Evaluation of phosphorous and nitrogen uptake by Phalaris arundinacea plants in a wastewater treatment wetland, Cooperstown, NY

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1 Evaluation of phosphorous and nitrogen uptake by Phalaris arundinacea plants in a wastewater treatment wetland, Cooperstown, NY S. Bouillon 1 BACKGROUND Two constructed wetlands were monitored during this study, one of which was a constructed wastewater treatment wetland. The purpose for monitoring both wetlands was to evaluate the differences in nutrient uptake by Phalaris arundinacea (reed canarygrass) plants between a wetland receiving treated wastewater, and one that does not. Treated wastewater from the Village of Cooperstown was the primary inflow of the constructed treatment wetland system. The other constructed wetland served as a control for this study. The control wetland lies adjacent to an active cow pasture. Cow manure can pollute surface water with phosphorous and nitrogen, indicating that the control wetland may be impacted by nutrient loading. Most likely, the control wetland does not receive nutrient loads to the degree of the treatment wetland, which is why it served as a control for this study. Both the treatment wetland and control wetland were created in The former began actively receiving treated wastewater in Since 2010, the Village of Cooperstown has been an active cosignatory of the SPDES/Chesapeake Bay Nutrient Reduction Strategy (Albright and Waterfield 2011). From 2014 on, the maximum amount of phosphorus released by the treatment plant has been set at 984 kg/year (or approximately 2 mg/l, given typical annual discharge volumes; Albright 2015). From the time when the treatment wetland began receiving effluent, retention rates for both total nitrogen and total phosphorus have consistently been near 30% (Albright 2015). INTRODUCTION Phosphorous and nitrogen are known to contribute to freshwater eutrophication (the overgrowth of cyanobacteria and algae that causes oxygen levels in the water to decline, ultimately changing freshwater ecosystems for the worse; EPA 2004). Since 1990, treatment wetlands have been established more frequently as the final filtering process for phosphorous and nitrogen in treated waste water (Albright 2014). According to the Environmental Protection Agency, constructed treatment wetlands are a sometimes cheaper option for wastewater nutrient removal processes, and they act like natural wetlands with their soil and vegetation characteristics (2004). The Cooperstown, NY, wastewater treatment wetland was intended to reduce nutrient loading to the Susquehanna River and, ultimately, to the Chesapeake Bay via plant uptake and soil absorption (Albright and Waterfield 2011). This study seeks to monitor and compare the 1 Biological Filed Station Intern, summer Funding provided by the Village of Cooperstown.

2 removal of nutrients via plant uptake in P. arundinacea plants between both wetlands. This was done by utilizing sample site locations and strategies from Gazzetti s (2012) study of phosphorous uptake in P. arundinacea plants, and comparing them to the phosphorous concentration data we collected. P. arundinacea plant leaves were evaluated because this common species is known to be abundant in wetlands locally, and it demonstrates the ability to effectively remove nutrients, like phosphorous and nitrogen, from water (Cronk and Fennessey 2001, Gazzetti 2010). Both the treatment wetland and nearby control wetland were constructed in 2003 by the Army Corps of Engineers and Duck s Unlimited. The design of the treatment wetland was atypical for treatment wetlands, in that the depths throughout most of the system exceeded those needed for the establishment of emergent plants (Robb 2012). However, the treatment wetland system has effectively removed approximately 30% of both the phosphorus and nitrogen loads discharged into it (Albright 2015). Concurrent with the collection of plant leaf material, water samples were collected in each sampling area so that comparisons between nutrient content in leaf material and the adjacent water samples could be evaluated. Nutrient content in leaf material was compared both between the wetlands and between plants immediately proximal to the surface water compared to plants growing further upland from the water s edge. Higher nutrient content from plants adjacent to nutrient rich waters (as in the treatment wetland) would imply that P. arundinacea plants can assimilate more nutrients than are physiologically necessary for growth (i.e., it demonstrates luxuriant uptake). This work follows that of Olsen (2010) and Gazetti (2012) who demonstrated conflicting evidence regarding excessive phosphorus uptake by P. arundincea plants growing in the phosphorus-rich treatment wetland. In addition, this study will compare P. arundinacea phosphorous concentrations to those found in Gazzettis study of phosphorous uptake levels in emergent wetland plants (2012). Water sampling METHODS At each primary plant sampling site located near water, a single water sample was collected in a 125 ml bottle to test for calcium content using the EDTA titrimetric method (EPA 1983). There were a total of 6 water samples collected at each wetland. On 20 August 2015, an additional five water samples were taken within the treatment wetland. Weather conditions made it difficult to collect water samples from each primary sampling site in the control wetland on 20 August. The control wetland is an ephemeral wetland, meaning that the water levels change based on current weather conditions (EPA 2014). In the treatment wetland, water samples were collected at the inflow and outflow (see Figure 1), and at sites 2,3,4, and 6.

3 Calcium analysis of water samples While Gazetti (2012) reported that there was no statistical significance between phosphorous uptake levels by Typha spp. and those of Phalaris arundinacea plants between the wetlands, he did report that the ash content of these plants was higher in the treatment wetland than in the control. Since this study was modeled after his study, it was necessary to analyze areas of potential differences regarding the use of the dry-ashing methods outlined by Bickelhaupt and White (1982). It is apparent that there may be various inorganic components within the dry-ashed plant material contributing to the overall dry weight of the ash used to calculate how much phosphorous was within the plant leaves collected. A suspected source of this difference was calcium. Given local geology, concentrations of this cation were expected to be higher in the treatment wetland than in the control (Albright 2015). Analyzing water samples for calcium in both wetlands, using the EDTA titration method (EPA 1983), was conducted to attempt to verify this relationship. Nutrient concentration analysis of water samples All water samples were analyzed for total phosphorous (persulfate digestion followed by single reagent ascorbic acid method; Liao and Marten 2001) and total nitrogen content (cadmium reduction following peroxodisulfate digestion; Pritzlaff 2003, Ebina et al. 1983) using a Lachet autoanalyzer. Plant Sampling All plant and water samples were collected on 21 July Primary sampling sites (1-6) were established for both near and far locations within each wetland (Figures 1 and 2). These primary sampling sites were divided into three sub-sampling sites (A-C) located closely around each primary sampling site. Primary sampling sites were established at locations near and far from the water to identify differences in nutrients in P. arundinacea plant tissue growing in soils having high ( near, treatment wetland) vs lower nutrient content concentrations ( far, treatment wetland and near and far, control wetland). All near sampling sites were taken from Gazetti s (2012) comparative study on the variation of nutrient concentrations between Typha spp. and P. arundinacea plants between both wetlands. This was done in order to create a comparison of nutrient concentration data in P. arundinacea plant tissue between his 2011 data and the current 2015 data. At each near sub-sampling site, 20 P. arundinacea leaves were collected by cutting them from the base of their ligules. Far sites were established at varying distances from the water s edge where distinct vegetation changes towards upland habitat preferences were visible. At each sub-sampling site located far from the water s edge in each wetland, 20 Phalaris arundinacea leaves were collected as well. Plant tissue samples were processed as described below. The rationale for creating near and far sites was to compare and analyze the movement of nutrients throughout the wetlands by analyzing the movement of

4 nutrients within P. arundinacea plants. To compare the differences between near and far site nutrient level concentrations, t-tests were applied (two sample, assuming equal variances). This was also used to evaluate the differences in mean nutrient concentrations between near and far sites within each wetland. Figure 1. Aerial photograph of the control wetland sampled in The sites denoted as CW indicate control wetland sites, whereas the sites prefixed by Far indicate sites adjacent to, but upland from, the CW sites. Note that the Google Earth image was taken in 2011, and these ephemeral wetlands have since experienced changes in overall hydrology.

5 Figure 2. Aerial photograph of the treatment wetland sampled in The sites denoted as TW indicate treatment wetland sites, whereas the sites prefixed by Far indicate sites adjacent to, but upland from, the TW sites. Note that the Google Earth image was taken in 2011, and these ephemeral wetlands have since experienced changes in overall hydrology.

6 Plant sample processing Upon returning to the Biological Field Station after field sampling was complete, plants were washed immediately with deionized water, patted dry, put into paper bags, and placed into an oven where they were dried at o C for about 24 hours. Next, the dried plant samples were ground with a Krup s coffee bean grinder. Samples were heated again to 65 o C in 50 ml beakers for about 1 hour to evaporate moisture before taking dry weights for nitrogen and phosphorous analyses (Bickelhaupt and White 1982, Gazzetti 2012). All water samples were placed into a refrigerated storage unit for subsequent nutrient concentration sampling. Phosphorous analysis of plant material Dry-ashing, acid extraction, and phosphorous concentration determination methods were from Bickelhaupt and White (1982), and applied to all sub-samples within each primary sampling site for both wetlands. To prepare plant samples for dry-ashing, about 0.5 g of oven dried, ground plant sample were weighed out and placed into crucibles that had been acid washed, dried, and weighed. All plant samples were dry-ashed within these crucibles for about 4 hours at 475 o C in a muffle furnace. For the acid extraction process, 10mL 5N HN03 and 5mL distilled water were slowly added to the crucibles, which were gently boiled until dry on a hotplate. Next, the crucibles were placed into the muffle furnace and heated at 475 o C for an additional hour. After being heated in the oven, 5 ml of distilled water and 10mL of 6N HCL were added slowly to each crucible, which were boiled gently until dry again on a hotplate. Crucibles were cooled and 5 ml of distilled water and 10 ml of 6N HCL were slowly added again. This time, polypropylene stirring rods were used to scrape all dry-ashed material from the sides and bottom of each crucible to ensure that all plant material was dissolved in the. This solution was passed through No. 42 Whatman filter paper folded into funnels that drained into a 100 ml volumetric flask. The crucibles were rinsed with 15 ml of deionized water and passed through the filters. Crucibles were rinsed with 15 ml of distilled water and filtered twice more. All solutions were brought up to a final volume of 100 ml with DI water within the volumetric flasks and stored in a plastic bottle for further determination. The vanadomolybdophosphoric acid colorimetric method was used to determine the phosphorous concentrations from each extraction (APHA 2012). To determine the absorbency, a Milton Roy Spectronic spectrophotometer 501 was used at 440 nm wavelength. The concentration of all acid extracted solutions was used to find percent phosphorous content for each sample (Gazzetti 2011). The equation outlined in Bickelhaupt and White (1982) used to determine the percentage of phosphorous from the dry-weight of each sample is displayed below. %P of sample = mg/l P in extract * vol. extract * Sample dry wt. (g)

7 Nitrogen analysis of plant material The peroxidisulfate total nitrogen determination method outlined by Ebina et al. (1983) was used to digest samples for total nitrogen analysis of the dried, ground plant material. About g of finely ground plant sample was added into test tubes with 5 ml distilled water. Five ml persulfate oxidizing agent was added to each test tube. The tubes were autoclaved and total nitrogen was determined using the cadmium reduction method (Pritzlaff 2003) using a Lachet autoanalyzer. Finally, the following formula was used to determine total nitrogen: %N of sample = mg/l N in extract * vol. extract * Sample dry wt. (g) This total nitrogen procedure is intended for the analysis of water rather than for plant extracts, but it was intended to serve as a means to compare nitrogen levels between both wetlands. So even if the actual concentrations reported are not precise, relative differences between samples should reflect real differences. This should help us gain an understanding of how these wetlands vary, if at all, in nutrient uptake. RESULTS AND DISCUSSION The overall total phosphorous concentrations (see Figure 3) in P. arundinacea tissue between the treatment wetland and control wetland show that the treatment wetland (mean=0.373) had higher total phosphorous levels than the control wetland (mean=0.293). These results are statistically significant (p value= ), and they support this study s hypothesis that nutrient values will be higher at near sites within the treatment wetland because of treated effluent inflows. While comparing the total phosphorous values between the treatment wetlands near (mean=0.369) and far (mean=0.323) sites, the near sites had higher total phosphorous concentrations (p value=0.014). As seen in Figure 4, the total nitrogen values of the near sites (mean=0.7578) were higher than those at the wetlands far sites (mean=0.587), and were statistically significant from the treatment wetland with a p value of This supports this study s hypothesis, which expected the treatment wetland to have higher total nitrogen values near the input sources of treated wastewater. For the comparison of total nitrogen between the control wetlands near (mean=0.293) and far sites (mean=0.333), there was a slight statistical significance (p value=0.0488). For the comparison of the total nitrogen values between the control wetlands near (mean=0.666) and far sites (mean=0.708), there was no statistically significant difference between the values (pvalue=0.156). In Figure 4, it is evident that far sites in both wetlands differed significantly (p value= ). The control wetland (mean=0.708) had higher total nitrogen values at its far sites than the treatment wetlands far sites (mean=0.589). Expecting the mean total nitrogen values to

8 be higher at the treatment wetland points out potential for error in the sample processing and testing phases (see conclusion). Errors may have been made during the sample processing phase of leaf material. The total nitrogen values for all near sites in the treatment wetland were nearly half the amount of all other total nitrogen values for sampling sites between both wetlands. For total nitrogen analyses, plant material dried in the convection oven (heated at 65 degrees Celsius) before dry weighting may not have been ground evenly enough, or possibly over dried/burnt. Figure 5 addresses the differences in total phosphorous content of P. arundinacea between both wetlands within my study and Gazzetti s (2012) work. Overall, this study s total phosphorous concentrations in plant tissue were higher and statistically significant for the treatment wetland (mean= 0.367, p value= ) and control wetland (mean= , p value= 0.003) Mean % phosphorus near far 0.00 control treatment Figure 3. The average phosphorous concentrations of P. arundinacea tissue at near and far sampling sites between both wetlands. Error bars represent standard error. See Figure 1 and 2 for site locations.

9 Mean % nitrogen near far control treatment Figure 4. The average total nitrogen concentration of P. arundinacea tissue in near and far sampling sites between both wetlands. Error bars represent standard error. See Figure 1 and 2 for site locations Mean % phosphorous control treatment 0 Bouillon Gazzetti Figure 5. The comparison of average phosphorous concentrations (as percentage) of P. arundinacea in the treatment and control wetland, between my study and Gazzetti s (2012) study.

10 Table 1 shows the total phosphorus and total nitrogen content in P. arundinacea leaves in the control and treatment wetlands. Table 2 provides the concentrations of nitrite+nitrate, total nitrogen and total phosphorus of the water proximal to the sampling sites (see Figures 1 and 2 for site locations). Some sites in the control wetland could not be sampled due to low water levels. Table 1. The concentrations of total phosphorus and total nitrogen in the far and near sites of the control and the treatment wetlands, p Control Wetland Treatment Wetland Site Near Far Near Far TP(%) TN (% ) TP(%) TN (% ) TP(%) TN (% ) TP(%) TN (% ) 1A B C A B C A * B C A B C A B C A * B C * * 0.27 * sample lost Table 2. Nitrite+nitrate, total nitrogen and total phosphorus concentrations of water near sampling sites. Date Sampling Site Nitrate+Nitrite (mg/l) Total Nitrogen (mg/l) Total Phosphorus (ug/l) 8/17/2015 T /17/2015 T /17/2015 T /17/2015 T /17/2015 T /17/2015 T /17/2015 C /17/2015 C inflow, 2 outlow

11 Table 3 highlights the percent phosphorous found in dry-ashed material. I compared these total phosphorous values to the amount of calcium found in water samples. I found that the treatment wetland had higher amounts of calcium in water samples (average calcium= 52.4 mg/l +/- 4.1) than the control wetland (average calcium= 35.7 mg/l =/- 11.6). This likely is responsible for the higher ash content (or, lower percent C lost on ignition) measured in the control wetland as compared to the treatment wetland (see Table 3). Lastly, Table 4 compares total phosphorus content in P. arundinacea leaves in the near wetland sites of both wetlands between Gazzetti s (2012) study and this current work. Concentrations at both wetlands were somewhat higher in 2015 than in 2011 (Gazzetti 2012), and in both wetlands the concentrations were somewhat higher in the treatment wetland than the control wetland. Table 3. Total phosphorus content of P. arundinacea leaves at each subsample site in the treatment and control wetlands and the percent carbon lost on ignition. Near Far Treatment Wetland Control Wetland Treatment Wetland Control Wetland Site % P of Dry Weight % C Loss on Ignition % P of Dry Weight % C Loss on Ignition % P of Dry Weight % C Loss on Ignition % P of Dry Weight % C Loss on Ignition 1a b c a b c a * b c a b c a b c a * * 6b c * * * sample lost

12 Table 4. Comparison of total phosphorus content in P. arundinacea leaves between this study and that conducted in 2011 (Gazzetti 2011)) for the control and treatment wetlands. y y Control Treatment % P of Dry weight % P of Dry weight Location Site Bouillon(2016) Gazzetti (2011) Bouillon(2016) Gazzetti (2011) Near 1a Near 1b Near 1c Near 2a Near 2b Near 2c Near 3a * Near 3b Near 3c Near 4a Near 4b Near 4c Near 5a Near 5b Near 5c Near 6a Near 6b Near 6c Mean Std error * sample lost In conclusion, this study found that total phosphorous levels were higher within the treatment wetland overall, even when compared to Gazzetti s study (2012), which conforms to our hypothesis. However, the total nitrogen values were higher within the treatment wetland near sites, which did not support our hypothesis. However, this study did find statistically significant differences between near and far site nutrient concentrations within both wetlands, which supported our hypothesis as well. All in all, the treatment wetland seems to be taking up nutrients from the treated wastewater and soil, making it a successful treatment for nutrients in wastewater before it is discharged into the Susquehanna River.

13 REFERENCES Albright, M.F Personal communication. SUNY Oneonta Bio. Fld. Sta. Albright, M.F Monitoring the effectives of the Cooperstown wastewater treatment wetland, In 47 th Ann. Rept. (2014). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta.. Albright, M.F Monitoring the effectiveness of the Cooperstown wastewater treatment wetland. In 45 th Ann. Rept. (2012). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta. Albright, M.F., and H.A.Waterfield Monitoring the effectiveness of the Cooperstown wastewater treatment wetland. In 44 th Ann. Rept. (2011). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta. Bickelhaupt, D.H. and E.H. White Laboratory manual for soil and plant tissue analysis. State University of New York, Environmental Science and Forestry. Chesapeake Bay Program. The Bay Ecosystem. Web. 28 August < Cronk, J.K. and M.S. Fennessey Wetland Plants, Biology and Ecology. Lewis Publishers. Boca Raton, London, New York, Washington, D.C. Eastwood, G.W. Calcium s role in plant nutrition. Web. 3 February < Ebina, J., T. Tsutsi, and T. Shirai Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Water Res.7(12): EPA Vernal Pools. Web. 16 August < EPA Methods for the analysis of water and wastes. Environmental Monitoring and Support Lab. Office of Research and Development, Cincinnati, OH. Gazzetti, E Efficacy of emergent plants as a means of phosphorus removal in a treatment wetland, Cooperstown, New York. In 45 th Ann. Rept. (2011). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta. USDA. National Resource Defense Council. New York and Chesapeake Bay Watershed. Web. 13 August < Olsen, B Phosphorus content in reed canary grass (Phalaris arundinacea) in a treatment wetland, Cooperstown, NY. In 43rd Ann. Rept. (2010). SUNY Oneonta Biol. Fld. Sta., SUNY Oneonta.

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