Pharmaceuticals from Wastewater in Streams Draining a Forested Watershed on the Cumberland Plateau

Erica Teasley1, Julie Bennett1, Callie Crider2, Deborah McGrath1 and Ken Smith3

Departments of Biology1, Chemistry2, Forestry and Geology3

IntroductionOver the past several decades, research on water pollution has focused on traditional organics such as pesticides and household chemicals. As these pollutants have decreased in developed countries through new water treatment methods, research has increasingly focused on the excretion of pharmaceuticals and their metabolites into the environment through wastewater effluent. These compounds are present in such small quantities that only techniques developed recently have allowed scientists to detect them. compound the problem, there is no legally-mandated screening of pharmaceuticals during the wastewater treatment process.

Pharmaceuticals have significant potential to cause human health problems because they enter streams and underground water reserves used as municipal water supplies. While these pharmaceuticals may be present in only small concentrations, continued consumption through drinking tap water can cause enough bioaccumulation to alter the physiology and biochemistry of animals and humans. Important considerations for monitoring their impact on environmental health include the amount of pharmaceutical that is prescribed each year, the concentrations required to create problems, long substance half-live, and the pharmacokinetic behavior of the chemical and its metabolites (Ternes 2001).

There are two primary pathways through which pharmaceuticals and their metabolites enter the environment, particularly aquatic ecosystems: (i) excretion through urine and feces and (ii) disposal of unused pharmaceuticals at residential areas, hospitals, and veterinary clinics. With the increasing use of pharmaceuticals, more metabolites are finding their way into aquatic ecosystems and possibly human drinking water supply.

Literature Cited Kidd, K.A., et al. 2007. Collapse of a fish population after exposure to a synthetic estrogen. PNSA. 104(21):8897-8901.Newbold, R.R., E, Padilla-Banks, W.N. Jefferson, and J.J Heindel. 2007. Effects of endocrine disruptors on obesity. Intern. J. Androl. 31:201-208.Ternes, T.A. 2001. Analytical methods for the determination of pharmaceuticals in aqueous environmental samples. Trends Anal. Chem. 8:419-434.

MethodsStudy SiteWaste water comes to the Sewanee Utility District (SUD) treatment plant from Bob Stewman Road and enters a concrete splitter box, which sends the water to three lagoons for approximately 40 days. In the lagoons, the sewage is treated by aerobic and anaerobic bacteria. Aerobic bacteria consume oxygen during respiration to convert organic nitrogen to inorganic nitrogen. Algae consume the inorganic nitrogen and when they die they sink to the bottom of the lagoons. Anaerobic bacteria on the bottom of the lagoons decompose organics and convert inorganic nitrogen to dinitrogen (N2). All three lagoons are clay-lined and cover approximately 4.5 acres each with a depth of 5 feet. The water is then treated with 8-20 ppm of chlorine after it exits the lagoons to kill any harmful bacteria. This water fulfills EPA and state regulations and is comparable to the water that other waste water treatment centers discharge straight into streams. After chlorination, the water is sprayed onto 60 acres divided into 19 spray fields. As water percolates through the soil, bacteria covert nitrogen so that it can be used by plants and solid particles, bacteria, and viruses are removed from the water through natural chemical and physical properties. Spraying this water (100 inches total precipitation per year including natural precipitation) on upland hardwood forests has changed the species composition from drought resistant species (oak and hickory) to more drought intolerant species (yellow poplar and maple).

DiscussionPharmaceutical uses has let to the presence of un-metabolized portions of the compound to find ways into water sources. This could alter aquatic ecosystems and potentially cause human health effects if these substances are present in drinking water. Kidd (2007) found that continuous exposure to natural and synthetic estrogens decreased overall reproductive fitness of fish. This could detrimentally affect the fisheries industry. Additionally, endocrine-disrupting compounds (EDCs) have been linked to the increase in human obesity (Newbold et al. 2007).

We determined that some pharmacological metabolites were present. However, the limitation to this methodology is that each compound is specifically looked for; this method does not screen for all types of pharmaceuticals that could potentially be present. There was no background levels of compounds in Lakes Jackson and O’Donnell, which was expected as these lakes do not have development around them and are rain-fed at the top of the watershed. We are unable to draw any conclusions about the ability of the current wastewater treatment process to remove any pharmaceuticals present due to the drying of the streams in the Fall 2010 samples and the need for further sampling. We are currently still comparing the samples between summer and semester months. This comparison is necessary because during the semester, approximately half of all wastewater treated by SUD is from college students, who most likely would be using different pharmaceuticals (i.e. estrogens from birth control) than the general population.

Future Work We are currently working to analyze estrogens at each site. Samples from Spring 2011 are in the process of being analyzed. This summer, a greenhouse experiment will be conducted to determine how well soil and vegetation remove pharmaceuticals from wastewater effluent. Future analyses will attempt to quantify the amount of pharmaceuticals present in each location.

AcknowledgementsTammy Boles, Jeff Boles, Vanaysa , Dennis George, Gene Mullins, and Martha Wells at TN Tech Cookeville, TN for analyses.

ObjectivesThe objectives of this study were to:1. Determine what pharmacological metabolites are found in municipal wastewater2. Determine how well the current system removes the chemicals from the water3. Determine if there are background levels of these compounds in Lakes Jackson and O’Donnell, which serve as the

municipal water supply for Sewanee and surrounding areas.4. Compare samples between summer and semester5. Provide preliminary data for future study

ResultsSummer 2010Caffeine, ephedrine, PMMA, and NFA were detected in the raw sewage. NFA was the only compound detected in the chlorination chamber. Methamphetamine, NFA, and ephedrine were present in Stream 3. No compounds were detected in Stream 1, Stream 2, Lake Jackson, or Lake O’Donnell.

Fall 2010Caffeine, Ephedrine, Methamphetamine, Amphetamine, Phentermine, Cocaine, Methylphenidate, and PMMA were detected in the raw sewage.

Figure 1: Aerial view of Sewanee Utility District Wastewater Treatment Facility, Sewanee, TN.

Figure 3: Erica (left) and Julie (right) installing POCIS in a stream weir, SUD, June 2010.

Figure 2: POCIS disk Figure 4: POCIS disk inside vegetable steamer from Lake before cleaning and extraction, July 2010.

Figure 5: Extraction of POCIS disk with methanol, TN Tech, July 2010.

MethodsPharmaceutical screening using passive samplersTo qualitatively determine the presence of pharmaceuticals for more intensive analysis, POCIS passive samplers (Figure 2) from the supply company ETS were installed in seven (7) locations (Figure 3): untreated sewage, treated affluent before it is sprayed on the spray fields, three streams draining the spray fields, Lake Jackson, and Lake O’Donnell. Three (3) POCIS were placed at each location for three (3) weeks; the three POCIS at a single location were combined to a single sample for analysis. The membrane collects any pharmaceuticals present as water flows through the membrane.

Sampler extraction, filtration, and analysis Upon removal from each site the POCIS disks were wrapped in aluminum foil. They were stored on ice for transport and then stored in the refrigerator until analysis. For extraction, each disk was cleaned with pure water (Figure 4). The sorbent was extracted from the POCIS with methanol into a 5 mL syringe packed with glass wool and capped with a stopcock (Figure 5). After all the sorbent was removed from the POCIS, 50 mL of methanol was added. The resulting solution was filtered through 1 mL Pasteur pipettes containing 0.25 in filter paper (Figure 6). The filtered solution was evaporated with nitrogen down to approximately 2 mL and was centrifuged. The samples were then analyzed using a HPLC coupled with a triple quadrupole MS/MS (Figure 7). Two methods were used, depending on the compound that was being examined: 16 or 18 minute gradient elution (Tables 1 and 2). Sample chromatograms are provided for each method (Figures 8 and 9). Eighteen compounds were examined (Table 3).

Figure 6: Filtration of extracted sample, TN Tech, July 2010.

Figure 7: LC/MS/MS used for sample analysis, TN Tech, July 2010.

Figure 10: Stacked individual ion chromatograms for amphetamine, methamphetamine, phentermine, ephedrine/pseudoephedrine, caffeine, cocaine and TIC of sewer sample fall 2010; along with their retention times and signal-to-noise (S/N) ratios. Method used: 18 minute gradient elution.

Figure 9: Stacked individual ion chromatograms for ephedrine/pseudoephedrine in sewer summer 2010 and fall 2010. Method used: 16 minute gradient elution.

Run Time

% A

% B

Flow Rate (mL/min)

0:00 95 5 0.2

8:00 65 35 0.2

10:00 10 90 0.2

12:00 10 90 0.2

14:00 95 5 0.2

16:00 95 5 0.2

Table 1: Gradient elution for 14 minutes with 2 min of equilibration time. Aqueous Phase (A) - 10mM Ammonium Formate + 0.2% Formic acid in LC-MS Grade Water (pH-3.0); Organic Phase (B) – Acetonitrile:Methanol=1:1

Run Time

% A

% B

Flow Rate (mL/min)

0:00 95 5 0.2

12:00 65 35 0.2

14:00 10 90 0.2

15:00 10 90 0.2

17:00 95 5 0.2

18:00 95 5 0.2

Table 2: Gradient elution for 17 minutes with 1 min of equilibration time. Aqueous Phase (A) - 10mM Ammonium Formate + 0.2% Formic acid in LC-MS Grade Water (pH-3.0) Organic Phase (B) –Methanol

Table 4: Compounds detected at each of 7 locations throughout the wastewater treatment process.

Cleaning the POCIS Disk Extraction Filtration Nitrogen

Evaporation Centrifugation

Analysis by HPLC coupled

with triple quadrupole

MS/MS

(±)-Methamphetamine MDBD [(±)-Methylbenzodioxolylbutanamine] NFM [N-formyl methamphetamine]

(±)-Amphetamine PMMA [p-Methoxymethamphetamine] NDMS [N-desmethylseligiline]

Ephedrine/Pseudoephedrine Phentermine D5-Methamphetamine (Internal Standard)

Ecstasy (MDMA) [(±)-3,4-Methylenedioxymethamphetamine]

Caffeine D3-Ephedrine (Internal Standard)

MDA [(±)-3,4-Methylenedioxyamphetamine] Methyphenidate Cocaine

MDEA [(±)-3,4-Methylenedioxyethylamphetamine] NFA [N-formyl amphetamine] Heroine

Table 3: Compounds

Site Summer 2010 Fall 2010

Raw Sewage Caffeine, Ephedrine, PMMA, NFA Caffeine, Ephedrine, Methamphetamine, Amphetamine, Phentermine, Cocaine, Methylphenidate, PMMA

Chlorination Chamber NFA Compounds not found

Stream 1 Compounds not found --------------Dry-----------------

Stream 2 Compounds not found --------------Dry-----------------

Stream 3 Meth, NFA, Ephedrine --------------Dry-----------------

Lakes O’Donnell and Jackson Compounds not found ----------Not sampled---------

AbstractResearch has increasingly focused on the excretion of pharmaceuticals into the environment through wastewater effluent. Wastewater treatment at the Sewanee Utility District involves biodegradation of sewage passing through open lagoons. In lieu of discharging the treated wastewater directly into streams, the effluent is discharged onto 65 acres of hardwood forest. Using Polar Organic Chemical Integrative Samplers, we sampled water from rain-fed lakes, raw sewage, treated effluent and streams draining the watershed to determine if the treatment process removes pharmaceuticals. The samplers were analyzed using HPLC triple quadrapole MS/MS. Twice as many pharmaceuticals were found in the raw sewage from Fall 2010 compared to Summer 2010. No pharmaceuticals were present the drinking water lakes nor in two of the streams draining the watershed. Methamphetamine and ephedrine were present in one stream. Further study is necessary to determine whether tertiary treatment through forest application reduces the presence of pharmaceuticals in stream water.

Figure 8: Method for analyzing POCIS disks