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A total maximum daily load (TMDL), which is the calculated total amount of pollutant that a waterbody can receive from point and non-point sources, is established for streams that do not meet their designated use criteria. Physical, chemical, and biological water quality parameters are used to attempt to identify pollution sources. Microbial enzyme activity (mg/mL) is used to monitor the changes in the microbial community by identifying changes in their metabolism. The health of a stream can be monitored by the presence and absence of microorganisms due to the response of the microbial community to prolonged pollution exposure. To fully understand the metabolic activity, MEA data are compared to other factors including biochemical oxygen demand (BOD), nitrogen, phosphate, total coliform and Escherichia coli concentrations. Dissolved oxygen refers to the amount of oxygen used by microorganisms to degrade organic carbon, which reduces the survivability of aerobic organisms. Nitrogen and phosphate compounds from anthropogenic sources are readily dissolved into water and are limiting nutrients for microorganisms. Total coliforms and E. coli determine the fecal contamination in the waterbody. All of these factors are used to determine point and nonpoint pollution sources. From February 2014 to January 2016, water and sediment samples were collected monthly from 16 sites along Sinking Creek in Northeast Tennessee. During the two-year study, physical and field parameters were measured. Water samples were analyzed for chemical parameters including alkalinity, hardness, and BOD. Using an ion chromatograph, the concentration of phosphates and nitrates in the samples was measured. For biological parameters, the water sample was used to obtain total coliform and E. coli data using the Colilert enzyme substrate test. MEA data were collected from triplicate sediment samples collected at each site. These sediment samples were tested for acid and alkaline phosphatase, glucosidase, galactosidase, and dehydrogenase enzymes. The substrate for these enzymes was added to the respective sample, incubated for approximately 24 hours, and analyzed by colorimetric spectrophotometry to obtain absorbance. The enzyme concentration was calculated by comparing the absorbance to a generated standard curve. The results for the measured parameters were compared to identify correlation between the MEA concentrations and other biological and chemical parameters. The microbial activity should show a holistic view of changes in the waterbody, which should correlate with the other factors. A direct relationship between E. coli, total coliforms, nitrate, phosphate concentrations, and MEAs was expected. A correlation should be seen between MEAs and BOD data, due to an increase in oxygen demand during microbial metabolism. By examining multiple parameters together, the results would provide the necessary information to determine remediation efforts for the waterbody.

Microbial metabolism reacts quickly to environmental conditions. These reactions are dependent on the need for nutrients and respiration and can be measured using an assay of individual microbial enzyme activities (MEA’s). In this study, we measured MEA’s in the sediments of a stream in northeast Tennessee that had an approved fecal coliform Total Maximum Daily Load (TMDL). These values were compared to biochemical oxygen demand (BOD), phosphate concentration and nitrate concentration in the water column of this stream. Comparisons were grouped by season and land use. Stream sediments and water were collected monthly for one year and then quarterly for an additional two years at 14 sites located in agricultural, urban and forest regions. Dehydrogenase (DHA), a measure of microbial respiration, along with acid phosphatase (AcidPA), alkaline phosphatase (AlkPA), galactosidase (GalA) and glucosidase (GluA) activities were measured using colorimetric assays. BOD was determined using the standard 5-day BOD test (BOD5). Nitrate and phosphate concentrations were measured using colorimetric procedures. There were significant positive and negative correlations (p5, DHA vs. nitrate concentration, and DHA vs. phosphate concentration. Also in the fall months there were significant negative correlations between GalA and GluA vs. BOD5, and concentrations of nitrate and phosphate. There was also a negative correlation between AcidPA and BOD5. In the warmer months of spring and summer, there were positive correlations between AcidPA, AlkPA, GalA and GluA vs. the BOD5 ’s, and the concentrations of nitrate and phosphate. The only negative correlation in a warmer season was in the summer between AlkPA vs. BOD5 and phosphate concentration. No significant correlations were found by land use type. Results indicate that significant relationships may exist between MEA’s and other water quality measures (e.g. BOD5, nitrate concentration, and phosphate concentration) that could make it possible to use MEA’s as another tool for water quality assessment.

Four creeks within the Watauga River watershed in Northeast Tennessee are routinely monitored for water quality assessments. To identify sources and monitor remediation, Sinking Creek, Cash Hollow Creek, Buffalo Creek and Boones Creek are monitored for chemical and microbial parameters. These parameters include phosphates, nitrates, BOD and fecal coliforms. Sinking Creek is a tributary of the Watauga River with 10 miles of impaired water. Cash Hollow Creek enters the Watauga River at river mile 11.4 with 3.4 miles of impaired water. Boones Creek contains 18.6 impaired miles while the status of water quality in Buffalo Creek is not yet determined. Agricultural input is a major source of pollution for Sinking and Boones Creek. Cash Hollow Creek is impacted by a combination of sources of which urban runoff is the largest due to storm sewers and land development. Boones, Cash Hollow and Sinking Creeks are considered impaired and are on the state’s 303(d) list due to pathogen loading but only Sinking and Cash Hollow Creek have TMDLs. The seasonal and spatial patterns are more obvious for microbial than for chemical parameters. From 2002 - 2005, 14 stations on Sinking Creek were sampled quarterly. Fecal coliforms were elevated and always greater than 200 CFU/100ml for stations 1 – 5. Due to agricultural land use adjacent to stations 1 – 4, this would be expected. There was also a seasonal trend with higher concentrations found in the fall and spring. Cash Hollow Creek’s 9 stations were sampled monthly from 2002 - 2005. Although very high fecal coliforms concentrations were found, there were no obvious patterns. The 12 stations on Buffalo Creek were sampled quarterly from June 2004 to June 2005. Fecal coliform concentrations were high at station 8, which is adjacent to agricultural land. Boones Creek was sampled monthly from March 2005 to present and no obvious trends have been noted. The objective of this research is to compare patterns in these geographically similar creeks to identify any common patterns associated with various pollution sources. We will discuss the preliminary results and conclusions about the usefulness of these data to accomplish this objective.

Microbial enzyme activities (MEA’s) are measurements of microbial metabolism. These activities are dependent on the need for nutrients and respiration. This extended study evaluated four streams in the same watershed that had an approved fecal coliform Total Maximum Daily Load. Sediment and water samples were collected monthly for the first year of each specific stream study, and then quarterly to the end of 2006. Dehydrogenase, a measure of microbial respiration, along with acid phosphatase, alkaline phosphatase, galactosidase and glucosidase activities were measured using colorimetric assays. Biochemical oxygen demand (BOD) was determined using the standard 5-day test (BOD5). Nitrate and phosphate concentrations were measured using colorimetric procedures. Sediment MEA values were compared to the BOD, nitrate concentration and phosphate concentration in the overlying water. Seasonal means of each parameter were not significantly different (p5, nitrate concentration, and phosphate concentration). This suggests to us that MEA’s may be an alternative tool for water quality assessments.