Surficial sediments
On each sampling date, we collected four replicate hypolimnetic sediment cores using a K-B gravity sediment corer (Wildlife Supply Company, Yulee, FL, USA). Cores were collected in the deepest part of each reservoir, approximately 20 m from where water samples were taken. In 2019, each core was capped and kept on ice while transported back to the lab, where the top 1 centimeter of sediment from each core was immediately extruded, collected, and frozen in scintillation vials for future analysis. In 2021, cores were extruded in the field, and the samples were kept on ice while being transported back to the lab.
Sediment traps
To determine the amount of Fe-OC and total OC in material sedimenting from the water column, we deployed 19-L buckets approximately 1 m above the sediments at the deepest point of each reservoir (8 m at FCR and 10 m at BVR). These sediment traps were deployed from June–December 2021 and sampled every two weeks by slowly bringing the bucket to the surface, decanting and discarding water from the bucket, collecting up to 5 L of the remaining water and sediment, and transporting this material back to the lab on ice. Upon arriving at the lab, we allowed the sediment to settle for approximately 5 minutes, before decanting and discarding as much water as possible and filling four 50-mL centrifuge tubes with the remaining material. The samples were centrifuged for 10 minutes at 3100 rpm, then combined into one vial and frozen for later analysis. No sediment traps were collected and analyzed for Fe-OC in 2019.
Fe-OC analysis
We analyzed the amount of Fe-OC in both the whole-ecosystem and microcosm sediment samples using the citrate bicarbonate dithionite (CBD) method. This method was first described for marine systems by Lalonde et al. (2012) and has since been adapted for freshwater lakes by Peter and Sobek (2018). It is important to note that our measurement of Fe-OC as the percentage of OC that is extractable using the CBD method is an operational definition (Fisher et al., 2021). CBD extractions have documented inefficiencies when extracting crystalline hematite (Thompson et al. 2019; Adhikiri & Yang, 2015) and carboxyl-rich compounds (Fisher et al. 2020). While Fe is the primary reducible metal that associates with OC, other metals, including aluminum (Al) and calcium (Ca), may also release OC during CBD extractions. However, previous work in soils found that CBD-extracted aluminum was approximately an order of magnitude lower than CBD-extracted Fe, and therefore quantitatively much less important (Sondheim and Standish, 1983). Moreover, we found that Fe was present in much (≥ 5 times) higher quantities than Al and Ca in water samples across all of our sediment incubation treatments, further justifying our use of the operational term Fe-OC. We used the CBD method to enable comparisons both between oxygen treatments and with other published work that used the same general approach (e.g., Lalonde et al., 2012; Peter & Sobek, 2018).
Following the CBD method, each sediment sample was freeze-dried and divided into three treatments: initial, reduction, and control. “Initial” samples received no treatment and were used to measure the OC content of the sediment. “Reduction” samples were treated with a metal-complexing agent (trisodium citrate) and reducing agent (sodium dithionite) in a buffered solution (sodium bicarbonate) to measure how much Fe and OC were released as a result of Fe reduction. Control samples were used to account for the release of OC in the reduction treatment that resulted from processes other than Fe reduction. They were treated with the same buffer (sodium bicarbonate) and sodium chloride in the same ionic strength as the trisodium citrate and sodium dithionite of the reduction treatment.
For both the control and reduction treatments, we measured 100 mg of homogenized, freeze-dried sediment into 15-mL polypropylene centrifuge tubes (Falcon Blue, Corning Inc., Corning, NY, USA). We then added 6 mL of either control or reduction buffer solution (0.11 M sodium bicarbonate) to each tube. The reduction buffer contained 0.27 M trisodium citrate, while the control buffer contained 1.6 M sodium chloride. After heating samples to 80ºC in an oven, 0.1 g sodium dithionite was added to the reduction samples and 0.088 g sodium chloride was added to control samples. Samples were kept at 80ºC for an additional 15 min, then centrifuged for 10 min at 3100 RPM. The supernatant was discarded. This extraction process was repeated two more times for both treatments, resuspending the sediment pellet each time by vortexing with buffer solution (Peter and Sobek, 2018).
Following the extraction step, samples were rinsed three times using OC- and Fe-free artificial lake water. Artificial lake water was prepared by diluting Artificial Hard Water from Marking and Dawson (1973) to 12.5% with Type I reagent grade water. We added 3 mL of artificial lake water to each tube and resuspended the sediment pellet using a vortex. Samples were then centrifuged for 10 min at 3100 RPM, and the supernatant was discarded.
After extraction and rinsing, all sediment samples (including those in the initial treatment) were dried and acid-fumigated for 48 hours to remove remaining citrate and bicarbonate (Harris et al., 2001). Samples were then run on a CN analyzer (Elementar VarioMax, Ronkonkoma, NY, USA) to determine the amount of OC per unit mass of sediment. In these calculations, we adjusted sediment mass to account for Fe loss during control and reduction treatments (Peter and Sobek, 2018). The amount of OC removed with Fe reduction (CBD-extractable OC) was calculated as the difference between the OC content of the control and reduction samples and expressed as a percentage of the initial OC content of the sediment.
Reservoir_sediment_clean_current.Rmd provides the calculations associated with these sediment properties. We adjusted sediment mass to account for Fe loss during control and reduction treatments. The amount of OC removed with Fe reduction (CBD-extractable OC) was calculated as the difference between the OC content of the control and reduction samples and expressed as a percentage of the initial OC content of the sediment.
References
Adhikari, D., & Yang, Y. (2015). Selective stabilization of aliphatic organic carbon by iron oxide. Scientific Reports, 5(1), 1–7. https://doi.org/10.1038/srep11214
Fisher, B. J., Faust, J. C., Moore, O. W., Peacock, C. L., & März, C. (2021). Technical Note: Uncovering the influence of methodological variations on the extractability of iron bound organic carbon, 20.
Fisher, B. J., Moore, O. W., Faust, J. C., Peacock, C. L., & März, C. (2020). Experimental evaluation of the extractability of iron bound organic carbon in sediments as a function of carboxyl content. Chemical Geology, 556, 119853. https://doi.org/10.1016/j.chemgeo.2020.119853
Harris, D., Horwáth, W. R., & Kessel, C. van. (2001). Acid fumigation of soils to remove carbonates prior to total organic carbon or CARBON-13 isotopic analysis. Soil Science Society of America Journal, 65(6), 1853–1856. https://doi.org/10.2136/sssaj2001.1853
Lalonde, K., Mucci, A., Ouellet, A., & Gélinas, Y. (2012). Preservation of organic matter in sediments promoted by iron. Nature, 483(7388), 198–200. https://doi.org/10.1038/nature10855
Marking, L. L., & Dawson, V. K. (1973). Toxicity of quinaldine sulfate to fish (Report No. 48) (pp. 0–8). La Crosse, WI. Retrieved from http://pubs.er.usgs.gov/publication/2001015
Peter, S., & Sobek, S. (2018). High variability in iron-bound organic carbon among five boreal lake sediments. Biogeochemistry, 139(1), 19–29. https://doi.org/10.1007/s10533-018-0456-8
Sondheim, M. W., & Standish, J. T. (1983). Numerical analysis of a chronosequence including an assessment of variability. Canadian Journal of Soil Science, 63(3), 501–517. https://doi.org/10.4141/cjss83-052
Thompson, J., Poulton, S. W., Guilbaud, R., Doyle, K. A., Reid, S., & Krom, M. D. (2019). Development of a modified SEDEX phosphorus speciation method for ancient rocks and modern iron-rich sediments. Chemical Geology, 524, 383–393. https://doi.org/10.1016/j.chemgeo.2019.07.003
