Microsoft word - dioxin 07 holly m bl1.doc

PERSISTENT ORGANIC COMPOUNDS IN WASTEWATER: AZITHROMYCIN
AND UROBILIN CONCENTRATIONS IN WASTEWATER TREATMENT
PLANT SAMPLES FROM MURRAY, KENTUCKY, USA
Mowery HR, Loganathan BG Department of Chemistry and Center for Reservoir Research, Murray State University, Murray, KY 42071-3346, USA. Abstract
The presence of pharmaceutical and personal care products (PPCPs) in the environment and their possible
impact on wildlife and human health is an emerging area of research. In this study, we collected influent
and effluent samples during summer, fall and winter months from a small wastewater treatment plant in
Murray, Kentucky USA and analyzed for azithromycin and urobilin. Azithromycin concentrations ranged
from 4.4 ng/L to 52.6 ng/L. Urobilin concentrations ranged from below detection limit (<1 ng/l) to 39,573
ng/L The highest concentrations were found in influent samples collected during the month of February
2007. In general, influents contained higher concentrations of the analytes than the effluents indicating that
the compounds degraded during the wastewater treatment processes. To our knowledge this is the first
report on the occurrence of azithromycin and urobilin in wastewater treatment plant samples from Murray,
Kentucky, USA.
Introduction
Pharmaceutical and personal care products (PPCPs) residues have been detected in environmental samples
including groundwater, surface water, and municipal wastewater1-4. Pharmaceutical drugs given to people
as well as to domestic animals include antibiotics, hormones, pain relievers, tranquilizers, and
chemotherapy chemicals given to cancer patients. Many drugs are designed to be persistent and lipophilic,
so that they can retain their chemical structure long enough to do their therapeutic work. These drugs are
excreted and distributed into the environment by flushing toilets as well as by spreading manure and
sewage sludge onto soil2. These chemicals persist in the environment, enter the food chain, bioaccumulate,
biomagnify, and cause harmful effects in wildlife and humans. Because of aquatic contamination by these
chemicals, bacteria and other microbes in the aquatic environment can become more resistant to these
chemicals. This results in the development of more antibiotic resistant and virulent pathogens in the
environment. Therefore, the persistence of pharmaceutical chemicals in the environment has become a
global problem. Azithromycin, a commonly used antibiotic and urobilin, a breakdown product of bilirubin
in intestines were selected as target compounds for this study. Chemical structures of azithromycin and
urobilin are shown in Figure 1 and 2. Contamination levels of these compounds were measured in water
samples collected from a wastewater treatment plant located in Murray, Kentucky, USA. Murray is a small
university town with a population of approximately 25,000 including 10,000 students. Murray wastewater
treatment plant processes about 5.2 million gallons (19.8 million liters) wastewater per day. Source of
wastewater for the plant is predominantly domestic along with small fraction from commercial business
and industries. The objectives of the study was to (i) determine the levels of azithromycin and urobilin in
influent and effluent samples collected from Murray Wastewater Treatment Plant, (ii) assess seasonal
variation in concentrations of the analytes and (iii) elucidate removal and/or degradation of analytes during
the wastewater treatment processes. Understanding current levels of persistent pharmaceuticals and other
organic compounds is important in order to identify the source of the pollutants, ‘chemicals of concern’ in
the region, and to prevent further contamination in order to protect wildlife and human health.

Materials and Methods
Water samples were collected from the Murray Wastewater Treatment Plant (MWWTP) during the months
of August, September, December 2006, February, and March 2007. Samples were collected using high
density polyethylene (HDPE) bottles, stored in ice during sampling, and upon arrival at laboratory the
samples were stored at -20°C until analysis. The samples were filtered using glass fiber filter (10µ pore
size) and pH was lowered to 3 or less using 12 M HCl. SPE-HBL (Water Oasis SPE-HBL 6cc, 200 mg)
cartridges were used for sample extraction. The cartridges were prepared by running 5 mL of methanol
through each cartridge, followed by a further rinse with 10 mL of deionized water. Each sample filtrate,
100-200 mL, was passed through an SPE-HBL cartridge using vacuum suction at a constant flow of 3-4 mL/min. Polar compounds were eluted using 20-25 mL 1% acetic acid in methanol at a rate of approximately 3-4 mL/min. The eluate was then microconcentrated down to 1.0 mL or less using N2 gas (< 25 degrees C). The samples were further concentrated using Zymark TurboVac II (21°C Bath tub, 4-8 psi) and then volume was made up to 0.5 mL using methanol. LC-MS analysis was performed in the Environmental Protection Agency National Exposure Research Laboratory, Las Vegas, NE. Varian Prostar model 410 auto sampler was used for sample injection. Mobile phase consisted of 0.5 % formic acid in 82% methanol/18% acetonitrile, 0.5% formic acid in DI water. Formic acid was present for proton transfer and soft ionization. Solvent Pump model was Varian Prostar: Solvent Delivery Model with 2 pumps (A&B, psi: 730). Mass Spectometer model was Varian 500-M-S IT Mass Spectrometer. Precursor ions 749.5 (M+H)+ and 591 (M+H-HCl)+ were used for detection of azithromycin and urobilin respectively. Results and Discussion
Concentrations of azithromycin and urobilin in influent and effluent samples from Murray wastewater
treatment plant samples were shown in Table 1. Azithromycin was detected in all samples analyzed.
Highest concentration of azithromycin recorded was 52.6 ng/L in influent sample collected during February
2007. Effluent contained relatively less azithromycin than influent samples. Urobilin was detected in all
influent samples. Concentrations of urobilin were several orders of magnitude higher than azithromycin
residues (Table 1).

Table 1. Concentrations of azithromycin and urobilin (ng/L) in influent and effluent water samples
collected from Murray Wastewater Treatment Plant samples during different months.
A notable observation is that when azithromycin and urobilin concentrations were compared, azithromycin
concentrations did not change greatly with influent and effluent samples, whereas urobilin concentration in
effluents were several orders of magnitude lower than influent samples. This remarkable difference in
concentrations shows that urobilin is degraded or lost during wastewater treatment processes, while
azithromycin is relatively more persistent and wastewater treatment processes rarely affect the
concentrations of azithromycin. Therefore, azithromycin will be transported more to the receiving waters
from the wastewater treatment plants. Azithromycin is a macrolide, similar to erythromycin, as well as
clarithromycin and roxithromycin. In a similar study conducted in Switzerland, concentrations of 131 ng/L
for clarithromycin and 35 ng/L for roxithromycin were detected in effluent samples. The degraded form of
erythromycin, which no longer showed antibiotic function, was also found3. As an antibiotic, azithromycin
is useful for treating many infections of the respiratory system and skin. It is effective against pathogens
such as Haemophilus influenzae, Chlamydia pneumoniae, Streptococcus pyogenes, Mycoplasms
pneumoniae, and Legionella species. It is also useful against most Streptococcus pneumoniae and
Staphylococcus aureus that are resistant to erythromycin5. By remaining in the environment, azithromycin
resistant strains of pathogens are likely to occur through natural selection, causing azithromycin no longer
to be effective in fighting infections of microbes for which it had been in the past. The results indicate that
azithromycin can build up in the environment without degradation, causing need for further study and
development of new, less environmentally persistent drugs and tracking the fate of currently used
antibiotics.
Urobilin is formed from the break down of bilirubin in the intestines. A study conducted on human
urobilin excretion in Scotland found healthy males under the age of 36 to excrete 1.19 mmol/kg in feces
while women of the same age excreted 1.04 mmol/kg in feces4. Urobilin has been shown useful as a
marker of human waste in water systems because it is found in human feces and urine6 This study provides
evidence that detectable levels of azithromycin and urobilin are found in Murray wastewater treatment
plant samples. Variations in concentrations of the analytes during different seasons indicate variation in
input of these chemicals during different months. Azithromycin is more persistent and not affected greatly
by the wastewater treatment processes. Future monitoring studies with greater number of samples is
needed in order to describe the distribution, mass loading, behavior and fate of these compounds in the
environment.

Acknowledgements
This research was supported by Murray State University’s Committee on Institutional Studies and Research
(MSU-CISR). MSU’s Undergraduate Research and Scholarly Activity (URSA) Office provided Research
Scholar Fellowship to HM. Analytical help provided by Miss. Malia Phillips (MSU) and Mrs. Tammy
Jones-Lepp at the United States Environmental Protection Agency (USEPA), Las Vegas, NE is gratefully
acknowledged.

References
1. Kolpin D, Furlong E, Meyer M, Thurman E, Zaugg S, Barber L, Buxton H. Environmental Science &
2. Drewes J, Shore L. Pharmaceuticals and Personal Care Products in the Environment 2001; 206-228. 3. Drewes J, Shore L. Pharmaceuticals and Personal Care Products in the Environment 2001; 56-69. 4. Benno P, Alam M, Collinder E, Norin E, Midtvedt T. Microbial Ecology in Health and Disease; 2003 5. Langtry, H, Balfour J. Drugs 1998; 56: 273. 6. Jones-Lepp T. J. Environ. Monit 2006; 8:472.

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