Transcript TeasleyPharmaceuticalsSS11x

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
Abstract
Methods
Results
Research 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.
Pharmaceutical screening using passive samplers
Summer 2010
To 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.
Caffeine, 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 2010
Caffeine, Ephedrine, Methamphetamine,
PMMA were detected in the raw sewage.
Amphetamine, Phentermine, Cocaine, Methylphenidate, and
Table 4: Compounds detected at each of 7 locations throughout the wastewater treatment process.
Introduction
Over 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).
Figure 2: POCIS disk
Figure 3: Erica (left) and Julie (right)
installing POCIS in a stream weir, SUD, June
2010.
Figure 4: POCIS disk inside vegetable
steamer from Lake before cleaning and
extraction, July 2010.
Figure 6: Filtration of extracted sample, TN
Tech, July 2010.
Figure 7: LC/MS/MS used for sample
analysis, TN Tech, July 2010.
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---------
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.
Objectives
The objectives of this study were to:
1.Determine what pharmacological metabolites are found in municipal wastewater
2.Determine how well the current system removes the chemicals from the water
3.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 semester
5.Provide preliminary data for future study
Methods
Study Site
Waste 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).
Figure 5: Extraction of POCIS disk with
methanol, TN Tech, July 2010.
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 8: Method for analyzing POCIS disks
Cleaning the
POCIS Disk
Extraction
Filtration
Nitrogen
Evaporation
% %
A B
0:00
95
Centrifugation
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 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
Analysis by
HPLC coupled
with triple
quadrupole
MS/MS
Flow Rate
(mL/min)
Run
Time
% %
A B
Flow Rate
(mL/min)
5
0.2
0:00
95
5
0.2
8:00
65 35
0.2
12:00
65 35
0.2
10:00
10 90
0.2
14:00
10 90
0.2
12:00
10 90
0.2
15:00
10 90
0.2
14:00
95
5
0.2
17:00
95
5
0.2
16:00
95
5
0.2
18:00
95
5
0.2
Table 3: Compounds
Figure 1: Aerial view of Sewanee Utility District Wastewater Treatment Facility, Sewanee, TN.
Figure 9: Stacked individual ion chromatograms for
ephedrine/pseudoephedrine in sewer summer 2010 and fall 2010.
Method used: 16 minute gradient elution.
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.
Discussion
Pharmaceutical 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 rainfed 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.
Literature Cited
Kidd, K.A., et al. 2007. Collapse of a fish population after exposure to a synthetic estrogen. PNSA. 104(21):88978901.
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.
(±)-Methamphetamine
MDBD [(±)-Methylbenzodioxolylbutanamine]
NFM [N-formyl methamphetamine]
(±)-Amphetamine
PMMA [p-Methoxymethamphetamine]
NDMS [N-desmethylseligiline]
Ephedrine/Pseudoephedrine
Phentermine
D5-Methamphetamine (Internal Standard)
Acknowledgements
Ecstasy (MDMA) [(±)-3,4-Methylenedioxymethamphetamine] Caffeine
D3-Ephedrine (Internal Standard)
MDA [(±)-3,4-Methylenedioxyamphetamine]
Methyphenidate
Cocaine
Tammy Boles, Jeff Boles, Vanaysa , Dennis George, Gene Mullins, and Martha Wells at TN Tech Cookeville, TN for
analyses.
MDEA [(±)-3,4-Methylenedioxyethylamphetamine]
NFA [N-formyl amphetamine]
Heroine