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We present an evaluation of a nested high‐resolution Goddard Earth Observing System (GEOS)‐Chem chemistry transport model simulation of tropospheric chemistry over tropical South America. The model has been constrained with two isoprene emission inventories: (1) the canopy‐scale Model of Emissions of Gases and Aerosols from Nature (MEGAN) and (2) a leaf‐scale algorithm coupled to the Lund‐Potsdam‐Jena General Ecosystem Simulator (LPJ-GUESS) dynamic vegetation model, and the model has been run using two different chemical mechanisms that contain alternative treatments of isoprene photo‐oxidation. Large differences of up to 100 Tg C yr−1 exist between the isoprene emissions predicted by each inventory, with MEGAN emissions generally higher. Based on our simulations we estimate that tropical South America (30–85°W, 14°N–25°S) contributes about 15–35% of total global isoprene emissions. We have quantified the model sensitivity to changes in isoprene emissions, chemistry, boundary layer mixing, and soil NOx emissions using ground‐based and airborne observations. We find GEOS‐Chem has difficulty reproducing several observed chemical species; typically hydroxyl concentrations are underestimated, whilst mixing ratios of isoprene and its oxidation products are overestimated. The magnitude of model formaldehyde (HCHO) columns are most sensitive to the choice of chemical mechanism and isoprene emission inventory. We find GEOS‐Chem exhibits a significant positive bias (10–100%) when compared with HCHO columns from the Scanning Imaging Absorption Spectrometer for Atmospheric Chartography (SCIAMACHY) and Ozone Monitoring Instrument (OMI) for the study year 2006. Simulations that use the more detailed chemical mechanism and/or lowest isoprene emissions provide the best agreement to the satellite data, since they result in lower‐HCHO columns.