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Environmental Pollution 158 (2010) 2258e2265

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Environmental Pollution

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Effects of ammonia from livestock farming on lichen photosynthesis

Luca Paoli a,b, Stergios Arg. Pirintsos b, Kiriakos Kotzabasis b, Tommaso Pisani a,Eleni Navakoudis b, Stefano Loppi a,*aDepartment of Environmental Science “G. Sarfatti”, University of Siena, via Mattioli 4, I-53100 Siena, ItalybDepartment of Biology, University of Crete, 71409 Heraklion, Crete, Greece

Ammonia from livestock farming affects lichen photosynthesis.

a r t i c l e i n f o

Article history:Received 2 September 2009Received in revised form11 February 2010Accepted 13 February 2010

Keywords:Air pollutionArid environmentsEvernia prunastriJIP-testPseudevernia furfuracea

* Corresponding author.E-mail address: loppi@unisi.it (S. Loppi).

0269-7491/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.envpol.2010.02.008

a b s t r a c t

This study investigated if atmospheric ammonia (NH3) pollution around a sheep farm influences thephotosynthetic performance of the lichens Evernia prunastri and Pseudevernia furfuracea. Thalli of bothspecies were transplanted for up to 30 days in a semi-arid region (Crete, Greece), at sites withconcentrations of atmospheric ammonia of ca. 60 mg/m3 (at a sheep farm), ca. 15 mg/m3 (60 m from thesheep farm) and ca. 2 mg/m3 (a remote area 5 km away). Lichen photosynthesis was analysed by thechlorophyll a fluorescence emission to identify targets of ammonia pollution. The results indicated thatthe photosystem II of the two lichens exposed to NH3 is susceptible to this pollutant in the gas-phase. Theparameter PIABS, a global index of photosynthetic performance that combines in a single expression thethree functional steps of the photosynthetic activity (light absorption, excitation energy trapping, andconversion of excitation energy to electron transport) was much more sensitive to NH3 than the FV/FMratio, one of the most commonly used stress indicators.

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1. Introduction

The atmospheric concentration of ammonia (NH3) is often highin countries with intensive agriculture and animal husbandry(Graedel et al., 1995). The major sources of atmospheric NH3 inEurope are livestock farming, fertilizers and some industrial activ-ities (Asman, 1992). Emissions from livestock farming containa cocktail of compounds: NH3, CO2, gaseous amines and dust,including nutrient-containing particles, such as N, P and K, whichmay modify plant communities (Cape et al., 2009). In the atmo-sphere, ca 30% of NH3 is converted to ammonium (NH4

þ), whichmayfurther react, be dispersed with aerosols or be removed from theatmosphere mainly by wet deposition (Asman and Janssen, 1987).

NH3 is the main source of dry deposition of atmospheric Naround livestock husbandries, and as a gas it may influence lichencommunities composition (Frati et al., 2008). Owing to the fact thatatmospheric NH3 has been widely measured in areas of northernEurope with intensive agriculture and livestock farming (e.g. vanHerk, 1999; Sutton et al., 2003, 2009), it has been possible torecognize that nitrophilous epiphytes are positively correlated withNH3 (Sparrius, 2007) and that NH3 enhances nitrophilous- anddecreases acidophilous epiphytes mainly indirectly by rising bark

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pH (van Herk, 2001). In particular, prolonged exposures (years) toatmospheric NH3 concentrations >3 mg/m3 promotes nitrophilouslichens (van Herk et al., 2003) and rise bark pH of local trees within2e3 km from a point source (van Herk, 2001).

In theMediterraneanarea, also dust anddryconditions lead to anincrease in bark pH enhancing nitrophilous lichens, most of whichare also species of xeric environments (Loppi and De Dominicis,1996; Loppi et al., 1997), complicating the detection of the effectsof N compounds (Frati et al., 2008).

Concern for the increasing deposition of NH3 is mainly focusedon biological effects at community level, i.e. when changes ordamages to the environment have already occurred. On the otherhand, monitoring changes at physiological level may help to detectearly stress symptoms. Lichen transplants proved to offer rapidinformation on occurring stress by the analysis of selected physi-ological parameters (Paoli and Loppi, 2008).

The aim of the present study was to test the hypothesis that NH3pollution in the Mediterranean area directly influences the photo-synthetic performance of lichens.

2. Materials and methods

2.1. Study area

A point source of NH3 (35�1804700N, 25�0104000E), represented by a sheep farm,was selected in the lowlands of Northern Crete (Greece), where climate is xero-thermic and more than 125 dry days occur per year. During the experiment (see

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e2265 2259

below) there was no rainfall, the weather was dry and warm, with temperatures upto 38 �C. Dueing to these conditions, lichens could only be partially hydrated by dewin the night or humidity from the sea.

Farming is a traditional and important sector of Crete's agricultural productionand the island as a whole hosts more than one million sheep and goats that aremostly farmed by small landholders (Volanis et al., 2007).

The selected farm has around 150 heads, sheep were kept outdoors for most ofthe year and feeding was based chiefly on the available spontaneous vegetation.Flocks were free to graze and regularly gathered in traditional small stocks, e.g. formilking.

2.2. Lichen material and experimental design

Twigs (ca. 30 cm in length) carrying respectively a minimum of 5 thalli of thelichens Evernia prunastri and Pseudevernia furfuracea were harvested in May 2005from a rural area inMt. Olympos (Northern Greece) far from any local pollution sourceand let to acclimate 3 weeks at ambient light and temperature in Northern Crete(Greece). The species were selected being extensively used in biomonitoring surveys.Both have Trebouxia algae as a photobiont and a fruticose growth form, but havea slightly different ecology: P. furfuracea is considered a xero-mesophytic acidophilousspecies and E. prunastrimore hygro-mesophytic and less acidophilous. On the 15th ofJune 2005 lichen samples were exposed in the neighbourhood of the sheep farm andretrieved respectively after 15 and 30 days of exposure.

Lichens were exposed at the farm, i.e. 0e5 m from the boundary of the stockingarea and at a radial distance of 60e80 m. Another batch of samples was exposed ina remote area 5 km from the farm and was used as a control. Lichens were trans-planted together with their carrying substrate using a fishing-net (mesh size 2.5 cm)containing twigs of both species, at 2m above ground on the north side of local trees,tied directly to the brunches or protected in plastic cages to prevent possible damageby grazing. Ten lichen-nets were exposed at each site as replicates. Experimentalconditions were therefore similar at all sites, except for distance from the farm.

2.3. Ammonia monitoring

Atmospheric NH3 was measured by passive samplers using diffusion tubes(Radiello�, Aquaria). At each site, 5 samplers were placed together with lichen thalliat a height of around 2 m above ground for one 7-day period in the middle of theexperiment (between June, the 30th and July, the 7th). Samplers contained a filterimpregnated with phosphoric acid which adsorbs gas-phase NH3 as NH4

þ, that canbe easily measured spectrophotometrically by the indophenol blue method (Allen,1989). The detection limit was 0.6 mg/m3; uncertainty was 6.5%.

2.4. Chlorophyll fluorescence analysis

After each retrieval, lichen samples were air dried and stored at �20 �C.A physiological recovery of the lichens was carried out prior to measurements. Toavoid any osmotic stress by air humidity after the freezing, samples were left 15 minin dry ambient conditions. They were subsequently sprayed with water until wetand the excess water was removed by hand-shaking. Samples were then stored at4 �C in the dark for 24 h. The outermost 2 cm of the thalli were then randomlyselected for measurements, but avoiding areas with excess of vegetative structures(isidia and soredia). Measurements were carried out with a Plant Efficiency Analyser(PEA, Hansatech Ltd, UK). After dark-adaptation for 10 min, samples were lightenedfor 1 s with a saturating excitation pulse (1800 mmol s�1 m�2) of red light (650 nm)from a LED into the fluorometer sensor and fluorescence emission recorded. All thefluorescence transients were recorded with a time span from 10 ms to 1 s. Data wereacquiredwith a time resolution of 10 ms for the first 2 ms and later on the instrumentautomatically turned to slower registration rate. Ten fluorescence emission curveswere recorded for each retrieval in each site for both species.

Chlorophyll a fluorescence emission was analysed as follows:

I) to assess if NH3 emitted from a sheep farm affects lichen photosynthesis, wefirst used the classical physiological indicator of photosynthetic efficiency FV/FM, representing the potential quantum yield of primary photochemistry(Maxwell and Johnson, 2000).

II) Secondarily, we described the effects of NH3 on fluorescence kinetics. Thefast fluorescence kinetic typically outlines a transient curve: when the curveis plotted on a log-time axis a sequence of steps called OeJeIeP, each cor-responding to its changing inclination, is apparent (Strasser et al., 2000).The minimal fluorescence F0 is measured at 50 ms and corresponds to O, theJ-step is recorded at 2 ms, the I-step at 30 ms and P at about 300 ms,generally in correspondence of maximal fluorescence FM. The value of fluo-rescence emission recorded at these points is used to calculate a series ofparameters, which serve to translate original data to biophysical parametersthat quantify energy fluxes and their ratios, physiological states, conforma-tion and overall performance of the samples (Strasser et al., 2000).

III) On a third step we therefore evaluated the effects of NH3 on the functioningof PSII through the JIP-test. Information concerning the theory and derivationof the formulae is described e.g. in Strasser et al. (2000, 2004).

The energy cascade from light absorption by PSII to electron transport involvesthe absorption of photon flux (ABS) by antenna pigments, creating excitedchlorophyll. The excitation energy is partly dissipated (DI0) as heating and fluo-rescence emission, and in part is profitably addressed to the reaction centre (RC)as trapping flux (TR0); in the RC the excitation is converted into redox energy byreducing the electron acceptorQA toQA�which is then reoxidised toQA leading tothe electron transport (ET0) and later to CO2 fixation (Strasser et al., 2004).To quantify energy fluxes we used the following parameters that refer to timezero, when all reaction centres of PSII are open:

e ABS/RC ¼ (M0/VJ)/[1 � (F0/FM)] is the equation representing the absorbedenergy per RC of PSII

e TR0/RC ¼ M0/VJ is the specific trapping flux at time zero per RCe DI0/RC ¼ ABS/RC � TR0/RC is the energy flux which is dissipated chiefly as

heate ET0=RC ¼ M0=VJ$ð1� VJÞET0/RC ¼ M0/VJ (1 � VJ) is the energy flux corre-

sponding to the effective electron transport per RCwhere VJ ¼ (F2 ms e F0)/(FM e F0) and M0 ¼ 4$ðF300ms � F0Þ=ðFM � F0Þ is theinitial slope of the curve, representing a measure of QA reduction in the first250 ms, multiplied by 4 to give the value at 1 ms.

To quantify phenomenological energy fluxes per excited cross-section (CS):e ABS/CS for light absorptione TR0/CS for excitation energy trappinge DI0/CS for heat dissipatione ET0/CS for electron transport.

The fraction of active RCs per excited cross-section (RC/CS) and the total numberof active RCs per absorption (RC/ABS) were also considered.To quantify flux ratios:e J0 ¼ ET0/TR0 expresses the probability that a trapped exciton, a quantum of

electronic excitation, enters the transport chain and moves an electronfurther than QA;

e 4P0 ¼ TR0/ABS expresses the probability that an absorbed photon will betrapped by the reaction centre of PSII, it represents the maximum quantumyield of primary photochemistry and roughly corresponds to FV/FM;

e 4D0 ¼ DI0/ABS, expresses the probability that excitation energy will bedissipated in the antenna chlorophyll, being 4P0 þ 4D0 ¼ 1;

e 4E0 ¼ ET0/ABS is the maximum yield of electron transport (¼j0�4P0).

Some technical parameters are referred to the area above the transient curve:e Sm ¼ Area/(FM�F0) is a measure of the energy needed to close all the RCs,

accounting themultiple turnover in the closure of the RCs, where Area is thearea growth between the fluorescence curve and the maximal fluorescencesignal;

e the expressionN¼ Sm$M0/VJ indicates howmany times QA has been reducedto QA in the time span from t0 to tFmax;

e the ratio Sm/tFmax expresses the average redox state of QA�/QA, that meansthe average fraction of open RCs during the time needed to complete theclosure of all the RCs.

IV) The performance index PIABS, a global indicator that resumes the contribu-tion of all parameters, was used to express the overall vitality of the samples:

PIABS ¼ RC=ABS$4P0=ð1� 4P0Þ$j0=ð1� j0Þ

2.5. Statistical analysis

Significance of differences (P < 0.05) among various treatments and controlswas checked by one-way analysis of variance (ANOVA), using the Bonferroni test forpost-hoc comparisons. Prior to analysis data not matching a normal distribution(KolmogoroveSmirnov test at the 95% confidence interval) were treated withBox-Cox transformation.

3. Results

3.1. Concentrations of NH3 around the sheep farm

Atmospheric concentration of NH3 recorded by passive samplersfor 7 days in the middle of the experiment was 62.4 � 4.3 mg/m3 atthe sheep farm, 15.0 � 1.5 mg/m3 at a radial distance of 60 m and1.3 � 0.9 mg/m3 at a neighbouring remote control area.

3.2. Effects of NH3 on the potential quantum yield of PSII

Lichens transplanted in close proximity of a point source of NH3showed a marked decrease in the potential quantum yield of PSIIalready after 15 days, as indicated by the low values of the

Table 1Mean (�SD, N ¼ 10) fluorescence parameters per site and exposure length: extremes of the curves (F0, FM) and ratios between the variable fluorescence and the extremes (FV/FM, FV/F0). Shaded values differ from controls (including the background area), bold at the farm differs from 60 m distance (t-test, P < 0.05).

Control Background area 60 m from the farm Farm

15 days 30 days 15 days 30 days

E. prunastriFV/FM 0.57 � 0.05 0.53 � 0.08 0.37 � 0.05 0.46 � 0.09 0.26 � 0.15 0.26 � 0.04F0 177 � 60 284 � 72 146 � 40 156 � 70 192 � 52 161 � 37FM 421 � 187 590 � 235 237 � 70 295 � 69 264 � 64 229 � 62FV/F0 1.18 � 0.29 1.08 � 0.28 0.56 � 0.11 0.85 � 0.27 0.39 � 0.32 0.39 � 0.12

P. furfuraceaFV/FM 0.61 � 0.03 0.53 � 0.11 0.43 � 0.07 0.39 � 0.10 0.34 � 0.08 0.15 � 0.04F0 212 � 6 276 � 33 136 � 44 148 � 63 139 � 74 122 � 70FM 543 � 34 569 � 173 250 � 109 260 � 167 213 � 114 147 � 93FV/F0 1.43 � 0.16 1.30 � 0.22 0.74 � 0.21 0.64 � 0.28 0.50 � 0.17 0.18 � 0.05

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e22652260

conventional physiological indicator of photosynthetic efficiency FV/FM, shown by both species (Table 1). Basal andmaximal fluorescence(F0 and FM) and the ratio between variable (FV ¼ FM�F0) and basalfluorescence (FV/F0) are also given. After 30 days of exposure to ca.60 mg/m3 NH3, P. furfuraceawas more affected than E. prunastri, withFV/FM in the range 0.1e0.2 owing to the drop of FM. The FV/FM ratio ofthalli exposed in the remote control areawas not different from thatmeasured in samples before the transplantation (P > 0.05).

3.3. Effects of NH3 on fluorescence kinetics

The exposure to NH3 altered the shape of the chlorophyll a fluo-rescence emission transient curves. Fig. 1 shows the fast Chl a fluo-rescence induction kinetics in control and transplanted thalli of E.prunastri and P. furfuracea after 30 days. When lichen thalli areexposed to a saturating light pulse, Trebouxia photobionts begin Chla fluorescence emission up to a peak (corresponding to the maximalfluorescence emission, FM) that normally reach after 150 ms. Fastfluorescence curves of control samples and transplanted thalli at theremote area showed a characteristic sequence of OeJeIeP steps,typical of unstressed samples (see Fig. 1). In samples exposed toambient NH3, especially at the highest concentration, a markeddecrease of FM is observed, and a clear peak of emission is notevident. In such a condition, fluorescence emission rapidly drop tozero in the time span of 0.2e1 s. Therefore, the samples in proximityto the point source of NH3 have shown a compressed fast fluores-cence curve and the characteristic sequence of OeJeIeP steps cor-responding to its changing inclination was less or not apparent.

3.4. Effects of NH3 on the functioning of PSII

NH3 has several targets on the photosynthetic process asdepicted by the JIP-test.

The effects of different concentrations of NH3 are presented inboth lichen species as radar plots as far as it concerns the specificenergy fluxes per reaction centre (RC) and excited cross-section(CS): i.e. light absorption (ABS), excitation energy trapping (TR),electron transport (ET), energy dissipated per cross-section of themeasured samples (DI0/CS) and density of reaction centres RC/CS0(Fig. 2). Parameters of control thalli before the exposure are nor-malised to the value of one and those of thalli exposed for 30 daysboth in the remote area and at the farm (to 15 and 60 mg/m3 NH3)are given for comparison as a fractional increase or decreaserelative to the controls. Deviation of selected parameters fromcontrol is the highest at the sheep farm and the lowest at theremote control area; both lichen species showed a similar responseto NH3 air pollution.

Energy fluxes through PSII were affected in several ways,especially at the highest concentration: the average energy

absorbed per active reaction centre (ABS/RC) increased as well asthe amount of energy dissipated chiefly as heat (DI0/RC, in Fig. 3)owing to the inactivation of some RCs. The inactivation of RCs is alsoconfirmed by the overall decrease of the density of active RCs perexcited cross-section (RC/CS0). On the contrary, the density of RCsper CS is higher at the remote area, where lichens acclimated risingenergy fluxes per CS.

Important information is provided by the decrease of j0(Table 2), the efficiency with which a trapped exciton (a quantum ofelectronic excitation) enters the transport chain and moves anelectron further than QA. In particular, the trapping flux is nega-tively influenced by the distance from the farm when analysed perexcited cross-section or “leaf area” (TR0/CS0), but stable whenconsidered per active reaction centre (TR0/RC), independently ofthe level of atmospheric NH3.

Amain consequence of NH3 pollution is the strong depression ofthe electron transport activity, 4E0 (Table 2). On the whole, theelectron transportflux decreased per active reaction centre (ET0/RC)and excited cross-section of the sample (ET0/CS0).

The maximum quantum yield of primary photochemistry, 4P0(zFV/FM) and the complementary flux of dissipated excitationenergy, 4D0 are summarised in Table 3. In short, the normal yield ofdissipated energy is around 40e50% for controls and samplestransplanted in the remote area, 50e70% where NH3 is 15 mg/m3

and more than 70% with NH3 around 60 mg/m3.A further influence of NH3 on late metabolic aspects of PSII

(electron transport beyond the oxidation of QA�) can be assessed bythe turnovernumber (N) and the average fractionof openRCsduringthe time needed to complete their closure (Sm/tFmax). A comparisonbetween control and transplanted thalli after 30 days at the studysites is shown inTable 4. The turnover number (N) decreased by 50%in both lichen species when exposed to 15 mg/m3 NH3 in the air anddropped drastically after the exposure to 60 mg/m3 at the farm.In particular at the farm, we calculated a significant decrease of theaverage redox state of QA�/QA, represented by the ratio Sm/tFmax,clearly reflecting a drop of electron transport beyond QA�.

3.5. Global photosynthetic performance (PIABS)

The global index of the photosynthetic performance (PIABS)decreased in close proximity to the sheep farm in both lichenspecies (Table 5). In Table 5 we also expressed PIABS in terms ofdeviation from the average value recorded in the remote area: theidea was to rule out any possible effect of the habitat duringthe experiment, allowing a field control for the effect of thetransplantation. On the whole, control thalli have the same PIABSafter one month in the remote area, indicating that the photosyn-thetic performance in a short-term exposure has not been signifi-cantly influenced by the transplantation itself (P > 0.05).

0,01 0,02 0,05 0,10 0,2 0,5 1,0 2 5 10 20 50 100 200 500 1000Time [ms]

100

200

300

400

500

600

700

800

]V

m[ ecnecseroul

F

0.01 0.02 0.05 0.10 0.2 0.5 1.0 2 5 10 20 50 100 200 500 1000

Time [ms]

100

200

300

400

500

600

]V

m[ ecnecsero

ulF

control

remote area, NH3 up to 2 g/m3

farm, NH3 ca 60 g/m3

60 m from the farm, NH3 ca 15 g/m3

control

remote area, NH3 up to 2 gµ

µ

µ

µ

µ

µ

/m3

farm, NH3 ca 60 g/m3

60 m from the farm, NH3 ca 15 g/m3

a

b

Fig. 1. Fluorescence induction kinetics: each transient curve outlines the typical behaviour recorded in control and transplanted thalli after 30 days; a) E. prunastri. b) P. furfuracea.

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e2265 2261

PIABS shows that under a regime of 15 mg/m3 NH3 E. prunastriwas affected after 15 days, but significantly recovers its photo-synthetic performance after a further 15 days, which is chieflyevident by the rise of electron transport activity, 4E0 (see Table 2).On the contrary, all thalli of P. furfuracea are influenced by NH3irrespective of exposure length and NH3 concentrations within60 m from the farm.

4. Discussion

4.1. Assessment of chlorophyll a fluorescence emission

The technique of chlorophyll a fluorescence analysis is widelyused to screen photosynthesis, assess the vitality of the samplesand provide early indication of physiological stress (Maxwell andJohnson, 2000). This technique has been used to show tolerance

and chronic effects of heavymetals and other xenobiotics in lichens(Dzubaj et al., 2008).

The analysis of the fast chlorophyll a fluorescence signalsrecordedwith high time resolution demonstrated that PSII of lichentransplants exposed to NH3 in the field is susceptible to thispollutant. The fast fluorescence transients of both lichen speciesexposed around the farm showed a decrease in maximal fluores-cence emission (FM), that, as a consequence, reduced the potentialquantum yield for primary photochemistry (FV/FM). P. furfuraceawas particularly sensitive, since thalli exposed to NH3 undergoa drop of FV/FM to the range 0.1e0.2, whereas the normal range forhealthy lichens is 0.5e0.76 (depending on the species) andvalues< 0.1 correspond to deadmaterial (Jensen and Kricke, 2002).Parameters measured in the present study in control lichens are inline with similar studies using both lichens and their photobiontscultivated in the laboratory (Ba�ckor and Fahselt, 2008).

0

0.4

0.8

1.2

1.6

2ABS/RC

TRo/RC

ETo/RC

ETo/CSo

TRo/CSo

ABS/CSo

RC/CSo

DIo/CSo

control

remote area

60 m from the farm

farm

0

0.4

0.8

1.2

1.6

2ABS/RC

TRo/RC

ETo/RC

ETo/CSo

TRo/CSo

ABS/CSo

RC/CSo

DIo/CSo

a

b

Fig. 2. Radar plot presentation of selected parameters after 30 days in the study sitesrelative to the controls before the transplantation: a) Pseudevernia furfuracea; b) Everniaprunastri. In a) ABS/RC at the farm has been divided by 2 to be included in the plot.

0

2

4

6

8

10

12

14

control remote area 60 m from the farm

farm

ID

0C

R/

Pseudeverniafurfuracea

Evernia prunastri

Fig. 3. Amount of the energy flux dissipated per active reaction centre of the PSII (DI0/RC) in control samples and after 30 days of exposure. For both species values at thefarm differ from control, background area and 60 m distance (t-test, P < 0.05).

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e22652262

Despite chlorophyll a fluorescence emission is widely investi-gated also in lichens, to date, a few information concerns lichensand the JIP-test (Ilík et al., 2006). For this reason, fast chlorophylla fluorescence emission curves were analysed in lichens to identifyNH3 targets in the photosynthetic apparatus by the so called JIP-test(Strasser et al., 2000).

Getting inmore detail with the impact of NH3 on photosystem II,the decrease of maximum quantum yield of primary photochem-istry 4P0 (zFV/FM) occurred together with an increase of energydissipation per reaction centre RC (DI0/RC), as shown in Fig. 3. Theparameter DI0/RC indicates the amount of energy that reaching thereaction centres of the photosystem II is dispersed mostly as heat(Strasser et al., 2000).

We also observed a strong depression of the electron transportactivity 4E0 (ET0/ABS) and of the efficiency of electron transport j0

Table 2Mean (�SD, N ¼ 10) energy flux ratios per site and exposure length: j0 (ET0/TR0), efficiefurther than QA; 4E0 (ET0/ABS), maximum quantumyield of electron transport. Shaded val60 m distance (t-test, P < 0.05).

Control Background area 60 m from th

15 days

E. prunastriJ0 0.376 � 0.093 0.442 � 0.067 0.254 � 0.0704E0 0.205 � 0.074 0.230 � 0.063 0.093 � 0.036

P. furfuraceaJ0 0.509 � 0.033 0.452 � 0.070 0.307 � 0.0744E0 0.299 � 0.009 0.238 � 0.079 0.131 � 0.047

(ET0/TR0), a lesseningof the trappingfluxper cross-section (TR0/CS0)combined with a certain stability of the trapping flux per active RCs(TR0/RC). The total number of active RC per CS (RC/CS0) alsodecreased. These results indicate a high level of energy dissipationafter the photon flux is absorbed by the antenna pigments creatingexcited chlorophylls (Krüger et al., 1997). As reported by Strasseret al. (2004), this could be due to the transformation of someactive RCs to silent RCs: i.e. RCs that act as efficient traps becausethey dissipate all the excitation energy and do not contribute to thevariable fluorescence, acting as a possible protection mechanism(Krause et al., 1990). While FV/FM refers to the whole sample, thetrapping flux (TR0/RC) is calculated from the kinetics of the variablefluorescence and thus it refers to the RCs that can reduce QA, beingphotosynthetically active (Strasser et al., 2004). Therefore,a concomitant increase of absorbed energy (ABS/RC) does not indi-cate a structural increase of the antenna chlorophylls, but a rise ofthe total absorption per active RCs (Strasser et al., 2004), as probablyoccurred to our species exposed to NH3. However, the results alsoindicated that NH3 affected the photosynthetic performance atsingle RC level, as shown by the low ET0/RC. Similar results werefound after treating Spirodela plants with increasing levels of chro-mate (Appenroth et al., 2001). These authors also demonstrated thecorrelation between O2 evolution and decreased ratios of energyfluxes (4P0, j0, and 4E0) and found a lower number of QB bindingcentres and a decrease in the turnover number. These flux ratioshave been similarly affected also in our transplantations; we cantherefore hypothesise that NH3 affect also later reactions of thephotosynthetic process, including O2 evolution.

The technical parameters referred to the area above the tran-sient curve (energy needed to close all the reaction centres, Sm;

ncy with which a trapped exciton enters the transport chain and moves an electronues differ from controls (including the background area), bold at the farm differs from

e farm Farm

30 days 15 days 30 days

0.368 � 0.121 0.302 � 0.126 0.081 ± 0.0450.173 � 0.080 0.089 � 0.092 0.023 ± 0.018

0.323 � 0.166 0.415 ± 0.055 0.019 � 0.0280.135 � 0.095 0.136 � 0.042 0.003 � 0.004

Table 3Mean (�SD, N¼ 10) energy flux ratios per site and exposure length: 4P0 (TR0/ABS), maximum quantumyield of primary photochemistry and 4D0 (DI0/ABS), the complementaryflux of dissipated excitation energy. Shaded values differ from controls (including the background area), bold at the farm differs from 60 m distance (t-test, P < 0.05).

Control Background area 60 m from the farm Farm

15 days 30 days 15 days 30 days

E. prunastri4P0 0.535 � 0.058 0.512 � 0.072 0.359 � 0.046 0.448 � 0.084 0.255 � 0.144 0.274 ± 0.064D0 0.465 � 0.058 0.488 � 0.072 0.641 � 0.046 0.552 � 0.084 0.745 � 0.144 0.726 ± 0.06

P. furfuracea4P0 0.588 � 0.026 0.513 � 0.106 0.418 � 0.065 0.376 � 0.102 0.326 � 0.074 0.152 ± 0.0334D0 0.412 � 0.026 0.487 � 0.106 0.582 � 0.065 0.624 � 0.102 0.674 � 0.074 0.848 ± 0.033

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e2265 2263

turnover number, N; average fraction of open RCs, Sm/tFmax) dependon the time necessary to reach FM (tFmax). This parameter makessense only if can be accurately measured, this means only if a clearFM appears in the fluorescence transient (Strasser et al., 2000).Since a few of our fluorescence curves did not show a clear FM andreached the highest fluorescence at 2 ms, in those cases, the aboveparameters have been excluded from the analysis.

Theoretically, the more the electrons from QA� are transferredinto the electron transport chain, the longer the fluorescenceemission remain lower than FM and the bigger the turnover numberwill result (Strasser et al., 2000). Our data indicated that thedecrease in the turnover number and the reduction of the fractionof open reaction centre are a consequence of NH3 treatment, up toa drastic drop in the thalli exposed at the farm.

The global index of the photosynthetic performance (PIABS)measured in both E. prunastri and P. furfuracea was much moresensitive to NH3 than FV/FM, the most commonly used indicator fora rapid screening of photosynthesis. A similar observation is repor-ted by van Heerden et al. (2007), which applied the JIP-test to assessthe effects of limestone dust from quarrying on the desert shrubZygophyllum prismatocarpum. In fact, the parameter PIABS combinesin a single expression the three functional steps of the photosyn-thetic activity (light absorption, excitation energy trapping, andconversion of excitation energy to electron transport), resulting ina very sensitive indicator of stress suitable to be applied for physi-ological and environmental screenings (Strasser et al., 2004).

4.2. Lichen transplants and ammonia pollution

Transplanting lichens from a site to another is useful to revealimpacts of polluted areas or, conversely, the benefits of cleanconditions. Lichen thalli were exposed ensuring similar conditionsbetween sites (cardinal exposure, irradiance, height from ground)except for NH3 level. For an appraisal of the influence of climate,both species were harvested from a remote area. Values of thetransplants were compared with control samples before theexposure and air temperature, light irradiance and humidity werechecked out for differences between sites, whichwere insignificant.

Table 4Mean values (�SD, N ¼ 10) of technical parameters referred to the area above thetransient curve in control and transplanted thalli after 30 days: N ¼ turnovernumber; Sm/tFmax ¼ average redox state of QA�/QA. Shaded values differ fromcontrols (including the background area), bold at the farm differs from 60m distance(t-test, P < 0.05).

Control Backgroundarea

60 m Farm

E. prunastri N 39 � 6 41 � 15 20 � 18 2 ± 1Sm/tFmax 0.14 � 0.03 0.12 � 0.03 0.11 � 0.04 0.09 � 0.03

P. furfuracea N 42 � 7 35 � 10 17 � 13 1 ± 1Sm/tFmax 0.16 � 0.02 0.15 � 0.03 0.11 � 0.03 0.06 � 0.06

Many stressors can alter chlorophyll fluorescence, our resultsshowed that in the short-term exposure a negative contribution tophotosynthesis was given by the impact of NH3 pollution and thatthe short duration of the transplantation probably reduced theinfluence of the dry climate. However, stressing environmentalconditions influence photosynthetic pigments also in short-termexposures: for example, a general depression of photosyntheticpigments was found in E. prunastri exposed to high irradiance anddifferent water treatments during a Mediterranean summer (Paoliet al., in press). A prolonged exposure to dry conditions affectslichen photosynthesis also in remote areas. During a study carriedout in Northern Crete, the same species were transplanted along analtitudinal gradient (manuscript in preparation): the overall effectwas a general depression of the photosynthetic performances andthe content of photosynthetic pigments from upland to lowlandand with increasing drought and length of the exposure. Pirintsoset al. (2009) showed under laboratory conditions that the photo-synthetic efficiency of E. prunastri is impaired by heavy loads ofammonium independently of light regime and that polyaminesreduce the sensitivity of this species to excess N.

Lichens with green algae depend on direct N deposition on thethallus surface to satisfy their N requirements (Honegger, 1991) andhence are sensitive to excess NH3 in the atmosphere.

NH3 is generally readily deposited within the first 200 m fromsource and reaches background level within 1000 m, but it can bealso converted in the atmosphere to aerosol NH4

þ particles that maydiffuse over larger areas (Krupa, 2003). The residence time of NH3in the atmosphere may vary from few hours to 4 days, dependingon the season, source and site characteristics (Krupa, 2003).

Atmospheric concentrations of NH3 around the selected sheepfarm were similar to the values measured around isolated pointsources: NH3 concentrations > 14 mg/m3 as an annual average aregenerally found within 100 m from similar sources, whereasbackground levels of 0.7e3.5 mg/m3 can be found within 0.5e3 km(Fangmeier et al., 1994). In Mediterranean Italy, Frati et al. (2007)found high NH3 levels at the centre of a pig stockfarm (peak of267 mg/m3), with a sharp decline of atmospheric level to theregional background of 0.7 mg/m3 within a distance of 2.5 km, andwith a 98% reduction (4.6 mg/m3) reached already in the first 200 mfrom the source. Pitcairn et al. (2002) reported a 95% reduction inNH3 at 650 m from a point source in the UK.

In a region with intensive farming the regional annual level riseup to 21 mg/m3 (Fangmeier et al., 1994), which is higher comparedwith thevalueswerecorded ina remoteareaofCrete (up to2mg/m3),where sheep farms are conducted traditionally with extensivesystems (De Rancourt et al., 2006). However, as part of the tradi-tional farming system, the numerous flocks are kept outdoors andgrazing is continuous and diffuse (Volanis et al., 2007). Land forextensive livestock production is grazed in most of the island andseveral flocks are grazed also on highmountain pastures during thewarm season (Hadjigeorgiou et al., 2005). As a consequence, all theregion is widely concerned by diffusion and deposition of N

Table 5Global indexes of the photosynthetic performance (PIABS, N ¼ 10) and % deviation from the samples exposed in the remote area (see text). Shaded values differ from controls(including the background area), bold at the farm differs from 60 m distance (t-test, P < 0.05).

Control Background area 60 m from the farm Farm

15 days 30 days 15 days 30 days

E. prunastriPIABS 2.377 � 2.005 2.318 � 1.774 0.386 � 0.224 1.587 � 1.218 0.080 ± 0.064 0.054 ± 0.062Deviation 2.5 e 83.3 31.5 96.5 97.7

P. furfuraceaPIABS 4.754 � 0.456 4.050 � 1.817 1.002 � 0.675 0.823 � 0.791 0.622 � 0.462 0.002 ± 0.003Deviation 14.8 e 75.3 70.1 84.6 100

L. Paoli et al. / Environmental Pollution 158 (2010) 2258e22652264

compounds and habitat eutrophication. This point is relevant inconsideration of the fact that in Europe the critical level for NH3 inthe air is set at 8 mg/m3 as annual average concentration, butspecific effects of NH3 have been observed on sensitive communitiesat much lower concentrations and recently the concentration of1mg/m3NH3hasbeenproposedasnewcritical level aftera long termexposure (years) for bryophyte and lichen communities (Cape et al.,2009). In the future, it would be of interest to test minimalconcentrations and exposure time needed to cause effects on lichenphotosynthesis for a betterdesignationof critical levels for ammoniato lichens in the Mediterranean region.

5. Conclusions

This study indicated that photosystem II of lichens exposed toNH3 in the field is susceptible to this pollutant in the gas-phase, asshown by the JIP-test. A main consequence of NH3 pollution is thestrong depression of the electron transport activity and the dissi-pation of absorbed energy, chiefly as heat. The parameter PIABS,a global index of photosynthetic performance that combines threefunctional steps of the photosynthetic activity (light absorption,excitation energy trapping, and conversion of excitation energy toelectron transport) was much more sensitive to NH3 than the FV/FMratio, themost commonly used stress indicator. P. furfuracea is moresensitive to NH3 than E. prunastri. The results suggest that fluo-rescence measurements applied to lichen transplants, as stressindicators, are a suitable tool to investigate the effects of NH3 fromlivestock husbandry.

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