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• Experimental design was implemented as shown in Fig. 2 • Three sampling strategies were used: 1. Direct measure (drift net samples) (Fig. 3) - Samples taken at 11:00 am, 12:00 pm, 1:00 pm (injection), 2:00 pm and 3:00 pm, and water column filtered for 15 min. At 12:55 pm, CO₂ tank opened to allow CO₂ to enter the stream for 1 hour 2. Indirect measure/benthic (mini-Surber samples) (Fig. 4) - 3 samples taken Upstream and at the First, Second and Third stations before and after acidification 3. Indirect measure/artificial substrate (leaf pack samples) (Fig. 5) - ~7 g leaf packs developed in stream for 5 days, 3 packs removed prior to CO₂ injection and 3 packs removed after. CO₂ tank opened for 1 hour • All insects were preserved in the field with 95% ethanol and identified to family level • This study shows a decline in stream pH along a gradient associated with the injection of CO 2 (Fig. 6) • No significant results were obtained from drift net analysis of macroinvertebrate composition, possibly because low stream flow inhibited effective macroinvertebrate drift (downstream displacement) • Leaf pack analysis showed that pH decline had a significant effect on macroinvertebrate composition in terms of abundance (Fig. 7A) and taxonomic richness (7B), with the most pronounced macroinvertebrate response observed at the injection site - These data may suggest a high sensitivity of organisms that use this resource to stream acidification (shredders in order Trichoptera) - Higher composition at the Upstream station after CO 2 injection (Fig. 7A, 7B) may possibly be explained by active upstream movement to avoid the environmental stressor in addition to macroinvertebrate drift downstream • Surber analysis showed no significance, but a trend may be present in macroinvertebrate abundance (Fig. 7C) and taxonomic richness (Fig. 7D) before and after CO 2 injection indicating a negative change in composition closest to the injection site that follows a gradient downstream • The results of this study can be used to understand the macroinvertebrate response connected with decreased pH and increased atmospheric CO₂ - One component of climate change is increased concentrations of atmospheric CO 2 - Increased CO 2 was shown by this study to be linked to decreased pH in tropical freshwater streams - Macroinvertebrates located at the site of CO 2 injection exhibited the greatest negative response 4 5 6 7 8 11:00 12:00 1:00 1:45 2:00 3:00 pH Time of Day CO 2 -Driven pH Change Upstream Injection First Second Third To experimentally manipulate the Buruquena Stream (Fig. 1) using CO₂ in order to measure the effects of acidification on macroinvertebrates in a tropical stream. Crystal C. Purcell¹ and Pablo E. Gutierrez-Fonseca² ¹Department of Entomology, Purdue University, 901 West State Street, West Lafayette, IN 47907 ²Department of Biology, University of Puerto Rico - Rio Piedras, PO Box 70377, San Juan, PR 00936-8377 Introduction Study Site Methods 0 3 6 9 12 15 Upstream Injection First Second Abundance (ind/g) Stations 0 2 4 6 8 Upstream Injection First Second Richness (#taxa/g) 0 1000 2000 3000 4000 5000 6000 7000 Upstream First Second Third Abundance (ind/m 2 ) Stations 0 2 4 6 8 10 12 Upstream First Second Third Richness (#taxa/sample) * p < 0.05 p < 0.005 Before CO₂ After CO₂ * * * Figure 4: Mini-Surber used for benthic sampling (indirect measure of macroinvertebrate response) Figure 3: Drift net used for direct sampling of macroinvertebrates in drift Figure 5: Leaf pack used as artificial substrate (indirect measure of macroinvertebrate response) * * Thank you to Pedro J. Torres and Bethany Vázquez for helping with sampling, as well as all participating 2014 REU students and mentors for their input. Figure 1: Location of El Verde Field Station, El Yunque National Forest, Puerto Rico (1A) and study site, Buruquena Stream (1B) Objective Mechanism • Streams have highly variable temperature, discharge and pH 1 • Of the factors affecting macroinvertebrates, stream acidification is a growing concern 2,3,4 • Stream acidification simplifies macroinvertebrate assemblages and reduces taxonomic richness 3,5 , and macroinvertebrates may use passive displacement (drift) to escape the unfavorable conditions of a particular area • In a natural stream system, decreased pH can be driven by: the dilution of acid neutralizing capacity (ANC) 2 , organic acids or dissolved organic carbon (DOC) from decomposing leaf matter 2 , precipitation events 2,3,4 , acid rain 6 , and increased dissolved CO₂ 2 • Some studies have attempted to demonstrate the effect of acidification on macroinvertebrates in tropical streams using HCl 7 or other strong acids • Because acidification is not due to HCl in nature, this study experimentally acidified a stream with the addition of gaseous CO₂ - Manipulates the carbonate-bicarbonate equilibrium present in streams 2,7 (see Mechanism) to model stream conditions resulting from an increased level of atmospheric CO₂ References: 1. Ramírez A, Pringle CM, Douglas M. 2014. Temporal and spatial patterns in stream physicochemistry and insect assemblages in tropical lowland streams. Am Benthol Soc. 25(1):108–125. 2. Small GE, Ardón M, Jackman AP, Duff JH, Triska FJ, Ramírez A, Snyder M, Pringle CM. 2012. Rainfall-driven amplification of seasonal acidification in poorly buffered tropical streams. Ecosystems.15: 974–985. 3. Lepori F, Ormerod SJ. 2005. Effects of spring acid episodes on macroinvertebrates revealed by population data and in situ toxicity tests. Freshw Biol. 50:1568–1577. 4. Lepori F, Barbieri A, Ormerod SJ. 2003. Effects of episodic acidification on macroinvertebrate assemblages in Swiss Alpine streams. Freshw Biol. 48:1873–1885. 5. Durance I, Ormerod SJ. 2007. Climate change effects on upland stream macroinvertebrates over a 25-year period. Glob Chang Biol.13:942–957. 6. Driscoll CT, Lawrence GB, Bulger AJ, Butler TJ, Cronan CS, Eagar C, Lambert KF, Likens GE, Stoddard JL, Weathers KC. 2001. Acidic deposition in the northeastern United States: sources and inputs, ecosystem effects, and management strategies. BioScience. 51:180-198. 7. Ardón M, Duff JH, Ramírez A, Small GE, Jackman AP, Triska FJ, Pringle CM. 2013. Experimental acidification of two biogeochemically-distinct neotropical streams: buffering mechanisms and macroinvertebrate drift. Sci Total Environ. 443:267– 77. Figure 2: Experimental design schematic showing the Buruquena Stream with CO 2 injection site, locations of each sampling station and sampling method used per station Results Discussion Acknowledgements 1B. Figure 6: Decline in pH over time at each sampling station associated with CO 2 injection Figure 7: Macroinvertebrate response to CO 2 injection for two sampling methods: abundance (7A) and richness (7B) in leaf pack samples, and abundance (7C) and richness (7D) in Surber samples before and after CO 2 injection 7B. 7A. 7C. 7D. CO 2 + H 2 O H 2 CO 3 H + + HCO 3 - H + + CO 3 2- carbon dioxide water carbonic acid bicarbonate ion hydrogen ion hydrogen ion carbonate ion Stations Stations Experimental acidification effects on aquatic macroinvertebrates in a tropical stream Leaf Pack Abundance Surber Abundance Leaf Pack Taxonomic Richness Surber Taxonomic Richness 1A. 1. Direct measure (drift net samples) (Fig. 3) - Samples taken at 11:00 am, 12:00 pm, 1:00 pm (injection), 2:00 pm and 3:00 pm, and water column filtered for 15 min. At 12:55 pm, CO₂ tank opened to allow CO₂ to enter the stream for 1 hour 2. Indirect measure/benthic (mini-Surber samples) (Fig. 4) - 3 samples taken Upstream and at the First, Second and Third stations before and after acidification 3. Indirect measure/artificial substrate (leaf pack samples) (Fig. 5) - ~7 g leaf packs developed in stream for 5 days, 3 packs removed prior to CO₂ injection and 3 packs removed after. CO₂ tank opened for 1 hour
