merlin et al., 2003). The relationship illustrated in this investiga-tion between mono- and sesquiterpenes is of obvious interest assesquiterpene emission sampling is recognized to be complex ow-ing to their high reactivity and short atmospheric lifetimes.

4.3. Compost fertilizing effect

N and P supply through compost was particularly investigated,since they are generally known to be the most limiting elementsfor plant growth in Mediterranean ecosystems and have a criticalimportance in plant nutrition. Consistently with results previouslynoted during the short-term study carried out on the same sam-pling site (Larchevêque et al., 2006), compost principally changedN and PE concentration in the organic soil layer. Although PE con-tent was also improved in the mineral soil compartment, this in-crease was by far less marked than in the humic organic horizon.As sewage–sludge contains large amounts of N and P, amendmentgreatly enriches humic organic fraction in these elements. How-ever, the mature compost used was a very stable humified product,limiting the mineral horizon enrichment in N and P by percolation.

Despite the low enrichment of the mineral horizon, composthad a clear nitrogen fertilizing effect, since leaf N availability forR. officinalis and Q. coccifera was still enhanced 4 years afteramendment, which could be related to durable soil enrichmentby compost amendment. Increasing N uptake by plants might be

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explained because most feeding roots can directly dip into humusnutrient reserves (Larchevêque et al., 2009), and sporadic leachingof compost could also have provided available forms of nutrientsfor deep roots.

Unlike N, important soil PE content only enhanced P content inleaves of R. officinalis, whereas leaves of Q. coccifera remained un-changed, giving evidence of a lower responsiveness to fertilizers.These results are in accordance with results reported in Larchevê-que et al. (2009) on the same site and for the same season one andtwo years after amendment. Over the seasonal cycle, Larchevêqueet al. (2009) showed that Q. coccifera P concentration was higher onD100 (+50%) compared to control plants only in October 2002, pos-sibly because nutrients became less available for the deep roots ofthis species.

4.4. Compost and nutrient effect on terpene content

Herein, no effect of compost or nutrients on terpene storage wasdetected. Only a few recent reports have dealt with the influence ofcompost fertilizer on terpene storage in recent years. For Draco-cephalum moldavica L., compost application seems to promote oilaccumulation (terpenes included) (Hussein et al., 2006). Similar re-sults were described by Tanu et al. (2004) for different varieties ofCymbopogon winterianus.

Besides, some studies have focused on the influence of N and Pon terpene concentration in terpene-storing species, but their re-sults prove evident discrepancies. As illustrated here, monoterpeneconcentration in needles of Douglas fir (Litvak et al., 2002) and R.officinalis (Ormeño et al., 2008) seems to be independent of thesemacronutrients. By contrast, a strong and positive relationship be-tween diterpene resin acids in resin ducts and N supply was notedfor Pinus sylvestris in Bjorkman et al. (1998), who argued that ter-pene pool formation could be favored under N supply. Likewise,monoterpene concentration in needles of Pinus halepensis increaseswith soil PE and N (Ormeño et al., 2008). Because some of thesestudies were performed on species belonging to the same func-tional group (i.e. conifers), they confirm that N and P impact on ter-pene concentration remains controversial.

and semi-volatile plant emissions through nitrogen supply and chlorophyll

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The absence of effect of compost-supplied nutrients on terpeneconcentration in glandular trichomes of R. officinalis could be dueto the fact that as biomass production is likely favored by increas-ing leaf N and P uptake, either the production of new terpene stor-age organs (Litvak et al., 2002), or the development of thepreexisting organs is not proportionately elicited as a means toavoid extra energetic costs required for their further maintenance.In other words, the terpene-storage pool density could diminish asthe fertilizer rate increases, as also suggested in Blanch et al.(2007). Indeed, the cost for building the cellulose walls of non-glandular trichomes is relatively lower in comparison with the costof building glandular trichomes, since the later contain secondarymetabolites – such as flavonoids and terpenes – produced byexcretory cells in glands (Gonzáles et al., 2008). This strategy couldhave been especially developed in Mediterranean species since theodds of plant survival during summer months may be reduced byan inadequate distribution of resources.

4.5. Compost, nutrient and some chlorophyll fluorescence parameterseffects on terpene emissions

Mono- and sesquiterpene emissions of R. officinalis and Q. coccif-era were enhanced in soils with intermediate compost rates. Incontrast, terpene emissions of plants located in plots with thehighest compost rates were as low as in control plots. Sinceenhancement of N supply up to intermediate leaf N concentrationsstimulated terpene emissions, while the highest leaf N concentra-tions did not cause any effect, in comparison to control plants, itis likely that sub and supra-optimal N conditions occurred innon- and highly amended plots, respectively, and that optimal fer-tilizing conditions were achieved in intermediate amended plots,allowing plant investment in terpenoids. The effect of leaf P wasmore punctual, since it was only involved in the increase ofmono- and sesquiterpene basal emissions of R. officinalis underD50 plots.

Variation in terpene emissions was more robustly explained bychanges in leaf chlorophyll fluorescence parameters than in nutri-ent supply. Changes in ETR and UPSII were however partially causedby N supply: while intermediate leaf N contents increased ETR andUPSII, resulting in higher terpene emissions (Fig. 2A and C), thehighest N rates lowered the value of these parameters (Fig. 2Band D), as low as in non-amended plots, and, as a result, mono-and sesquiterpene emission rates were reduced (Fig. 6).

In agreement with our results, Niinemets et al. (2002a) demon-strated that foliar photosynthetic electron transport is highlycorrelated with monoterpene emissions of Quercus sp., strengthen-ing the argument of the possible dependence of plant emissions onETR. These authors also reported a less consistent correlationbetween terpene emissions and the photosynthetic activity of Q.coccifera, therefore revealing that taking into account such biolog-ical controls on terpene emission could be especially relevant forsimulation of terpene emissions during stress periods, for whichempirical models may significantly overestimate terpene emissionrates. Finding that terpene emission rates of R. officinalis showedthe same trend than ETR and UPSII and were correlated with thesefluorescence parameters is even more surprising, since R. officinalispossesses specific terpene storage pools in leaves that generallymitigate the impact of the physiological state of the plant on therate of terpene emission. In other words, although any carbonbased secondary metabolite ultimately depends on the photosyn-thetic functioning of the plant, terpene emissions of R. officinalisare assumed to be controlled by its terpene content alone (Hansenet al., 1997). The possible link between plant photosynthetic func-tioning and terpene emissions of R. officinalis could then elucidatewhy monoterpene emissions of R. officinalis could not be estimatedfrom the monoterpene content.

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Although no study has dealt with the effect of nutrients sup-plied through compost on terpene emissions, some reports haveemphasized the effect of fertilizers supplied through mineral solu-tions on terpene emissions. On the one hand, concurring with thisstudy, Gouinguene and Turlings (2002) showed low inducedmonoterpene and sesquiterpene emissions from N unfertilizedplants (fertilizer composition and leaf N concentrations not shown)and Blanch et al. (2007) did not report any relationship betweenleaf P (ranging from �0.8% to �1.8%) and monoterpene emissionsof P. halepensis and Q. ilex, a storing and non-storing species. More-over, as shown here, the Growth Differentiation Balance Hypothe-sis (Herms and Mattson, 1992), states that as the photosyntheticactivity decreases or increases under low and intermediate N sup-ply, respectively, production of secondary metabolites, such asterpenes, is limited or stimulated, correspondingly. However, thetheory also expects that as a high photosynthetic efficiency occursfor high levels of N (which presumably did not occur in this study)terpene production is weakened (as in this study), because carbonsynthesized through photosynthesis would be allocated rather togrowth than to terpene production.

On the other hand, it has been reported that (i) there is no rela-tionship between leaf N (between <0.01% and �0.04%) and mono-terpene emissions (Blanch et al., 2007), (ii) there is a strongnegative effect of high leaf P concentrations on isoprene emission(Fares et al., 2008), which is generally expected to vary similarlyto monoterpene emissions of non-storing species (Niinemetset al., 2002a), and (iii) high leaf N concentrations are accompaniedby high emissions of monoterpenes, during the most importantpart of the seasonal cycle of Pseudotsuga menziesii (Mirb) (Lerdauet al., 1995, under leaf N content of �1%DM, 1.5%DM and 2%DM),and isoprene of Pseudotsuga menziesii (Litvak et al., 2002: N treat-ments, �0.8%, 0.9%, 1.1% and 1.3%). All these studies were per-formed on juvenile plants whereas this study was performed onmature plants. This could be at the origin of differences found, be-cause in mature plants, production of plant defenses, such as terp-enes, can be more constrained than in juvenile plants (Boege andMarquis, 2005).

5. Conclusions

There are environmental, political as well as economical incen-tives to increase the application of sludge. Yet, such usage shouldbe performed with care as there are also ways whereby sludge fer-tilization could harm the environment and human health (Latur-nus et al., 2007). In this study we show the effect of compost onterpene emissions through nitrogen supply and chlorophyll fluo-rescence. If these results are corroborated for other species, it islikely that the intermediate compost rates applied in this studyprompt terpene emissions from plants, thus favoring plant defensemechanisms, but also having a potential impact in air quality, ow-ing to the recognized high reactivity of numerous terpenes in thetroposphere.

Further studies are required before a conclusive statementregarding compost effect on terpene production can be made. Wesuggest the need to conduct this kind of experiment both in ashorter (1 or 2 years) and a longer term experiment (8–10 years).Experimentation with other types of compost (e.g. solid-wastecompost) and organic amendments different from compost (e.g.poultry manure) could also be interesting, since their impact in ter-pene emissions might be different.

Although the main aim of this study was to test whether com-post affects terpene emissions from plants, we have also illustratedthat monoterpene emissions and content are mostly not corre-lated, suggesting that a fraction of these emissions does not comefrom terpene pools. The application of 13CO2 is however necessary

and semi-volatile plant emissions through nitrogen supply and chlorophyll

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to discern which compounds are emitted in the atmosphere imme-diately after their synthesis and which are released from preexist-ing material stored in specific terpene pools.

Acknowledgements

The authors thank S. Greff, C. Lecareux and S. Dupouyet for theircollaboration in measurement campaigns and for their help inchemical analyses. We also thank Phyllis Bischof and Prof. MarkRosenzweig, for their help regarding English correction. This studywas funded in part by the Environmental agency (ADEME) (No.0575C0004) and the PACA (Provence Alpes Côte-d’Azur) region.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.chemosphere.2009.05.014.

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and semi-volatile plant emissions through nitrogen supply and chlorophyll