Fluxes of N2O, CH4 and CO2 in a meadow ecosystem exposed to elevated ozone and carbon dioxide for three years

Environmental Pollution 145 (2007) 818e828 www.elsevier.com/locate/envpol

Fluxes of N2O, CH4 and CO2 in a meadow ecosystem exposed to elevated ozone and carbon dioxide for three years Teri Kanerva a,*, Kristiina Regina b, Kaisa Ra¨mo¨ a, Katinka Ojanpera¨ b, Sirkku Manninen a a

Department of Biological and Environmental Sciences, University of Helsinki, P.O. Box 27, 00014 Helsinki, Finland b MTT Agrifood Research Finland, 31600 Jokioinen, Finland Received 17 March 2005; received in revised form 27 March 2006; accepted 30 March 2006

The soil fluxes of N2O, CH4 and CO2 in a meadow ecosystem changed in response to elevated O3 and CO2 in an OTC experiment. Abstract Open-top chambers (OTCs) were used to evaluate the effects of moderately elevated O3 (40e50 ppb) and CO2 (þ100 ppm) and their combination on N2O, CH4 and CO2 fluxes from ground-planted meadow mesocosms. Bimonthly measurements in 2002e2004 showed that the daily fluxes of N2O, CH4 and CO2 reacted mainly to elevated O3, while the fluxes of CO2 also responded to elevated CO2. However, the fluxes did not show any marked response when elevated O3 and CO2 were combined. N2O and CO2 emissions were best explained by soil water content and air and soil temperatures, and they were not clearly associated with potential nitrification and dentrification. Our results suggest that the increasing O3 and/or CO2 concentrations may affect the N2O, CH4 and CO2 fluxes from the soil, but longer study periods are needed to verify the actual consequences of climate change for greenhouse gas emissions. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Elevated carbon dioxide; Elevated ozone; Greenhouse gases; Meadows; Open-top chambers

1. Introduction The concentrations of tropospheric ozone (O3) and carbon dioxide (CO2) have been progressively increasing over the past century (Houghton et al., 1996; Runeckles and Krupa, 1994), which has given rise to a discussion about the effects of these two gases on below-ground processes. Of particular interest in the context of global climate change is the modification of soil processes involved in the production and consumption of greenhouse trace gases (Houghton, 1993; Ineson et al., 1998). While much is known about the above-ground plant responses to elevated CO2 and O3, comparatively little information is available on below-ground processes (Andersen, 2003; Baggs and Blum, 2004). CO2 enrichment may lead to an

* Corresponding author. Tel.: þ358 505229423. E-mail address: [email protected] (T. Kanerva). 0269-7491/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2006.03.055

increase in soil carbon (C) input via enhanced plant biomass productivity and root C losses (Paterson et al., 1997; Zak et al., 1993). Elevated O3 is known to decrease C allocation to roots (Cooley and Manning, 1987; Gorissen and van Veen, 1988; Gorissen et al., 1994; Rennenberg et al., 1996; Spence et al., 1990) and to alter C flux to soils through modified rhizodeposition, root turnover and changes in leaf litter quality or quantity (Kim et al., 1998; King et al., 2001; McCrady and Andersen, 2000). These indirect effects of elevated O3 and CO2 through plants may alter soil microbial processes in a number of ways, principally by modifying the soil physical conditions and changing the availability of C substrates (Andersen, 2003; Hungate et al., 1997a,b; Islam et al., 2000). These modifications can then influence the emissions of trace gases, such as nitrous oxide (N2O), methane (CH4) or carbon dioxide (CO2). The impact of elevated CO2 and O3 on the production and consumption of these trace gases is not well understood and has not been assessed in natural or semi-natural grasslands with herbaceous

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

species. Actually, to our knowledge, no long-term fumigation experiments with both O3 and CO2 on trace gas fluxes have been made within any ecosystem. The possible effects of CO2 and O3 on the soil C and N cycles as well as the trace gas fluxes may have a vital influence on the global atmospheric budgets of these gases and ultimately on the sustainability of grasslands (Mosier et al., 2002). A study of natural meadows shows that this particular, nitrogen-poor ecosystem has a role as a consumer of atmospheric CH4 and a producer of N2O (Kanerva et al., 2005). Nitrification and denitrification (Knowles, 1982) as well as fungal transformations (Laughlin and Stevens, 2002) are the main processes causing N2O production from soils. CO2 enrichment could lead to enhanced N2O emissions by increasing the availability of C for denitrifying bacteria. However, C addition can decrease nitrification due to increased N immobilization (Phillips et al., 2001b), and thus the availability of N significantly regulates these processes. The influence of elevated CO2 on soil N2O emissions is relatively well known in a variety of ecosystems (e.g. Ambus and Robertson, 1999; Baggs et al., 2003; Ineson et al., 1998; Phillips et al., 2001b), but the effects of O3 on N2O flux are not known. Atmospheric CH4 is oxidized in aerated soils (Hu¨tsch, 2001). Several studies from different ecosystems have indicated that CH4 consumption is reduced at elevated levels of CO2 (Ambus and Robertson, 1999; Ineson et al., 1998; Phillips et al., 2001a,c), but the causative mechanisms are not well known. It is possible that CO2 enrichment affects the size or activity of the CH4-oxidizing microbial community or causes a higher soil C concentration and competition for O2 and therefore suppression of the CH4-oxidizing microbial community (Phillips et al., 2001a). Increased soil moisture under elevated CO2 reduces the rate of diffusion (Castro et al., 1995) and therefore decreases CH4 oxidation in the soil (Ambus and Robertson, 1999; Phillips et al., 2001a). However, if the rising temperature due to the global climate change makes the soil drier, CH4 oxidation may be enhanced (Del Grosso et al., 2000). In this experiment, we were unable to assess the effect of soil moisture since the soil was kept at a certain moisture level to optimize plant growth and to minimize the chamber effect. In contrast to the impacts of elevated CO2 on CH4 emissions, which have been studied previously, information on the effects of O3 on CH4 emissions is still lacking. Elevated CO2 has been found to enhance net photosynthesis and to modify the C cycle in soil ecosystems by increasing the C input into soil as a result of increased litter deposition, root turnover and rhizodeposition (Karberg et al., 2005; Pregitzer et al., 1995) as well as increased fungal and bacterial activity and substrate degradation (Phillips et al., 2002). Therefore, increases in soil C input may cause greater CO2 emissions from soil. Elevated O3, in turn, is known to decrease photosynthesis via oxidative damage to cell membranes, which may have several negative effects on meadow productivity, which will eventually diminish ecosystem C accumulation (Karberg et al., 2005). The synergistic effects of O3 and CO2 have been reported to have a major influence on the ecosystem C storage. Loya et al. (2003), for instance, found that a combination of

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O3 and CO2 caused forest plots to produce less new soil C compared to plots with only elevated CO2. We examined the responses of the N2O, CH4 and CO2 fluxes of meadow mesocosms on elevated O3 and CO2 and their combination with open-top chambers for three summers of exposure. The gas fluxes and their correlations with microbial potential activities, mineral N and climatic factors were also studied. The specific aims were to investigate: i) if elevated O3 would decrease N2O and CO2 emissions and CH4 consumption; ii) if elevated CO2 would increase N2O and CO2 emissions and decrease CH4 consumption; and iii) whether the negative effects of O3 would be recognizable regardless of the CO2 level used. 2. Material and methods 2.1. Description of the experimental set-up An open-top chamber (OTC) experiment was undertaken in 2002e2004 at Rehtija¨rvi in Jokioinen (60  490 N, 23  280 E) in south-western Finland. Each study plot (3.10 m in diameter, 0.25 m in depth) was dug in natural soil, lined with polypropene root barrier fabric (230 g/m2) and filled with a mixture of sand and low-fertility peat (1:1). The soil was sieved through a 2-cm sieve to remove stones, roots and large pieces of organic residue and mixed properly before planting. Artificial soil formation was used to ensure the homogeneity of the soil and to avoid weeds and unwanted plant residues. Inside each plot there was planted a 2.25 m2 mesocosm at the beginning of the experiment in June 2002. The mesocosms were designed to hold the major functional types of plants that occur on lowland hay meadows, including: (1) grasses (Agrostis capillaris L. and Anthoxanthum odoratum L.); (2) herbs (Fragaria vesca L., Campanula rotundifolia L. and Ranunculus acris L.); and (3) legumes (Trifolium medium L. and Vicia cracca L.). They are all perennial species that grow in open, dry habitats and tolerate disturbance. All plants were grown from seeds of southern Finnish origin. The grasses are archaeophytes, and the herbs are native and common in Finland. The mesocosms consisted of two different but consistent parts; a larger round meadow mimic and a smaller square plot to enable gas flux measurements. For more details and illustrations, see Kanerva et al. (2005). The mesocosms were harvested once a year in early September, except in the first year 2002, when the plant coverage was not fully developed yet. The mesocosms overwintered naturally during the first winter 2002e2003, and before the second winter 2003e2004 the collar used for gas flux measurements was covered with a frost cover blanket to prevent the formation of ice inside the collar. The mesocosms were fertilized right after the transplantation of the seedlings in early July 2002. Soluble NPK fertilizer (N-P2O5-K2O 19-10-24), conþ sisting of N 19%, NO 3 -N 7.2%, NH4 -N 1.8% and urea 10.0%, was used, and the addition resulted in N 2.38 mg l1 in each mesocosm. The fertilization was repeated once in late July. The soil in the mesocosms was coarse sand (sand 86.5%/silt 11.9%/clay 1.6%). The soil chemical properties are shown summarized in Table 1 (see also Kanerva et al., 2005; Manninen et al., 2005). The mesocosm soil did not differ from that of natural meadows except for its concentration of mineral N, which was significantly lower (1.55e4.11 mg N g1 d.w.1) (Kanerva et al., in press) than that in similar Finnish natural meadows (5.05e18.53 mg N g1 d.w.1) (Kanerva et al., 2005). OTCs (3 m in diameter, 2.8 m in height) of Raleigh design (Heagle et al., 1973) with added frustum were used. All chambers were equipped with blowers to exchange 3 air volumes per minute. The chambers were removed in the winter. The OTCs were placed on the experimental field in a completely randomised design. The treatments included unchambered open-field plots (AA) and the following OTC treatments: (i) non-filtered ambient air (NF); (ii) non-filtered air þ elevated O3 (NF þ O3); (iii) non-filtered air þ elevated CO2 (NF þ CO2); and (iv) non-filtered air þ elevated O3 þ CO2 combined (NF þ O3 þ CO2). Each treatment included three replicates. O3 was generated by electric discharge from pure oxygen (Oy AGA Ab) with an O3 generator (Fischer 502), and it was bubbled through ultra-pure

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

820

Table 1 Average main nutrient concentrations (mg/l), organic C (%), C/N ratio and pH in the mesocosm soil (NF) after each growing season (n ¼ 3), seasonal O3 (ppb, 9-h average) and CO2 (ppm, 9-h average) concentrations from the OTCs and AAs measured 1 m above the ground during the fumigation periods (n ¼ 2) Year 2002a

2003b

2004c

P K Ca Mg C org. C:N ratio pH

6.1 61 1474 392 2.9 34 6.9

7.1 65 1414 380 2.8 47 6.8

7.1 80 1324 347 3.1 28 6.9

AA NF NF þ O3 NF þ CO2 NF þ O3 þ CO2 AA NF NF þ O3 NF þ CO2 NF þ O3 þ CO2

33 31 47 31 41 353 351 355 455 448

31 25 40 24 40 354 352 357 455 449

31 28 50 28 51 411 409 412 530 527

Soil characteristics

Air quality O3 (9-h)

CO2 (9-h)

a b c

Between July 1e28 August. 3 Junee31 August. 18 Maye23 August.

water to remove the harmful compounds other than O3 possibly generated from oxygen before it was fed to the OTCs. The concentrations of CO2 (Oy AGA Ab) in the OTCs were measured with a CO2 meter (Vaisala GM 12 A) and distributed to the air circulation system of each CO2 OTC using rotameters for CO2 (Kyto¨la¨ EK-8937). O3 was monitored with Environment O341M (O3) s/n 606 and Thermo Environmental Instruments Model 49 UVphotometric O3 monitors at approximately 1 m above the soil surface. The monitors were cross-calibrated with the EMEP monitor (European Monitoring and Evaluation Programme) at the Finnish Meteorological Institute. An instrument trailer was located at the site, with data acquisition and control equipment. Daily precipitation (mm), relative air humidity (%), temperature (  C) and global radiation (Wm2) were measured from two of the OTCs and one open-field plot (unchambered) with Adcon Telemetry (A730MD). We aimed to keep the elevated O3 and CO2 concentrations quite moderate, close to the predicted ambient concentrations in the year 2050, with a yearly increase of 0.5e2% in O3 (Vingarzan, 2004) and a moderate 0.5% increase in CO2 (IPCC, 2001). Daily exposure time was 9 h, from 10:00 to 19:00 h, during each growing season (Table 1).

2.2. Soil moisture and temperature Soil temperature at the depth of 5 cm in one mesocosm was recorded every four hours by a temperature logger (Optic StowAway Temp, Onset computer corp., MA, USA). With every gas flux measurement, soil temperature was measured from the air and the soil at depths of 5 and 20 cm (Fluke 52 Thermometer, Fluke Corporation, USA). In the first growing season 2002, the mesocosms were watered with the same amount of water regularly, and the soil water content (5 cm depth) was determined with a portable capacitance sensor (TDR) (Theta Meter, type HH1, Delta D Devices, Cambridge, England). The plots received ambient rainfall and were additionally watered with tap water as needed (TDR readings lower than 0.20 m3 m3). From the second growing season 2003 onwards, the mesocosms were equipped with tensiometers (Soil Moisture Equipment Corp., Santa Barbara, CA) to maintain the desired (5e30 kPa) moisture regimes. The

water content in the soil was determined with gypsum blocks. For details, see Kanerva et al. (2005).

2.3. Soil sampling and laboratory analyses Soil samples were taken at the beginning of the experiment in May 2002 and in mid-September after each exposure period, in the years 2002, 2003 and 2004. Soil samples for mineral N were taken before and after each growing season (May and September) to monitor the amount of N available to plants. Soils were sampled using a 2.0-cm diameter auger. On each sampling occasion, 20 cores were taken from the depth of 0e20 cm. All of the cores from each plot were bulked and stored in polyethylene bags at 18  C. Soil pH and nutrients were analysed as described in Kanerva et al. (2005). The soil total N and C concentrations were determined by combustion of dried (60  C) samples with a LECO (St. Joseph, MI, USA) CN-2000 analyzer. For  mineral N (Nmin ¼ NHþ 4 -N þ NO3 -N) analysis, soil samples (100 g) were shaken for 2 h with 250 ml of 2 M KCl to extract exchangeable inorganic N.  Concentrations of NHþ 4 -N and NO3 -N in the KCl extracts were determined with a Lachat Autoanalyzer (Lachat Instruments, Milwaukee, Wisconsin, USA). Potential nitrification was assayed with the chlorate inhibition method (Pell et al., 1998) using a 14 h shaken soil-slurry technique. Samples were analysed for NO2-N with a Skalar autoanalyzer SA 400. For a detailed description of the method, see Kanerva et al. (2005). The soil denitrification potential was determined as in Klemedtsson et al. (1988) and Henault et al. (1998) with several modifications. The 10 g soil (d.w.) samples (moisture at 80% of WHC) were treated with KNO3 and glucose solutions and analysed with a gas chromatograph. For details, see Kanerva et al. (2005).

2.4. Ecosystem N2O, CH4 and CO2 fluxes The ecosystem N2O, CH4 and CO2 fluxes (net ecosystem dark respiration) were measured twice a month within each OTC and AA during each growing season. One steel base frame was installed in each mesocosm. During the flux measurements, the frames were covered with an opaque aluminum chamber (60  60  40 cm, 0.144 m3). A water-filled groove at the upper edge of the frame ensured the gas-tightness of the chambers. The chambers were equipped with 1 m vents (diameter 1 mm) for pressure equilibration. Gas samples were taken with polypropylene 20 ml syringes at 0 and 40 min after the chambers were closed and stored in 12 ml glass vials (Exetainer, Labco, UK). The gas samples were analysed within 48 h with a gas chromatograph (HP 6890 Series, GC System, Hewlett Packard, USA) equipped with flame ionization (FID) and electron capture detectors (ECD) and a nickel catalyst for converting CO2 to CH4. The precolumn and analytical columns were 1.8 m and 3 m long steel columns packed with Hayesep Q (80/100 mesh). The GC had a 10-way valve with a 2 ml sample loop and a backflush system for flushing the precolumn between the runs. A 6-way valve passed the flow to either the FID or the ECD. The temperatures of the GC oven, FID and ECD were 70  C, 300  C and 350  C, respectively. Nitrogen was used as the carrier gas and a mixture of argon and methane (5%) as a make-up gas (1.4 ml min1) to increase the sensitivity of the ECD. A standard gas mixture (AGA) of known concentrations of N2O, CH4 and CO2 was used for calibration. An autosampler (222 XL Liquid handler, Gilson Medical Electronics, France) fed the samples to the loop of the GC. The coefficients of variation for the analysis of the atmospheric concentrations of N2O, CH4 and CO2 were 0.3%, 0.6% and 0.8%, respectively. Total cumulative fluxes of N2O, CH4 and CO2 over the period from May 15 to Sep 10 for the growing seasons of 2003 and 2004 were calculated by linear interpolation between the daily fluxes. Cumulative fluxes were not calculated for the growing season 2002, since only four measurements were made during the first exposure season, and the small number of measurements did not allow the calculation of values comparable to those of the other growing seasons.

2.5. Statistical analyses Time-repeated observations of daily gas fluxes were analyzed using repeated-measures Anova (22 e Anova) (SPSS Windows software 11.0, SPSS Inc., 2001). When necessary, data were log-transformed to meet the

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There was variation in the greenhouse gas fluxes and their responses to elevated O3 and/or CO2 during the three summers and among the years. As the fluxes often depended markedly on climatic factors, we will start by briefly presenting and discussing the climatic conditions during the experiment.

higher mean daily temperature) and dryer (46 mm lower precipitation) compared to the 30-year averages measured for the site (Fig. 1, Table 2a). The summer 2003 was also slightly warmer, but rainier than the long-term average for the site, whereas the mean daily air temperature in the summer 2004 was normal for the site, but rainfall was significantly more abundant (145 mm higher precipitation) than the long-term average (Nordlund et al., 2002a,b,c,d; 2003a,b,c,d; 2004a,b,c,d). Soil water content (recorded with gypsum blocks) was, in turn, significantly higher in 2003 than in 2004 ( p ¼ 0.001, all treatments included). The lower density of the plants in 2003 (Ra¨mo¨ et al., 2006) may have resulted in a higher soil water content compared to the season 2004. In other words, the increased plant density probably prevented some of the rain from reaching the soil, while the enhanced growth of plants simultaneously demanded more water in 2004. Also, from 2003 onwards, irrigation was regulated by tensiometers, and the soil water content was therefore not clearly regulated by rain. The mean air temperatures and precipitation in the OTCs did not differ from those measured on the AA plots in all summers ( p > 0.05) (Table 2b), but relative humidity was higher ( p ¼ 0.048) in the AA plots than in the OTCs in the summer 2002. Soil water content was higher in the AA plots than in the NF in the summers 2003 and 2004 ( p ¼ 0.025 and p ¼ 0.001, respectively). This parallels our previous OTC observations (Kanerva et al., 2005). Daily fluxes of CO2 were significantly higher in the AA than in the NF only in 2002, but the fluxes of N2O and CH4 did not differ between the AA and NF mesocosms in any of the growing seasons (Figs. 1e3).

3.1. Climate and the chamber effect

3.2. N2O emissions

The first experimental summer (MayeAugust) 2002 was the warmest and dryest of the three, and warmer (2.3  C

The meadow mesocosms acted as sources of N2O, i.e. we measured positive fluxes of N2O (Fig. 2). The daily N2O fluxes

assumption of normal distribution. The possible difference in cumulative fluxes between the growing seasons 2003 and 2004 and the differences between the AAs and OTCs were estimated with t-test, and whenever there was a problem due to unequal variances that could not be resolved with simple transformations, Mann-Whitney non-parametric test was used. To test the correlations between the climatic, soil and plant parameters and the N2O, CH4 and CO2 fluxes, Spearman’s correlation analysis was performed. The values of CH4 fluxes were log-transformed by first adding the value 10 to all data to turn all values positive. The values of daily fluxes were used in the analysis with the variables that were measured daily (air and soil temperature, soil water content) and the cumulative fluxes were used in the correlation analysis with variables measured only once or twice during the growing season. Stepwise multiple regression analysis was performed to test the possible dependency of the gas fluxes on environmental and soil factors, such as air and soil temperatures, soil water content, potential denitrification and nitrification and mineral N. The results at p < 0.10 were considered significant if not otherwise noted. The significance level of p < 0.10 was chosen, because of the low number of replicates (n ¼ 3) and the consequently low statistical power. The use of the p < 0.10 significance level in the FACE (Free Air Carbon dioxide Enrichment) experiments has been thoroughly discussed by Filion et al. (2000), and it can also be applied to the OTC experiments. Our experiment provides novel information about the effects of O3 on below-ground processes, and it is therefore important to report even the possible effects with less statistical power. In addition, the significance level of p < 0.10 has been used in other fumigation experiments (e.g. Holmes et al., 2003; Montealegre et al., 2002).

3. Results and discussion

temperature (º C)

30 25 20 15 10 5 0

50

0.8 0.7 0.6

40

0.5

30

0.4

20

0.3 0.2

10 0 15-May 22-Jun 30-Jul 10-Sep 15-May 22-Jun 02 02 02 02 03 03

0.1

soil water content (bar)

precipitation (mm)

60

0 30-Jul 10-Sep 15-May 22-Jun 30-Jul 10-Sep 03 03 04 04 04 04

Fig. 1. Air temperature, soil water content (line) and rainfall (bars) in the open-field plots in the growing seasons 2002e2004.

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

822

Table 2 a) Precipitation (mm) for four months (MayeAugust) and monthly mean temperatures (  C) in Jokioinen (data from Finnish Meteorological Institute, Jokioinen) 2002e2004, and b) means (SD) and p-values (t-test) of air temperatures (  C) and precipitation (mm) from the OTCs (NF) and from the open-field plots (AA) during the summers 2002e2004 a)

Year 2002

2003

2004

Precipitation Air temperature May June July August

207

302

397

b)

2002a Mean

11.3 15.6 18.2 17.9

9.7 12.9 19.7 15.0 N

9.6 12.2 15.5 15.7 2003b Mean

p

n

p

Precipitation AA NF

1.5 (3.4) 1.2 (2.6)

52 52

0.300

2.2 (3.4) 1.9 (3.4)

85 85

0.342

Air temperature AA NF

17.5 (3.1) 18.2 (3.1)

53 53

0.162

17.5 (4.2) 18.3 (4.0)

82 82

0.111

a b c

2004c Mean

n

p

1.7 (5.3) 1.7 (4.3)

103 103

0.485

15.6 (3.5) 16.2 (4.1)

65 65

0.166

Between 28 Junee28 August. 3 Junee31 August. 19 Maye23 August.

varied during each growing season and were decreased in the NF þ O3 treatment after three seasons of exposure (Table 3). The highest average daily N2O fluxes (1729 mg N2O m2 d1 in the NF treatment) were recorded during the first exposure period in JulyeAugust 2002 following the N fertilization, after which they declined. During the second exposure period in JuneeAugust in 2003, the daily N2O emissions were highest in May (655 mg N2O m2 d1 in the AA treatment) and then continued to decline towards the end of the summer. In 2004, the daily fluxes of N2O ranged between 311 and 32 mg N2O m2 d1. The emission rates were less variable in the summer and much more variable in the fall (Fig. 2). Even though the daily fluxes of N2O changed over time in each growing season, there were no time*treatment interactions, except that observed in the summer 2002 for time* O3 þ CO2 (Table 3). These results of decreasing N2O emissions towards the end of each growing season and/or the experiment may partly be related to the soil N concentrations. For example, the studies on Lolium perenne swards show that elevated CO2 only increased the emissions of N2O from the highly fertilized swards (Baggs et al., 2003; Baggs and Blum, 2004), and Phillips et al. (2001b) reported that N2O production was strongly limited by the availability of N. In our experiment, no fertilizers were applied after the beginning of the experiment, and it therefore seems that, in low-N soils, the greater C availability under elevated CO2 does not lead to greater N2O emissions. At any rate, the finding that CO2 enrichment did not have an increasing effect on the N2O fluxes is contradictory to our hypothesis and to the findings of Arnone and Bohlen (1998), Baggs et al. (2003) and Baggs and Blum (2004). Plant biomass was also unaffected in the NF þ CO2 treatment in this experiment (Ra¨mo¨ et al., 2006), and this may, in part, help to explain why elevated CO2 did not affect the N2O fluxes, either. In

addition, the CO2 concentration was increased only by 100 ppm, while other studies with similar systems have reported markedly more elevated levels, occasionally even twofold CO2 concentrations (e.g. Islam et al., 2000; Holmes et al., 2003; Kasurinen et al., 1999). Nevertheless, as stated previously, the concentrations used here were in agreement with those projected for 2050 (IPCC, 2001). The cumulative fluxes of N2O were markedly lower in the NF þ O3 and NF þ O3 þ CO2 treatments in 2004 than in 2003 ( p ¼ 0.01 and p ¼ 0.046, respectively). Although there was no statistically significant correlation between the cumulative N2O emissions and the above-ground plant biomass, the decreases in N2O fluxes under elevated O3 are presumably plant-mediated, as O3 does not penetrate soil (Blum and Tingey, 1977; Turner et al., 1973). It is known that plants exhibit physiological toxicity to even low concentrations of O3 (Karnosky et al., 1996). In our experiment, O3 reduced the above-ground biomass of the community (up to 40%), and four out of seven species (including N2-fixing T. medium and V. cracca) showed significant growth reduction and/or visible injuries under elevated O3 (Ra¨mo¨ et al., 2006). The average root biomass was also decreased (34%) in the NF þ O3 treatment compared to the NF treatment, suggesting decreased C allocation to the roots under elevated O3 (Manninen et al., 2005). In addition to causing a reduction in above-ground biomass and C allocation to the roots, O3 has been found to alter the quantity and quality of root exudates (Andersen and Rygiewicz, 1995; McCrady and Andersen, 2000). The difference in the cumulative N2O emissions between the exposure years might also be linked to the warmer soil and air temperatures in 2003 than in 2004 as well as to the increase of plant biomass from 2003 to 2004 (Ra¨mo¨ et al., 2006), which means that the plants required more N for their growth in 2004 than 2003. The daily fluxes of N2O rose with

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828 AA

NF+O3

NF

NF+CO2

823 NF+CO2+O3

N2O flux (µg m-2 d-1)

2500

2002 2000 1500 1000 500 0

N2O flux (µg m-2 d-1)

1600

2003 1200 800 400 0

N2O flux (µg m-2 d-1)

800

2004 600 400 200 0 ay

-M

15

ay

-M

26

n

-Ju

10

n

-Ju

23

ul

7-J

l

-Ju

22

ug

4-A

g

-Au

19

ep

2-S

ct

2-O

Fig. 2. Daily N2O fluxes from the meadow mesocosms under different treatments in the growing seasons 2002e2004. Arrows indicate the times of beginning and ending the fumigations in each growing season. Stars indicate the times of harvest. Error bars represent the positive standard deviation of the means (n ¼ 3).

the rising air and soil temperatures and soil water content. The stepwise multiple regression analysis indicated that 30% of the variation in the daily N2O (loge) fluxes from the mesocosms was explained by the air and soil temperatures, soil water content and mineral N concentration. The strong correlation between N2O emissions and soil water content suggests that denitrification, rather than nitrification, was the main process contributing to N2O emissions from the mesocosms, as was also pointed out by Mu¨ller and Sherlock (2004). However, the total cumulative N2O emissions did not relate to the measured nitrification or denitrification potentials, as the fluxes only correlated with the pH of the tested soil variables (Table 5). The actual rate of N2O production depends more on the soil conditions than on the potential microbial activity (Henault et al., 1998). The higher N2O emissions were associated with an increase in soil and air temperatures, but appeared to be mainly attributable to higher soil moisture levels. Such a response is well documented (Arnone and Bohlen, 1998; Baggs et al., 2003; Baggs and Blum, 2004). 3.3. CH4 fluxes The mesocosms acted mainly as sinks of CH4 (Fig. 3). In 2002, both net production and oxidation of CH4 took place

in the first exposure period. Net CH4 production may result from lowered activity of the bacteria consuming CH4 after the planting of the mesocosms. CH4 oxidizers are known to be highly sensitive to soil disturbance (Ambus and Robertson, 1999; Hu¨tsch, 2001), and the soil of the mesocosms was thoroughly mixed before planting. Elevated O3 and CO2 alone or combined did not have any significant effect ( p > 0.10) on the daily fluxes of CH4 analysed with repeated-measures Anova during the first exposure season (Table 3). In 2003, the daily fluxes of CH4 were negative, indicating net consumption of CH4. The treatments had no significant main effect ( p > 0.10) on the daily fluxes of CH4 during the second exposure season, but the CH4 fluxes changed significantly over time in the NF þ O3 þ CO2 treatment. The response was positive in most cases, but no clear pattern was detected. In 2004, both CH4 production and oxidation were seen. CH4 consumption was highest at the beginning of the summer and lower during the summer, but the temporal differences in uptake rates were insignificant (Fig. 3, Table 3). The daily fluxes of CH4 were lower in the NF þ O3 treatment compared to the NF treatment in the third year of exposure (Table 3). This may be at least partly related to the decreased plant biomass and/or soil N, although the correlations between them and N2O fluxes were not statistically significant (Table 5).

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

824

AA

CH4 flux (µg m-2 d-1)

2.5

NF

NF+O3

NF+CO2

NF+CO2+O3

2002

2 1.5 1 0.5 0 -0.5 -1

CH4 flux (µg m-2 d-1)

0.5

2003 0.25 0 -0.25 -0.5 -0.75

CH4 flux (µg m-2 d-1)

1.5

2004 1 0.5 0 -0.5 -1 ay

-M

15

ay

-M

26

n

n

-Ju

10

-Ju

23

ul

7-J

l

-Ju

22

ug

4-A

g

-Au

19

ep

2-S

ct

2-O

Fig. 3. Daily CH4 fluxes from the meadow mesocosms under different treatments in the growing seasons 2002e2004. Arrows indicate the times of beginning and ending the fumigations in each growing season. Stars indicate the times of harvest. Negative values represent the net CH4 consumption. Error bars represent the positive standard deviation of the means (n ¼ 3).

The lack of responsiveness of CH4 fluxes to CO2 fumigations was contrary to our expectations, which were based on earlier studies. Soils that normally function as CH4 sinks, such as aspen stands (Ambus and Robertson, 1999), grassland soils (Ineson et al., 1998) and loblolly pine soils (Phillips et al., 2001a,c), are known to undergo marked reductions in atmospheric CH4 consumption under elevated CO2. However, the availability of growth resources, such as N, may strongly affect the response to CO2 (e.g. Daepp et al., 2000; Fischer

et al., 1997; Gloser et al., 2000; Jackson and Reynolds, 1996; Zanetti and Hartwig, 1997), and previous studies have shown that, in low-N soils, the CH4 flux response to CO2 may be inhibited (Baggs and Blum, 2004; Mosier et al., 2002). Given this, the lack of a CO2 effect could be explained by the low N status in our soil, though the CH4 fluxes did not show any correlation with soil N variables. Indeed, the variables used in stepwise multiple regression analysis did not explain any of the variation in the daily

Table 3 Main effects ( p-values) of elevated O3 and/or CO2 and time and treatment (O3 and CO2) interactions on the daily fluxes of N2O, CH4 and CO2 in the growing seasons 2002e2004 Source

N2O

CH4

CO2

2002

2003

2004

2002

2003

2004

2002

2003

2004

O3 CO2 O3 þ CO2

n.s. n.s. n.s.

n.s. n.s. n.s.

0.076 n.s. n.s.

n.s. n.s. n.s.

n.s. n.s. n.s.

0.081 n.s. n.s.

n.s. n.s. n.s.

0.022 0.076 n.s.

0.016 n.s. n.s.

Time Time*O3 Time*CO2 Time*O3 þ CO2

<0.001 n.s. n.s. 0.070

0.093 n.s. n.s. n.s.

0.034 n.s. n.s. n.s.

n.s. n.s. n.s. n.s.

n.s. n.s. n.s. 0.037

n.s. n.s. n.s. n.s.

0.004 n.s. n.s. n.s.

<0.001 0.040 n.s. n.s.

<0.001 0.023 0.092 n.s.

n.s. not significant ( p > 0.10).

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

825

Table 4 Means (SD) of total cumulative N2O, CH4 and CO2 fluxes during 4 months in 2003e2004 from the mesocosms under different treatments Treatment

mg N2O m2

All treatments AA NF NF þ O3 NF þ CO2 NF þ CO2 þ O3

25.7 32.2 21.7 28.9 22.1 23.6

mg CH4 m2

2003

2004 (12.2) (23.9) (6.26) (5.75) (7.00) (7.24)

a a a a a a

15.5 20.9 19.0 12.3 14.0 11.4

kg CO2 m2

2003 (5.04) (4.94) (4.81) (2.45) (6.63) (1.24)

27.6 28.5 31.2 28.3 24.4 25.4

b a a b a b

2004 (10.6) (15.8) (16.2) (4.14) (7.98) (12.7)

2003

20.3 15.9 11.9 29.6 19.8 24.5

a a a a a a

(17.2) (33.6) (18.5) (3.20) (11.7) (12.2)

a a a a a a

2.38 2.36 2.38 2.13 2.72 2.28

2004 (0.29) (0.34) (0.35) (0.23) (0.09) (0.18)

a a a a a a

2.94 3.11 2.97 2.48 3.46 2.68

(0.53) (0.61) (0.43) (0.37) (0.64) (0.15)

b b b a b b

Letters indicate statistically significant differences ( p < 0.10) between the years within each group of fluxes.

CH4 (loge) fluxes from the mesocosms ( p ¼ 0.576). This lack of correspondence between CH4 flux and soil moisture may be explained by the constant monitoring of water content with tensiometers, which helped to keep soil moisture between 10 and 30%. As in our study, others have also found that temperature has only a small or no effect on the CH4 oxidation rate (Born et al., 1990; Bowden et al., 1998; Crill, 1991). Moreover, the cumulative fluxes of CH4 did not differ between the exposure seasons 2003 and 2004 ( p ¼ 0.177) and did not significantly correlate with any of the measured parameters (Tables 4 and 5).

AA

mg CO2 (m-2d-1)

36000

NF

3.4. CO2 emissions The mesocosms also acted as sources of CO2, and the CO2 emissions varied during the growing seasons and between the years (Fig. 4, Tables 3 and 4). In 2002, the CO2 emissions (net ecosystem dark respiration) decreased from the beginning towards the end of the growing season. Elevated O3 and CO2 alone or combined did not affect daily fluxes during the first exposure season. In the summer 2003, the daily CO2 fluxes from all treatments were consistently small at the beginning of the growing season, much more variable in the summer

NF+O3

NF+CO2

NF+CO2+O3

2002

30000 24000 18000 12000 6000 0

mg CO2 (m-2 d-1)

42000

2003

36000 30000 24000 18000 12000 6000 0

mg CO2 (m-2 d-1)

60000

2004

50000 40000 30000 20000 10000 0

ay

-M

15

ay

-M

26

n

-Ju

10

n

-Ju

23

ul

7-J

l

-Ju

22

ug

4-A

ug

-A

19

ep

2-S

ct

2-O

Fig. 4. Daily CO2 fluxes (soil respiration and plant dark respiration) from the meadow mesocosms under different treatments in the growing seasons 2002e2004. Arrows indicate the times of beginning and ending the fumigations in each growing season. Stars indicate the times of harvest. Error bars represent the positive standard deviation of the means (n ¼ 3).

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

826

Table 5 Spearman’s correlation coefficients of the fluxes of N2O, CH4 and CO2 with soil and environmental variables N2O r a

Mineral N spring Mineral N fallb Total Nc Organic C pHc Denitrification potentialc Nitrification potentialc Total plant biomassd Plant biomass inside the collard Air temperature Soil temperature Soil water content

CH4 N

r

CO2 N

r

N

0.12 0.29 0.35 0.16 0.41* 0.11 0.16 0.28

45 45 30 30 30 30 30 30

0.15 0.72 0.70 0.13 0.16 0.20 0.15 0.59

45 45 30 30 30 30 30 30

0.45** 0.63** 0.50** 0.32 0.32 0.21 0.08 0.70**

45 45 30 30 30 30 30 30

0.71

15

0.26

15

0.73**

15

237 210 120

0.06 0.02 0.14

237 210 120

0.50** 0.69** 0.18*

237 210 120

0.27** 0.34** 0.53**

Asterisks denote two-tailed significances (*p < 0.05; ***p < 0.001). a Samples taken before each growing season in May. b Samples taken in mid-September after the fumigations. c Data from Kanerva et al. in press. d Data from Ra¨mo¨ et al. (2006).

**p < 0.01;

(Edwards, 1991; Coleman et al., 1996) and positive (Andersen and Scagel, 1997; Kasurinen et al., 2004; Scagel and Andersen, 1997). All of these studies were conducted with forest trees, which are highly different ecosystems compared to meadows and thus a possible reason for the contradictory results. Other possible reasons for the ambiguous results could be the different exposure times and amounts of enrichment. Stepwise multiple regression analysis indicated that 40% of the variation in the daily CO2 (loge) fluxes from the mesocosms was explained by air temperature, soil water content and mineral N concentration (see also Table 5). Soil CO2 flux is known to correspond to temperature and soil moisture, since extremely dry and wet conditions usually reduce soil CO2 fluxes (Do¨rr and Mu¨nnich, 1987). The cumulative fluxes of CO2 were significantly higher in all the other treatments except NF þ O3 in 2004 than in 2003, and the daily fluxes of CO2 increased with increasing air and soil temperatures and soil water content (Tables 4 and 5). Cumulative CO2 fluxes also correlated positively with soil mineral N (spring and fall), total organic N and above-ground plant biomass. The increase in CO2 fluxes from 2003 to 2004 may be explained by increased plant biomass in general and consequent increases in C flow to the soil. 4. Conclusions

and less variable in the autumn. The highest daily CO2 fluxes were detected in mid-July. Elevated O3 increased and CO2 decreased the daily CO2 fluxes when given alone, but no such response was detected when these two gases were combined (Table 3). A significant interaction with time was only observed in the NF þ O3 treatment. The findings of stimulated microbial respiration under elevated CO2 are in agreement with the study of Hungate et al. (1996) on annual grassland. They are, however, different from the observations on Lolium perenne swards, where Ineson et al. (1998) observed a decrease in CO2 emissions under elevated CO2, and on Colorado short grass steppe, where Mosier et al. (2002) reported that ecosystem respiration, measured as dark chamber CO2 flux, was not affected by elevated CO2. When both plants and microbes contribute to the measured flux of CO2, as in our study, it is impossible to say which process causes the observed change. In 2004, the CO2 fluxes followed the previous growing season’s pattern, showing the lowest values in the spring and fall and the highest in late July and early August (Fig. 4). The daily fluxes of CO2 were significantly reduced under elevated O3 (main effect), but were not influenced by CO2 alone or combined with O3 (Table 3). Nevertheless, there was a significant change over time in the NF þ O3 and NF þ CO2 treatments. However, no such interaction was seen in the combination treatment. The reduction in the soil CO2 flux beneath the mesocosms under elevated O3 suggests that O3 altered the C flux from soils. O3 is known to decrease root biomass and growth as well as root carbohydrate concentrations (Andersen and Rygiewicz, 1991; Edwards, 1991; Cooley and Manning, 1987; Kasurinen et al., 2004; King et al., 2001; Rennenberg et al., 1996; Spence et al., 1990), which may lead to diminished C flux. However, the effects of elevated O3 on soil CO2 flux have been reported to be both negative

The fluxes of N2O, CH4 and CO2 from the soils of an OTC experiment in a meadow ecosystem appeared to be somewhat modified under elevated CO2 and/or O3. Elevated O3 decreased N2O and CO2 emissions and CH4 fluxes after three years of fumigation. Elevated CO2 caused effects only on CO2 fluxes, but not consistently during every growing season. However, even though elevated O3 had effects on all of the measured fluxes, we did not recognize the effects when CO2 was present, and that is contrary to what was predicted. The effects of elevated O3 or CO2 seemed to be linked to above-ground growth and visible injuries, as elevated O3 decreased plant biomass and elevated CO2 had no effect on above-ground growth (Ra¨mo¨ et al., 2006). Soil water content as well as air and soil temperatures explained best the N2O and CO2 emissions, whereas CH4 fluxes could not be linked to any of the measured variables in this experiment. The chamber effect was quite moderate in this experiment, as soil water content and relative humidity were only modified by the chamber. In addition, the fact that the daily fluxes of CO2 were significantly higher in the AA than in the NF only in 2002, and that the fluxes of N2O and CH4 did not differ between the AA and NF mesocosms in any of the growing seasons also shows that OTCs may be used in meadow ecosystem studies of this kind. It can be concluded that moderately enhanced CO2 and/or O3 may have effects on the N2O, CH4 and CO2 fluxes, but longer measurement periods are required to verify the interactions between elevated CO2 and O3 at the soil ecosystem level. Acknowledgements We would like to thank Maj and Tor Nessling Foundation, Helsinki University Environmental Research Centre and

T. Kanerva et al. / Environmental Pollution 145 (2007) 818e828

University of Helsinki for funding of the work. We wish to thank all the staff at the MTT/MPY laboratory for their invaluable assistance. Special appreciation is dedicated to Mr. Peter Huhtala for site maintenance throughout the study. Thanks also to Ms. Sirkka-Liisa Leinonen for the revision of English.

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