Processing of Particulate Organic Carbon Associated with Secondary

Nov 8, 2013 - Centre for Coastal Biogeochemistry, Southern Cross University, Military Road, Lismore, New South Wales 2480, Australia. ‡. Institute f...
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Processing of Particulate Organic Carbon Associated with Secondary-Treated Pulp and Paper Mill Effluent in Intertidal Sediments: A 13C Pulse-chase Experiment Joanne M. Oakes,*,† Donald J. Ross,‡ and Bradley D. Eyre† †

Centre for Coastal Biogeochemistry, Southern Cross University, Military Road, Lismore, New South Wales 2480, Australia Institute for Marine and Antarctic Studies, University of Tasmania, Private Bag 49, Hobart, Tasmania 7001, Australia



S Supporting Information *

ABSTRACT: To determine the benthic transformation pathways and fate of carbon associated with secondary-treated pulp and paper mill (PPM) effluent, 13C-labeled activated sludge biomass (ASB) and phytoplankton (PHY) were added, separately, to estuarine intertidal sediments. Over 28 days, 13C was traced into sediment organic carbon, fauna, seagrass, bacteria, and microphytobenthos and into fluxes of dissolved organic carbon (DOC) and dissolved inorganic carbon (DIC) from inundated sediments, and carbon dioxide (CO2(g)) from exposed sediments. There was greater removal of PHY carbon from sediments (∼85% over 28 days) compared to ASB (∼75%). Although there was similar 13C loss from PHY and ASB plots via DIC (58% and 56%, respectively) and CO2(g) fluxes (500 μm), seagrass roots, and seagrass shoots. Calculations. We calculated the total flux of DO, DIC, alkalinity, DOC across the sediment-water interface and the flux of CO2(g) across the sediment−air interface, as well as fluxes of 13 C in DOC, DIC, and CO2(g) that were in excess of those from control plots (i.e., the flux of 13C derived from the added PHY or ASB). We also determined the total uptake of added 13 C (incorporation) into sediment OC, bacteria, MPB, seagrass shoots and roots, and fauna. The rate of 13C loss from sediment OC was determined by fitting 1-G models to the data.15 Separate models were generated for each experimental plot, allowing calculation of means and standard errors for model parameters. To account for differences in quantities of OM added to PHY and ASB plots, and to allow comparison of treatment types, excess 13C flux and incorporation data was converted to a percentage of the 13C initially added to sediments.



EXPERIMENTAL SECTION The methods outlined in the following text and the data analysis methods are detailed in the Supporting Information (SI). Site description. The study site was an intertidal mudflat in the Derwent estuary, Tasmania (42°48′55″S, 147°15′36″E), ∼23 km downstream of a PPM (Norske-Skog) using mechanical pulping, followed by secondary treatment of effluent with ASB. The site had an even cover of short seagrass (Zostera tasmanica, < 5 cm height, ∼1.3 g dry wt m−2) and benthic microalgae (22.3 mg chlorophyll-a m−2). Salinity varied from 10.7 to 24.6 during the study, depending on river flow. Organic Carbon Addition. Twelve experimental plots (1 m × 1 m) were established on the mudflat. Three plots were haphazardly allocated to each of four treatments: control, procedural control (PC), PHY addition, and ASB addition. Quantities of 13C-labeled OC equivalent to estimated daily loadings4 (see Supporting Information) were added to PHY (1.5 g dry phytoplankton per m−2) and ASB plots (8.5 g dry biomass per m−2), allowing for direct comparison of these two alternate sources of particulate OC associated with secondarytreated PPM effluent discharge. Importantly, 13C label was primarily incorporated into the metazoa and protozoa within ASB (∼64% of biomass). The processing of this component of ASB is therefore the focus of this study. The procedures for



RESULTS Fluxes of DO, DIC, alkalinity, DOC, and CO2(g) from PC and control plots were not significantly different (p > 0.05), indicating that OM addition did not affect inherent carbon cycling rates (see SI Figure S1). Organic carbon addition did not significantly affect dark or light fluxes of DO, DIC, alkalinity, or CO2(g), or light fluxes of DOC. However, DOC fluxes in the dark were significantly more positive for control plots than for either of the OM addition plots (two-way ANOVA: F2,36 = 9.800, p < 0.001). There was significant temporal variability in dark and light fluxes of DO, DIC, alkalinity, CO2(g), and dark fluxes of DOC (see SI Figure S2). 13259

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h (Figure 2). However, 13C remained in sediment OC at 2−10 cm in ASB plots throughout the study, whereas little 13C was in 2−10 cm sediment OC in PHY plots after 2.5 d, and none by 27.4 d after OM addition. 13 C Incorporation into Heterotrophs. The OM added to ASB plots contained 13C-labeled bacterial biomass, but this represented only 1.6% of the 13C in bacteria within ASB plots 1 day after OM addition (when this was first measured). PHY was free of bacteria. The 13C within bacterial biomass in experimental plots therefore represents uptake and transfer by the sediment bacterial community. In both PHY and ASB plots, bacteria only incorporated 13C in 0−2 cm sediments but were far more important for processing ASB (Figure 2). Across all times, the average contribution of bacteria to the 13C within ASB sediment OC was 48 - 90%. In comparison, although bacteria accounted for all of the 13C within 0−2 cm sediment OC in PHY plots after 26 h, their contribution to the 13C within sediment OC decreased markedly thereafter, with no 13C in bacteria in PHY plots by 9.3 d after label addition (Figure 2). Due to considerable variation, no statistically significant difference was detected in 13C incorporation in bacteria across sampling times or treatment types. The contribution of fauna to 13C within sediment OC in both PHY and ASB plots was greatest in surface sediments, reflecting the low biomass of fauna in deeper sediments (Figure 2). Fauna in surface sediments was represented by nine taxa: molluscs (Tatea ruf ilabris and Nassarius pauperatus), a bivalve (Arthritica semen), amphipods (Paracorphium sp., a species in the family Phoxocephalidae, and two unidentified species), polychaetes, and ostracods. In contrast, few taxa were in deeper sediment layers: A. semen and polychaetes in 2−5 cm sediments and polychaetes in 5−10 cm sediments. However, given the size and mobility of polychaetes, it is likely that not all individuals were captured. This is likely to have led to faunal biomass being underestimated, particularly in deeper sediments. Excluding polychaetes, fauna in 2−10 cm sediment accounted for 0.05), due to considerable variability among replicates (Figure 1).

Figure 1. Budget showing the cumulative excess 13C lost via fluxes of DIC and DOC from the addition of (A) activated sludge biomass and (B) phytoplankton until each sampling time, and the excess 13C within sediment organic carbon, seagrass leaves, and other sediment compartments at each sampling time, as a percentage of the 13C initially added to experimental plots (mean ± SE). Arrows indicate predicted 13C content of sediment OC at each sampling time based on 2-G models that best fitted the data.

However, there was substantial loss of added 13C from sediment OC during the study, particularly for PHY plots (∼85% of added 13C over the study period, compared to ∼75% for ASB plots, Figure 1). A significantly larger proportion of added 13C remained in ASB plots compared to PHY plots (three-way ANOVA: F1,58 = 0.015, p = 0.01). For both PHY and ASB plots, 13C loss from sediment OC was fitted using 1-G models (R2 = 0.92 and 0.88, respectively, Figure 1). The reactive fraction (G1) accounted for a greater portion of the sediment OC in PHY plots (85.61 ± 4.78%, k = 0.026 ± 0.007 h−1) than in ASB plots (74.82 ± 0.99%, k = 0.013 ± 0.003 h−1). Over the duration of the study 14.39 ± 4.78% and 25.17 ± 0.99% of the sediment OC in PHY and ASB plots, respectively, was nonreactive (GNR fraction). Although the quantity of excess 13C in sediment OC per area was significantly lower in deeper sediments (three-way ANOVA: F3,58 = 5.860, p = 0.001), there was considerable downward transport of 13C in both plot types. This was more rapid in PHY plots, with a maximum of ∼38% of added 13C within 2−10 cm sediments within 26 h of label addition (Figure 2), compared to ASB plots, for which the maximum 13C incorporation into sediment OC at 2−10 cm (∼17%) was at 36 13260

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Figure 2. Excess 13C incorporation into sediment organic carbon, bacteria, MPB, fauna, and seagrass roots at sediment depths of 0−2 cm, 2−5 cm, and 5−10 cm throughout the study period as a percentage of the 13C initially added to experimental plots (mean ± SE). Note that some bars are too small to be seen. Sediment organic carbon was not measured at the first time period (0.5 days after 13C addition).

ANOVA: time × darklight interaction, F1,48 = 5.378, p = 0.001). Except on days 2.5 and 3.4, the flux of excess 13C as DIC was significantly greater from ASB than from PHY plots (three-way ANOVA: time × treatment interaction, F5,48 = 6.147, p = 0.01, p < 0.03 for all days except 2.5 and 3.4). Although 11.9% of 13C was lost from ASB plots over 27.4 d via DOC effluxes, this pathway was far more important for 13C loss from PHY plots (40.9% over 27.4 d). This was driven by differences in the 13C efflux as DOC during the dark. Dark fluxes of excess 13C as DOC were greater than light fluxes for both ASB and PHY plots (PHY, F1,34 = 15.251, p = 0.001; ASB, F1,34 = 8.031, p = 0.008) but were significantly greater from PHY plots (darklight × treatment interaction, F1,48 = 10.106, p = 0.003) Very little 13C was lost from ASB or PHY plots via CO2(g) fluxes from exposed sediments during the study, with only 0.8% (PHY) and 0.4% (ASB) of added 13C lost via this pathway. The only significant difference in CO2(g) fluxes across light condition, times, and treatments was for dark fluxes of CO2 at time 2, when the flux of 13C-labeled CO2(g) from PHY plots was significantly greater than from ASB plots (F1,6 = 9.116, p = 0.023).

ASB plots. The highest uptake of 13C by MPB was toward the beginning of the study for both ASB (up to 37 h) and PHY plots (up to 60 h). There was 13C incorporation by seagrass roots and shoots in both ASB and PHY plots. In both ASB and PHY plots, the greatest incorporation of 13C into seagrass roots was in surface sediments (up to 9.7% and 1.7% of added 13C, Figure 2), but 13 C was also in seagrass roots at 2−5 cm (up to 5.5% and 0.5%). There was considerable excess 13C in seagrass roots in ASB and PHY plots from the first sampling time, and the excess 13 C content of seagrass roots was also high in both plots toward the end of the study. Overall, there was greater uptake of 13C into seagrass roots in 0−5 cm sediments in PHY plots than in ASB plots. The levels of 13C incorporation into seagrass shoots were similar in both ASB and PHY plots (up to 2.3 and 1.54% of added 13C, respectively), but whereas there was excess 13C in seagrass shoots at most sampling times in ASB plots, excess 13C was only detected in PHY plots in the later sampling times (i.e., from 84 h). Pathways for Loss of 13C. DIC effluxes were the major pathway for loss of 13C from both ASB (58.2% of added 13C) and PHY plots (55.8% of added 13C). Dark and light fluxes of 13 C generally decreased over time in both plots (three way 13261

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Figure 3. Conceptual model showing pathways for transfer of carbon derived from activated sludge biomass (top) or phytoplankton detritus (bottom) when sediments are inundated. Thicker arrows indicate dominant pathways.



with the current study, where 13C was transported below 2 cm within 0.5 d and below 5 cm within 1.0 d. Transport by polychaetes, with or without ingestion, could therefore easily account for the 13C observed in deeper sediments. Given that little of this 13C was within fauna, bacteria, seagrass roots, or MPB, and a large quantity (∼40%) of added PHY was transported below 2 cm within only 1 d (Figure 2), active transport without ingestion appears most likely. Greater downward transport of 13C may reflect the preference of fauna for PHY due to its high lability and/or the presence of toxins associated with ASB.6 This is supported by the higher degradation rate and larger reactive fraction (based on 1-G modeling) of PHY, as well as the persistence at the end of the study of much of the ASB that had been transported to deeper sediments. The preference of fauna for PHY may have contributed to differences in 13C incorporation by bacteria and MPB (Figure 3). Bacteria dominated 13C incorporation in ASB plots throughout the study but were only important in PHY plots early in the study, and 13C incorporation into MPB was lower in PHY plots than ASB plots. This reflects the rapid removal of

DISCUSSION Differences in the quality of OM added to PHY and ASB plots were reflected in marked differences in carbon incorporation, transformation, and loss pathways (summarized in Figure 3). Incorporation and Transfer of 13C. Two main differences in OM processing within PHY and ASB plots were the greater downward transport of carbon in PHY plots and the importance of bacterial uptake for ASB processing. The greater downward transport of 13C in PHY plots is most likely related to the activity of fauna, particularly polychaetes, which were found throughout all sediment depths. Given the high clay content of the sediment studied, and the presence of stabilizing vegetation and MPB,16,17 physical mixing by water movement would contribute little to downward transport. This also would not account for the different rates in PHY and ASB plots. Benthic fauna, however, could rapidly transport OC to deeper sediments.18 Although fauna can rapidly consume PHY,7,19,20 active transport can occur without ingestion. Polychaetes, for example, can transport Thalassiosira pseudonana, the phytoplankton species used in the current study, to depths of over 10 cm within 1.5 d.21 This compares favorably 13262

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was similar, there was greater loss of 13C via DOC fluxes from PHY plots. This may relate to differences in lability,11 with lower C:N ratios for PHY (∼7) indicating that it is a higher quality food source. However, C:N ratios for ASB were also relatively low (∼10) and may have been even lower for the component of ASB that acquired 13C-label, depending on the contribution of residual pulp fiber (C:N ∼304) to the biomass that was analyzed. It therefore seems likely that residual toxins in ASB (e.g., tannins and/or resin acids)6 may have inhibited its processing. DOC fluxes were more important for 13C loss from PHY plots than from ASB plots. Although few studies have been done, considerable DOC fluxes immediately following phytoplankton addition have been attributed to accidental addition of 13C-labeled DOC with phytoplankton material.10 In the current study, however, fluxes of 13C-labeled DOC from PHY plots were sustained over 28 d and can therefore be attributed to processing of PHY. Given that bacteria can rapidly utilize DOC,25,28 the loss of 13C from PHY plots via DOC fluxes suggests that bacterial access to DOC was limited. This may relate to the activity of fauna. The apparent importance of polychaetes in PHY processing suggests that polychaetes released DOC from PHY via cell lysis during grazing, and/or excreted DOC following consumption of 13C-labeled PHY. Water flowing from fauna burrows would have removed this DOC, limiting its availability to sediment-bound bacteria (Figure 3). Given that little 13C remained in deeper sediments beyond 2.5 d (Figure 2), it seems likely that surface grazers influenced DOC fluxes from PHY plots in the longer term, reflecting the lack of toxins in PHY compared to ASB. Through grazing, surface fauna may exude or release 13C-labeled DOC that fluxes directly to the overlying water, rather than being available to sediment bacteria. Alternatively, the DOC produced in ASB plots may have been more labile, enhancing its utilization by sediment bacteria . Given that both bacteria and MPB exude extracellular DOC, the relatively high 13C content of MPB and bacteria in ASB plots could have resulted in considerable loss of added 13C via DOC fluxes. The relatively small fraction of added 13C that was lost from ASB plots via fluxes of DOC therefore indicates that bacteria rapidly consumed the ASB-derived DOC and that this DOC is therefore relatively labile. Similar efficient DOC uptake by bacteria has been reported previously14 and is likely to have contributed to the slower rate of 13C loss from ASB plots compared to PHY plots. Implications. Secondary-treated PPM effluent has a considerably lower oxygen demand in the receiving environment than primary-treated effluent. However, given that ∼32% of the daily load of ASB carbon remained in sediments after 28 d, ASB is likely to accumulate and may therefore result in sediment anoxia, although to a lesser extent than that associated with primary-treated PPM. Although there was greater loss of PHY carbon from sediments (89% over 28 d), approximately 41% of this was released as DOC, offsetting reductions in DOC made through secondary-treatment of PPM effluent. Conversion of this DOC to DIC may result in the receiving environment becoming more heterotrophic,2 particularly as PHY production is likely to coincide with ASB release, which is also ultimately respired to DIC. This highlights the need to control the quality and quantity of OC associated with secondary-treated PPM effluent, but these findings must be considered in the context of the following caveats:

OC from surface sediments in PHY plots, limiting its availability to MPB and bacteria. Some PHY OC may have been incorporated by bacteria, but this was not detected, possibly due to the 3−6 fold lower biomass of bacteria at these depths. Alternatively, the composition of bacterial communities, and their ability to process PHY, may vary with sediment depth. In ASB plots, bacteria dominated 13C incorporation, reflecting limited competition with fauna for OC. Uptake of 13C into MPB in ASB plots may therefore reflect utilization of the 13C made available as DIC following bacterial respiration. Seagrass incorporated 13C in both ASB and PHY plots. This carbon may have been acquired as DIC22 or DOC23 from the water column or the sediment and may have been subsequently translocated within the plant.24 Lack of replication makes it difficult to draw conclusions about differences in 13C incorporation by seagrass in ASB and PHY plots. However, the greater incorporation of 13C into seagrass roots in PHY plots hints at 13C uptake via seagrass roots following downward transport. Seagrasses exude carbon from their roots as DOC. The 13C content of extracellular DOC in sediments was not measured, but may account for some of the uncharacterized 13C in deeper sediments, particularly in PHY plots, where the 13C content of seagrass roots was highest. However, it is unlikely that this made a large contribution, as seagrass exudates are rapidly assimilated by bacteria,25 but PLFA biomarkers showed no evidence of this. Loss of 13C from Sediments. Regardless of plot type, 13C was lost from sediments primarily as inorganic carbon. Similar to previous studies of unvegetated temperate sediments,26,27 inorganic carbon was lost during inundation (as DIC) rather than exposure (as CO2(g)), but the difference was more pronounced than has been reported, with CO2(g) fluxes at least an order of magnitude lower than fluxes of DIC. This may be because the 13C-labeled DIC and CO2(g) fluxes considered in the current study were ultimately derived from the added PHY and ASB and therefore represent inorganic carbon production in the time since OM addition. In contrast, DIC and CO2(g) fluxes in previous studies were derived from existing OC and reflect inorganic carbon production over a longer period. Differences in CO2(g) and DIC fluxes are discussed further in the Supporting Information. Respiration to DIC was an important 13C loss pathway in both PHY and ASB plots. The rate of respiration of PHY to DIC (∼10% over 24 h) was similar to that reported for sediments from estuaries to the deep sea (∼15% over 24 h).9 The higher C:N ratio, and slower degradation, of ASB suggested that it should be respired to DIC more slowly than PHY. However, ASB was initially respired more rapidly than PHY (∼20% within 24 h), and a similar fraction of ASB and PHY was respired over the 28 d study (∼60%). The initial rapid respiration of ASB may reflect the utilization of a more labile component of ASB by sediment microbes. However, macrofauna can also reduce phytoplankton respiration through downward transport of OC.10 This may account for the slower initial respiration of OC in PHY plots, where greater downward transport of 13C appeared to be linked to subduction by fauna (Figure 3). Overall, a greater fraction of added 13C was lost from PHY plots than from ASB plots during the study, reflecting the higher degradation rate of PHY (based on 1-G modeling), and differences in carbon processing pathways (Figure 3). Although the loss of 13C as inorganic carbon from PHY and ASB plots 13263

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• OC was added as a single pulse representing. Long-term input of PPM material may alter sediment conditions and carbon processing. However, the sediments studied were within the zone of influence of a PPM and would already be subject to such conditions. • Bacteria represent ∼36% of ASB but their processing is not considered here due to low 13C incorporation. However, it is likely that the processing of bacterial carbon would be similar to that of the protozoa and metazoan that primarily acquired 13C label in the current study; the low C:N ratio of bacteria should result in rapid processing, but this is likely to be inhibited by ASBassociated toxins. • The load of ASB in secondary-treated PPM effluent can be considerably below that we assumed and, at the PPM we studied, has since reduced by ∼66% (D. Richardson, personal communication). This would reduce the accumulation of ASB within sediments and any associated impacts on the receiving environment. • This study considered sediment within the main settling zone for PPM particulate OC. Pathways for processing PHY and ASB would likely be similar in sediments further from the PPM (i.e., with reduced OC input), but lower ASB loading should reduce its accumulation. Processing may also be affected by different sediment conditions and faunal communities. • The study was done intertidally, but OM also deposits subtidally. Subtidal processes are likely to be similar to those in inundated intertidal sediments. Autotrophs would be affected by reduced light conditions but played a relatively minor role in processing. This was the first study to investigate the transformation and ultimate fate of carbon associated with secondary-treated PPM effluent. Stable isotope labeling is a valuable tool for exploring and comparing the processing of anthropogenic inputs to aquatic environments and, in this study, revealed fundamental differences in the processing and fate of two relatively labile forms of OC.



anonymous reviewers for feedback that improved this manuscript. Des Richardson supplied activated sludge and background pulp mill information. This work was supported by grants to D.J.R. and B.D.E. (ARC Linkage LP0770222), B.D.E. and J.M.O. (ARC Discovery DP0878568), B.D.E. and J.M.O. (ARC LIEF grants LE0989952, LE0668495), and J.M.O. (ARC DECRA DE120101290). Norske-Skog Boyer and the Derwent Estuary Program provided financial and in-kind assistance.



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ASSOCIATED CONTENT

S Supporting Information *

Details of experimental methods; tesults for DO, alkalinity, CO2(g), DIC, and DOC fluxes; discussion of differences in 13Clabeled DIC and CO2 fluxes; table of characteristics for PHY and ASB; figures showing similar dark and light fluxes of DO, DIC, alkalinity, DOC and CO2(g) in control and PC plots; figure comparing dark and light fluxes of DO, DIC, alkalinity, DOC, and CO2(g) from control, PHY, and ASB plots. This material is available free of charge via the Internet at http:// pubs.acs.org.



REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Telephone: +61 2 6620 3092. Fax: +61 (2 6621 2669. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank John Keane, Iain Alexander, Nicholas Ceely, and Ryuji Sakabe for laboratory and field work assistance, Matheus Carvalho and Melissa Bautista for isotope analysis, and three 13264

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(19) Blair, N. E.; Levin, L. A.; DeMaster, D. J.; Plaia, G. The shortterm fate of fresh algal carbon in continental slope sediments. Limnol. Oceanogr. 1996, 41, 1208−1219. (20) Witte, U.; Aberle, N.; Sand, M.; Wenzhofer, F. Rapid response of a deep-sea benthic community to POM enrichment: an in situ experimental study. Mar. Ecol.: Prog. Ser. 2003, 251, 27−36. (21) Levin, L. A.; Blair, N. E.; DeMaster, D. J.; Plaia, G.; Fornes, W.; Martin, C.; Thomas, C. Rapid subduction of organic matter by maldanid polychaetes on the North Carolina Slope. J. Mar. Res. 1997, 55, 595−611. (22) Penhale, P. A.; Thayer, G. W. Uptake and transfer of carbon and phosphorus by eelgrass (Zostera marina L.) and its epiphytes. J. Exp. Mar. Biol. Ecol. 1980, 42, 113−123. (23) Van Engeland, T.; Bouma, T. J.; Morris, E. P.; Brun, F. G.; Peralta, G.; Lara, M.; Hendriks, I. E.; Soetaert, K.; Middelburg, J. J. Potential uptake of dissolved organic matter by seagrasses and macroalgae. Mar. Ecol.: Prog. Ser. 2011, 427, 71−81. (24) Collier, C. J.; Prado, P.; Lavery, P. S. Carbon and nitrogen translocation in response to shading of the seagrass Posidonia sinuosa. Aquat. Bot. 2010, 93, 47−54. (25) Kaldy, J. E.; Eldridge, P. M.; Cifuentes, L. A.; Jones, W. B. Utilization of DOC from seagrass rhizomes by sediment bacteria: 13Ctracer experiments and modelling. Mar. Ecol.: Prog. Ser. 2006, 317, 41− 55. (26) Gribsholt, B.; Kristensen, E. Benthic metabolism and sulfur cycling along an inundation gradient in a tidal Spartina anglica salt marsh. Limnol. Oceanogr. 2003, 48, 2151−2162. (27) Cook, P. L. M.; Butler, E. C. V.; Eyre, B. D. Carbon and nitrogen cycling on intertidal mudflats of a temperate Australian estuary. I. Benthic metabolism. Mar. Ecol.: Prog. Ser. 2004, 280, 25−38. (28) Oakes, J. M.; Eyre, B. D.; Middelburg, J. J.; Boschker, H. T. S. Composition, production, and loss of carbohydrates in subtropical shallow subtidal sandy sediments: Rapid processing and long-term retention revealed by 13C-labeling. Limnol. Oceanogr. 2010, 78, 685− 693.

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