Article pubs.acs.org/est
Pitfalls and New Mechanisms in Moss Isotope Biomonitoring of Atmospheric Nitrogen Deposition Xue-Yan Liu,†,‡ Keisuke Koba,*,‡ Cong-Qiang Liu,† Xiao-Dong Li,† and Muneoki Yoh‡ †
State Key Laboratory of Environmental Geochemistry, Institute of Geochemistry, Chinese Academy of Sciences, Guiyang, 550002, China ‡ Institute of Agriculture, Tokyo University of Agriculture and Technology, Fuchu 1838509, Japan S Supporting Information *
ABSTRACT: Moss N isotope (δ15Nbulk) has been used to monitor N deposition, but it remains questionable whether inhibition of nitrate reductase activity (NRA) by reduced dissolved N (RDN) engenders overestimation of RDN in deposition when using moss δ15Nbulk. We tested this question by investigation of δ15Nbulk and δ15NO3− in mosses under the dominance of RDN in N depositions of Guiyang, SW China. The δ15Nbulk of mosses on bare rock (−7.9‰) was unable to integrate total dissolved N (TDN) (δ15N = −6.3‰), but it reflected δ15N-RDN (−7.5‰) exactly. Moreover, δ15N-NO3− in mosses (−1.7‰) resembled that of wet deposition (−1.9‰). These isotopic approximations, together with low isotopic enrichment with moss [NO3−] variations, suggest the inhibition of moss NRA by RDN. Moreover, isotopic mixing modeling indicated a negligible contribution from NO3− to moss δ15Nbulk when the RDN/NO3− reaches 3.8, at which maximum overestimation (21%) of RDN in N deposition can be generated using moss δ15Nbulk as δ15 N-TDN. Moss δ15N-NO3− can indicate atmospheric NO3− under distinctly high RDN/NO3− in deposition, although moss δ15Nbulk can reflect only the RDN therein. These results reveal pitfalls and new mechanisms associated with moss isotope monitoring of N deposition and underscore the importance of biotic N dynamics in biomonitoring studies.
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INTRODUCTION Since the 19th century, anthropogenic N emissions attributable to increasing urbanization, agricultural, and industrial activities have more than doubled the input of reactive N into terrestrial ecosystems.1 Most atmospherically deposited N takes one of two main forms: oxidized N (e.g., NO3−, nitric acid, and NOx) or reduced N (e.g., NH3, NH4+, and dissolved organic nitrogen (DON) although atmospheric DON probably comprises few oxidized N species; hereinafter RDN).2 Consequences of elevated N deposition include water quality degradation, lacustrine and estuarine eutrophication, ecosystem N saturation, and reduced diversity of vegetation.3 Therefore, considerable interest has arisen in tracing sources and partitioning components of N deposition.4−8 Because of receiving N exclusively and passively from atmospheric deposition, mosses are extremely sensitive and important plants in monitoring N sources and recording N-deposition effects.9,10 Plant δ15Nbulk inherently provides a measure integrating N sources and physiological processes during plant N utilization.11 Therefore, measuring moss δ15Nbulk can elucidate atmospheric N sources and N chemical compositions.9,12−14 To date, two substantial contributions have been made from moss Nbulk monitoring. First, moss Nbulk concentration and δ15Nbulk signature provide a rapid and integrated method to identify the influence of anthropogenic N © 2012 American Chemical Society
sources at a given site because the dominant N sources are usually differentiable on the basis of their δ15N signatures.4−6 For example, although both kinetic and equilibrium isotope fractionations remain largely uncertain in NH3 volatilization from different sources and subsequent conversion to NH4+ in atmosphere,15−17 the δ15N of rain NH4+ originating from human/animal excretory wastes and urban sewage appeared to be generally lower than that from man-made fertilizers in rural areas (see a data review in Supporting Information Table S6). Gaseous NOx can have positive and negative δ15N signatures. For instances, Heaton 18 reported negative δ15N values for NOx from vehicle engines (−13 to −2‰). Widory 19 found negative δ15N values (−19.4 to +2.9‰, mean = −7.5‰) for fuel-oil particles. Differently, positive δ15N values have been reported for tailpipe NOx (+3.7 to +5.7‰),9,20,22 roadside NOx (mean = +4 to +6‰),9,21 and particles from unleaded and diesel oils (+3.9 to +5.4‰).19 Clearly, the δ15N of gaseous and particulate N depends on the sources and isotopic fractionations during combustion and after-emission processes. In addition, the Received: Revised: Accepted: Published: 12557
February 26, 2012 October 10, 2012 October 10, 2012 October 10, 2012 dx.doi.org/10.1021/es300779h | Environ. Sci. Technol. 2012, 46, 12557−12566
WO = wet deposition in open fields; TF = throughfall in broadleaf−conifer mixed forests; DIN = dissolved inorganic N; EB = epilithic mosses on bare rock. bData calculated according to the mean percentage of DON in TDN measured at the same site (U1 in Figure 1) in 2009. cData cited from ref40. dSample collected in July 2010; − = data not available. Values in parentheses show the data range.
oxidation of atmospheric N2 during the combustion of fuels may also complicate δ15N differences between NOx and fuel sources. Even so, the δ15N of NOx from traffic sources generally exhibits lower values than that reported for NOx from coal-fired power plants (+6 to +26‰),4−6,18,21 and particulate N is generally more 15N-enriched than N in wet deposition.15−22 Second, moss Nbulk and δ15Nbulk enable the high-resolution monitoring of anthropogenic N distribution at geographic scales that are difficult to achieve through direct sampling of the atmosphere.12−14 On the basis of the generally higher δ15N of NO3− than RDN in deposition,4−6,15−22 moss Nbulk and δ15Nbulk were applicable to differentiate the dominant N form in deposition, facilitating a link between inputs of specific N species with ecosystem responses.12−14 Because N deposition in most regions of Europe and eastern Asia has a higher RDN than NO3−−N deposition,1,23 a general pattern of decreasing moss δ15Nbulk with an increasing ratio of RDN or NH4+−N to NO3−−N has been established.12,13,24,25 Such a relation is now extended to evaluate N deposition in eastern Asia, where RDN is markedly higher than NO3−−N in deposition (e.g., NH4+−N/NO3−−N = 3.1−21.5 in China).24,25 Nevertheless, many studies have revealed that moss species prefer RDN (NH4+ and DON) over NO3− for energetic economy and have revealed the inhibition of NRA by coexisting NH4+−N or amino acids.26−30 Even the high N deposition (>10 kg N ha−1 yr−1) and ambient NOx were found to suppress moss NO3− assimilation severely.31,32 Moss NRA, which can be inhibited by end products (NH4+ and amino acids) and which can be induced by substrate NO3−, appears to be sensitive to the fractional compositions of dissolved N species in deposition.29,32 For these reasons, the contributions of RDN and NO3− to moss Nbulk are inherently difficult to regard as consistent with those in deposition. These results, however, raise the uncertainty of whether the interpretation of N deposition using moss δ15Nbulk underestimates the contribution of NO3− in deposition and overestimates the contributions and influences of RDN, which dominates N deposition. Alternatively, because of the inducibility of plant NRA by substrate NO3−, low ratios of RDN to NO3−−N in deposition (the dominance of NO3−−N) might cause more assimilation of NO3− than estimated from deposition data.26−29 In this case, moss δ15Nbulk might overestimate the contribution of NO3− and underestimate RDN in deposition. Therefore, investigating the aberrations between moss δ15Nbulk and that of bioavailable N in deposition and verifying the reliability of moss δ15Nbulk for expressing the actual contributions of bioavailable N in deposition are valuable tasks, given the effect of moss N utilization on δ15Nbulk. Recently, we applied the denitrifier method for measuring NO3− concentration ([NO3−]) and stable isotopes (δ15N and δ18O) in natural plants.33,34 These parameters provided novel insights into plant NO3− sources and dynamics, which is more straightforward than traditional methods (e.g., 15N-labeling) because they entail no disturbance to N pools and metabolic dynamics in target organisms. A preliminary survey in western Tokyo showed that moss NO3− isotopes can change with isotopes of accessible NO3− sources.33 Differently, vascular plant organs in which NRA was conducted showed isotopic compositions of tissue NO3− much higher than that of soil NO3−.34 These benefits underscore the potential of tissue NO3− isotopes for tracing external NO3− sources and for elucidating plant NO3− dynamics. This study was designed to extend our knowledge of NO3− isotopic records in natural mosses and to promote the development of moss isotope biomonitoring techniques. The
a
35, 36 −
−3.5 ± 1.0, 24, 38 (−5.5 to −1.4) −
− −
− −
− −
− 10.1
10.9 −
− 8.2
8.3
4.2 ± 1.0
1.9
3.2
−
0.7 0.2
Sept.−Oct. 2006 rural
WO (920)
1982−1984 rural
WO (1174)
0.2, (0.0−0.4) 0.9, (0.6−1.2)
−
2.6
39 − − − − − 3.7 − 2.4 2.3 TF (322) 2001−2003
0.3, (0.1−1.6) 0.7, (0.1−2.6)
−
1.3
39 − − − − − 8.1 − 6.2 3.2 WO (854) 2001−2003 suburban
0.3, (0.1−0.8)
0.8, (0.04−2.9)
−
1.9
35, 36 − − − − − 12.2 − 8.1 1.9 1982−1984 suburban
WO (894) Dec. 2008−Sept. 2009 urban
WO (1174)
0.4
0.7
−
4.2
this study −7.9 ± 3.3d, (−12.6 to −4.4) −6.3 ± 5.5 (−18.0−7.4) −7.5 ± 7.2 (−22.9−9.7) −15.9 ± 3.3c (−19.8 to −10.4) −1.9 ± 3.0 (−8.3−2.9) 30.7 9.8 15.6 5.3 4.6 ± 2.3, (0.7−13.0) 1.8 ± 1.7, (0.04−8.1) 0.6 ± 0.3, (0.1−1.4)
1.1 ± 1.1, (0.4−5.9)
−8.9 ± 1.7, 24, 38 (−12.5 to −6.0) − − − − 26.0 6.3 16.3 3.4 8.2 ± 6.2, (2.1−29.0) 1.6 ± 0.8, (0.4−3.8) 0.3 ± 0.2, (0.1−0.8) Sept.−Oct. 2006 urban
WO (920)
1.0, (0.2−1.7) June−July, 2001 urban
WO (983)
0.2, (0.1−0.9)
0.7 ± 0.3b, (0.2−1.6)
37 − − − −12.2 ± 6.7 2.0 ± 4.4 11.4 − 9.5 1.9 5.1 −
ref moss (EB)
− −
TDN RDN
− − − 16.4 − 13.0 3.5 3.8 1982−1984
WO (1174)
Article
urban
site
period
type (annual amount, mm)
0.3
NO3−
1.1
NH4+
DON −
NO3−
NH4+
DON
DIN or TDN
NO3−
NH4+
δ15N/‰ wet N deposition (kg N ha−1 yr−1)
RDN or NH4+− N: NO3−−N concentration (mg-N L−1)
Table 1. Concentrations and Isotopic Signatures of Wet N Deposition Across the Urban, Suburban and Rural Area at Guiyang, SW Chinaa
35, 36
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Figure 1. Map showing the Guiyang area and sampling sites of natural mosses. R, S, and U denote rural, suburban, and urban sites, respectively.
dissolved nitrogenous species in wet deposition were measured from Dec. 2008 to Sep. 2009 (Table 1, materials and methods are explained in the Supporting Information).
main hypothesis to be tested in this study is the following: in a case in which N deposition is dominated by RDN (high RDN/ NO3−), moss δ15Nbulk records the δ15N of RDN more exactly, whereas tissue NO3− will preserve isotopic signals of atmospheric NO3− because of the inhibition of moss NRA by RDN. We chose the Guiyang area (SW China) because of (1) the marked dominance of RDN in N deposition and (2) wellcharacterized information related to N deposition sources and levels, and moss δ15Nbulk across the urban−rural gradient (Table 1). Briefly, the δ15N of NH4+ in wet deposition was found to be much lower than that of NO3−, the δ15Nbulk in epilithic mosses showed lower values in the urban than in rural areas.24,37,40 Inorganic N deposition was dominated by NH4+− N, with major sources from sewage/waste NH3 emissions in urban and fertilizer NH3 in rural areas.24,37 For a synoptic survey of mechanisms of moss N utilization and isotopic records, explicit concentrations and isotopic compositions of
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MATERIALS AND METHODS The Guiyang area, which has a subtropical monsoon climate, has mean an annual relative humidity and temperature of 86% and 15.3 °C, respectively. A decrease in the N levels and the dominance of RDN have been found in atmospheric depositions across the urban−rural transect (Table 1). Sampling sites were located along the northeastern urban− suburban−rural transect in the Guiyang area (Figure 1). In July 2010, sampling was conducted on saxicolous mosses on bare rock and with thin soil layers and on terricolous mosses in open fields and on the floor of pine forests (Pinus massoniana Lamb.). To avoid possible interspecific differences, we collected moss species with uniform or similar morphological traits. Each 12559
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Figure 2. Concentrations and δ15N ratios of bulk N (a, b) and tissue NO3− (c, d) in natural mosses in the Guiyang area. EB and ES denote epilithic species on bare rock (n = 6 in urban and suburban, n = 5 in rural) and on rocks with organic soils (n = 3), respectively. TO and TF denote terricolous species in open fields (n = 3) and on floors of pine forests (n = 3), respectively. Supporting Information Tables S1−S4 present results of the statistical analysis (multiple comparisons among mean values).
Procedures of moss NO3− extraction, and for the measurements of tissue [NO3−] and stable isotopes were the same as those described in earlier reports.33,34 Detailed experimental procedures for N deposition data (concentrations and δ15N analysis) are presented in the Supporting Information. The analytical precision for δ15N-NO3− was better than ±0.2‰ (SD) and ±0.5‰ (SD) for δ18O-NO3−. Following the eighth Stable Isotope Brochure,41 the extraneous factor of 1000 in the traditional delta definition was avoided, and the natural abundances of 15N and 18O (δ15N and δ18O) were expressed in parts per thousand (per mille) by multiplying them by 1000:
sample of epilithic mosses was a mixture of Erythrodontium (mainly E. julaceum and H. plumaeforme), Eurohypnum (E. leptothallum) and Haplocladium (mainly H. microphyllum), whereas terricolous moss sample included species of Hypnum (mainly H. plumaeforme), Thuidium (mainly T. cymbifolium), and H. microphyllum. All of these species are pleurocarpous and are commonly distributed in many parts of the world. Mature and green tissues at 5−10 subsampling sites were collected. They were then mixed as composite samples for each site marked in Figure 1. Epilithic mosses on bare rock (EB) had thickness of
