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Increase of nitrous oxide flux to the atmosphere upon nitrogen addition to red mangroves sediments
Marine Pollution Bulletin 44 (2002) 992–996 www.elsevier.com/locate/marpolbul
Increase of nitrous oxide flux to the atmosphere upon nitrogen addition to red mangroves sediments ~oz-Hincapie *, Julio M. Morell, Jorge E. Corredor Milton Mun Department of Marine Sciences, University of Puerto Rico, P.O. Box 908, Lajas, PR 00667, Puerto Rico
Abstract Response of nitrous oxide N2 O sediment/air flux to nitrogen addition was assessed in mangrove (Rhizophora mangle) sediments. Fluxes were enhanced with both ammonium and nitrate loading. Greatest fluxes (52 lmol m2 h1 ) were obtained with ammonium addition and saturation was achieved with additions of 0.9 mol m2 . Maximum flux following ammonium addition was 2785 times greater than control plots and 4.5 times greater during low tide than with equivalent ammonium addition at high tide. Nitrate enrichment resulted in exponential growth, with maximal mean flux of 36.7 lmol m2 h1 at 1.9 mol m2 ; saturation was not achieved. Differential response to ammonium and nitrate, and to tide and elevation, indicate that microbial nitrification is responsible for most of the observed gas flux. Mangrove sediments constitute an important source of global atmospheric N2 O and increases in nitrogen loading will lead to significant increases in the flux of this atmospherically active gas. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nitrous oxide; Mangrove; Sediment flux
1. Introduction Nitrous oxide N2 O is a greenhouse gas, with a direct global warming potential 170–290 times that of carbon dioxide (Wang et al., 1976). Although chemically inert in the troposphere, it is readily transported to the stratosphere where it is involved in destruction of protective ozone (Hutchinson et al., 1993; Hahn and Crutzen, 1982; Delwiche, 1981; Crutzen, 1970). Nitrous oxide also absorbs outgoing planetary IR radiation at wavelengths not removed by atmospheric CO2 or H2 O and has been estimated to contribute 6% of the overall anthropogenic greenhouse effect (Hutchinson et al., 1993; Libes, 1992; Rhode, 1990). It is a stable compound that does not decay readily in the atmosphere, with an atmospheric lifetime of over 121 years (Crutzen, 1970; Khalil and Rasmussen, 1983); even if emissions were held constant, it would be decades before concentration stabilized. By 1994 concentrations of atmospheric N2 O increased from a pre-industrial level of about 275–312 ppbv. Global anthropogenic emissions of N2 O presently *
Corresponding author. Tel.: +1-787-899-2048; fax: +1-787-8995500. E-mail address: [email protected] (M. Mu~ noz-Hincapie).
amount to 4:5 0:6 Tg N yr1 , increasing atmospheric concentrations by 0.25% per year (Khalil and Rasmussen, 1992; Schlesinger, 1991). Nitrous oxide is an intermediate product of both nitrification, the aerobic oxidation of ammonium to nitrite and nitrate, and denitrification, the suboxic reduction of nitrate and nitrite, principally to molecular nitrogen (Corredor et al., 1999a; Firestone and Davidson, 1981; Smith and Zimmerman, 1981). Increased anthropogenic N inputs are known to increase these microbial processes in soils (Bouwman, 1996; Mosier, 1994) and aquatic environments (Seitzinger, 1988). Marine ecosystems are known to constitute a net source of N2 O to the atmosphere (Cohen and Gordon, 1979; Butler et al., 1989) but these appear to be underestimated in current global nitrous oxide N2 O budgets (Nevisson et al., 1995; Capone, 1991). The increasing atmospheric N2 O concentration and the imbalance in its global budget have triggered interest in quantifying N2 O fluxes from various ecosystems (Corre et al., 1999). While the magnitude and global distribution of N2 O emissions from natural soils and from agricultural soils have been investigated, there has not been a comparable analysis of the magnitude or global distribution of N2 O emissions in aquatic ecosystems due to natural and/or anthropogenic processes.
0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 2 ) 0 0 1 3 2 - 7
M. Mu~noz-Hincapie et al. / Marine Pollution Bulletin 44 (2002) 992–996
Mangrove forests occupy a large fraction of the tropical coastline and receive high nitrogen inputs (Corredor and Morell, 1994); such ecosystems dominate the intertidal zone of diverse environmental settings in the Caribbean, from carbonate platforms to terrigenous deltas that exhibit different patterns of nutrient cycling (Twilley, 1997). Mangrove litter fall is made up of leaves, flowers, propagules, twigs and branches, and the percentage of each varies throughout the year. Generally, mangrove leaves are the most important source of detritus and their degradation allows significant energy transfer from mangroves to higher trophic levels via the detritus food web (Gonz alez-Farias et al., 1997). The high productivity of mangroves and the subsequent detrital fall lead to vigorous microbial recycling of nitrogenous products. Corredor et al. (1999a,b) reported nitrous oxide fluxes from mangrove sediments proportional to the availability of inorganic nitrogenous compounds in porewaters. We evaluate the response of N2 O flux to experimental nitrogen loading in sediments within a red mangrove (Rhizophora mangle) forest, southwest coast of Puerto Rico, enriching sediments with oxidized (nitrate) and reduced (ammonium) inorganic nitrogen substrate and quantifying N2 O production under nitrogen loading. Results of these experiments confirm our hypothesis that N2 O flux from the sediments increases dramatically with nitrogen addition.
2. Materials and methods The red mangrove fringe area selected is located at about 17°580 N and 67°030 W on the southwest coast of Puerto Rico in the northeastern Caribbean Sea. Two stations were established at the sampling site. ‘‘Wet’’ sediments correspond to sediments closest to the shoreline and submerged during high tide, ‘‘dry’’ sediments correspond to sediments further from the shoreline. Sediments were manipulated by injecting different doses of ammonium sulphate and potassium nitrate to 0.054 m2 plots of both ‘‘wet’’ and ‘‘dry’’ sediments and compared with unenriched control plots. The nutrient solution was injected with plastic syringes (60 ml) directly into the sediments through plastic pipettes introduced to different depths. The treatment began by inoculating the nutrient solution into the sediments twice a day, three days before samples were taken. Final enrichment was performed while the incubation was carried out. Inoculation doses injected into the sediments were 9:3 103 mol m2 (equivalent to a final theoretical porewater concentration of 0.5 mM), 1:9 102 mol m2 (1.0 mM), 2:8 102 mol m2 (1.5 mM), 1:1 101 mol m2 (6.0 mM), 9:3 101 mol m2 (50 mM) and 1.9 mol m2 (100 mM). In all cases, the vol-
993
ume of the nutrient solution inoculated into the sediments was two liters. Nitrous oxide exchange between sediments and atmosphere was quantified using a closed chamber technique. The incubation chambers are made of rigid polyvinyl chloride pipe, with a height of 15 cm, diameter of 7.5 cm, an internal volume of 2650 ml, and a sediment area covered of 0.018 m2 . The top of the chambers is fitted with an air sampling port and a vent constructed from a 0.6 cm and a 1.2 cm Swagelok bulk-head union respectively (Keller and Reiners, 1990). Samples were collected with a 5 ml glass syringe through the air sampling port. The incubation chamber was inserted 3 cm into the sediments for each series of measurements. Samples were collected at an initial time or time zero, at 30, 60 and at 90 min at both ‘‘wet’’ and ‘‘dry’’ fringe sediment layer in triplicate, and transported to the laboratory for same-day analysis. Concentration of nitrous oxide was determined by gas chromatography using a 63 Ni electron capture detector (Hewlett–Packard 5890 series II) equipped with a MolSieve capillary 5A column 80/100 (mesh). The carrier gas was a mixture of 95% argon and 5% methane at a column flow rate of 21 ml/min. Temperatures were 240 °C in the oven, 350 °C in the detector. Nitrous oxide concentrations were quantified by comparing retention times and peaks areas for samples and certified standards of 0.510 and 0.484 ppm with an analytical accuracy of 2% (Scott Specialty Gases). A calibration standard was injected into the gas chromatograph after every two samples. Field samples were analyzed only when the coefficient of variation of the N2 O standard readings was <1%. Nitrous oxide fluxes were calculated from the linear regression of the rate of concentration change at the four different incubation times in the microatmosphere of the incubation chambers. Only regressions with r2 > 0:5 at the 90% confidence level (P ¼ 0:1) were used. Method detection limit (MDL) was calculated at 6.91 ppb, using seven readings obtained directly from the gas chromatograph as follows: MDL ¼ tS
ð1Þ
where, t is Student’s t value for a 99% confidence level and an S is standard deviation (with n 1 degrees of freedom) of the replicates. Porewater nutrients were analyzed in the lower range of ammonium inoculations (9:3 103 and 1:9 102 mol m2 ) targeted to achieve theoretical porewater concentrations of 0.5 and 1 mM; 5–10 times the observed concentrations in porewaters of control plots. Nutrient content was not analyzed during the nitrate addition experiments. Sediment cores were taken in triplicate at each station with a perforated piston-type corer (30 cm long; 6.5 cm; 3.0 cm diameter). The cores were sealed at the bottom and transported to the laboratory for sameday analyses. Sediment samples were transferred to 50 ml
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M. Mu~noz-Hincapie et al. / Marine Pollution Bulletin 44 (2002) 992–996
centrifuge plastic tubes excluding the root portions. Interstitial porewater samples were obtained from sediment cores by centrifugation at 7000 rpm (15 min). Samples obtained were poisoned with 50 ll mercuric chloride. Ammonium was determined using a modification of the Sol orzano (1969) phenol–hypoclorite colorimetric technique. Nitrite and nitrate were quantified using the method of Strickland and Parsons (1972). The extinction of the diazo compound was measured at 543 nm. All absorbance measurements were performed using a Shimadzu UV-1601 UV–visible spectrophotometer. 3. Results Nitrous oxide fluxes from untreated control plots averaged 0:05 0:03 lmol m2 h1 during low tide and 0:04 0:01 lmol m2 h1 during high tide (Table 1). At the highest levels of inoculation, ammonium addition enhanced N2 O flux by factors of up to 2784 and 245.8 during low tide and high tide respectively compared with control plots. A maximum mean flux of 55.7 lmol m2 h1 was observed during the ammonium enrichment experiments at low tide. Ammonium addition resulted in a sigmoidal increase in N2 O flux with saturation occurring at enrichment levels of 1 mol m2 (Fig. 1a). Nitrate addition was less effective in stimulating N2 O flux. The highest rate enhancements from these additions increased fluxes 1834-fold during low tide with a maximum mean flux of 36.7 lmol m2 h1 . Nevertheless, saturation was not observed during these experiments indicating that higher fluxes might be achieved with further nitrate addition. In contrast to the ammonium additions, sigmoidal behavior was not apparent upon nitrate enrichment. During these experiments, exponential increase of N2 O flux was observed from the lowest to the highest inoculation levels (Fig. 1b). Compared to fluxes obtained with equivalent ammonium concentrations, N2 O fluxes were higher under nitrate addition at the lower range of enrichment (6.6 times at 0.028 mol m2 and 3.2 times at 0.11 mol m2 nitrate addition respectively). Porewater ammonium, nitrite and nitrate concentrations measured after incubation with ammonium
Fig. 1. Nitrous oxide fluxes in response to nutrient inoculation to the sediment during low tide in ‘‘dry’’ sediments. (a) Ammonium addition and (b) nitrate addition. J ¼ N2 O flux.
amendments at 0.5 and 1.0 mM were statistically indistinguishable from those of the control (Table 2).
4. Discussion Our findings confirm the argument that inorganic nitrogen loading of mangrove sediments results in enhancement of N2 O flux to the atmosphere. As noted
Table 1 Nitrous oxide flux rates (lmol m2 h1 ) for ammonium and nitrate inoculation assays in ‘‘dry’’ sediments (each value is a mean, n ¼ 3) Nutrient dose (mol m2 ) 9:3 103 1:9 102 2:8 102 1:1 101 9:3 101 1.9
Low tide
High tide
NHþ 4
NO 3
inoculation
inoculation
0.2 0.14 0.29 1.42 51.90 55.68
– – 1.91 4.51 9.38 36.68
Control 0.04 0.1 0.05 0.05 – 0.02
NHþ 4 inoculation 0.11 0.02 – 0.43 – 12.29
NO 3 inoculation
Control
– – – 0.1 – –
0.03 0.05 – 0.03 – 0.05
M. Mu~noz-Hincapie et al. / Marine Pollution Bulletin 44 (2002) 992–996
995
Table 2 Sediment porewater nutrient content in the ammonium inoculation to sediments (each value is a mean, n ¼ 3) NH4þ dose (mol m2 )
LT ‘‘Dry’’
Ammonium (lM) 9:3 103 118.0 1:9 102 54.0 Average 86.0
SD
Control
SD
HT ‘‘Dry’’
SD
Control
SD
19.3 15.4 17.4
123.1 66.2 94.6
16.8 13.4 15.1
113.0 61.7 87.4
10.6 16.8 13.7
107.5 55.1 81.3
15.9 31.6 23.8
Nitrite (lM) 9:3 103 1:9 102 Average
3.5 2.5 3.0
0.4 0.0 0.2
2.53 5.81 4.17
0.05 0.03 0.04
2.9 2.0 2.5
0.0 0.4 0.2
2.14 5.81 4.0
0.03 0.03 0.03
Nitrate (lM) 9:3 103 1:9 102 Average
1.5 1.6 1.6
0.0 0.1 0.05
1.60 3.83 2.72
0.15 0.12 0.14
2.2 6.9 4.6
0.5 0.9 0.7
1.70 7.33 4.5
0.00 1.36 0.68
‘‘Dry’’ ¼ ‘‘dry’’ sediment stations. SD ¼ standard deviation.
previously (Corredor et al., 1999a,b), anthropogenic pressure is increasing the rate of nitrogen loading to mangroves throughout the tropical belt and globally significant increases in N2 O flux to the atmosphere may be expected as a result of these pressures. While mangroves serve as an important buffer to eutrophication resulting from anthropogenic nitrogen loading (Corredor and Morell, 1994) the negative consequences of enhanced N2 O flux must be considered. Differences in the response of ‘‘wet’’ and ‘‘dry’’ sediments and of responses during low and high tide provide strong evidence for the argument that N2 O flux results mainly from the activity of nitrifying bacteria present in the mangrove sediments which oxidize the ammonium substrate; a process that benefits from air exposure during low tide. Nitrous oxide production, a by-product of microbial nitrification, is thus enhanced at low tide under augmented conditions of reduced nitrogen loading. Our experiment shows that bacterial populations in mangrove sediments exhibit a rapid response capacity to reduce oxidized nitrogen species. However, nitrification would seem to be the principal mechanism for N2 O production since ammonium is the most abundant dissolved inorganic nitrogenous species in these mangrove sediments (Corredor et al., 1999a,b). Porewater concentrations measured 2 h after inoculation were indistinguishable from those of untreated control plots although treatments were designed to theoretically elevate ammonium concentrations to 0.5 and 1.0 mM. This contrasts with our previous observations (Corredor et al., 1999a) where moderately elevated porewater nutrient concentrations were found in mangrove porewaters subjected to sewage treatment plant effluent and more significant enhancement was found in the naturally enriched environment of a cattle egret rookery. We believe that slow release from bird guano may in large part explain the difference with the latter, and long-term exposure to very high nitrate effluents may explain the
former. The experiments here described demonstrate the very rapid consumption of substrate when added in the readily available form of dissolved inorganic salts as result of both microbial and mangrove metabolism. Nitrous oxide flux under nitrate addition must be attributed to denitrification alone. Under ammonium addition however, both nitrification and denitrification can contribute to the total N2 O flux. Thus, in the nutrient loading experiments, denitrification must contribute up to 38% of the total nitrous oxide flux registered during the nitrification process (Fig. 1a and b). Isotopic analysis of the nitrogen cycle in the red mangrove sediments, particularly that of the gaseous products of microbial metabolism, should further improve our understanding of the relative contribution of the nitrification and denitrification process to N2 O flux. Acknowledgements This research was supported by NASA-UPR-EPSCoR grant no. NCCW-56 and the Department of Marine Sciences, University of Puerto Rico. Diana Marıa Mora-Pinto, Maribel Velez-Rivera, Martha Prada and Gladys Lissette Cintr on assisted in field sampling.
References Bouwman, A.F., 1996. Direct emissions of nitrous oxide from agricultural soils: nutrient cycling in agroecosystems. Fert. Res. 46, 53–70. Butler, J.H., Elkins, J.W., Thompson, T.M., 1989. Tropospheric and dissolved N2 O of the West Pacific and East Indian Oceans during the El Ni~ no southern oscillation event of 1987. J. Geophys. Res. 94, 14865–14877. Capone, D.G., 1991. Aspects of the marine nitrogen cycle with relevance to the dynamics of nitrous and nitric oxide. In: Rogers, J.E., Whitman, W.B. (Eds.), Microbial Production and Consumption of Greenhouse Gases: Methane, Nitrogen Oxides, and
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Halomethanes. American Society of Microbiology, Washington, DC, pp. 255–275. Cohen, Y., Gordon, L.I., 1979. Nitrous oxide production in the ocean. J. Geophys. Res. 84, 347–353. Corre, M.D., Pennock, D.J., Van Kessel, C., Elliot, D.K., 1999. Estimation of annual nitrous oxide emissions from a transitional grassland-forest region in Saskatchewan, Canada. Biogeochemistry 44, 29–49. Corredor, J.E., Morell, J.M., Bauza, J., 1999a. Atmospheric nitrous oxide fluxes from mangrove sediments. Mar. Poll. Bull. 38 (6), 473– 478. Corredor, J.E., Howarth, R.W., Twilley, R.R., Morell, J.M., 1999b. Nitrogen cycling and anthropogenic impact in the tropical interamerican seas. Biogeochemistry 46, 163–178. Corredor, J.E., Morell, J.M., 1994. Nitrate depuration of secondary sewage effluents in mangrove sediments. Estuaries 17 (18), 295–300. Crutzen, P.J., 1970. The influence of nitrogen oxides on the atmospheric ozone content. Quart. J. Royal Meteorol. Soc. 96, 320–325. Delwiche, C.C., 1981. Denitrification, nitrification and atmospheric nitrous oxide. In: Andreae, M.O., Chimel, D.S. (Eds.), Exchange of Trace Gases between Terrestrial Ecosystems and the Atmosphere. Wiley, New York, pp. 1–15. Firestone, M.R., Davidson, E.A., 1981. Microbiological basis of NO and N2 O production and consumption in soil. In: Andreae, M.O., Chimel, D.S. (Eds.), Exchange of Trace Gases between Terrestrial Ecosystems and the Atmosphere. Wiley, New York, pp. 7–23. Gonz alez-Farias, F., Fl orez-Verdugo, F.F., Hernandez-Garza, M., 1997. Annual variation of particulate organic carbon and mangrove detritus in Agua Brava lagoon, Nayarit, Mexico. In: Kjerfve, B., de Lacerda, L.D., Salif Diop, E.H. (Eds.), Mangrove Ecosystem Studies in Latin America and Africa. United Nations Educational, Scientific and Cultural Organization, UNESCO, France, pp. 47–63. Hahn, J., Crutzen, P.J., 1982. The role of fixed nitrogen in atmosphere photochemistry. Philos. Trans. Royal Soc. London, Ser. B Biol. Sci. 296, 521–541.
Hutchinson, G.L., Livingston, G.P., Brams, E.A., 1993. Nitric and nitrous oxide evolution from managed subtropical grassland. In: Oremland, R.S. (Ed.), Biogeochem of Global Change: Radiatively Active Trace Gases. Chapman and Hall, New York, pp. 290–316. Keller, M., Reiners, W.A., 1990. Emissions of nitrous oxide and nitric oxide from wet tropical forest soils. EOS 71, 1259. Khalil, M.A.K., Rasmussen, R.A., 1983. Increase and seasonal cycles of nitrous oxide in the Earth’s atmosphere. Tellus 35, 161–169. Khalil, M.A.K., Rasmussen, R.A., 1992. The global sources of nitrous oxide. J. Geophys. Res. 97, 14651–14660. Libes, S.M., 1992. An Introduction to Marine Chemistry. Wiley, New York. Mosier, A.R., 1994. Nitrous oxide emissions from fertilized soils. Fert. Res. 37, 191–200. Nevisson, C.D., Weiss, R.F., Erickson III, D.J., 1995. Global oceanic emissions of nitrous oxide. J. Geophys. Res. 100 (C8), 15809– 15820. Rhode, H., 1990. A comparison of the contribution of various gases to the greenhouse effect. Science 248, 1217–1219. Schlesinger, W.H., 1991. Biogeochem: An Analysis of Global Change. Academic Press, San Diego, CA. Seitzinger, S.P., 1988. Denitrification in freshwater and coastal marine ecosystems: ecological and geochemical significance. Limnol. Oceanogr. 33, 702–724. Smith, M.S., Zimmerman, K., 1981. Nitrous oxide production by nondenitrifying soil nitrate reducers. Soil Sci. Soc. Am. J. 45, 865– 871. Sol orzano, L., 1969. Determination of ammonia in natural waters by the phenol hypochlorite method. Limnol. Oceanogr. 14, 799–801. Strickland, J.D.H., Parsons, T.R., 1972. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Ottawa, Canada, 311 pp. Twilley, R.R., 1997. Mangrove wetlands. In: Messina, M., Connor, W. (Eds.), Southern Forested Wetlands: Ecology and Management. CRC Press, Boca Raton, FL, pp. 445–473. Wang, W.C., Yung, Y.L., Lacis, A.A., Mo, T., Hansen, J.E., 1976. Greenhouse effects due to man made perturbation of trace gases. Science 194, 685–690.
Increase of nitrous oxide flux to the atmosphere upon nitrogen addition to red mangroves sediments ~oz-Hincapie *, Julio M. Morell, Jorge E. Corredor Milton Mun Department of Marine Sciences, University of Puerto Rico, P.O. Box 908, Lajas, PR 00667, Puerto Rico
Abstract Response of nitrous oxide N2 O sediment/air flux to nitrogen addition was assessed in mangrove (Rhizophora mangle) sediments. Fluxes were enhanced with both ammonium and nitrate loading. Greatest fluxes (52 lmol m2 h1 ) were obtained with ammonium addition and saturation was achieved with additions of 0.9 mol m2 . Maximum flux following ammonium addition was 2785 times greater than control plots and 4.5 times greater during low tide than with equivalent ammonium addition at high tide. Nitrate enrichment resulted in exponential growth, with maximal mean flux of 36.7 lmol m2 h1 at 1.9 mol m2 ; saturation was not achieved. Differential response to ammonium and nitrate, and to tide and elevation, indicate that microbial nitrification is responsible for most of the observed gas flux. Mangrove sediments constitute an important source of global atmospheric N2 O and increases in nitrogen loading will lead to significant increases in the flux of this atmospherically active gas. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Nitrous oxide; Mangrove; Sediment flux
1. Introduction Nitrous oxide N2 O is a greenhouse gas, with a direct global warming potential 170–290 times that of carbon dioxide (Wang et al., 1976). Although chemically inert in the troposphere, it is readily transported to the stratosphere where it is involved in destruction of protective ozone (Hutchinson et al., 1993; Hahn and Crutzen, 1982; Delwiche, 1981; Crutzen, 1970). Nitrous oxide also absorbs outgoing planetary IR radiation at wavelengths not removed by atmospheric CO2 or H2 O and has been estimated to contribute 6% of the overall anthropogenic greenhouse effect (Hutchinson et al., 1993; Libes, 1992; Rhode, 1990). It is a stable compound that does not decay readily in the atmosphere, with an atmospheric lifetime of over 121 years (Crutzen, 1970; Khalil and Rasmussen, 1983); even if emissions were held constant, it would be decades before concentration stabilized. By 1994 concentrations of atmospheric N2 O increased from a pre-industrial level of about 275–312 ppbv. Global anthropogenic emissions of N2 O presently *
Corresponding author. Tel.: +1-787-899-2048; fax: +1-787-8995500. E-mail address: [email protected] (M. Mu~ noz-Hincapie).
amount to 4:5 0:6 Tg N yr1 , increasing atmospheric concentrations by 0.25% per year (Khalil and Rasmussen, 1992; Schlesinger, 1991). Nitrous oxide is an intermediate product of both nitrification, the aerobic oxidation of ammonium to nitrite and nitrate, and denitrification, the suboxic reduction of nitrate and nitrite, principally to molecular nitrogen (Corredor et al., 1999a; Firestone and Davidson, 1981; Smith and Zimmerman, 1981). Increased anthropogenic N inputs are known to increase these microbial processes in soils (Bouwman, 1996; Mosier, 1994) and aquatic environments (Seitzinger, 1988). Marine ecosystems are known to constitute a net source of N2 O to the atmosphere (Cohen and Gordon, 1979; Butler et al., 1989) but these appear to be underestimated in current global nitrous oxide N2 O budgets (Nevisson et al., 1995; Capone, 1991). The increasing atmospheric N2 O concentration and the imbalance in its global budget have triggered interest in quantifying N2 O fluxes from various ecosystems (Corre et al., 1999). While the magnitude and global distribution of N2 O emissions from natural soils and from agricultural soils have been investigated, there has not been a comparable analysis of the magnitude or global distribution of N2 O emissions in aquatic ecosystems due to natural and/or anthropogenic processes.
0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 2 ) 0 0 1 3 2 - 7
M. Mu~noz-Hincapie et al. / Marine Pollution Bulletin 44 (2002) 992–996
Mangrove forests occupy a large fraction of the tropical coastline and receive high nitrogen inputs (Corredor and Morell, 1994); such ecosystems dominate the intertidal zone of diverse environmental settings in the Caribbean, from carbonate platforms to terrigenous deltas that exhibit different patterns of nutrient cycling (Twilley, 1997). Mangrove litter fall is made up of leaves, flowers, propagules, twigs and branches, and the percentage of each varies throughout the year. Generally, mangrove leaves are the most important source of detritus and their degradation allows significant energy transfer from mangroves to higher trophic levels via the detritus food web (Gonz alez-Farias et al., 1997). The high productivity of mangroves and the subsequent detrital fall lead to vigorous microbial recycling of nitrogenous products. Corredor et al. (1999a,b) reported nitrous oxide fluxes from mangrove sediments proportional to the availability of inorganic nitrogenous compounds in porewaters. We evaluate the response of N2 O flux to experimental nitrogen loading in sediments within a red mangrove (Rhizophora mangle) forest, southwest coast of Puerto Rico, enriching sediments with oxidized (nitrate) and reduced (ammonium) inorganic nitrogen substrate and quantifying N2 O production under nitrogen loading. Results of these experiments confirm our hypothesis that N2 O flux from the sediments increases dramatically with nitrogen addition.
2. Materials and methods The red mangrove fringe area selected is located at about 17°580 N and 67°030 W on the southwest coast of Puerto Rico in the northeastern Caribbean Sea. Two stations were established at the sampling site. ‘‘Wet’’ sediments correspond to sediments closest to the shoreline and submerged during high tide, ‘‘dry’’ sediments correspond to sediments further from the shoreline. Sediments were manipulated by injecting different doses of ammonium sulphate and potassium nitrate to 0.054 m2 plots of both ‘‘wet’’ and ‘‘dry’’ sediments and compared with unenriched control plots. The nutrient solution was injected with plastic syringes (60 ml) directly into the sediments through plastic pipettes introduced to different depths. The treatment began by inoculating the nutrient solution into the sediments twice a day, three days before samples were taken. Final enrichment was performed while the incubation was carried out. Inoculation doses injected into the sediments were 9:3 103 mol m2 (equivalent to a final theoretical porewater concentration of 0.5 mM), 1:9 102 mol m2 (1.0 mM), 2:8 102 mol m2 (1.5 mM), 1:1 101 mol m2 (6.0 mM), 9:3 101 mol m2 (50 mM) and 1.9 mol m2 (100 mM). In all cases, the vol-
993
ume of the nutrient solution inoculated into the sediments was two liters. Nitrous oxide exchange between sediments and atmosphere was quantified using a closed chamber technique. The incubation chambers are made of rigid polyvinyl chloride pipe, with a height of 15 cm, diameter of 7.5 cm, an internal volume of 2650 ml, and a sediment area covered of 0.018 m2 . The top of the chambers is fitted with an air sampling port and a vent constructed from a 0.6 cm and a 1.2 cm Swagelok bulk-head union respectively (Keller and Reiners, 1990). Samples were collected with a 5 ml glass syringe through the air sampling port. The incubation chamber was inserted 3 cm into the sediments for each series of measurements. Samples were collected at an initial time or time zero, at 30, 60 and at 90 min at both ‘‘wet’’ and ‘‘dry’’ fringe sediment layer in triplicate, and transported to the laboratory for same-day analysis. Concentration of nitrous oxide was determined by gas chromatography using a 63 Ni electron capture detector (Hewlett–Packard 5890 series II) equipped with a MolSieve capillary 5A column 80/100 (mesh). The carrier gas was a mixture of 95% argon and 5% methane at a column flow rate of 21 ml/min. Temperatures were 240 °C in the oven, 350 °C in the detector. Nitrous oxide concentrations were quantified by comparing retention times and peaks areas for samples and certified standards of 0.510 and 0.484 ppm with an analytical accuracy of 2% (Scott Specialty Gases). A calibration standard was injected into the gas chromatograph after every two samples. Field samples were analyzed only when the coefficient of variation of the N2 O standard readings was <1%. Nitrous oxide fluxes were calculated from the linear regression of the rate of concentration change at the four different incubation times in the microatmosphere of the incubation chambers. Only regressions with r2 > 0:5 at the 90% confidence level (P ¼ 0:1) were used. Method detection limit (MDL) was calculated at 6.91 ppb, using seven readings obtained directly from the gas chromatograph as follows: MDL ¼ tS
ð1Þ
where, t is Student’s t value for a 99% confidence level and an S is standard deviation (with n 1 degrees of freedom) of the replicates. Porewater nutrients were analyzed in the lower range of ammonium inoculations (9:3 103 and 1:9 102 mol m2 ) targeted to achieve theoretical porewater concentrations of 0.5 and 1 mM; 5–10 times the observed concentrations in porewaters of control plots. Nutrient content was not analyzed during the nitrate addition experiments. Sediment cores were taken in triplicate at each station with a perforated piston-type corer (30 cm long; 6.5 cm; 3.0 cm diameter). The cores were sealed at the bottom and transported to the laboratory for sameday analyses. Sediment samples were transferred to 50 ml
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centrifuge plastic tubes excluding the root portions. Interstitial porewater samples were obtained from sediment cores by centrifugation at 7000 rpm (15 min). Samples obtained were poisoned with 50 ll mercuric chloride. Ammonium was determined using a modification of the Sol orzano (1969) phenol–hypoclorite colorimetric technique. Nitrite and nitrate were quantified using the method of Strickland and Parsons (1972). The extinction of the diazo compound was measured at 543 nm. All absorbance measurements were performed using a Shimadzu UV-1601 UV–visible spectrophotometer. 3. Results Nitrous oxide fluxes from untreated control plots averaged 0:05 0:03 lmol m2 h1 during low tide and 0:04 0:01 lmol m2 h1 during high tide (Table 1). At the highest levels of inoculation, ammonium addition enhanced N2 O flux by factors of up to 2784 and 245.8 during low tide and high tide respectively compared with control plots. A maximum mean flux of 55.7 lmol m2 h1 was observed during the ammonium enrichment experiments at low tide. Ammonium addition resulted in a sigmoidal increase in N2 O flux with saturation occurring at enrichment levels of 1 mol m2 (Fig. 1a). Nitrate addition was less effective in stimulating N2 O flux. The highest rate enhancements from these additions increased fluxes 1834-fold during low tide with a maximum mean flux of 36.7 lmol m2 h1 . Nevertheless, saturation was not observed during these experiments indicating that higher fluxes might be achieved with further nitrate addition. In contrast to the ammonium additions, sigmoidal behavior was not apparent upon nitrate enrichment. During these experiments, exponential increase of N2 O flux was observed from the lowest to the highest inoculation levels (Fig. 1b). Compared to fluxes obtained with equivalent ammonium concentrations, N2 O fluxes were higher under nitrate addition at the lower range of enrichment (6.6 times at 0.028 mol m2 and 3.2 times at 0.11 mol m2 nitrate addition respectively). Porewater ammonium, nitrite and nitrate concentrations measured after incubation with ammonium
Fig. 1. Nitrous oxide fluxes in response to nutrient inoculation to the sediment during low tide in ‘‘dry’’ sediments. (a) Ammonium addition and (b) nitrate addition. J ¼ N2 O flux.
amendments at 0.5 and 1.0 mM were statistically indistinguishable from those of the control (Table 2).
4. Discussion Our findings confirm the argument that inorganic nitrogen loading of mangrove sediments results in enhancement of N2 O flux to the atmosphere. As noted
Table 1 Nitrous oxide flux rates (lmol m2 h1 ) for ammonium and nitrate inoculation assays in ‘‘dry’’ sediments (each value is a mean, n ¼ 3) Nutrient dose (mol m2 ) 9:3 103 1:9 102 2:8 102 1:1 101 9:3 101 1.9
Low tide
High tide
NHþ 4
NO 3
inoculation
inoculation
0.2 0.14 0.29 1.42 51.90 55.68
– – 1.91 4.51 9.38 36.68
Control 0.04 0.1 0.05 0.05 – 0.02
NHþ 4 inoculation 0.11 0.02 – 0.43 – 12.29
NO 3 inoculation
Control
– – – 0.1 – –
0.03 0.05 – 0.03 – 0.05
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995
Table 2 Sediment porewater nutrient content in the ammonium inoculation to sediments (each value is a mean, n ¼ 3) NH4þ dose (mol m2 )
LT ‘‘Dry’’
Ammonium (lM) 9:3 103 118.0 1:9 102 54.0 Average 86.0
SD
Control
SD
HT ‘‘Dry’’
SD
Control
SD
19.3 15.4 17.4
123.1 66.2 94.6
16.8 13.4 15.1
113.0 61.7 87.4
10.6 16.8 13.7
107.5 55.1 81.3
15.9 31.6 23.8
Nitrite (lM) 9:3 103 1:9 102 Average
3.5 2.5 3.0
0.4 0.0 0.2
2.53 5.81 4.17
0.05 0.03 0.04
2.9 2.0 2.5
0.0 0.4 0.2
2.14 5.81 4.0
0.03 0.03 0.03
Nitrate (lM) 9:3 103 1:9 102 Average
1.5 1.6 1.6
0.0 0.1 0.05
1.60 3.83 2.72
0.15 0.12 0.14
2.2 6.9 4.6
0.5 0.9 0.7
1.70 7.33 4.5
0.00 1.36 0.68
‘‘Dry’’ ¼ ‘‘dry’’ sediment stations. SD ¼ standard deviation.
previously (Corredor et al., 1999a,b), anthropogenic pressure is increasing the rate of nitrogen loading to mangroves throughout the tropical belt and globally significant increases in N2 O flux to the atmosphere may be expected as a result of these pressures. While mangroves serve as an important buffer to eutrophication resulting from anthropogenic nitrogen loading (Corredor and Morell, 1994) the negative consequences of enhanced N2 O flux must be considered. Differences in the response of ‘‘wet’’ and ‘‘dry’’ sediments and of responses during low and high tide provide strong evidence for the argument that N2 O flux results mainly from the activity of nitrifying bacteria present in the mangrove sediments which oxidize the ammonium substrate; a process that benefits from air exposure during low tide. Nitrous oxide production, a by-product of microbial nitrification, is thus enhanced at low tide under augmented conditions of reduced nitrogen loading. Our experiment shows that bacterial populations in mangrove sediments exhibit a rapid response capacity to reduce oxidized nitrogen species. However, nitrification would seem to be the principal mechanism for N2 O production since ammonium is the most abundant dissolved inorganic nitrogenous species in these mangrove sediments (Corredor et al., 1999a,b). Porewater concentrations measured 2 h after inoculation were indistinguishable from those of untreated control plots although treatments were designed to theoretically elevate ammonium concentrations to 0.5 and 1.0 mM. This contrasts with our previous observations (Corredor et al., 1999a) where moderately elevated porewater nutrient concentrations were found in mangrove porewaters subjected to sewage treatment plant effluent and more significant enhancement was found in the naturally enriched environment of a cattle egret rookery. We believe that slow release from bird guano may in large part explain the difference with the latter, and long-term exposure to very high nitrate effluents may explain the
former. The experiments here described demonstrate the very rapid consumption of substrate when added in the readily available form of dissolved inorganic salts as result of both microbial and mangrove metabolism. Nitrous oxide flux under nitrate addition must be attributed to denitrification alone. Under ammonium addition however, both nitrification and denitrification can contribute to the total N2 O flux. Thus, in the nutrient loading experiments, denitrification must contribute up to 38% of the total nitrous oxide flux registered during the nitrification process (Fig. 1a and b). Isotopic analysis of the nitrogen cycle in the red mangrove sediments, particularly that of the gaseous products of microbial metabolism, should further improve our understanding of the relative contribution of the nitrification and denitrification process to N2 O flux. Acknowledgements This research was supported by NASA-UPR-EPSCoR grant no. NCCW-56 and the Department of Marine Sciences, University of Puerto Rico. Diana Marıa Mora-Pinto, Maribel Velez-Rivera, Martha Prada and Gladys Lissette Cintr on assisted in field sampling.
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