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Mangroves and Salt Marshes 3: 127–134, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.

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Seasonal variation of inorganic nitrogen and net mineralization in a salt marsh ecosystem P. Cartaxana1 , I. Caçador1 , C. Vale2 , M. Falcão2 & F. Catarino1 1 Instituto

de Oceanografia, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1700 Lisboa, Por´ tugal (E-mail: [email protected]); 2 Instituto de Investigação das Pescas e do Mar (IPIMAR), Av. Brasilia, 1400 Lisboa, Portugal (Received August 1998; accepted in revised form December 1998)

Key words: ammonium, availability, cycling, nitrate, Tagus estuary

Abstract Inorganic nitrogen pools and net mineralization were estimated in three sites of a Tagus estuary salt marsh in Portugal throughout 1 year. Ammonium (NH+ 4 ) was the major form of inorganic nitrogen found in the salt marsh concentrations showed a marked seasonal pattern with a concentration peak during the hotter soil. Extractable NH+ 4 months of July/August. The great majority (> 99%) of the total nitrogen in the soil was found in sedimented organic matter, not readily available for plant uptake. Net nitrogen mineralization, determined using a field incubation method, showed a peak during the months of June/July which resulted in an increase on nitrogen availability. With the exception of the lower salt marsh, estimated rates of in situ net nitrogen mineralization in the soil during summer were well related to the increase in plant above-ground biomass and plant nitrogen pools, indicating that the process is an important source of available nitrogen for plant uptake and growth. Annual net nitrogen mineralization ranged between 2.4 and 4.5 g N m−2 yr−1 being significantly higher for the lower salt marsh site. Rates of net nitrogen mineralization were relatively low during most of the year with a particularly active period from June to August, possibly due to an effect of temperature on soil microbial activity.

Introduction The nitrogen cycle is rather complex, involving processes such as N2 -fixation, denitrification, uptake by plants, immobilization by micro-organisms and mineralization. Nitrogen mineralization is the conversion of bound nitrogen in soil organic matter to ammonium, and the nitrogen released by this process constitutes a large fraction of the plant available pool. Availability of nitrogen is one of the environmental factors that has a major impact upon primary production and species composition of plant communities (Berendse, 1990). This is of particular importance in salt marshes since there is considerable evidence that production is limited by nitrogen availability (e.g. Mendelssohn, 1979; Buresh et al., 1980; Osgood and Zieman, 1993). Several field and laboratory methods have been used to quantify net nitrogen mineralization in a variety of ecosystems. Laboratory methods usually in-

volve soil disturbance (e.g. sieving, mixing, drying and rewetting) and measured rates are unlikely to be quantitatively similar to those found in the field (Raison et al., 1987). Moreover, environmental conditions, which markedly affect rates of soil nitrogen mineralization, are difficult to reproduce in the laboratory. Factors such as temperature and soil moisture, which usually vary on a diurnal scale, strongly affect mineralization rates (Macduff and White, 1985; Raison et al., 1987). Therefore, field measurements of mineralization are a much better index for naturally occurring rates. The aim of this study was to quantify inorganic nitrogen pools and in situ net mineralization in a salt marsh ecosystem throughout 1 year. The data available on nitrogen mineralization in salt marshes is scarce (e.g. Abd. Aziz and Nedwell, 1986; Langis et al., 1991) and based on laboratory incubation methods (which significantly alter the naturally occurring rate

128 of mineralization) or single measurements in the field (which do not consider seasonal variation). Since the availability of nitrogen might be an important factor controlling primary production, we hypothesize that net nitrogen mineralization in the soil during summer is related to the increase in plant above-ground biomass and plant nitrogen pools. Methods Study site This study was conducted at Vasa Sacos salt marsh of the Pancas area, located in the Tagus estuary, Portugal (38 ◦500 N, 8 ◦ 570 W). Salt marshes occupy about, 1,300 ha of this 32,000 ha estuary (Catarino et al., 1985). The studied salt marsh is part of a nature reserve to which access is restricted. Three sites in the salt marsh were selected for this study. Site 1, hereafter called lower marsh, was dominated by the grass Spartina maritima (Curtis) Fernald in the pioneer zone; site 2 was covered with Halimione portulacoides (L.) Aellen, which dominates the mid marsh, especially along creek banks; site 3 had an uniform stand of Arthrocnemum perenne (Miller) Moss, which dominates the upper marsh. Soil characteristics On October 19, 1994, February 28, 1995 and July 12, 1995, five samples of the upper 0–10 cm soil layer were collected from each salt marsh location for general characterization of the soil using 5 cm diameter PVC (inert polyvinyl chloride) tubes. The soil pH was determined using a UNICAM 9455 pH ISE meter after adding 50 ml of deionised water to 10 g of fresh soil and shaking for 1 h. Soil microbial nitrogen was determined by the fumigation–extraction method as described by Brookes et al. (1985). Triplicate portions (approx. 10 g) of moist soil were extracted for 30 min with 0.5M K2 SO4 . Simultaneously, further triplicate portions (approx. 10 g) of moist soil were fumigated with ethanol-free chloroform (CHCl3 ) for 24 h at 25 ◦ C and then extracted with 0.5M K2 SO4 as above. The fumigant increases extractable nitrogen by lysing living soil organisms (Brookes et al., 1985). The filtered extracts − − were then analyzed for NH+ 4 , NO3 + NO2 and total nitrogen using an autoanalyzer (Bran and Luebbe, Traacs 800). The organic nitrogen fraction is oxidized to nitrate by sulphate radicals produced by the photolytic decomposition of peroxydisulphate within the

automated continuous flow analyzing system avoiding prior digestion of the extracts. The amount of nitrogen released by CHCl3 (CHCl3 —N) after 24 h fumigation was calculated subtracting the total nitrogen in K2 SO4 extracts of non-fumigated from that of fumigated soil. Microbial biomass nitrogen was calculated from CHCl3 —N/0.54 (Brookes et al., 1985). Part of each soil sample was dried (at 60 ◦C for 48 h), macro-organic matter removed and ground to a fine powder. Total nitrogen and carbon concentrations were determined using a CHNS/O analyser (Fisons Instruments Model EA 1108). Soil organic matter content was determined by percent weight loss after ignition (at 550 ◦C for 5 h). − − Extractable NH+ 4 and NO3 + NO2 and net nitrogen mineralization − − Inorganic nitrogen (NH+ 4 and NO3 + NO2 ) and in situ net nitrogen mineralization were measured in the upper 10 cm soil layer in each salt marsh location from October 1994 to October 1995. On each sampling date, ten pairs of undisturbed soil samples were collected from each location using sharpened PVC tubes (φ = 5 cm). One sample of each pair was kept cool and brought to the laboratory within 2 h of collection. The other tube was put back into the soil after being closed with plastic lids in order to measure the accumulation of mineral nitrogen during the following incubation period. Cores for incubation were perforated (four 5 mm diameter holes) to enhance the continuity between the enclosed core and the surrounding soil, allowing moisture and temperature equilibration. At approximately 6 weeks’ intervals the incubated cores were removed and sampling procedure repeated. In the laboratory, 20 g of moist sample of both initial and field incubated soil were extracted with 50 ml of 1M KCl for 1 h, centrifuged, filtered and frozen − − for later analysis. The NH+ 4 and NO3 + NO2 concentrations were measured colorimetrically in these extracts using a Technicon Autoanalyzer (detection − − limits: NH+ 4 – 0.1 µM; NO3 + NO2 – 0.05 µM). Approximately 20 g of each soil sample was dried at 60 ◦ C for at least 48 h to determine moisture content. The soil bulk density was determined at each site from the average soil dry mass in all mineralization tubes. − − NH+ 4 and NO3 + NO2 concentrations were expressed on a volume basis (nmol N cm−3 dry soil). Net nitrogen mineralization was considered to be the − − increase in NH+ 4 and NO3 + NO2 between initial and incubated soil samples and was expressed in

129 daily rates (nmol N cm−3 d−1 ) during the 6-week incubation. Annual rates of net nitrogen mineralization (g N m−2 yr−1 ) were derived by summing the amounts of nitrogen mineralized or immobilized during each incubation period. The proportion of total nitrogen mineralized was calculated by dividing the annual mineralization rates by the soil total nitrogen pool. Plant nitrogen pools To quantify above-ground biomass, four quadrat samples (0.25 m × 0.25 m) were clipped from each location on May 26, 1995 and August 29, 1995. The plant material was rinsed with water, dried (at 60 ◦ C for 48 h), weighed and ground to a fine powder. The total nitrogen concentration of each sample was determined using a CHNS/O analyser (Fisons Instruments Model EA 1108). Above-ground plant nitrogen pools were calculated taking into account the values of biomass and tissue element composition. Statistical analysis Significant differences in soil characteristics between sites were tested using one-way analysis of variance (ANOVA). Significant differences in extractable NH+ 4 and NO− 3 and net mineralization between sites and dates were tested using two-way ANOVA. Multiple comparisons among pairs of means were done using Tukey’s honestly significant difference method when a significant ANOVA result occurred. Normality was tested using Kolmogorov–Smirnov test. Homogeneity of variances was tested using the Bartlett’s test. Data were logarithmically transformed when necessary to comply with the assumptions of ANOVA. Results Soil characteristics General soil characteristics of the upper 0–10 cm layer in the three studied salt marsh locations are shown in Table 1. Total soil nitrogen and carbon were significantly (p < 0.01) higher in the mid marsh (0.37% and 4.1%, respectively) whereas for soil C/N ratio there were no significant differences between sites (average soil C/N ratio of 10.4). Microbial biomass nitrogen was significantly (p < 0.05) lower at the low salt marsh location with an average value of 50 µg g−1 dw. Organic matter was significantly (p < 0.001) higher in the mid marsh with an average value of 12.6%.

Table 1. Soil (upper 0–10 cm layer) characteristics for the three studied locations in a Tagus estuary salt marsh

pH Moisture (%) Bulk density (g cm−3 ) Organic matter (%) Total N (%) Total C (%) C/N ratio Microbial N (µg g−1 dw)

Lower marsh

Mid marsh

Upper marsh

8.0a 44a 0.74a 9.3a 0.24a 2.2a 9.7a 50a

7.5b 45a 0.56b 12.6b 0.37b 4.1b 11.2a 80b

7.1c 34b 0.88c 9.5a 0.25a 2.6a 10.3a 100b

Different letters indicate significant (p < 0.05) differences between sites (n = 15, except for moisture and bulk density where n = 270).

− − Extractable NH+ 4 and NO3 + NO2

Since nitrite (NO− 2 ) represented less than 1% of the − − NO3 + NO2 fraction, we will hereafter refer to this fraction as NO− 3 . Quantification of soil mineral nitrogen revealed distinctly higher NH+ 4 concentrations in the three studied sites of the salt marsh than NO− 3 + (Figure 1). Average NH4 concentrations ranged from 75 to 1408 nmol N cm−3 (Figure 1a), while NO− 3 ranged from 2 to 50 nmol N cm−3 (Figure 1b). Extractable NH+ 4 concentrations showed a marked seasonal pattern (Figure 1a). Concentrations remained fairly constant from October 1994 until May 1995 (75–197 nmol N cm−3 ) in all three sites. On July 12 the concentrations of extractable NH+ 4 significantly (p < 0.01) increased at all three studied sites, being significantly (p < 0.001) higher in the mid marsh location (1050 nmol N cm−3 ). On August 29 there was again a significant (p < 0.01) increase of the concentrations of extractable NH+ 4 in all three studied sites, reaching an overall maximum of 1408 nmol N cm−3 in the mid marsh location. On October 10 the concentrations of extractable NH+ 4 decreased significantly (p < 0.001) with the exception of the lower salt marsh site where concentrations remained relatively high (676 nmol N cm−3 ) and exceeded the values in the other two sites. NO− 3 concentrations showed no apparent seasonal pattern, although higher NO− 3 concentrations were found at the lower salt marsh from January to April (Figure 1b). Inorganic nitrogen pools were significantly (p < 0.01) greater during July/August 1995 when the highest temperatures were registered (Figure 2).

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− −3 Figure 1. Extractable NH+ 4 (a) and NO3 (b) concentrations (nmol N cm ) in the soil (upper 0–10 cm layer) at different salt marsh locations throughout the year of 1994/1995. Bars represent the standard deviation of the mean (n = 10).

Net nitrogen mineralization Net nitrogen mineralization, measured by the in situ incubation procedure, showed a marked seasonal pattern (Figure 3). Rates were similar in the three studied sites of the salt marsh from October 1994 until May 1995 ranging from 1.2 to 4.2 nmol N cm−3 d−1 . Dur-

ing June/July 1995 the rates increased significantly (p < 0.001) in the three sites reaching values of 28, 35 and 23 nmol N cm−3 d−1 for lower, mid and upper salt marsh, respectively. The value of net mineralization in the lower salt marsh (26 nmol N cm−3 d−1 ) was similar within the following 6-week incubation period, but rates at the other two studied sites decreased sig-

131 of net mineralization for the mid and upper salt marsh were well related to the increase on the above-ground biomass and nitrogen pools of H. portulacoides and A. perenne, respectively. For the lower salt marsh, where rates of net mineralization were higher, the above-ground nitrogen pool of S. maritima declined.

Discussion

Figure 2. Maximum and minimum air temperature (◦ C) throughout the year of 1994/1995. Data collected at Montijo meteorological station.

nificantly (p < 0.001). In September/October 1995 negative rates of net mineralization (i.e. net immobilization or denitrification) were found in all three sites of the salt marsh. Annual net nitrogen mineralization rate in the lower salt marsh (4.5 g N m−2 yr−1 ) was significantly (p < 0.05) higher than in the two other locations (Table 2). Approximately 80% of this annual mineralization occurred between May 26 and August 29. Yearly values of net nitrogen mineralization rates were 2.4 and 2.9 g N m−2 yr−1 in upper and mid salt marsh, respectively. The proportion of total nitrogen mineralized was 1.1%, 1.3% and 2.6% in upper, mid and lower salt marsh sites, respectively (Table 2). Nitrogen pools − Values of the inorganic nitrogen (NH+ 4 + NO3 ) pool, which constitutes the nitrogen readily available for plant uptake, were between 0.47 and 0.59 g N m−2 (Table 2), about 0.2–0.3% of the total nitrogen in the soil. Thus, the largest part (> 99%) of the total soil nitrogen was present in sedimented organic matter. Within this pool microbial biomass accounted for 3.7–8.8 g N m−2 (Table 2), that is 2–4% of soil total nitrogen. In Table 3, the net nitrogen mineralization from May 26 until August 29 is compared with the variation in plant above-ground biomass and above-ground nitrogen pools in the same period. The estimated rates

Ammonium was the major form of inorganic nitrogen found in the salt marsh soil (Figure 1). According to DeLaune et al. (1983), NH+ 4 is the predominant form of inorganic nitrogen found in saturated salt marsh sediments. Insufficient oxygen diffusion through the waterlogged soil may reduce rates of nitrification and explain the low NO− 3 concentrations found. Denitrification of NO− to gaseous forms of nitrogen (N2 3 and N2 O) may also have contributed to the low NO− 3 levels found. The values of extractable NH+ 4 reported here (1.3–33 µg N g−1 dry weight) are comparable to those reported for other salt marsh sediments. Buresh et al. (1980) found 5–45 µg N g−1 in a Louisiana salt marsh and Langis et al. (1991) reported values of 1–5 µg N g−1 in a San Diego Bay salt marsh. The great majority (> 99%) of the total nitrogen in the soil was found in sedimented organic matter (Table 2), not readily available for plant uptake, which is in agreement with the results of Buresh et al. (1980) for a Louisiana salt marsh. According to them, organic nitrogen comprised at least 99% of the total soil nitrogen. There was a clear pattern of high early summer net nitrogen mineralization rates in the three studied sites (Figure 3) which resulted in an increased nitrogen availability during summer, especially in the hotter months of July/August (Figure 1). For the mid and upper salt marsh, the estimated rates of net nitrogen mineralization during summer were well related to the increase in plant above-ground biomass and plant nitrogen pools (Table 3). Hence, nitrogen mineralization at these two sites contributed not only to the build up of the soil inorganic nitrogen pool, but it was also an important source of available nitrogen for plant uptake and growth. In the lower salt marsh, the above-ground biomass of S. maritima did not increase during summer (Table 3) when nitrogen availability was higher. This suggests that other factors, apart from nitrogen availability, are limiting plant production. Osgood and Zieman (1993), although concluding that nitrogen was

132

Figure 3. Daily net nitrogen mineralization (nmol N cm−3 d−1 ) in the soil (upper 0–10 cm layer) for incubation periods of 6 weeks in the field and at different salt marsh locations throughout the year of 1994/1995. Bars represent the standard error of the mean (n = 10).

Table 2. Average nitrogen pools and annual rates of net mineralization in the soil (upper 0–10 cm layer) for the three studied locations in a Tagus estuary salt marsh

Net N mineralization (g m−2 yr−1 ) Proportion of N mineralized (%) Total N pool (g m−2 ) Mineral N pool (g m−2 ) Microbial N Pool (g m−2 )

Lower marsh

Mid marsh

Upper marsh

4.5a 2.6a 176a 0.52ab 3.7a

2.9b 1.3b 222a 0.59a 4.8a

2.4b 1.1b 220a 0.47b 8.8b

Different letters indicate significant (p < 0.05) differences between sites.

the primary limiting nutrient in Virginia salt marshes, reported that sediment nitrogen stocks did not directly control the spatial pattern of nitrogen content or production of Spartina alterniflora. These authors suggested that other stresses, such as high porewater salinity and low sediment redox potential (EH ) interact with nitrogen availability to control tissue nitrogen content and plant production. In addition, Pezeshki et al. (1988) found that the primary factor limiting growth of S. alterniflora in Louisiana salt marshes was the low sediment EH , which could result in excess

ion availability and potentially cause sulphide-induced toxicity. Furthermore, the values of nitrogen concentration in the above-ground tissues of S. maritima decreased during summer, resulting in a reduction of the aboveground nitrogen pool (Table 3). This indicates a translocation of nitrogen to the roots, which represent approximately 90% of the total biomass of this plant (Cartaxana and Catarino, 1997). Our estimation of net nitrogen mineralization for the lower salt marsh is not supported by the variation of S. maritima aboveground nitrogen pool possibly because of a great investment of resources on below-ground growth. Annual net nitrogen mineralization rate was significantly higher in the lower salt marsh location (Table 2). At this site we often observed deposition of microalgae which constitutes an input of labile organic matter. This may trigger decomposition and increase the production of NH+ 4 , suggesting a link between organic production in the water and inorganic nitrogen production in the soil at this site. A similar in situ incubation method to quantify rates of net nitrogen mineralization has been used in other ecosystems (e.g. Adams and Attiwill, 1986; Adams et al., 1989; Berendse, 1990; Zak and Grigal,

133 Table 3. Variation in above-ground biomass (g m−2 ), above-ground nitrogen pools (g N m−2 ) and net nitrogen mineralization (g N m−2 ) from May 26 until August 29 for the three locations in a Tagus estuary salt marsh

Lower marsh Mid marsh Upper marsh

Above-ground biomass

Above-ground N pool

N mineralization

−29 +389 +373

−1.8 +3.0 +1.1

+3.6 +2.3 +1.8

1991; Olff et al., 1994). The soil samples were isolated from root uptake, leaching losses were prevented, and conditions inside the incubation tubes were similar to the surrounding soil. There were no significant differences between the moisture content of incubated soil samples and initial soil samples collected at the same sampling date (data not shown), which shows that the method allowed moisture equilibration within the core during the incubation period. It is necessary that this period is sufficiently long to allow a measurable change in the pools of soil mineral nitrogen. Incubation periods of 5–12 weeks have been used elsewhere (Berendse, 1990; Zak and Grigal, 1991; Olff et al., 1994). Although the method potentially allows the movement of NO− 3 , a much more mobile ion than , through the core holes, its contribution to total NH+ 4 mineral nitrogen is low. Moreover, the area of the holes is extremely small compared with sample size. Thus, losses/gains of NO− 3 through the core holes are very likely negligible. This incubation method cuts off living plant roots during core sampling whose subsequent decay may lead to an overestimation of the actual rates of nitrogen mineralization. On the other hand, fresh detritus cannot enter the incubated samples which may cause an underestimation of the in situ rates. The average daily rates reported here for nitrogen mineralization (0.07–0.17 mg N Kg−1 d−1 ) are considerably lower than the ones referred by Langis et al. (1991) for a San Diego Bay salt marsh using the buried bag technique. These authors found values of approximately 0.8 and 1.6 mg N Kg−1 d−1 for a constructed and a natural salt marsh, respectively. These rates were based on a single 21-day incubation period and therefore did not consider seasonal variations. If we consider the period of June/July 1995, mineralization rates ranged between 0.37 and 0.84 mg N Kg−1 d−1 and are comparable to the ones reported by Langis et al. (1991). Abd. Aziz and Ned-

well (1986), studying the nitrogen cycle of an U.K. salt marsh, reported nitrogen mineralization rates of approximately 18 and 22 g N m−2 yr−1 in areas dominated by Puccinellia maritima/Spartina townsendii and P. maritima/H. portulacoides, respectively. These values, which are considerably higher than the values reported in our study (2.4–4.5 g N m−2 yr−1 ), were estimated from rates measured in the laboratory at constant temperature. The disadvantages of estimating naturally occurring rates based on laboratory incubations have been discussed previously (see Introduction) and may explain some of the differences on nitrogen mineralization rates between these two studies. To our knowledge, this is the first study to report seasonal variations on nitrogen mineralization in a salt marsh ecosystem using a field incubation method and we suggest that annual rates are usually overestimated using either laboratory incubation methods or single measurements in the field. In conclusion, rates of net nitrogen mineralization were relatively low during most of the year with a particularly active period from June to August, possibly due to an effect of temperature on soil microbial activity. In the mid and upper salt marsh, nitrogen mineralization during summer contributed not only to the build up of the soil inorganic nitrogen pool, but it was also an important source of available nitrogen for plant uptake and growth.

Acknowledgements This study was funded by a PRAXIS XXI grant (BD/3602/94) to P. Cartaxana and was carried out in the framework of the project ‘Disturbance of European salt marsh ecosystems: the impact of environmental pollution (eutrophication) in relation to sedimentation patterns’ financed by the EU programme ‘Environment’, contract EV5V-CT93 0265.

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