REGULAR ARTICLE

Differences in yield, Ellenberg N value, tissue chemistryand soil chemistry 15 years after the cessation of nitrogen addition

Carly J. Stevens & J. Owen Mountford &

David J. G. Gowing & Richard D. Bardgett

Received: 12 October 2011 /Accepted: 30 January 2012 /Published online: 3 March 2012# Springer Science+Business Media B.V. 2012

AbstractBackground & Aims The consequences of fertiliseraddition to semi-natural grasslands are well under-stood, but much less is known about the consequencesof cessation of nitrogen fertiliser regimes, includingrates of recovery. This study aimed to investigatewhether the effects of nitrogen (N) additions to amesotrophic grassland were still apparent 15 yearsafter the cessation of N inputs.Methods A long-term experiment at Tadham Moor,UK, received N additions at rates of 0, 25, 50, 100

and 200 kg N ha−1 yr−1 between 1986 and 1994.Fifteen years after the cessation of N additions soilchemistry, plant tissue chemistry, plant biomass andEllenberg N values were assessed.Results KCl-extractable ammonium-N, total soil N,total organic carbon and microbial biomass N differedbetween the controls and the higher historic levels ofN addition. Plant tissue chemistry showed no signif-icant effects of previous N addition. Above-groundbiomass was higher where N had been added, al-though this response was only weakly significant.The species composition of the vegetation showedeffects of the N addition with mean Ellenberg Nvalues significantly higher than the control in mosttreatments.Conclusion The effects of long-term N addition canbe seen for many years.

Keywords Carbon . Nitrogen .Mesotrophicgrassland . Plant tissue chemistry . Recovery . Soilchemistry

Introduction

Semi-natural grasslands in England and Wales de-clined considerably in their extent during the secondhalf of the twentieth century. Fuller (1987) estimatedthat in the fifty years prior to 1984, semi-natural grass-lands had declined in area by 97%. One of the reasons

Plant Soil (2012) 357:309–319DOI 10.1007/s11104-012-1160-4

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C. J. Stevens (*) :D. J. G. GowingEnvironment, Earth and Ecosystems,The Open University,Walton Hall,Milton Keynes MK7 6AA, UKe-mail: c.j.stevens@open.ac.uk

C. J. Stevens : R. D. BardgettLancaster Environment Centre, Lancaster University,Lancaster LA1 4YQ, UK

J. O. MountfordCentre for Ecology & Hydrology,MacLean Building, Benson Lane, Crowmarsh Gifford,Wallingford, Oxfordshire OX10 8BB, UK

for this decline has been agricultural intensification,and especially the increasing use of inorganic fertil-isers to improve plant production (Blackstock et al.1999). The consequences of fertiliser addition to semi-natural grasslands are well established, and include areduction in species richness and diversity, and anincrease in dominance of a few agriculturally desirableand competitive species such as Lolium perenne andTrifolium repens (Kirkham et al. 1996). The ParkGrass experiment at Rothamsted, England, the world’slongest running ecological experiment, has clearly dem-onstrated these changes. Here, over 150 years of fertil-iser additions have led to reduced species richness and adomination by grasses (Silvertown et al. 2006). Nitro-gen additions can result in marked changes in soil chem-istry. For example, long-term experimental additions ofN in a range of habitats have resulted in increasedconcentrations of nitrate, ammonium and dissolved or-ganic N (DON) in the soil (e.g. Britton and Fisher 2008;Horswill et al. 2008; Pilkington et al. 2005a), total Ncontent and reduced C:N ratio (Gundersen et al. 1998;Pilkington et al. 2005a), changes in soil microbial bio-mass and community structure (Johnson et al. 1998),and increased turnover of soil organic matter includingN mineralisation and nitrification (e.g. Gundersen et al.1998; Morecroft et al. 1994; Pilkington et al. 2005b).Reductions in soil pH and associated changes in cationconcentrations (Blake et al. 1999; Horswill et al. 2008)have also been observed.

Although there is a good understanding of theeffects of N addition on the plant species compositionand soil chemistry, much less is known about the longterm consequences of cessation of N fertiliser regimes,including rates of recovery and the potential for rever-sion to a diverse grassland community. The few stud-ies that have investigated recovery from N additionreport slow effects being apparent in both vegetationspecies composition and nutrient cycling, even manyyears after the cessation of N inputs (Clark et al. 2009;Královec et al. 2009; Mountford et al. 1996; Olff andBakker 1991; Olff et al. 1994; Power et al. 2006;Strengbom et al. 2001). For example, plant communi-ty composition failed to show recovery from theeffects of N addition eight years after N inputs ceasedin heathland (7.7 kg N ha−1 yr−1 for seven years)(Power et al. 2006) and after 19 years in boreal forest(N addition 34, 68, 108 kg N ha−1 yr−1 for 28 years)(Strengbom et al. 2001). Effects on above-groundbiomass are more mixed, with higher productivity

observed in the heathland experiment (Power et al.2006) and non-significant trends observed in an ex-periment in Minnesota prairie grassland after 12 years(N addition 10, 20, 34, 54, 95, 170 kg N ha−1 yr−1 for10 years) (Clark et al. 2009). However, in a chronose-quence of hay meadows that had not received fertiliserinputs for 2, 6, 19 and 45 years (following long-term Naddition at rates between 100–250 kg N ha−1 yr−1) nopersistent treatment effects were apparent (Olff et al.1994). Soils also seem to be slow to recover from longterm N addition. For example, Clark et al. (2009)reported elevated nitrate levels and rates of N miner-alisation in prairie grassland 12 years after cessation ofN addition, whilst Power et al. (2006) found thatalthough soil pH recovered rapidly, effects on totalsoil N and microbial biomass N persisted.

The overall aim of this study was to test the hy-pothesis that N addition in mesotrophic grasslandresults in changes to the plant community and soilchemistry that can be observed many years after Naddition has ceased. There are very few recoveryexperiments where treatments have been long-termand monitoring after the cessation of the experimenthas taken place after a long time period. In order toaddress this knowledge gap we report the results of along-term N addition experiment at Tadham Moor,Somerset, UK (Mountford et al. 1993), 15 years afterthe cessation of N additions.

Previous studies at this site have demonstrated thatthese grasslands are very sensitive to N addition, andapplication of N at relatively low levels can lead toadverse effects, 25 kg ha−1 N yr−1 encouraged thespread of agriculturally productive grasses withintwo years. At 50 kg N ha−1 yr−1 species richness wassignificantly reduced within three years (Mountford etal. 1993). Moreover, after five years of fertiliser appli-cation, the balance of plant species in the seed bankhad changed in favour of species that were morecompetitive under fertile conditions (Kirkham andKent 1997). Only a limited assessment of soil chemistrywas made at Tadham Moor between 1986 and 1994when the fertiliser experiment ceased to be activelymanaged, but there were clear impacts of N additionon nitrogen cycling. Kirkham and Wilkins (1993)showed that between 1987 and 1990 soil nitrate con-centrations and total mineral N (nitrate+ammonium),assessed on a monthly basis, frequently increased withthe level of N addition (the N25 treatment was notassessed), and rates of nitrification were also higher in

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plots receiving larger amounts of N. Here, we investi-gate whether the effects of N addition on vegetation andsoil are still evident 15 years after fertiliser additionceased.

Methods

Field experiment

The field experiment is located at Tadham Moor in theSomerset Levels, in the south west of England (lati-tude 50.199°, longitude −7.007°) on a peat soil, pH is6–6.5. The soils are earthy, eu-fibrous peats withoutclear horizons, belonging to the Altcar 1 series withsmall areas of the Adventurers’ and Blackland seriestogether with oligo-fibrous peats of the Turbury Moorseries (Mountford et al. 1993). Atmospheric N depo-sition is currently approximately 21 kg N ha−1 yr−1

(estimated using www.apis.ac.uk). The Somerset Lev-els are internationally important for conservation andsupport large populations of wading birds, as well aslarge areas of grassland on peat soils that support highplant species diversity (Kirkham et al. 1996). Prior tothe initiation of the experiment, the grassland had beentraditionally managed for many years with a hay cuttaken in mid-summer followed by aftermath grazingby cattle. The vegetation was a mosaic of the UKNational Vegetation Classification communities MG5Cynosurus cristatus-Centaurea nigra grassland and

MG8 Cynosurus cristatus-Caltha palustris flood pas-ture (Rodwell 1992). The experiment was initiated in1986 following concerns that extensive areas of old,species-rich grassland were at risk of reductions infloristic diversity as a result of agricultural intensifica-tion (Mountford et al. 1993). The experiment aimed toexamine the effects of N application on plant speciesrichness and productivity. The experiment consists ofthree randomised blocks with five N treatments ran-domly allocated to treatments within each block.Nitrogen was added to replicated field size plots(between 0.56 and 1.13 ha) twice a year in mid-April and early July as ammonium nitrate at rates of0, 25, 50, 100 and 200 kg N ha−1 yr−1 (referred toas N0, N25, N50, N100 and N200) (Fig. 1). Phos-phorus (P) and potassium (K) removed in hay werereplaced (P as triple superphosphate and K as muri-ate of potash). Nitrogen application rates cover therange of fertiliser additions typically added to grass-lands in the Somerset Levels and Moors (Mountfordet al. 1994). Plots were subdivided in 1990 withfertilisation ceasing on half of each plot. This inves-tigation considers only the half of the plot wheretreatments were continued until 1994, with the lastfertiliser application being in July 1993.

The meadow is cut annually for hay in the summerusing a reciprocating hay mower and the aftermath isgrazed with cattle. During the period when the exper-iment was running, this aftermath grazing was man-aged to maintain the same amount of herbage on each

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Fig. 1 Plan of the experi-ment at Tadham moor,Somerset, UK. Treatmentsare: 0, 25, 50, 100, 200 kg Nha−1 yr−1

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plot (12 month old Hereford x Friesian steers with twoanimals per plot until herbage reached 5.5 to 6.5 cmheight (Tallowin and Smith 1994)). Since the cessationof fertiliser additions, fences between plots were re-moved so that animals can graze freely. During thesecond phase of the experiment water levels wereraised. The changes in N turnover as a consequenceof raised water levels are likely to have had an impacton N cycling with enhanced denitrification in wetareas or the meadow (Tallowin and Smith 1994). Nadditions were completely ceased in 1994. Full detailsof the experiment are provided in Mountford et al.(1996, 1993) and images of the plots can be found intable S1.

Sampling

Plant species composition was surveyed in May 2009.Ten 2×2 m quadrats were located on two transects toform an ‘X’ across each of the plots. In each quadrat,all vascular plants and bryophytes were identified tospecies level. Mean Ellenberg values for N were cal-culated based on species presence using Ellenbergvalues recalculated for the UK and for bryophytes(Hill et al. 1999, 2007). Ellenberg N values describethe realised niche of plant species in terms of theirpreference for soil fertility. The scale runs from 1(indicator of extremely infertile sites) to 9 (indicatorof extremely rich situations) (Ellenberg 1979).

Soil and plant tissue samples were collected inJune, September and December 2009 and March2010, to capture seasonal variation in soil chemicalproperties. Four randomly located samples were col-lected within each plot. Areas that showed clear signsof animal congregation were avoided. Vegetation sam-ples for analysis of tissue chemistry were collected bycutting an area approximately 10 cm2 to a height of1 cm above the soil surface. Soil samples were col-lected from the same four locations as vegetationsamples at a depth of 0 to 10 cm using a 5 cm diameterDutch auger. Samples were bagged and kept coolbeing returned to the laboratory for analysis.

Above-ground biomass samples were collected inJune only, prior to the hay cut. Four samples werecollected per plot in 10×100 cm strips. Data are pre-sented per m2. Root biomass samples were collected inSeptember only in 5 cm diameter metal cores to a depthof 30 cm at the same locations as samples were collectedfor soil chemistry (four replicate samples per plot).

Laboratory analysis

KCl-extractable nitrate-N and ammonium-N, total or-ganic nitrogen (TON) and total organic carbon (TOC),N and C in microbial biomass, pH, and N and C invegetation were determined on four replicate samplesper plot for each of the seasonal visits. For all analysesstandard curves were determined, measurement blankswere analysed and measurement replicates were made.

Soils were stored in cool boxes for transit and thenwere kept refrigerated prior to analysis within 72 h.Soil samples were disaggregated and large roots andstones were removed by hand. Soil moisture wasdetermined by drying approximately 10 g of soil inthe oven at 105°C for 24 h. KCl extracts were per-formed using 5 g of fresh soil and shaking for 1 hourwith 25 ml 1 M KCl then filtering through a WhatmanNo 1 filter paper (MAFF 1986). Organic N was deter-mined using water extracts (5 g of fresh soil andshaking for 10 min with 35 ml deionised water thenfiltering through a Whatman No 1 filter paper) fol-lowed by a digest of the supernatant using 0.165 Mpotassium persulphate followed by autoclaving.Nitrate-N and ammonium-N concentrations were de-termined colorimetrically using an autoanalyser(Braun Lubbe AutoAnalyzer 3). The organic fractionwas determined as the difference between the sum ofnitrate-N and ammonium-N in the water extract andthe sum of nitrate-N and ammonium-N after the per-sulphate digest (Ameel et al. 1993). TOC analysis wasperformed on water extracts using 5 g of fresh soil andshaking for 10 min with 35 ml deionised water thenfiltering through a Whatman No 1 filter paper fol-lowed by analysis using a total carbon analyser (Shi-madzu TOC 5000A). Microbial biomass C and N weredetermined using 5 g fresh soil fumigated with ethanolfree chloroform for 24 h followed by extraction with0.5 M K2SO4 (Brookes et al. 1985). Extracts wereanalysed for nitrate-N and ammonium-N using theautoanalyser, and C using the total carbon analyser.All measurements were corrected for soil moisturecontent and expressed on a μg per g dry soil basis.pH was determined using a Mettler Toledo pH probewith 10 g fresh soil in 50 ml deionised water shakenfor 30 min.

Soil total C and N and extractable P were deter-mined only on the soil samples collected in June. TotalC and N were determined using an elemental analyser(Elementar Vario El III). Extractable P was determined

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using an Olsen extract followed by colorometric anal-ysis (MAFF 1986).

Vegetation N and C were determined using anelemental analyser. For plant-tissue P, the four repli-cate samples per plot were bulked for each plot. Planttissue P, K, Ca, Mg, and Na was analysed using a dryashing method (Chapman and Pratt 1985) followed byacidification with 1% HNO3 and analysis with ICP-AES (Leeman Prodigy).

Aboveground biomass samples were dried in anoven at 55°C for three days and then weighed. Rootbiomass samples were washed thoroughly to removeall soil and then dried at 55°C for three days andweighed.

Data analysis

Log corrections were applied to data where necessary.Data were analysed using general linear models in R(package lme4) with N treatment, block and month(where applicable) as factorial independent variables.

Results

Ellenberg values and plant biomass

Fifteen years after the cessation of N addition, assess-ment of the mean Ellenberg N values showed a sig-nificant positive effect of N treatment on Ellenberg Nvalue (p<0.001; Fig. 2a). Mean Ellenberg N values inall treatments except the N25 treatment were signifi-cantly higher than the control; mean Ellenberg valueswere 5.6 % higher than controls in the N50 treatment,3.0 % higher in N100 treatment and 9.2 % higher inN200 treatment (table S1).

Above-ground biomass also increased with increas-ing N addition (p00.05; Fig. 2b) but although theoverall model showed a significant effect of N noindividual treatments were significantly higher thanthe control (table S1). There was no significant effectof N addition on root biomass in any of the treatmentswhen compared to the control (p00.75) .

Soil chemistry

Results for soil chemistry showed some statisticallysignificant differences between treatments and controlswith many variables showing strong seasonal effects

(Table 1. and table S1). KCl extractable ammonium-Nshowed a significant effect of N addition (p<0.05),although values were significantly greater than the con-trol only in the N100 treatment where mean extractableammonium concentrations (averaged across four sea-sonal measurements) were 10 % higher than in thecontrol. This measure also varied with season, beinghighest in September and lowest in December. KClextractable nitrate-N showed no significant effect of Naddition (p00.16), but did show a significant effect ofseasonality with values being highest in December andMarch and lowest in June. There was no significanteffect of N addition (p00.33) on TON, although therewas a seasonal effect (p<0.01) with values being signif-icantly lower in June. TOC also showed a significanteffect of N additions (p<0.05) with the N200 plotsshowing significantly lower (12 %) TOC levels thanthe control. Olsen extractable P showed no significanteffect of N addition (p00.41).

Microbial biomass N showed a very strong sea-sonal effect with lower values in March, June andDecember, but higher values in September (p<0.001).There was also a significant effect of N addition onmicrobial biomass N; however, values were quitevariable and only the N100 treatment was signifi-cantly higher than the control (Fig. 2c). Microbialbiomass C showed no significant effect of N addi-tion (p00.29) although there was a strong seasonaleffect with values being highest in March and allother months showing significantly lower microbialbiomass C values (p<0.001).

Percentage soil C and N were only measured duringone sampling date; however, there was a significanteffect of N addition on total N (p<0.001). All Naddition treatments had significantly higher total Ncontents than the control, but the largest effect wasseen in the N100 treatment (29 %). The mean Ncontent in the N25 treatment was 5 % higher thanthe control whilst the N50 was 15 % higher and theN200 was 19 % higher than the control. Total Cshowed no significant effect of N addition (p00.21).Soil C:N was significantly affected by N addition (p<0.05), although only the N50 treatment was signifi-cantly different from the control (7 % lower) (Fig. 2d).

Soil pH also showed no significant effect of Naddition (p00.36), but did show a seasonal effect(p<0.001), with samples collected in June showinga significantly higher pH than samples collected inMarch.

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Plant tissue chemistry

Plant tissue chemistry showed no significant effectsof N addition the elements or element ratios analysedalthough seasonal variability in the results was apparent(Table 2).

Discussion

In this study, which was done 15 years after thecessation of N applications, effects of N addition werestill apparent. There were some differences in the soilchemistry between the N addition treatments (KClextractable ammonium, microbial biomass N, totalsoil N, C:N and TOC), although the majority of these

were only apparent, as might be expected, at thehigher levels of N addition (N100 and N200). Atlower levels of N addition most of these soil variablesdid not differ significantly from the control suggestingthat there is potential for recovery where low levels ofN are added but at higher levels of N addition recoverytime may be very long or recovery may not be possi-ble. In contrast, other soil variables, including nitrate,TON, and pH, appear to have recovered from theeffects of eight years of N addition but significantdifferences in microbial biomass N and ammoniumindicate that some soil processes are still affected bythe N additions. These results are consistent with otherstudies: Clark et al. (2009) found elevated inorganicammonium after 12 years of recovery, and Power et al.(2006) found elevated microbial biomass after 8 years

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N treatment (kg N ha-1 yr-1) N treatment (kg N ha-1 yr-1)

N treatment (kg N ha-1 yr-1)N treatment (kg N ha-1 yr-1)

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Fig. 2 Mean and standard error for (a) Ellenberg N scoresampled in May 2009, (b) above-ground biomass (g m−2) sam-pled in June 2009, (c) soil microbial biomass N (μg g−1 dry soil)

sampled in June, September and December 2009 and March2010, and (d) Soil C:N sampled in June 2009 following eightyears of N addition and fifteen years of recovery

314 Plant Soil (2012) 357:309–319

of recovery. Recovered soil variables are likely to havebeen changed through declines in above-ground bio-mass, above-ground N concentrations and, conse-quently, declines in the N content and biomass ofdecomposing litter (Clark et al. 2009), as well asleaching, elevated rates of N turnover processes andbiomass removal (Power et al. 2006). Both regularflooding and the movement of grazing animals be-tween plots (Tallowin and Smith 1994) could have

contributed to the homogenisation of the plots throughthe dispersal of seed and the redistribution of nutrientsin urine and dung. This suggests that recovery may beeven slower in some circumstances. Regular floodingof the grassland may also have assisted in the recoveryof soil pH through the introduction of base cationsfrom the floodwaters emanating from the surroundingbasic geology (BGS 1984). Susceptibility to floodingis also the most likely explanation for the block effects

Table 1 Mean above- and below-ground biomass and seasonalsoil concentrations for Olsen P, total N, total C, KCl extractablenitrate-N and ammonium-N, TON, TOC, microbial biomass N

and C, and soil pH each N treatment (kg Nha−1 yr−1). F valuesand significance (*p<0.1; **p<0.05; ***p<0.01) are shown forN treatment and seasonality for plant tissue chemical analysis

N treatment F value

0 25 50 100 200 N treatment Seasonality

Root biomass (g core−1) Sept. 4.9 4.8 4.5 4.1 4.6 0.48

Olsen P (μg g−1) June 27.6 27.2 26.7 31.4 24.2 0.41

Soil N (%) June 2.2 2.3 2.3 2.4 2.1 1.68 ***

Soil C (%) June 32.2 34.7 31.8 34.2 30.7 1.50

KCl extractable nitrate-N (μg g−1) June 5.1 3.7 4.1 5.0 5.6 1.66 41.08***Sept. 9.1 7.2 6.2 8.5 7.2

Dec. 11.1 10.1 10.7 11.4 13.7

March 15.2 10.5 20 13.8 12.0

KCl extractable ammonium-N (μg g−1) June 5.9 5.3 4.7 5.5 4.9 4.43*** 96.62***Sept. 7.2 5.3 5.7 8.9 7.3

Dec. 2.0 2.5 2.2 3.0 1.8

March 4.1 4.4 3.5 4.0 2.9

TON (μg g−1) June 32.3 22.3 23.8 40.1 35.4 1.14 3.87***Sept. 47.3 41.7 50.9 51.9 54.6

Dec. 44.7 43.1 37.7 40.4 51.7

March 47.9 35.2 93.5 40.8 53.9

TOC (μg g−1) June 690.4 635.7 635.5 734.2 634.4 3.35** 35.73***Sept. 855.3 763.4 962.9 877.7 826.5

Dec. 1045.9 1349.9 957.9 1144.4 919.6

March 1074.0 911.2 877.7 1087.6 833.1

Microbial Biomass N (μg g−1) June 397.6 403.3 420.3 450.5 397.0 2.91** 211.42***Sept. 929.8 1016.1 1124.0 1266.7 1204.1

Dec. 432.8 408.1 480.3 601.3 529.3

March 240.0 217.8 215.4 248.7 197.6

Microbial Biomass C (μg g−1) June 3565.0 3393.3 3662.6 3455.8 2432.8 1.24 411.15***Sept. 2432.8 1790.4 2220.4 2338.4 2233.3

Dec. 429.1 323.3 373.7 475.4 381.1

March 5471.9 6529.9 4462.0 5079.9 4065.3

pH June 6.6 6.6 6.5 6.5 6.3 0.36 4.15***Sept. 6.3 6.4 6.4 6.3 6.5

Dec. 6.4 6.2 6.5 6.4 6.5

March 6.3 6.4 6.3 6.2 6.4

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observed in many of the analyses (Tallowin and Smith1994), the water table and soils of block 3 differ fromblocks 1 and 2 (Mountford et al. 1994).

Plant-tissue chemistry showed no significant effectsof N addition despite inorganic ammonium availabil-ity still being higher in the highest N treatments. Thisindicates recovery from the situation between 1986

and 1993 when N and K content were significantlyincreased by the N addition treatments (Tallowin andSmith 1994). Investigations across a range of habitatshave demonstrated changes in tissue nutrient concen-trations with increasing N input (e.g. Arroniz-Crespoet al. 2008; Gordon et al. 2001; Leith et al. 1999;Magill et al. 1997). Several studies have found that

Table 2 Mean content (%) or element ratio for plant tissue chemical analysis for each N treatment (kg Nha−1 yr−1). F values andsignificance (*p<0.1; **p<0.05; ***p<0.01) are shown for N treatment and seasonality

N treatment F value

0 25 50 100 200 N treatment Seasonality

C June 41.72 42.11 41.81 41.67 41.28 2.39 35.82 ***Sept. 42.35 42.45 43.09 42.33 41.75

Dec. 43.26 42.45 43.09 42.33 41.75

March 43.67 43.39 43.20 43.37 43.05

N June 1.48 1.16 1.48 1.52 1.48 1.92 83.40 ***Sept. 2.22 2.37 2.42 2.15 2.27

Dec. 2.34 2.12 2.32 2.17 2.74

March 2.08 1.80 2.06 1.96 2.01

P June 0.10 0.10 0.11 0.10 0.12 0.86 16.99 ***Sept. 0.16 0.16 0.18 0.14 0.16

Dec. 0.21 0.17 0.14 0.15 0.13

March 0.13 0.10 0.12 0.15 0.13

K June 0.82 0.80 0.79 0.81 0.79 0.25 19.25 ***Sept. 1.11 1.11 1.30 1.02 1.01

Dec. 0.10 0.90 0.66 0.72 0.73

March 0.50 0.51 0.65 0.59 0.48

Ca June 0.63 0.66 0.63 0.59 0.74 0.60 0.015 **Sept. 0.64 0.71 0.72 0.65 0.71

Dec. 0.65 0.70 0.75 0.69 0.64

March 0.54 0.54 0.50 0.52 0.63

Mg June 0.19 0.21 0.20 0.17 0.21 0.86 24.44 **Sept. 0.23 0.24 0.22 0.22 0.21

Dec. 0.17 0.20 0.23 0.18 0.16

March 0.12 0.11 0.11 0.12 0.12

Na June 0.28 0.21 0.36 0.26 0.40 1.87 4.069 **Sept. 0.21 0.25 0.27 0.18 0.35

Dec. 0.43 0.32 0.29 0.24 0.20

March 0.15 0.12 0.16 0.17 0.31

N:P June 14.59 15.21 13.79 15.54 12.77 0.79 1.467Sept. 14.04 15.40 13.61 15.60 13.61

Dec. 11.32 13.07 16.21 15.70 20.32

March 15.69 19.17 16.92 13.59 15.94

P:K June 0.13 0.13 0.14 0.13 0.15 0.574 21.83 ***Sept. 0.16 0.15 0.15 0.14 0.14

Dec. 0.21 0.19 0.21 0.25 0.27

March 0.29 0.21 0.21 0.25 0.27

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plant tissue nutrient concentrations recovered rapidlyfollowing the cessation of N additions (Boxman et al.1998; Clark et al. 2009; Power et al. 2006) and,consistent with our findings, this has been shown tobe a relatively plastic trait responding rapidly toincreases and decreases in nutrient inputs (Clark etal. 2009; Dise and Gundersen 2004).

Above-ground biomass also increased with N addi-tion in the majority of years between 1986 and 1993(Kirkham and Wilkins 1994). This effect was still ap-parent in our study although the response was onlyweakly significant. Clark et al. (2009) found very sim-ilar results after 10 years of N addition at rates between10 and 270 kg N ha−1 yr−1 and 12 years of recovery in aprairie grassland, with a non-significant trend for in-creased biomass. Differences in species compositionbetween the plots, as discussed below, may account forsome of the differences in above-ground biomass aswell as an increase in the productivity of individualspecies. These effects were not apparent in the below-ground root biomass which showed no significant re-sponse to previous N addition. The addition of N cantypically be expected to reduce below-ground biomass(Bardgett et al. 1999; Ennick et al. 1980) and in theirmeadow chronosequence, Olff et al. (1994) found thattotal belowground biomass and fine root biomass in-creased with time as fields progressed from two to sixand then 19 years since fertilisation. In the field with45 years since last fertilisation root biomass was lowerthan in the field with 19 years since last fertilisation.Unfortunately there is no root biomass data availablefrom the pre-recovery stage so it is not possible todetermine whether root biomass has decreased and thenrecovered. Vegetation Ellenberg N values showedeffects of N addition. Mean Ellenberg N values weresignificantly higher than the control in the N50, N100and N200 treatments, although values in the N25 treat-ment were not significantly different from the control.Although difference in mean Ellenberg scores betweentreatments were small (mean 4.49 for the control and4.91 for 200 kg N ha−1 yr−1) in their calibration ofEllenberg scores, Ertsen et al. (1998), showed that thiscould indicate an increase in above-ground standingbiomass of 1.2 tonnes ha−1. These differences are of asimilar magnitude to those reported by Critchley et al.(2007) who found mean Ellenberg N scores of 4.54 forspecies rich mesotrophic grasslands and 4.89 for de-graded mesotrophic grasslands where grasslands werecategorised based on similarity to British National

Vegetation Classification communities. The resultsdemonstrate that 15 years after the cessation of fertiliseraddition the N25 plots have recovered from the addi-tional N inputs, at least in terms of the mean Ellenberg Nvalue. The apparent recovery of most soil chemistryvariables in the N50 plots, but not in the Ellenberg Nvalue, suggests that there is a lag in the response of thevegetation. This is in agreement with the results ofPower et al. (2006) who found vegetation recoveredmore slowly from the cessation of N inputs than soilchemical variables.

After five years of N addition, Mountford et al.(1996) used trend lines based on individual species,community variables and groups of species and simi-larity to control plots to predict recovery times forvegetation of three, five, seven and nine years respec-tively for the N25, N50, N100, and N200 treatments.This assessment, 15 years after the cessation of Nadditions, demonstrates that recovery times are muchlonger. Indeed, Mountford et al. (1996) suggested thatin a closed sward with few regeneration niches, suchas that found at Tadham Moor, the recruitment of newspecies or the return of those species lost with fertiliseraddition is likely to be slow. This may explain thelack of recovery in the vegetation species composition,but invites the question ‘will the vegetation speciescomposition ever recover’? After 15 years without Nadditions and impacts on soil chemistry still beingapparent, albeit mostly at higher application rates, itcertainly seems possible that the vegetation hasreached an alternative steady state with an increaseddominance of species typically found in higher nutri-ent situations. Reversion may not occur because thenew dominant species have a tendency to be morecompetitive and may support more rapid cycling ofN and other nutrients. Species such as Agrostis capillaris,Holcus lanatus, Rumex acetosa and Lolium perenneincreased in response to N addition (Mountford et al.1993), but had always formed a component of the grass-land sward though previously at lower frequency andcover. Given the lack of regeneration niches, it seemslikely that the less competitive species will find fewopportunities to ‘invade’.

Conclusion

The results of this study, and of a number of otherrecovery studies (Clark et al. 2009; Královec et al.

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2009; Olff and Bakker 1991; Olff et al. 1994; Power etal. 2006; Strengbom et al. 2001), show that the effectsof long-term N addition can be seen for many yearsafter the cessation of N inputs. In the grassland atTadham Moor, lower levels of fertiliser input (i.e.25 kg N ha−1 yr−1) showed signs of recovery in boththe Ellenberg N value and soil chemistry, suggestingthat recovery from similar levels of N addition oratmospheric deposition may be possible. But, at pastapplication rates of 50 kg N ha−1 yr−1 and above,effects on the Ellenberg N value and soil chemistrywere still apparent, demonstrating that at these levelsof N addition recovery is considerably slower. How-ever, N addition through atmospheric N deposition ischronic and has had cumulative impacts over manyyears (Duprè et al. 2010), so recovery may not occuras readily as in this experiment.

Acknowledgements This work was funded by a LeverhulmeEarly Career Fellowship awarded to Carly Stevens. Thanks goto B. Cookson, V. Van Velzen, C. Long, G. Howell and H. Quirkfor assistance in the field and laboratory. The authors are alsograteful to the many people who established and maintained theexperiment and contributed to data collection at Tadham Moorover many years, especially colleagues within Natural Englandand its forebears and from the North Wyke research station ofBBSRC. Thanks to C. Field and three anonymous referees forcomments on this manuscript.

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