Exhibit 017

Cooling Tower "Gray Water" Emissions as as Source of Health Concerns

September 16. 2002

STATE OF NEW YORK DEPTnrPto iC SERVICE

CAST-HO ,on-y-? '' KEVIN M. BERNSTEIN BOND, voice mall ext. 329 [email protected] SCHGENEClC: •• ^KlN^FLLC September 16,2002 Attorneys at Law/Since 1'897 VIA ELECTRONIC AND REGULAR MAIL OneUncolnCenter , i; : Syracuse, NY I32ffi-1355;. Hon. Jaclyn Brilling .Ifyne:-:315i422-0121 Voice Maa:3l542^0104- r' Administrative Law Judge Fax: 315422-3598, 'i: ••;• New York State Department of Public Service www.bsk.com Office of Hearings and Alternative Dispute Resolution ; Albany,NY: ' _ : ; Three Empire State Plaza Buffiio,NY ;:. "•, : '::, • • Albany, NY 12223-1350 Oswego.NY > Utica,NY • ' ';/'', OveriaiidPa*,KS , ;• Hon. P. Nicholas Garlick Administrative Law Judge Ld, Schoaicck & Kbig, PA •, nita Springs, FL New York State Department of Naples, ft Environmental Conservation Office of Hearings and Mediation Services 625 Broadway, 1st Floor Albany, NY 12233-1500

Re: Case No. 00-F-205 7 - Application ofBesicorp-Empire Development Company, LLC (BEDCO) - Gray Water - Biological Agents

Dear Judge Brilling and Judge Garlick:

At the Technical Conference, Dr. Christopher Long made a presentation on this agenda item. During the discussion that followed, there were some questions about his presentation. As a result. Dr. Long and his colleague. Dr. Peter Valberg, have prepared the attached paper to respond to the questions raised, all in an effort to show that this should not be an issue for the upcoming hearings. Also provided are two (2) of the primary references cited in the body of the paper.

Thank you.

Sincerely,

BOND. SCHOENECK & KING, PLLC

Kevin M. Bernstein

/ajh Enclosure cc: Active Party List (w/enclosure)

825833.1 9/18/2002 September 16,2002 Gradient Corporation Prepared by: Peter A. Valberg, Ph.D./Ghristopher M. Long, Sc.D.

Cooling Tower "Gray Water" Emissions as a Source of Health Concerns

The Comments of the Sierra Club regarding the Besicoip Project included a question (#11.) on whether there may be agents in the "gray water" from the waste-water treatment plant (WWTP) that could be of health concern when aerosolized and emitted as a component of the drift droplets that are produced during normal operation of the cooling tower. Responses to these concerns are given in the discussion below. If further site-specific information becomes available, our analysis can be refined.

Cooling Towers as a Source of Air Emissions

Cooling towers represent a well-established and safe technology wherein heat is carried away • through the evaporation of water. The major emission of a cooling tower is water vapor, a gaseous form of water which is 100% pure water. A small fraction of the water loss from a cooling tower takes place in the form of a water mist composed of "drift droplets." Modem cooling towers are constructed so that the emission of drift-droplet "mist" occurs at a minimal rate, typically at a rate less than 0.001% of the water re-circulation rate.

Because the normal operation of a cooling tower requires that water continuously evaporate, this loss must be replenished with "makeup" water. Possible sources of makeup water include either potable ! (drinking) water, surface water, or treated effluent from a WWTP. Generally, any effluent water from a ' WWTP must meet rigorous standards in order to allow its discharge to surface waters, and the quality of i the discharged water must be comparable to that of existing surface waters. I

The effluent from a WWTP is treated and released into waterways under specific quality-control '' guidelines. Treated wastewater may also be recycled for useful purposes, and a beneficial reuse is as makeup water in cooling towers. Reclamation of wastewater for cooling tower use has widespread i application in the U.S. (Rogozen et al., 1981; EPRI, 1987), and it is only one of the many applications for | reclaimed water. The Environmental Protection Agency's document on Guidelines for Water Reuse \ (1992) gives standards that must be maintained for the quality of reclaimed water, depending upon the | use to which the water is applied. One of the technical requirements for the use of reclaimed water is to | assure that the use will not result in exposure of the public to unsafe concentrations of infectious i microbes. It has been found that various disinfecting agents can ftdfill this requirement The use of ! reclaimed water in a manner that is consistent with public safety is not only feasible, but is practiced | throughout the United States on a scale of billions of gallons per day.

The possibility of health risk hinges on the concentration of agents of potential concern in the drift droplets, which are primarily water. It is only the nonaqueous dissolved and suspended substances that need to be evaluated for toxicity. Because the proper operation of utility cooling towers is crucial to the electric generation output, both the quality of the source water and the quality of the re-circulating water are carefully monitored so as to limit the buildup of dissolved and suspended substances. c G y, Mr

Microbial agents are present throughout our environment, and we are regularly exposed to infectious microbes through skin contact, food and water ingestion, and air inhalation. Our bodies have multiple and extensive lines of defense against potentially pathogenic microbes, and exposure to the microbes does not generally cause disease unless the amount is sufficient to overwhelm our defense mechanisms. Microbes are normally present in ambient air, and the contribution of a utility cooling tower to viable microbes in the air will typically be far below background levels when properly operated and maintained (Adams et a/., 1978). Potential microbial contamination of the cooling-tower water is avoided through the use of disinfectant (anti-nricrobial) agents such as chlorine. Disinfection procedures reduce bacterial and viral pathogens in cooling-tower water to levels below concern. Similar treatment procedures are used to assure the safety of our drinking water (NRC, 1980).

Many studies have been carried out to characterize how disinfectants neutralize bacteria (typically coliform bacteria) and viruses (typically poliovirus). For example, experiments have shown that chlorine concentrations of 0.5 ppm can neutralize these pathogens in about 1 to 2 minutes of contact t time (NRC, 1980). Bacteria aside from fecal coliform, and viruses other than poliovirus are considered more sensitive to killing by chlorination, and thus these two species are used as markers of the efficiency of the disinfectant process. The water treatment procedures used in utility cooling towers have been shown to effectively reduce viable pathogens to very low levels.1 Thus, cooling-tower water quality is comparable or better than that allowable for human contact situations such as swimming, public fountains, landscape irrigation, and crop irrigation. Cooling tower drift droplets are thus not expected to cause detectable exposure to infectious pathogens due to effective removal of pathogens by disinfection.

Metabolic Products and Human DNA Fragments

Deoxyribonucleic acid (DNA) is a molecule ubiquitous both in our bodies and in all living organisms around us.2 In animal species, DNA makes up between 0.1 to 1% of the body weight. DNA is used in our bodies to synthesize "proteins," which are key molecules in maintaining the function of living organisms. That is, the DNA molecule, the structure of which is inherited from our parents, provides a "template," from which the sequence of amino acids in proteins is determined.

Fragments of DNA have no biological activity outside the nucleus of a living cell. Inside the nucleus of a living cell, there are molecular mechanisms that translate the information encoded in the sequence of DNA bases into a messenger molecule called RNA (ribonucleic acid). This RNA then moves out of the nucleus of the cell into the cytoplasm, where other specialized structures called ribosomes translate the message into newly synthesized protein molecules. A cell relies on the DNA molecules that it inherited from progenitor cells, and even if fragments of DNA were somehow inserted into the nucleus, the cell would not incorporate them into its normal DNA unless the fragments had been treated so that the strands terminated in special "ligase" sequences that allow insertion into endogenous DNA.

Potential "metabolic products" in gray water may include not only DNA fragments, but also proteins, carbohydrates, amino acids, sugars, phospholipids, polysaccharides, triglycerides, and other

1 Appendix D-5B of Section S1-4-E of die Application provides specifics on the proposed use of filtration, chlorination, and microbiocide additions to disinfect ACSD gray waters. O: 2 Aside from "vinises," for which genetic infomiation is stored in strands of RNA, ribonucleic acid. &v•ur*>c 2 Gradient CORPORATION ( ) normal constituents of the food we eat. The mere presence of such biological molecules is not a health concern because, after all, the food we eat is of biologic origin and hence contains all of these constituents plus others. The ambient air also contains biologic components (dander, exfoliated cells, spores, pollen, insect and animal tissue fragments, mold, endotoxins, pheromones, fragrances, etc.). Aside from well-known biological toxins, "metabolic products" are not considered a source of adverse health impacts.

Animal species survive by ingesting either plants or other animals, and consequently are exposed to large quantities of DNA fragments in food. Because life forms are found everywhere, DNA can also be found in drinking water and ambient air. The air in our homes and offices contains cells shed from our friends and co-workers, and hence we are inhaling small quantities of DNA on a regular basis.

The DNA that is part of our food supply is completely degraded (broken down) in our digestive tract by en2ymes, and a similar process occurs in the lungs. That is, when we breathe particles into our lungs, some of the particles become deposited on airway and lung surfaces. The airway surfaces are kept clean by a moving blanket of mucus that carries deposited particles to the junction of die airways and t esophagus, at which point they are swallowed and pass into the digestive tract. For particles that land deeper in the lung, on gas-exchange surfaces, a specialized cell, called the "alveolar macrophage" comes along and ingests the particle into a vacuole inside the cell. This process is similar to how a single-celled organism like an amoeba would ingest food. Once the particle is in the vacuole, the macrophage secretes emymes into the vacuole to digest and break down the particle. If the particle is of biological origin, such as a DNA fragment, this digestive process degrades the particle into elementary chemical compounds.

In summary, typical "metabolic products" and DNA fragments are easily dealt with by our body's digestive processes, both in the lungs and the gastrointestinal tract These substances are not expected to have any biological activity aside from being a source of food energy.

Hormones and Hormone Mimics

Recent water quality studies have shown that human hormones can be found in many natural surface waters that receive human, industrial, and agricultural wastewaters. Kolpin et al (2002) published the results of a comprehensive U.S. Geological Survey (USGS) investigation of organic wastewater contaminants in U.S. streams that included a number of hormones as analytes. Table 1 provides concentrations for a number of hormones that have been detected in U.S. streams, and these data show that concentrations are generally on the order of a few nanograms per liter to a few hundred nanograms per liter. These streams represented areas considered susceptible to contamination from human, industrial, and agricultural wastewater - most are located short distances downstream from expected sources. It is important to note that the detection frequencies were relatively low, and that when detected, concentrations were very small. The results of this study indicate that people can be exposed to low levels of human honnones in their everyday lives - such as from incidentally ingesting surface waters when swimming in streams and water bodies that receive waste water treatment plant (WWTP) effluent, or from ingesting drinking water. In addition, these data indicate that cooling towers that use existing surface waters may also result in airborne exposures to small quantities of human hormones.

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Ont]rwater.doc Gradient CORPORATION Table 1 Levels of Hormones In U.S. Streams (Kolpin et aJ., 2002)-.3

Hormone Number of DetFreq Median4 Maximum Samples (%) (HE/L) (WJ/L) 17a-ethynyl estradiol 70 5.7 0.094 0.273 17a-estradiol 70 5.7 0.03 0.074 17P-cstradiol 70 10 0.009 0.093 Estriol 70 21.4 0.019 0.051 Estrone 70 7.1 0.027 0.112 Mestranol 70 10 0:074 0.407 19-nore this terone 70 12.8 0.048 0.872 Progesterone 70 4.3 0.11 0.199 Testosterone 70 2.8 0.116 0.214

# The levels of hormones in the gray water to be used in the Besicorp cooling tower are not known, and it is thus not possible to calculate the specific level of exposures that may occur. Because hormones tend to be lipophilic substances, they are largely removed via sorptive processes during waste-water treatment, when they partition into sludge. Hormone concentrations in Albany County Sewer District (ACSD) gray waters may be below the limit of detection. By making conservative assumptions, screening-level calculations can be performed to assess the potential magnitude of inhalation exposures to hormones in cooling tower droplets when gray waters are used. These calculations indicate that potential hormone exposures via airborne droplets firom a cooling tower using gray water are likely to be insignificant and smaller than those that may be received from other exposure pathways (e.g., incidental ingestion of surface water). For example, annual inhalation doses of the hormones in Table 1 were estimated for cooling tower droplets by assuming that (1) gray water concentrations of the hormones are equal to the maximum detected concentrations measured by USGS in the recent study of U.S. streams5; (2) hormone concentrations are increased six-fold in the cooling tower due to water evaporation; (3) droplets form at a drift rate that is 0.0005% of the water recirculation rate of 117,500 gallons per minute; and (4) a maximally exposed individual is present 24 hours per day with a typical adult ventilation rate of 0.9 cubic meters per hour. Air modeling results provided in Tables 4-14D and 4-62 of the Application were used to convert hormone emission rates into maximum annual air impacts. These calculations yielded annual inhalation doses from breathing cooling tower droplets that ranged from 0.2 to 3.8 nanograms (billionths of a gram). Table 2 indicates that potential doses from breathing the cooling tower droplets over an entire year would be less than those that might be received by incidentally ingesting 50- 100 mL of surface water while swimming in a stream or lake with hormone concentrations equal to the

3 Note that 17a-cthynyl estradiol (EE2) data have been corrected from the values published in the Environmental Science & Technology journal article based on an errata release available on the USGS website (http://toxics-usgs.gov/ regiona]/est_errataJitnil). As discussed in die errata release, seven concentrations of EE2 were erroneously published that should have been reported as nondetections due to the presence of potential analytical interferences. The errata release reports the updated frequency of detection (5.7%), maximum detected concentration (0.273 ng/L), and median detected concentration (0.094 Hg/L)forEE2. 4 Median concentration is median detectable concentration. 3 In the conservative, screening-level calculations from the August 28, 2002 Technical Conference presentation, hormone concentrations equal to 100 times the median USGS concentrations were used. Based on further review of literature data on hormone levels in sewage effluent (Table 3) as well as the stream-specific USGS data (see discussion above concerning New York State streams), it was determined that die maximum detected concentrations from die USGS study were more representative of potential upper-limit bounds for hormone levels in the ACSD gray waters. As upper-limit bounds, these values remain conservative estimates of potential hormone levels, but they are more realistic than die presentation values which were generally orders of magnitude higher than the highest measured values in the literature.

Q w!,tt toc ^ '< 4 Gradient CORPORATION n median concentrations in Table 1 (note that an incidental ingestion rate of 50 niL per swimming event is a typical default value used in human health risk assessments).

It is important to note that the above calculation represents a hypothetical worst-case exposure scenario rather than an actual exposure scenario. This is because this calculation combines together several conservative assumptions, including the levels of gray water hormones, the concentration factor of six times, the use of maximum annual impact air modeling results, and the presence of a maximally exposed individual. In particular, ACSD gray waters potentially have lower hormone concentrations than the maximum detected concentration in the USGS study. The maximum detected concentrations from the USGS study are generally at least an order of magnitude higher than typical sewage effluent hormone concentrations reported in the literature (Table 3). This is likely a result of the fact that streams included in the USGS study were affected by not only human wastewaters but also industrial and agricultural wastcwaters. Furthermore, the USGS study included 12 sampling locations for New York State streams (stream-specific data available from the USGS website at http://toxics.usgs.gov/regional/emc.html), and concentrations of most hormones were below detection limits in nearly all samples.*

Even for this worst-case calculation, potential inhalation doses of hormones via cooling tower droplets are very small and far below biologically effective levels. For example, the estimated daily inhalation dose for 17a-ethynyl estradiol (EE2) of 0.003 ng/day is nearly 30,000-fold below a worst-case daily intake of 85 ng/day that was estimated for multi-pathway exposures (i.e., exposures via drinking water, leaf crops, root crops, fishes, dairy productions, mean, and inhalation) to EE2 by Christensen (1998). Christensen (1998) determined that this dose, 85 ng/day, posed insignificant human health risk. Also, as shown in Table 4, endogenous production of natural sex hormones (e.g., 17a-estradiol, or E2) in both boys and adult men - two populations at risk from environmental estrogens- are far greater than the conservative screening-level inhalation doses. Furthermore, oral contraceptives provide daily doses of several of the synthetic hormones (e.g., 17a-ethynyl estradiol and 19-norethisterone) that are more than a million-fold higher than the results of our conservative screening-level calculations.

In summary, even though some level of human hormones may exist in WWTP effluent, consideration of the actual dose that can be inhaled due to cooling-tower drift droplets suggests that the dose will be far below biologically effective levels.

References:

Adams, A.P., M. Garbett, H.B. Rees, and B.G. Lewis. 1978. Bacterial aerosols from cooling towers. J. Water Pollution Control Federation 50:2362-2369.

Baronti, C, R. Curini, G. D'Ascenzo, A. Di Coroia, A. Gentili, and R Samperi. Monitoring natural and synthetic estrogens at activated sludge treatment plants and in receiving river water. Environ. Set. Technol. 34: 5059-5066.

Christensen, F.M. 1998. Pharmaceuticals in the environment- a human risk? Regulatory Toxicology and Pharmacology 28:212-221.

6 17a-estiadiot, 17{J-cstradiol, estrone, mestianol, 19-norethisterone, progesterone, and testosterone were not detected in any samples from New York State streams. 17a-etfaynyl estradiol was detected in 1 of 12 samples at a concentration of 31 ng/L, while estriol was detected in two samples at concentrations of 3 and 6 ng/L. J Gt.>«itcr.

Johnson, A.C. and J.P. Sumpter. 2001. Removal of endocrine-disrupting chemicals in activated sludge treatment works. Environ. Sci. Technol. 35:4697-4703.

Electric Power Research Institute (EPRl). 1987. Wastewater Reuse as Cooling-Tower Makeup. EPRI CS-5373. Prepared by Water General Corporation, Lexington, MA. August 1987.

Kolpin, D.W., E.T. Furlong, M.T. Meyer, EM. Thurman, S.D. Zaugg, L.B. Barber, and H.T. Buxton. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999- 2000: a national reconnaissance. Environ. Sci. Technol. 36:1202-1211.

National Research Council (NRC). 1980. John Doull, Chairman, Safe Drinking Water Committee, Drinking Water and Health, Volume 2. National Academy Press, Washington, D.C., 1980, 392 pp. (See Chapter II, "The Disinfection of Drinking Water," p. 29-35.)

Rogozen, M.B., A.R. Phillips, M.A. Guttman, R.F. Shokes, L. Fargo, and J.L. Hahn. 1981. Emission Characteristics of Cooling Towers Using Reclaimed Wastewater in California. ARB Contract No. A8- 126-31. August 11,1981. Available at: www.arb.ca.gov/research/abstracts/a8-126-31.htm.

Spengler, P., W. Komer, and J.W. Metzger. 2001. Substances with estrcgenic activity in effluents of sewage treatment plants in southwestern Germany. 1. Chemical analysis. Environmental Toxicology and Chemistry 20: 2133-2141.

Temes, T.A., M. Stumpf, J. Mueller, K. Haberer, R.-D. Wilken, and M. Servos. 1999. Behavior and occurrence of estrogens in municipal sewage treatment plants-1. Investigations in Germany, Canada, and Brazil. The Science of the Total Environment 225: 81-90. .

United States Environmental Protection Agency (USEPA). 1992. Guidelines for Water Reuse. US EPA Office of Technology Transfer and Regulatory Support. EPA/625/R-92/004. September 1992.

&****** 6 Gradient CORPORATION Table 2. Conservative Screening-Level Analysis of Potential Inhalation Doses of Hormones Via Cooling Tower Droplets

o •• _, * , Maximum Water Scaling Factor of . , Daily Annual Hormone Concentration Emission it >, .. Annual Inhalation Hormone 1 Drift Rate Recycle Air Cone, to . , .. Inhalation Inhalation Cone Factor Rate ,1 Impact Air Rate Rate Emissions Dose Dose Cone.

(Hg/L) (gpm) (Ib/hr) (^g/m3)/(lb/hr) (ng/m3) (m3/hr) (ng/day) (ng/yr)

17a-ethynyl estradiol 0.273 0.0005% 117,500 6 4.81E-07 0.311 1.50E-07 0.9 3.23E-03 1.18E+00

17a-cstradiol 0.074 0.0005% 117,500 6 1.30E-07 0.311 4.05E-08 0.9 8.76E-04 3.20E-01 17P-cstradiol 0.093 0.0005% 117.500 6 1.64E-07 0.311 5.09E-08 0.9 1.10E-03 4.02E-01 Estriol 0.051 0.0005% 117,500 6 8.98E-08 0.311 2.79E-08 0.9 6.03E-04 2.20E-01 Estrone 0.112 0.0005% 117,500 6 1.97E-07 0.311 6.14E-08 0.9 1.33E-03 4.84E-01 Mcstranol 0.407 0.0005% 117,500 6 7.17E-07 0.311 2.23E-07 0.9 4.82E-03 1.76E+00 19-norethisterone 0.872 0.0005% 117,500 6 1.54E-06 0.311 4.78E-07 0.9 1.03B-02 3.77E+00 Progesterone 0.199 0.0005% 117,500 6 3.51E-07 0.311 1.09E-07 0.9 2.35E-03 8.59E-01 Testosterone 0.214 0.0005% 117,500 6 3.77E-07 0.311 1.17E-07 0.9 2.53E-03 9.24E-01

(1) Equal maximum concentrations detected in USGS study of U.S. streams (Kolpin et al, 2002)- See Table 1. (2) Scaling factor based on ratio of maximum annual impact/annual average emission for particulate compounds from data in Tables 4-14D and 4-62, respectively- sodium dodecylbenzensulfonate. was used to calculate scaling factor since it is a chemical additive to the cooling waters and thus its only source is the cooling waters.

Ortywuerdoc Gradient CORPORATION Table 3. Comparison of Assumed Gray Water Hormone Concentrations with Reported Literature Concentrations for Sewage Treatment Plant Effluent

Hormone1 Hormone Published Sewage Treatment Effluent Concentrations (ng/L) Cone Assumed in Hypothetical Calculations1

(Hg/L) Typical Effluent Median (and Ranges for 7 Median cone Median cone (and Median cone Cone (Based on range) cone for British STPs- (and range) for range) for 16 (and range) for published studies)- 18 German STPs- Desbrow et ah, 6 Italian STPs- German STPs- 10 Canadian Johnson et al^ Spengler rf a/., 1998 Baronti e* a/., Ternes <*«/., 1999 STPs-Ternes «* 2001 2001 2000 aL 1999 17a-cthynyl 0.273 0.0005 0.0004 (ND-0.012) Generally

17p-estradiol 0.093 0.0015 0.0016 (ND-0.015) 0.003-0.048 0.001 (0.00035-

(1) These hormones are considered to be among the most biologically active estrogens. (2) Equal maximum concentrations detected in USGS study of U.S. streams (Kolpin et al, 2002) to account for lack of data specific to Albany County Sewer District gray water. (3) ND=not detectable; LOD=liniit of detection.

Oreywnerdoc Gradient CORPORATION Table 4. Comparison of Screening-Level Calculation Results with Hormone Levels of Human Health Significance

Hormone Conservative Daily Dose in Oral Endogenous Production Worst-case Estimate Screening-Level Contraceptives (ng/day)' Human Daily Intake Estimates of (ng/pill) (ng/day) from Daily Inhalation Christensen (1998)2 Doses (ng/day) Prepubescent Adults Boys 17a-ethynyl estradiol (EE2) 0.003 30.000-35,000 i 85 17cw5Stradiol(E2) 0.001 6,000 45,000- i 48,000 17P-estradiol 0.001 EstrioI(E3) 0.001 Estrone (El) 0.001 Mestranol 0.005 IP-norethisterone 0.010 500,000-1,000,000 Progesterone 0.002 Testosterone 0.003

(1) As cited by Christensen (1998). (2) Considered by Christensen (1998) to constitute insignificant human health risk.

Orajnvitcrdoc Gradient CORPORATION o Environ. Sci. Technol. 2002, 36, 1202-1211 Phamiaceiiticals, Hormones, and rarely exceeded drinking-water guidelines, drinking-water health advisories, or aquatic-life criteria. Many compounds, Other Organic Wastewater however, do not have such guidelines established. The detection of multiple OWCs was common for this study, with Contaminants in U.S. Streams, a median of seven and as many as 38 OWCs being 1999-2000: A National found in a given water sample. LitUe is known about the potential interactive effects (such as synergistic or Reconnaissance antagonistic toxicity) that may occur from complex mixtures of OWCs in the environment In addition, results of this DANA W. KOLPIN* study demonstrate the importance of obtaining data on U.S. Geological Survey, 400 S. Clinton Street, Box 1230, metabolites to fully understand not only the fate and transport Iowa City. Iowa 52244 of OWCs in the hydrologic system but also their ultimate overall effect on human health and the environment EDWARD T. FURLONG OS. Geological Survey, Box 25046, MS 407, Denver, Colorado 80225-0046 Introduction MICHAEL T. MEYER The continued exponential growth in human population has U5. Geological Survey, 4500 SW 40th Avenue, created a corresponding increase in the demand for the Ocala. Florida 34474 Earth's limited supply of freshwater. Thus, protecting the integrity of our water resources is one of the most essential E. MICHAEL THURMAN environmental issues of the 21st century. Recent decades US. Geological Survey, 4821 Quail Crest Place, have brought Increasing concerns for potential adverse Lawrence, Kansas 66049 human and ecological health effects resulting from the production, use, and disposal of numerous chemicals that STEVEN D. ZAUGG offer improvements in industry, agriculture, medical treat- ment, and even common household conveniences (/). US. Geological Survey. Box 25046. MS 407. Research has shown that many such compounds can enter Denver, Colorado 80225-0046 the environment, disperse, and persist to a greater extent LARRY B. BARBER than first anticipated. Some compounds, such as pesticides, are intentionally released in measured applications. Others, D U.S. Geological Survey, 3215 Marine Street. such as industrial byproducts, are released through regulated Boulder, Colorado 80303 and unregulated industrial discharges to water and air resources. Household chemicals, pharmaceuticals, and other HERBERT T. BUXTON consumables as well as biogenic hormones are released US. Geological Survey, 810 Bear Tavern Road, directly to the environment after passing through wastewater West Trenton, New Jersey 08628 treatment processes (via wastewater treatment plants, or domestic septic systems), which often are not designed to remove them from the effluent {ft. Veterinary pharmaceu- ticals used in animal feeding operations may be released to To provide the first nationwide reconnaissance of the the environment with animal wastes through overflow or occurrence of pharmaceuticals, hormones, and other leakage from storage structures or land application (3). As organic wastewater contaminants (OWCs) in water resources, a result, there are a wide variety of transport pathways for the U.S. Geological Survey used five newly developed many different chemicals to enter and persist in environ- analytical methods to measure concentrations of 95 OWCs mental waters. in water samples from a network of 139 streams across Surprisingly, little is known about the extent of environ- 30 states during 1999 and 2000. The selection of sampling mental occurrence, transport, and ultimate fate of many sites was biased toward streams susceptible to contami- synthetic organic chemicals after their intended use, par- nation (i.e. downstream of intense urbanization and livestock ticularly hormonally active chemicals (4). personal care products, and pharmaceuticals that are designed to stimulate production). OWCs were prevalent during this study, a physiological response in humans, plants, and animals (7, being found in 80% of the streams sampled. The compounds 5). One reason for this general lack of data is that, until detected represent a wide range of residential, industrial, recently, there have been few analytical methods capable of and agricultural origins and uses with 82 of the 95 detecting these compounds at low concentrations which OWCs being found during this study. The most frequently might be expected In the environment (Q. Potential concerns detected compounds were coprostanol (fecal steroid), from the environmental presence of these compounds cholesterol (plant and animal steroid), /V,AMiethy1toluamide Include abnormal physiological processes and reproductive (insect repellant). caffeine (stimulant), tridosan (antimicrobial impairment (7-12), increased incidences of cancer (75), the disinfectant), tri(2-chloroethy0phosphate (fire retardant). development of antibiotic-resistant bacteria (14-17), and the potential increased toxicity of chemical mixtures (IS). and 4-nonylphenoI (nonionic detergent metabolite). Measured For many substances, the potential effects on humans and concentrations for this study were generally low and aquatic ecosystems are not clearly understood (/, 2, 19). The primary objective of this study is to provide the first 'Correspondingauthorphone (319)3S8-3614:&x: (319)358-3606: nationwide reconnaissance of the occurrence of a broad suite O e-mail [email protected]. of 95 organic wastewater contaminants (OWCs), Including 1 tOZ-ENVtRONM£NTAl_SaENC£S, TECHNOLOGY fVOC 36. NO. 6.2002 10.ia21fes0110SS| Hex subject to US. copyrlgln. PuM. 2002 Am. CtMnLSoc PutHisha) an Web 0^13/2002 •••":-.

TABLE 1. Sunmaij ot Analytical Results of Streams Sampled for 95 Organic Wastewater Contaminants'

lowest LCa for the o most sensitive MCI or indicator spedes RL freq max mcd /Ml (23) Vig/U/na. of aquatic chemical (meUwd) CASftN « (f.grti (%) O-SA) bam use HAJ studies identified (24 Veterinary and Human Antibiotics carbodox(l) 6804-07-5 104 0.10 0 NO ND antibiotic - -n chlortetracycline (1) 57:62-5 115 0.05 0 ND ND antibioUc - SBOOCfl chlortetracycline (2) 57-62-5 84 0.10 2.4 0.69 0.42 antibiotic — 88000«/3 clprofloxacin (1) 85721-33-1 115 0.02 2.6 0.03 0.02 antibiotic -^ -/O doxycyciine (1) 564-25-0 115 0.1 0 ND ND antibiotic - -10 enrofloxadn (1) 93106-60* 115 0.02 t) NO ND antibiotic - 40»/29 erythromydn-HzO (1) 114-07-8 104 0.05 21.5 1.7 0.1 erythromydn - 665000^/35 iiR-uiuviiusmptahnlitft lincomydn (1) 154-21-2 104 0.05 19.2 0.73 0.06 antibiotic _ -10 noffloxacln(l) 70458-9&7 115 0.02 0.9 0.12 0.12 antibioUc - -16 oxytetracycline (1) 79-57-2 . 115 0.1 0 ND ND antibiotic — 1O20O0'/46 oxytetracycline (2) 79-57-2 84 0.10 1.2 0.34 0.34 antibiotic - 102000»/46 roxlthromycln (1) 80214-83-1 104 0.03 4.8 0.18 0.05 antibiotic - -/D sarafloxacin (1) 98105-99-8 115 0.02 0 NO ND antibiotic - -rt) sulfachloropyrldaiine (2) 80-32-0 84 0.05 0 NO ND antibiotic — -A) 1 sulfadimethoxine (1) 122-11-2 104 0.05 0 NO ND antibiotic - -IS sulfadimethoxine (2) 122-11-2 84 0.05 1.2 0.06 0.06 antibiotic - -'5 i ^^^ sulfameraiine (1) 127-79-7 104 0.05 0 ND ND antibiotic - loooocm ' mm sutfamerazlne (2) 127-79-7 84 0.05 0 ND ND antibiotic - 100000917 sulfamethazine (1) 57-68-1 104 0.05 4.8 0.12 0.02 antibiotic - lOOOOO^ , ^^^ sulfamethazine (2) 57-68-1 84 0.05 1.2 0.22 0.22 antibiotic - 100(XXW17 sulfamethlzole (1) 144-82-1 104 0.05 1.0 0.13 0.13 antibiotic - -10 sulfamcthoxazole (1) 723-46-6 104 0.05 12.5 1.9 0.15 antibiotic - -10 sutfamethoxazole (3) 723-46-6 84 0.023 19.0 0.52 0.066 antibiotic - -K) sulfathlazole (1) 72-14-0 104 0.10 0 ND ND antibiotic - . -10 ! sutfathiazole (2) 72-1*0 84 0.05 0 ND ND antibioUc - -10 tetraeydlne (1) 60-54-8 115 0.05 0 ND ND antibiotic - SSOOOO*} tetracycline (2) 60-54-8 84 0.10 1.2 0.11 0.11 antibiotic - 55(KXKW3 Wmethoprim (1) 738-70-5 104 0.03 12.5 0.71 0.15 antibiotic — 30(KW4 trlmethpprim (3) 738-70-5 84 0.014 27.4 0.30 0.013 antibiotic — 300074 tytosin(1) 1401-69* 104 0.05 13.5 0.28 0.04 antibiotic — -m virglniamycin (1) 21411-53-0 104 0.10 0 ND ND antibiotic - -m Prescription Drags •3 albuterol (salbutamol) (3) 18559-94-9 84 0.029 0 ND ND antlasthmatic -. -m cimetidine (3) 51481-61-9 84 0.007 9.5 0.58« 0.074" antacid - -10 codeine (3) 76-57-3 46 0.24 6.5 0.019 0.012 analgesic — -10 codeine (4) 76-57-3 85 0.1 10.6 1.0* 0.2" analgesic — -AD dohydronlfedipine (3) 67035-22-7 84 0.01 14.3 0.03 0.012 antianglnal - -10 digoxln (3) 20830-75-5 46 0.26 0 ND" ND" cardiac stimulant — 10000000-/24 dlgoxlgenln (3) 1672-46-4 84 0.008 0 ND NO digoxln metabolite - -A) diltiazem (3) 42399-41-7 84 0.012 13.1 0.049 0.021 antihypertenslve - -/o enalaprllat (3) 76420-72-9 84 0.15 1.2 0.046" 0.046" enalaprll mateate — -10 (antihypertenslve) metabolite ^fl^k fluoxetlne (3) 54910-89-3 84 0.018 1.2 0.012" 0.012" anUdepressant — -10 HH gemfibrozll (3) 25812-300 84 0.015 3.6 0.79 0.048 antihypefiipidemic — -10 ^^^F metfonmln (3) 657-24-9 84 0.003 4* 0.15" 0.11" antidlabetic — -10 paroxetlne metabolite (3) 84 0.26 O ND" ND" paroxetlne -10 (anUdepressant) " metabolite rartttldine (3) 66357-35-5 84 0.01 1.2 0.01" 0.01" antadd - -to warfarin (3) 81-81-2 84 0.001 0 ND ND anticoagulant - 16000733 Nonpf escription Drugs 1 acetaminophen (3) 103-90-2 84 0.009 23.8 10 0.11 antipyretic — 6000714 caffeine (3) 58-08-2 84 0014 6^3 6.0 0.081 stimulant — 40000777 caffeine (4) 58*8-2 85 0.08 70.6 5.7 0.1 stimulant - 40000777 1 cotinine(3) 486-56-6 84 0.023 38.1 0.90 0.024 nicotine metabolite — -to ' cotlnlne (4) 486-56* 54 0.04 315 0.57 0.05 nicotine metabolitB - -to . V-dimcthylxantWne (3) 611-59* 84 0.018 28.6 3.1" 0.11" caffeine metabolite - -10 1 ibuprofen(3) 15687-27-1 84 0.018 9.5 1.0 0.20 antiinflammatory - -10

Other Wastewater-Related Compounds • 1.4-dlchtorobenzene (4) 106-46-7 85 0.03 25.9 4.3 0.09 deodorizer 75 11007190 2,6-di-tefMHitylphenol (4) 128-39-2 85 aba 3.5 0.11" 0.06" anttoxldant — -/2 (4) 719-22-2 85 0.10 9.4 0.46 0.13 antioxldant - -to ; 5-methyl-1H-benzotHazole (4) 136*5-6 54 0.10 31.5 2.4 0.39 antiocorrosive - -10 ' acetophenone (4) 98*6-2 85 0.15 9.4 0.41 0,15 fragrance - 155000721 anthracene (4) 120-12-7 85 0.05 4.7 0.11 0.07 PAH — 5.47188 benzoMpyrene (4) 50-32* 85 0.05 9.4 0.24 0.04 PAH 0.2 1.57428 25013-16-5 85 0.12 2.4 0.2" 0.1" antioxldant - 870714 butylated hydroxy toluene (4) 128-37* 85 0.08 2.4 0.1" 0.1" antioxldant - 1440715 bls(2-ethyfhexyt) adipate (4) 103-23-1 85 2.0 3.5 10' 3' plastidzer 400 48079 o bls(2-ethylhexy0 phthalate (4) 117*1-7 85 2.5 10.6 20' T ptasticlzer 6 75007309 1204 • ENVIRONMENTAL SCIENCE « TECHNOLOGY / VOL 36. NO. 6. 2002 • . • i •"•••• •••:••..-• .:::...' •.

• TABLE 1. (Continued)

lowest LCs* for the f) most sensitive MCL or indicator species RL freq max med H4i{23) (^g/l)/no. of aquatic chemical (method) CASRN N (pg/U (%) ipgiU fcg/i) use (fSM studies identified m Other Wastewater-Related Compounds bisphenol A (4) 80-05-7 85 0.09 41.2 12 0.14 plasticizer — 360CW26 eartoaryl (4) 63-25-2 85 0.06 16.5 Ol" 0.04" insecticide 700 0.4V1541 rfs-chlordane (4) 5103-71-9 85 0.04 4.7 0.1 0.02 Insecticide 2 7.4''/28 cMorpyrifos (4) 2921-88-2 85 0.02 15.3 0.31 0.06 insecticide 20 0.1»/1794 333-41-5 85 0.03 25.9 0.35 0.07 insecticide 0.6 0.56^1040 dieMrln(4) 60-57-1 85 0.08 4;7 0.21 0.18 insecticide 0.2 2.6Steroids and Hormones £/«-androsterone (S) 53-41-8 70 0.005 -14.3 0.214 0.017 urinary steroid — -A) chototerol (4) 57-88-5 85 1.5 55.3 10" 1" plant/animal steroid — -A) cholesterol (S) 57-88-5 70 0.005 84.3 60" 0.83 plant/animal steroid — -JO coprostanol (4) 360-68-9 85 0.6 35.3 9.8" 0.70" fecal steroid - -JO i coprostanol (5) 360-68-9 70 0.005 85.7 150" 0.088 fecal steroid — ~J0 equftenin (5) 517-09-9 70 0.005 2.8 0.278 0.14 estrogen replacement - -JO equDin(5) 474-86-2 70 0.005 1.4 0.147 0.147 estrogen replacement — -JO • 57-63-6 70 0.005 15.7 0.831 0.073 ovulatton Inhibitor — -J22 ! 17a-e$tradiol (5) 57-91-0 70 0.005 5.7 0.074 0.03 reproductive hormone — -JO ^^^ IT^estradiol (4) 50-28-2 85 0.5 10.6 0.2- 0.16" reproductive hormone - -JO fl B IT^-estradiol (5) 50-28-2 70 0.005 10.0 0.093 o.tm reproductive hormone — -JO ^| ^F cstrtal(S) 50-27-1 70 0.005 21.4 0.051 0.019 reproductive hormone — -JO • estrone(5) 53-16-7 70 0.005 7.1 0.112 0.027 reproductive hormone - -m 72-33-3 70 0.005 10.0 0.407 0.074 ovulatton inhibitor — -J0 68-22-4 70 0.005 12.8 0.872 0.048 ovulatlon Inhibitor — -J0 yV ^^^•^^W^^p* ^^^ ^^r \*'j 57-83-0 70 0.005 4.3 0.199 0.11 reproductive honnone - -J0 stigmasianol (4) 19466-47-8 54 2.0 5.6 4* 2" plant steroid - ~J0 testosterone (S) 58-22-0 70 0.005 2.8 0.214 0.116 reproductive honnone - -/4 • Daphnla magna (water flea) - 48 h exposure LCs.. * Other species and variable conditions. • Oncorhynctttis mykiss (rainbow trout) - 96 h exposureLC^'Cooceiiiratkwestimated-averogerecove^ estbnated - confound routinely detected in laboratoiy Wanks. « Concentration estimaled - rrftere^ *Cencan>raBon estimated -value greater than highest point on calibration a^ve. 'Compounds suspected of being hormonany active are In bold (< ^. CASRN, Chemical Abstracts Servk» Registry Numben M number erf samples; RL repo^ 1 maximum concentration; med. median detectable concentration; MCL, maximum contaminant level: HA1, health advisory level; lAo. lethal i concentration with 50% mortality: ND, not detected: -, not available; PAH, potycyclfc aromatic hydrocarbon.

i compounds. Target compounds within each method were spectra and Ion abundance ratios were required to match selected ftom the large numbCTofchemlcal possibilities based that of the reference standard compounds. The base-peak • upon usage, toxidiy. potential hormonal activity. and ion was used for quantltatlon, and. If possible, two qualifier i

• persistence in the environment Some compounds that fit ions were used for confinnation. After qualitative criteria 1 the above criteria, however, could not be included (such as were met. compound concentrations were calculated from 1 amoxidllin, roxarsone, polybrominated diphenyl ethers) S to 8 point calibration curves (generally from 0.01 to 10.0 1 because they were either Incompatible with the correspond- yWg/L) using internal standard quantltatlon. Methods 1 and 1 ,- •., ing method or reference standards were not available. Positive 2 process calibration standards through the extraction 1 Identification of a compound required ekidon within the procedure . whkh generally corrects concentrations for 1 U expected retention time window. In addition, the sample method losses but not matrix effects. Methods 3-5 do not

VOL 36. NO. 6. 2002 / ENVIRONMENT At SCIENCE & TECHNOLOGY • 1205 •

!

1 extract calibration standards, thus the reported concentra- 1 L water samples using SPE cartridges that contain 0.5 g of tions are not corrected for method losses. Reporting levels HLB (flow rate of 15 mL/min). After extraction, the adsorbed (RLs) were determined for each method by either an compounds were eluted with CHaOH followed by CH3OH o evaluation of instrument response, calculation of limit of acidified with CiHCbOj. The two fractions were reduced detection, or from a previously published procedure [25}. under Nj to near dryness and then combined and brought RLs were adjusted based on experience with the compounds to a final volume of 1 mL in 10% C2H3N:90% HjO buffered In each method, known interferences, or known recovery with NH^Oj/CHzOz. problems. Compounds were separated and measured by high- The following descriptions are intended to provide a brief performance liquid chromatography (HPLC) using a polar overview of the five analytical methods used for this study. . (neutral silanol) reverse-phase octylsUane (C8) HPLC column More comprehensive method descriptions are provided (Metasil Basic 3/«n, 150x 2.0 mm: Metachem Technologies). elsewhere (26-28) or will be available In subsequent pub- The compounds were eluted with a binary gradient of mobile lications. phase A (aqueous NH^OJ/CHJOZ buffer, 10 mM, pH 3.7) Method 1. This method targets 21 antibiotic compounds and mobile phase B (100% C2H3N). (Table 1) in 500-mL filtered water samples using modifica- Method 4. This method (27, 28) targets 46 OWCs (Table tions from previously described methods (26, 29). The 1) in unfiltered water. One-liter whole-water samples were antibiotics were extracted and analyzed by tandem SPE and extracted using CLLE with CHzCfc. Distilled solvent was single quadrapole, LC/MS-ESI(+) using SIM. To prevent the 2 2 recycled through a microdroplet dispersing frit to improve tetracydlne antibiotics from complexing with Ca * and Mg * extraction efficiency. Samples were extracted for 3 h at ions and residual metals on the SPE cartridges, 0.5 mg of ambient pH and for an additional 3 h at pH 2. The extract disodium ethylenediaminetetraacetate (NajEDTA; CioHuOt- - was concentrated under N2 to 1 mL and analyzed by capillaiy- NazNi H2O) was added to each water sample. Sample pH column GC/MS. Available standards for the 4-nonyIphenol was adjusted to 3 using concentrated ftSO*. The tandem compounds were composed of multiple Isomers, and thus, SPE included an Oasis Hydrophilic-Llpophllic-Balance laboratory standards for these compounds as well as oc- (HUB) cartridge (60 mg) followed by a mixed mode, HLB- tylphenol ethoxylates were prepared from technical mixtures. cation exchange (MCX) cartridge (60 mg) (Waters Inc., Milford, MA). The HLB and MCX cartridges were conditioned Method 5. This method (28) targets 14 steroid compounds with ultrapure H2O. CHjOH, and CH3OH with 5% NH,OH: including several biogenic and synthetic reproductive hor- The HLB cartridge was attached to the top of the MCX mones (Table 1). The CLLE extracts from the previously cartridge, and the sample was passed through the SPE analyzed samples of Method 4 were derivatlzed and reana- cartridges using a vacuum extraction manifold. The cartridges lyzed. Analysis of steroid and hormone compounds by GC/ were eluted with CH3OH, and the MCX cartridge was eluted MS Is enhanced by derivatization to deactivate the hydroxyl separately using CH3OH with 5% NH4OH. The eluate was and keto functional groups. The technique used in this study spiked with 500 ngof "Q-sulfamethazlne (internalstandard), is the formation of trimethylsllyl (TMS) ethers of the hydroxyl groups and oximes of the keto groups. Samples were stored vortexed, and evaporated to 20/sulfathiazole; 98.8% for target compounds 0.5 85.1 11.6 oxytetracycline, sulfadimethoxlne, sulfamethazine, and tet- Ctj-phenacetin 1.0 96.8 14.0 racydlne; 98.6% for cholesterol and coprostanol; 97.6% for Method 4 chlortetracyline; 95.7% for 17^-estradlol: 94.4% for cotinlne: target compounds 1.0 81.0 11.0 94.0% for trlmethoprim; 89.1% for sulfamethoxazole; 86.4% d2,-BHT 2.0 63.0 25.0 for codeine: and 83.3% for caffeine. The comparisons for o-nonylphenol 2.0 83.0 20.0 codeine, caffeine, and cotinlne may have been affected by Methods the differing extractions (SPE versus CUE) as well as differing target compounds NA NA NA types of sample (filtered versus whole water). d^-estradiol* 0.047 128.8 42.0 An Interlaboratory comparison of Methods 1 and 3 was di-testosterone' 0.051 148.5 47.3 dT-cholesterol* 0.053 116.9 55.9 conducted using two reagent water blanks and 24 reagent water spikes prepared at concentrations ranging from 0.5 to 'Surrogate standard added after CCLE extraction but prior to 1.1 fig/t for two frequently detected antibiotics (sulfamethox- dertvltteaUon. »RSD, relative standard deviation; NA. not currently available. azole and trlmethoprim). The results demonstrated that both methods are accurately confirming the presence of sul- famethoxazole and trlmethoprim in water, with the measured a detection of naphthalene, 4-nonylphenol, phenol, 4-rert- concentrations being within a factor of 3 or better of Jhe octylphenol monoethoxylate, and ethanol,2-butoxy-phos- actual concentrations for these compounds. No false positives phate. Most of these detections were near their respective or false negatives occurred for this experiment

100 T—i—r T—r 1—r—i—r -i—r

10

1 85 as c o I 1 "1

0.001 r : 85.7 84J 74.1 70JS 57J 57J 50.8 45.9 45.9 415 41.2 38.1 XSSIJS 29.4 28.8 282 27.4 254 25.9 24.7 218 2&S 235 S 19.0 17.8 185 i J 1 1 1 1 1 1 1 1 I 1 I I 1 I I I I 1 I < • • 1 L

FIGURE2. Measured concentrations for the 30 most frequently detected org^cwastewater contaminant ,- •-. distribution truncated at the reporting level Estimated values below the reporting level are shown. Estimated maxhrnm values tor { ) <»prostanol and choiesterol obtained from MedMd 5 (Table 1) are not sh^ ^— at the end of each compound name. An explanation of a boxpiot is provided in Figure J.

VOL X, NO. 6. 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY •1207 n i—r

10 84 gs

104 EXPLANATION 70 Number of observations Maximuni value 1 ' 84 o . 7Siti percemie 1 Medbn o s- 2Sth peroentBo c 8 i * Mlnknum value -"• Reportingi'level

o Estimated vatues T : 70.6 Detection frequency

613 70.8 55.3 84.3 38.1 31.5 1ZJ 19.0 I I I ' I l_ I L_ ^^ x> ,* /: .^.^ .* <* c/c/ ^ yy // FIGURE 3. Comparison of concentrations of select compounds that were measured using two different methods with significantly different reporting levels. Boxplots show concentration distribution truncated at the reporting level Estimated values below the reporting level are shown. Estimated maximum values for chloesterol and coprostanol obtained from Method S (Table 1) are not shown. The analytical method number is provided On parentheses) at the end of each compound name. .

Results and Discussion ronmental exposure to select OWCs appear to be of much One or more OWCs were found In 80% of the 139 streams greater concern (I). Such chronic effects have been docu- sampled forthis study. Theliigh overall frequency of detection mented in the literature (34—38). In addition, because for the OWCs is likely influenced by the design of this study, antibiotics are specifically designed to reduce bacterial which placed a focus on stream sites that were generally populations in animals, even low-level concentrations in the considered susceptible to contamination (i.e. downstream environment could increase the rate at which pathogenic of intense urbanization and livestock production). In addi- bacteria develop resistance to these compounds (15—17. tion, select OWCs (such as cholesterol) can also be derived 39). from nonanthropogenic sources. Furthermore, some of the The 30 most frequently detected compounds represent OWCs were selected because previous research (2S) Identified a wide variety of uses and origins including residential. them as prevalent in the environment. Thus, the results of industrial, and agricultural sources (Figure 2, Table 1). Only this study should not be considered representative of all about S% of the concentrations for these compounds streams in the United States. A previous-investigation of exceeded 1 fig/t. Over 60% of these higher concentrations streams downstream of German municipal sewage treatment were derived from cholesterol and three detergent metabo- plants also found a high occurrence of OWCs (3/). lites (4-nonyphenol. 4-nonyIphenoI monoethoxylate, arid A large number of OWCs (82 out of 95) were detected at 4-nonyIphenol dlethoxylate). The frequent detection of least once during this study (Table 1). Only eight antibiotics cotlnine, 1,7-dimethyixanthine, erythromydn-HzO, and other . and five other prescription drugs were not detected in the OWC metabolites demonstrate the Importance of obtaining samples analyzed (Table I). Measured concentrations were data on degradates to fully understand the fete and transport generally low (median detectable concentrations generally of OWCs in the hydrologic system. In addition, their presence < 1 /ig/L, Table 1), with few compounds exceeding drinking- suggests that to accurately determine the overall effect on water guidelines, health advisories, or aquatic-life criteria human and environmental health (such as pathogen resis- (Table 1). The concentration of benzo(a]pyrene exceeded its tance and genotoxidty) from OWCs, their degradates should maximum contaminant level (MCL) of 0.2 /ig/L at one site also be considered. The presence of the parent compound and bls(2-ethylhexyl)phthalate concentrations exceeded its and/or their select metabolites in water resources has MCL of 6.0/(g/L at five sites. In addition, aquatic-life criteria previously been documented for OWCs (40, 41) as well as were exceeded for chlorpyrifos (Table 1) at a single site. other classes of chemicals such as pesticides (42, 43). However, many of the 95 OWCs do not have such guidelines Manyof the most frequently detected compounds (Figure or criteria determined (Table 1). In feet, much is yet to be 2) were measured In unflltered samples using Method 4. known about the potential toxlcological effects of many of Thus, their frequencies of detection may be somewhat higher the OWCs under investigation (I). For many OWCs. acute because concentrations being measured include both the effects to aquatic biota appear limited because of the low dissolved and patticulate phases, whereas concentrations concentrations generally occurring In the environment (24. measured by Methods 1-3 include Just the dissolved phase. u 32—34). More subtle, chronic effects from low-level envi- For example, about 90% of the coprostanol discharged from 1208 • ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL 36, NO. G. 2002 100 r -r -r- 4 A o 5 e so 1 5 3 7 2 60 1 22 7 6 o 11 40 J? 14 c 5 o 2 tr3 1 f> 20 u- / 0 J1 1 1 I 40 1 1 1 I

^ 30

a c 20 o c o U s p 10 -^y^yyyx^ -r // FIGURE 4. Frequency of detection of organic wsstewater contaminants by general use category (4A), and percent of total measured concentration of organic wastewater contaminants by general use category (48). Number of compounds in each category shown above bar.

sewage effluents has been shown to be associated with determinations: frequency of detection (Figure 4A) and partlculate matter (44)- Thus, the concentration and fre- percent of total measured concentration (Figure 4B) for each quency of detection for select compounds would likely have group of compounds. These two views show a vastly different been reduced if sample filtration had taken place. representation of the data. In relation to frequency of Variations in RL also influence the frequency of OWC detection, there were a number ofgroups that were frequently detection (Figure 2). For example, the detection of 4-non- detected, with seven of the IS groups being found in over yiphenol would Ukely have been much greater if an order of 60% of the stream samples (Figure 4A). However, three groups magnitude lower RL (similar to other OWCs) could have been (detergent metabolites, plasddzers, and steroids) contributed achieved. The effect of RL on frequencies of detection is more almost 80% of the total measured concentration (Figure 4B). dearly demonstrated by comparison of concentrations of For those groups of compounds that have received recent select compounds that were measured using multiple public attention—namely antibiotics, nonprescription drugs, analytical methods (Figure 3). As expected, the frequency of other prescription drugs, and reproductive hormones (/, 2, detection for a given compound was higher with the lower /Q—nonprescription drugs were found with greatest fre- RL. The only exception being caffeine, where filtration of quency (Figure 4A). Antibiotics, other prescription drugs, Method 3 may have reduced caffeine concentrations com- and reproductive hormoneswere found at relatively similar pared to that of the unflltered Method 4. Figures 2 and 3 also frequencies of detection. The greater frequency of detection demonstrate the importance of estimated values (45) below for nonprescription drugs may be at least partially derived the RL Clearly the numerous estimated concentrations from their suspected greater annual use compared to these illustrate that the current RLs are not low enough to accurately other groups of compounds. When toxlctty Is considered, characterize the total range of OWC concentrations in the measured concentrations of reproductive hormones may stream samples and that the frequencies of detection for this have greater implications for health of aquatic organisms study are conservative. than measured condentrations of nonprescription drugs. To obtain a broader view of the results for this study, the Previous research has shown that even low-level exposure. o 95 OWCs were divided into 15 groups based on their general (<0.001/

VOL 36, NO. 6. 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY • 1200 • • """i • ' '•• •! additional research also needs to be focused on those OWCs • • • •,• •': 5<5 median concentration - 0.91 uglt not frequently detected in this stream sampling. Select OWCs o may be hydrophobic and thus may be more likely to be 30 _ » present in stream sediments than in streamwater (65, 6S). For example, the low frequency of detection for the tetra- oIf E •..' U « cycline (chlortetracycllne. doxycycllne, oxytetracycllne. tet- - £ racycllne) and quinolone (ciprofloxadn, enrofloxadn. nor- - 20 ••* *• • • floxadn, sarafloxadn) antibiotics is not unexpected given :: •. . their apparent affinity for sorptlon to sediment (66). In O TJ .. v. • • addition, select OWCs may be degrading Into new, more persistent compounds that could be transported into the «»- so 10 - .j. • •a o environment instead of (or in addition to) their associated § » parent compound. 2° n Acknowledgments 0.001 0.01 0.1 10 100 Total Concentration (ug/L.) The authors wish to acknowledge the USGS sdentlsts and field technidans who provided essential assistance to this FIGURE S. Relation between total concentration (summation from project by Identilylng candidate stream sites across the United all detections) and number of organic wastewater contaminants States and In collecting and processing stream samples. In found per water sample (Spearman's rank correlation coefficient addition, the authors thank Michele Lindsey, Jeff Cahill, and = CM, P < 0.001). Greg Brown for their Important contributions to developing the analytical methods being used. The authors also ac- Mixtures of various OWCs were prevalent during this knowledge Steffanie Keefe for her efforts in compiling the study, with most (75%) of the streams sampled having more existing ecotoxicological data, Jessica Hopple for her as- than one OWC Identified. In fact, a median of seven OWCs sistance in generating select figures for this paper, and Kymm were detected In these streams, with as many as 38 Barnes for her assistance in compiling the water-quality data compounds found In a given streamwater sample (Figures). for this study. This project was supported by the U.S. Because only a subset of the 95 OWCs were measured at Geological Survey, Toxic Substances Hydrology Program. The most sites collected during the first year of study. It Is use of trade, firm, or brand names In this paper is for suspected that the median number of OWCs for this study identification purposes only and does not constitute en- Is likely underestimated. Although Individual compounds dorsement by the U.S. Geological Survey. were generally detected at low-levels, total concentrations of the OWCs commonly exceeded 1 /ig/L (Figure 5). In addition, 33 of the 95 target OWCs are known or suspected Literature Cited to exhibit at least weak hormonal activity with the potential (1) Daughton, C. G.; Temes, T. A. Environ. Health Petspect 1999. to disrupt normal endocrine function (•#, 7, 8,10,12,22,36. J07 (Supplement G), 907-938. 37,48-50), all of which were detected in at least one stream (2) Halllng-Soremen, B.; Nielson. S. N.; Lanzky, P. p.; Ingerslev, F.; HohenLutzhoft.J.;Jorgensen,S.E. Chemospherel998.35.357- sample during this study fTable 1). The maximum total 393. concentration of hormonally active compounds was 57.3^g/ (3) Meyer. M T.: Bumgamer. J. E.: Vams. J. L: Daughtridge, J. V.: L. Aquatic species exposed to estrogenic compounds have Thurman. E. M; Hostetkr. K. A. Sd. TotalEnvimt 2000,243, been shown to alter normal hormonal levels (7,4& 51). Thus, 181-187. the results of this study suggest that additional research on (4) National Research Council. Hormonally active agents In die environmencNaUona] Academy Press: Washington, DC, 1999: the toxldty of the target compounds should include not only 430 pp. the individual OWCs but also mixtures of these compounds. (5) Jorgensen. S. E.: Halling-Sorensen, B. Chemosphere 2000. 40, The prevalence of multiple compounds in water resources 691-699. has been previously documented for other contaminants (6) Sedlak, D. U: Gray. J. L: Pinlcston. K. E. EnvrUm. ScL TechnoL (52,53). In addition, research has shown that select chemical 2000. 34. 509A-515A. combinations can exhibit additive or synergistic toxic effects (7) Purdom, C. E.; Hardiman, P. A; Bye, V. J.; Eno. N. C; Tyler, C. (54—56), with even compounds of different modes of action R; Sumpter, J. P. Chera. Ecol 1994, 8.275-285. (8) White, R; Jobling. S.; Hoare, S. A.: Sumpter. J. P.; Paiker. M. G. having interactive toxicological effects (57). Endocrinology 1994.135,175-182. The results of this study document that detectable (9) Shaipe. R M: Skakkebaek. N. E tanccf 1993.341.1392-1395. quantities of OWCs occur in U.S. streams at the national (10) Panter, G. H: Thompson. R S.; Sumpter, J. P. Environ. Sd. scale. This Implies that many such compounds survive Technol 2000. 34.2756-2760. wastewater treatment {1, 6, 58) and biodegradatlon (59). (11) Harrison, P. T. C; Holmes. P.: Humfrey, C. D. N. 5d. Total Future research will be needed to identify those factors (i.e. Environ. 1997. 205. 97-106. (12) Jobling. S.; Nolan, M; Tyler, C. R; Brighty. G.; Sumpter, J. P. high use and chemical persistence) that are most important Environ. Set Technol 1998. 32. 2498-2506. in determining the occurrence and concentration of OWCs (13) Davis, D. L.: Bradlow, H. L &£. Ant 1995, 273.166-172. in water resources. (14) DuPont. H. L: Steele, J. H Rev. Infect Dis. 1987, * 447-460. Although previous research has also shown that antibiotics (15) GilBver. M A.; Bennett, M; Begon. M: Hazel, S. M: Hart. C A (60), other prescription drugs (/, 2, 19, 61-63), and non- Mature 1999. 401, 233-234. (16) Khachatourians, G. G. Can. Wed i4ssoc / 1998. 159,1129- prescription drugs (/, 40, 62,64) can be present in streams, 1136. this study Is the first to examine their occurrence in a wide (17) Smith.RE.:Besser.J.M;Hedberg.C.W.;Leano,F.T.:Bender. variety of hydrogeologic, climatic, and land-use settings J. B.; WkMund, J. R; Johnson, B. P^ Moore, K. A; Ostethobn. across the United States. Much is yet to be learned pertaining M T. N. EngL J. Med. 1999. 340. ISZ5-1532. to the effects (particularly those chronic in nature) on (18) Sumpter. J. P.;JobBng. S. Environ. Health Perspect 1995, J03. humans, plants, and animals exposed to low-level concen- 174-178. trations of pharmaceuticals and other OWCs. Furthermore. (19) Ayscough, N. J.; Fawell, J.; Franklin, G.: Young. W. Review of . human pharmaceuticals in the environment: Environment little is known about the potential Interactive effects (syn- Agency. R&D Technical Report P390; 2000. o ergistic or antagonistic toxidty) that may occurfirom complex (20) http;//toxics.usgs.gov/regtonal/emchtmL mixtures of these compounds in the environment Finally. (21) Shehon. L R Open-File Rep.. US. CeoL Surv. 1994. No. 94-455.

1210 • ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 36. NO. E. 2002 (22) Foran, C. M-; Bennett E. Jt; Benson. W. H. Mar. Environ. Res. (44) Venkatesan, M. I.; Kaplan, 1. R Environ. ScL Tedmol 1990,24 2000, SO. 153-156. 208-213. (23) U.S. Environmental Protection Agency. U.S. EPA No. 822-B- (45) Childress, C. J. O.; Foreman, W. T.; Connor B. F.; Maloney, T. o 00-001; US. Government Printing Office; Washington, DC, 2000. J. Open-File Rep.-U.S. Ceol Surv. 1999, No. 99-193. (24) U.S. Environmental Protection Agency Ecotoxlcology database. (46) BaronU, C; Curini, R; D'Ascenro, G.; Di Corcia, A.; Gentili, A.; http7/www.epa.gov/medecotx/quicksearcKhtm. (accessed May Samperi, R Environ. Sd. Technol 2000. 34. 5059-5066. 2001). (47) Rbutledge. B. J.; Sheahan. D.; Desbrow. C; Sumpter, J. P.; (25) U.S. Environmental Protection Agency. Guidelines establishing Waldock, M Environ. Sd. Technol 1998, 32,1559-1565. test procedures for the analysis of pollutants (App. B to Part (48) Lye, C. M.; Frid. C. L J.; GiR M. E.; Cooper. D. W.; Jones, D. M. 136, Definition and procedure for the determination of the Environ. ScL Technol 1999, 33.1009-1014. method detection limit) US. Code of Federal Regulations, Title (49) Swarm, J. M; Schuhz, T. W.; Kennedy, J. R Arch. Environ. 40, revised as of July 1,1992. Contam. Toxicol 1996, 30,188-194. . (26) Lindsey. M. E.; Meyer, M.; Thurman, E. M. Anal. Chem. 2001. (50) Keith, T. L; Snyder, S. A.: Naylor. C. G.; Staples, C A.; Sumer. 73, 4640-4646. C; Kannan, IC; Giesy. J. P. Environ. Sd. Tedmol 2001,35,10- (27) Brown, G. K.; Zaugg. S. D.; Barber. L B. Water-Resour. Invest 13. Rep.-UJ. GeolSurv. 1999. JVo. 99-4018B. pp 431-435. (28) Barber, L. B.; Brown. G. K.; Zaugg, S. D. In Analysis of (51) Fobnar^L C; Denslow, N. D.; Kroll, K.; Orlando, E. F.; Enblom, Environmental Endocrine Dismptorr. Keith, L H.,]ones-Lepp, J.; Mardno, J.: Metcalfe, C; Gulllette. L J., Jr. Arch. Environ. T. L, Needham, L. U. Eds.; ACS Symposium Series 747; American Contam. Toxicol. 2001, 40, 392-398. Chemical Society: Washington. DC. 2000; pp 97-123. (52) Kolpin, D. W.; Barbash. J. R; GOUom, R J. Ground Water 2000, (29) Hlrsch,R.;Temes,T.A.;Haberer.K.;Mehlich,A.;Ballwanz,F.; 38,858-863. Kratz. K. L / Chromatogr. A 1998. 815. 213-223. (53) Stackelberg. P. E.; Kauffman, L J.; Ayers, M. A.; Baehr. A. U (30) Mitscher, L. A. The Chemistry of the Tetracycline Antibiotics Environ. Toxicol. Chem. 2001, 20, 853-865. Mated Dekker. New York. Basel. 1978. (54) Marinovich, M; Ghilardi, F.; Galll. C L Toxicology 1996. f OS. 01) Heberer, T.; Schmldt-Baumler, K.; Stan, R J. Acta Hydmchlm. 201-206. HydrobioL 1998. 2S. 272-278. (55) Thompson. R M. Ecotoxlcology 1996,5, 59-81. (32) Baguer, A. ].; Jensen, J.; Krogh, P. H. Chemosphere 2000, 40, (56) Thorpe, K. L.; Hutchinson. T. R; Hetheridge. U. J.; Scholze. U.; 751-757. Sumpter, J. P.; T^ler, C. R Environ. Sd. Technol 2001,35,2476- (33) Lutzhofl. H. C; Halling-Sorensen, B.; Joigensen. S. E. Arch. 2481. Environ. Contam. Toxicol 1999, 36,1-6. (57) Porter, W. P.; Jaeger, J. W.; Carlson, I. R Toxicol tod Health (34) Wollenberger. L; Halling-Sorensen, B.; Kusk. K. O. Chemosphere 1999, 15, 133-150. 2000. 40, 723-730. (58) Kuch, R M.: Ballschmiter, K. Environ. Sd. Tedmol. 2001,35, (35) Hartmann, A.; Golet E M.; Gartiser, S.; Alder. A. C; Koller, T.'; 3201-3206. Widmer, R. M. Arch. Environ. Contam. Toxicol. 1999, 36,115- (59) Al-Ahmad, A.; Daschner, F. D.; Kummerer, R Arch. Environ. 119. Contam. Toxicol 1999,37,158-153. (36) Fong, P. P. Bio/. Bull 1998. 194. 143-149. (60) Hirsch, R; Terries, T.; Haberer, R: Kratz, R Sd. Total Environ. (37) Sohonl, P.: Tyler, C. R.; Hurd. K.: Gaunter, J.; Hetheridge, M.: 1999. 225, 109-118. Williams. T.; Woods. C; Evans, M; Toy. R; Gargas, M.; Sumpter. (61) Koenlg, B. G.; Metcalfe, C D.; Temes, T.; Hirsch, R SETAC J. P. Environ. 5a. Tedmol 2001. 35. 2917-2925. Proceedings 2000, 76. (38) Harris. C. A.; Santos. E.M.;Ianbakhsh. A.; Pottinger,T.G.;TVier. (62) Stumpf,M;Temes,T.A.; Wilken, R; Rodriques, S. V.; Baumann. . aR.;Sumpter,J.P.£m*tOT.Sd.rechnal2001,35.2909-2916. W. Sd. Total Environ. 1999, 225, 135-141. (39) Chee-Sanford. J. C; Aminov. R. I.; Krapac. L J.; Carrigues- (63) Temes. T. A. Water Kes. 1998, 32, 3245-3260. Jeanjean, N.; Mackie. R. L Appl. Environ. MicrobioL 2001, 67, 1949-1502. (64) Seiler.RL:Zaugg,S.D.:Thomas,J.M.;Howcroft,D.UCmimd Water 1999. 37, 405-410. (40) Baser, H.-R.; Poiger, T.; MuIIer, M. D. Environ. Sd. Tedmol 1998.32, 3449-3456. (65) Heberer, T. J. Hydrol 2002 On press). (41) Buser, R-R.; Poiger. T4 Muller, M D. Environ. ScL Tedmol (66) Tolls, /. Environ. ScL Technol 2001, 35, 3397-3406. 1999, 33. 2529-2535. (42) Kolpin, D. W.; Thurman. E M; Linhart, S. M- Set TotalEmirxm. Received for review June 12, 2001. Revised manuscript re- 2000. 248, 115-122. ceived November 26, 2001. Accepted December 12, 2001. (43) Clark, G. M.; Goolsby, D. A. Sd. Total Environ 2000.243.101- 113. ES011055J

o

V01_ 36. NO. 6. 20021 ENVIRONMENTAL SCIENCE & TECHNOLOGY •1211 REGULATORY TOXICOLOGY AND PHARMACOLOGY 28, 212-221 (1998) o ARTICLE NO. RT98i253

Pharmaceuticals in the Environment—A Human Risk?

F. M. Christensen Danish Toxicology Centre (DTC),2 KogUAlli, 2970 Htrsholm, Denmark

Received July 20,1998

ously is a part of their nature. Furthermore, pharma- Pharmaceuticals in the environment and their po- ceuticals are usually lipophilic and often have a low tential toxic effects are emerging research areas, biodegradability. These intrinsic properties pose a po- which is also reflected in the drug approval regula- tential for bioaccumulation and persistence in the en- tion. This far, focus has mainly been directed toward vironment. potential effects on nature and wildlife. In this paper, A growing body of literature dealing with fate and human risk as a consequence of exposure via the en- effects of pharmaceuticals in the environment is vironment has been addressed and assessed. The syn- emerging, and extensive reviews can be found in R5m- thetic estrogen 17a-ethinylestradiol (EE2), the antibi- bke et al. (1996) and Halling-Sorensen et al. (1998). otic phenoxymethylpenicillin {Pen V), and the However, from these reviews it is clear that the main antineoplastic drug cyclophosphamide (CP) were cho- attention has been paid to potential effects on nature sen as modeling substances based on criteria of recep- and wildlife. Only few investigations have looked into tor specificity, elevated risk for human population the exposure of humans via the environment. This groups for which the pharmaceuticals are not thera- tendency is also reflected in the regulation of pharma- peutically intended, different modes of action, and prescription frequency. Attention has been focused on ceuticals. Since January 1, 1995, an environmental assess- emissions from the use phase and subsequent diffuse release via the sewer systems. A reasonable worst-case ment has been demanded in connection with applica- environmental fate and human exposure were esti- tion for approval of new drugs in the European Union mated using the software BUSES on worst-case emis- (CPMP, 1996). The draft versions of guidelines on how sion quantities. The results indicate a negligible hu- to carry out such assessments do not deal with humans man risk connected to the environmental exposure for as end points in the environment (EU, 1994a,b). In the these substances. Danish conditions have been used as United States, the National Environmental Policy Act the modeling area, but the results are assumed to be of 1969 (NEPA) requires all federal agencies to assess valid for regions with similar drug consumption pro- the environmental impacts of their activities. A guide files. C 1998 Academic Press on how to carry out such assessments for drugs was Key Words: EUSES; risk assessment; pharmaceuti- released in 1995 (FDA, 1995). In this guide, humans cals; drugs; human health; environment; exposure; are also not addressed as end points for the assess- ITa-ethinylestradiol; EE2; phenoxymethylpenicillin; ment: Pen V; cyclophosphamide; CP. The purpose of the present paper is to evaluate whether the extensive use of pharmaceuticals and the subsequent release of the substances into the environ- INTRODUCTION ment may pose a potential human risk. Human drugs In recent years a growing attention has been di- are addressed and attention has been focused on the rected toward the discharge, presence, and potential use phase. The entry route to the environment will adverse effects of pharmaceuticals in the environment. consequently be via the sewer systems. This paper Discharges of human drugs and their metabolites from therefore deals with the diffuse release of these sub- production facilities, hospitals, and private household stances into the environment and not with point effluent as well as the disposal of nonused drugs pose a sources like production facilities, where reduction mea- load on the environment. Veterinary drugs may enter sures can be and often are utilized to reduce the emis- the environment more directly than does human drugs, sion and thus the potential impacts. for instance via growth promoters used in fish farming Danish conditions are used as a modeling area, as or field application of manure from animals treated drug consumption data are relatively easily obtainable for this region. However, the results are assumed to be with drugs. o Pharmaceuticals are suspicious environmental con- an indicator for other countries and regions with drug taminants as they are biologically active, which obvi- consumption profiles similar to Danish conditions. 212 6273-230(V98 $25.00 Copyright O 1998 by Academic Press All rights of reproduction in any form reserved. PHARMACEUTICALS IN THE ENVIRONMENT—A HUMAN RISK? 213

METHODS used in oral contraceptives is mainly 17a-ethinylestra- o diol (EE2), but also to a small extent its 3-methyl ether, Identification and Quantification of Pharmaceuticals mestranol (Juul, 1997). However, mestranol has no estrogenic action itself, but is metabolized to EE2 in Initially, a number of broad pharmaceutical catego- the body (Bolt, 1974; Hardman and Limbird, 1996); ries were identified for subsequent quantification. A therefore, mestranol is considered to be EE2 in the number of criteria were put up for the selection of these following. Based on detailed statistics on the use of oral categories. With a few exceptions, pharmaceuticals are contraceptives in Denmark (Juul, 1997), it can be cal- not high-production-volume chemicals, and the ex- culated that 3.64 kg EE2 was used in Denmark in pected environmental concentrations may conse- 1996. Oral contraceptives contain 20-35 /ig/pill (Hard- quently be assumed low. The chosen substances should man and Limbird, 1996; Juul, 1997). The consumption therefore be expected to be able to cause effects at low figures for sex hormones reflect sales from the private exposure levels, i.e., preferably have a high receptor pharmacies, i.e., use in hospitals is not included as it is affinity. In connection with this, choosing pharmaceu- assumed that these drugs are mainly domestically tical groups assumed to cause efTects on individuals used. more sensitive to the drug than individuals therapeu- By far the most prescribed antibiotic in the primary tically treated with the drug was tried. Finally, differ- sector in Denmark is phenoxymethylpenicillin (ATC ent modes of action were searched for. em J01CE02)—also named penicillin V or Fen V. The sale ased on the above criteria, the following pharma- 1 for human use from private pharmacies reached W*tical groups were chosen: 8,590,282 DDD in 1996 (Laegemiddelstyrelsen, 1997). • ATC'-group G03: Sex hormones and modulators of One DDD equals 2 g (WHO, 1995), which gives a yearly the genital system; amount of approximately 17,200 kg. From Eager • ATC^group J01: Antibacterials for systemic use; (1997), it can be extracted that penicillin use in hospi- and tals is about 10% of use in the primary sector. The total • ATC^-group L01: Antineoplastic agents. use of Pen V in Denmark is therefore assumed to be in the range of 19 ton per year. All three categories reflect receptor-specific modes of Antineoplastics differ from the other groups by the action and may have the potential of causing effects on fact that they are mainly utilized in the hospitial sector, j ^individuals which they are not intended for. Sex hor- and that it makes no sense to deal with DDD as treat- PM nones-^ especially estrogens, which are used to a great ments are very individual. The most used substance extent as the active substance in oral contraceptives within this group is cyclophosphamide (ATC and remedies for postmenopausal hormone replace- LOlAAOl)—abbreviated CP. About 13-14 kg is used at ment therapy—are expected to pose a risk to exposed Danish hospitals per year (T0nnesen, 1997). In addi- men and boys. Antibacterials and especially penicillins tion, 5.969 kg is prescribed for sale at private pharma- may trigger an effect in allergic individuals at very low cies (Laegemiddelstyrelsen, 1997). A total annual use of doses. Finally, antineoplastics are cytotoxic if origin 20 kg is assumed. and a group of substances for which even a high degree Chemical names, abbreviations, identification num- •side effects is accepted in cancer treatment. There- bers, DDDs, and the annual use of the identified sub- e, these genotoxic substances may also pose a poten- stances are summarized in Table 1. tial problem, especially because it has been shown that antineoplastics are persistent in the environment Application, Metabolism, and Excretion (Kummerer et al., 1996; Steger-Hartmann et al.. 1997). The use within the three broad pharmaceutical E2 is a natural female horinone. In Denmark E2 is groups was identified based on statistics from the Dan- therapeutically used for replacement therapy in defi- ish Medicine Agency (Laegemiddelstyrelsen, 1996; ciency states. These include primary amenorrhea iand Juul, 1997; Laegemiddelstyrelsen, 1997) and contact delayed onset of puberty as well as management of the with the pharmacy at the Danish University Hospital— menopausal syndrome (Kristensen et al, 1995). A no- Rigshospitalet (Tonnesen, 1997). table difference to American conditions is the fact that Based on DDD (defined daily doses), 17/J-estradiol in the United States, conjugated estrogens have histor- (ATC GO3CA03) and estrogens used in oral contracep- ically been most commonly used as menopausal drugs tives (several ATC numbers for the different combina- (Hardman and Limbird, 1996). tion preparations) are the most used sex hormones E2 is administered orally. After intake, an extensive (Laegemiddelstyrelsen, 1997). 22,376,236 DDD 17/3-es- first-pass hepatic metabolism to the less potent estro- tradiol (E2) was used in Denmark in 1996 (Laegemid- gens estrone and estriol takes place with the latter delstyrelsen, 1997). This equals approximately 45 kg/ being the major urinary metabolite. A variety of sulfate ... ,year as one DDD is 2 mg (WHO, 1995), The estrogen and glucoronide phase II metabolites are also excreted along with a minor part of nontransformed E2 (Hard- U 1 Anatomical Therapeutic Chemical (ATC) classification index. man and Limbird, 1996). 214 F. M. CHRISTENSEN

TABLE 1 o Annual Use of the Pharmaceuticals Selected for Further Investigation Dnig name Abbreviated ATC code CAS No. DDD* Annual use (kg) Reference year

17p-EstradioI E2 G03CA03 50-28-2 2mg 45 1996 17a-Ethinylestradiol EE2 —° 57-63-6 20-35 p.g 3.64 1996 PhenoxymethylpeniciUin Pen V J01CE02 87-08-1 2g 19.000 1996 Cydophosphamide CP L01AA01 50-18-1 .20 1996/1997 ° Several ATC codes as EE2 are present in different combination formulations * DDD, defined daily dose. e Not relevant for antineoplastics.

As E2 and its metabolites are already naturally ex- thus very low. However, it has been shown that the creted to the environment due to the natural produc- conjugates may undergo bacterial hydrolysis (Kulkami tion of E2 in women and female animals, it was decided and Goldzieher, 1970). It has thus been speculated by early in the study to compare the amount of E2 used other authors that free EE2 may be reformed by bac- therapeutically with the "natural" estrogen excretion. terial hydrolysis in the sewer system, in waste water Based on statistics on the number of children (Dan- treatment plants, or in nature [see for instance Kalbfus marks Statisitk, 1997) and foals (Stambrugskontoret, (1997) and Rurainski et al. (1977)]. 1997) bom per year and the excretion of estrogens from Pen V is administered orally for infections with fertile, pregnant, and menopausal women (Griffen, gram-positive bacteria and can be given as the calcium 1996; Fotsis et al.. 1980, 1987; Turan, 1995) and from salt, the potassium salt, or a combination of the acid pregnant mares (Reaside and Liptrap, 1975; Hardman and the potassium salt (Kristensen et al., 1995). Pen V and Limbird, 1996), the following estimated excretion of estrogens can be calculated: fertile women, 55 kg/ is readily hydrolyzed by penicillinase (Hardman and year; pregnant women, 560 kg/year; menopausal Limbird, 1996). Penicillins are eliminated rapidly women, 7 kg/year; and pregnant mares, 540 kg/year. causing short half-lives in the body (typically 30-60 On top of these figures comes estrogen excretion min) and high concentrations of these substances in from other animal species and even men produce some the urine (Hardman and Limbird, 1996). It is reported E2. When it is assumed that the therapeutically ad- that up to 40% (25.9% on average for six individuals) of ministered E2 is metabolized in the same manner as Pen V is excreted unchanged in the urine (Cole et al. endogenous E2, it can be concluded that the extra 1973). estrogen load due to 45 kg E2 used therapeutically CP is administered orally or intravenously and has a contributes less than 5% compared with the natural broad spectrum of activity in cancer treatment (Hard- excretion. Though there is a difference between the man and Limbird, 1996). CP is not active in itself, but metabolism of endogenous E2 in humans and mares, undergoes metabolism with the first step leading to the above calculations are still assumed to be justified 4-hydroxycyclophosphamide, which dependent on the by considering that the estrogenic activity of urine conditions can be further converted by a number of from pregnant mares used for production of meno- pathways, among others leading to the formation of pausal drugs is well known. Altogether, it was there- phosphoramide mustard, which is probably responsi- fore decided not to consider E2 further in the study. ble for the antitumor alkylating effect, and acrolein EE2 is a synthetic estrogen primarily used in oral (IARC, 1981; Hardman and Limbird, 1996). Acrolein contraceptives. EE2 is much more potent than E2 as it and possibly the phosphoramide mustard are believed is not first-pass metabolized to the same extent in the to be responsible for the toxic effects of CP. Among liver (Hardman and Limbird, 1996). The doses, used in others, CP has been shown to be genotoxic [see for oral contraceptives are therefore very low—in the instance Matney et al. (1985)]. (See IARC (1981) and range of 20 to 35 /xg (Hardman and Limbird, 1996; Hardman and Limbird (1996) for further details on Juul, 1997). metabolism and toxicity.) Some CP is excreted un- The metabolism of EE2 is rather complex and in- changed: 5-35% according to Cohen etal. (1971) and up volves both phase I and II metabolism. The primary to 20% as indicated by Bagley et al. (1973). Ensslin et phase I route has been shown to be the 2-hydr6xylation al. (1994) assume an excretion rate of 11.3% unmetabo- (Bolt, 1974,1979; Bolt et al.. 1974). However, far from lized CP in their interpretation of urinary excretion iall EE2 is phase I metabolized (Maggs et al., 1983). from hospital personnel occupationally exposed to CP. Before excretion, EE2 and its phase I metabolites are Sometimes CP is administered as the monohydrate. conjugated to a high degree to water-soluble com- The physical-chemical properties of the monohydrate o pounds (Helton et at. 1976; Maggs et al., 1983; Park are, however, very similar to those of nonhydrated CP and Maggs, 1986). The rate of excretion of free EE2 is (HSDB; Verschueren, 1996), and it is therefore as- PHARMACEUTICALS IN THE ENVIRONMENT—A HUMAN RISK? 215 f ) sumed not to considerably influence the calculations of the introduction, only the use phase is considered in • the environmental fate. this study. Therefore only the "private use" module of EUSES has been applied, and therefore the local sce- Assessment of Environmental Fate nario models the situation close to a public sewage treatment plant based on some conservative assump- General. A conservative approach is used for the tions as for instance the dilution factor for which a assessment of the biological fate of the drugs using the value of 10 is used. computer program BUSES (EC, 1997). BUSES is de- veloped as a support to The Technical Guidance Docu- Fate calculations. The fate calculations of the pro- ment (EC-TGD, 1996), the guide on how to carry out gram are based on level 3 fugadty models which ac- risk assessments of new and existing substances in the count for interphase resistance and describe a steady- European Union. The fate models used in the program state system with nonequilibrium distribution of the are aimed at estimating reasonable worst-case scenar- chemical. Emissions may be into one or more phases. ios and should be used for screening purposes only. The Degradation reactions and advection are accounted for. results obtained by using the program may therefore Partition coefficients and bioconcentration factors can highly overestimate the risk and demand a more de- be entered directly into the program. If no data on tailed assessment [see for instance Murin et al. (1997)]. partition and/or bioconcentration are available, EU- the other hand, an estimated little or no risk may SES utilizes QSAR methods to predict these, mainly on en be interpreted as if there is no or negligible risk. •' the basis of log Pow. The applied QSAR methods are Human exposure. The program gives estimates on applicable to nonpolar organic compounds, while polar human exposure via the environment. The environ- and ionic substances are poorly modeled. Neither par- mental fate of the emitted substance/drug, including tition coefficients nor bioconcentration factors have uptake in foodstuffs and distribution to air and drink- been found for the pharmaceuticals investigated in this ing water, is modeled and the daily human exposure is study. calculated by assuming certain daily intakes of drink- This especially causes a problem in relation to mod- ing water, leaf crops, root crops, fishes, dairy products, eling the fate of Pen V, which with a pKa value of 2.73 meat, and inhalation of air (EC-EUSES, 1996). (HSDB) will be highly dissociated in the aqueous me- Geographical scenarios. EUSES operates with lo- dia in the environment. However, as a worst case (with Vcal, regional, and continental conditions. The regional respect to human uptake) it is assumed that all of the scenario represents an average background level; substances will be in the undissociated state. This is a while the local scenario is included to estimate the worst case because the undissociated Pen V acid must situation at hot spots differing considerably from the be assumed to have the higher tendency to bioaccumu- average background. The regional scenario is used as a late. Physicochemical properties applied for the fate background for the local scenario and the continental modeling are listed in Table 2. conditions as a background for the regional scenario. The fate of the drugs in effluent treatment plants is Calculations on environmental fate and human ex- modeled using the default figures and assumptions in ure are therefore carried out for both local and EUSES. This part of the modeling is of course very ^eg:egional emission scenarios. The default regional sce- relevant for the present study as the entry route to the nario in BUSES is assumed to be an area of 200 X 200 environment is assumed to be via the sewer systems, km2 with 20 million inhabitants. Typical or represen- 90% of which is assumed—^representing Danish condi- tative values for a standard European region have tions—to be connected to effluent treatment plants. been selected. The regional default values relating to population and area have for the calculations carried Assumptions on drug emission. The same problem out in this study been changed to Danish conditions as with Pen V arises for the modeling of excreted drug according to Fredenslund et al. (1995). The local sce- metabolites which are usually water soluble. On top of narios are dependent on some emission assumptions that, it is usually very difficult to find physicochemical connected to local conditions. These are again related data on the metabolites in the literature. In general, to the "Industry Category" (IC), which in this project however, the parent compounds are more toxic than has been set to "Personal/Domestic Use" and the "Use the excreted metabolites. It is therefore as a worst case Category" (UC), which has been assigned "Pharmaceu- assumed that only the parent compound is emitted to ticals." Based on the entered values of IC and UC, the environment. This assumption is partly justified EUSES assumes some typical properties for the local considering that conjugated metabolites—as described emission scenario, which for the present study includes for EE2 and speculated for other compounds as well— the assumptions that 2% of the regional emission will may be bacterially hydrolyzed to the parent compounds ^ake place from one point source and that discharge to (Kulkarni and Goldzieher, 1970; Berger et al., 1986; ic sewer takes place 365 days per year. The latter Henchel et al., 1997). Therefore, as a conservative seems reasonable for the use of drugs. As explained in starting point, it is assumed that the entire amounts 216 F. M. CHRISTENSEN

TABLE 2 Physicochemical Properties of the Pharmaceuticals

Vapor Molecular weight Melting point Log Pow° Water solubility pressure (g/mol) (°C) (-) (mg/liter) (Pa)

b c EE2 296.46 U2-146 (144) 3.67 (4.75? 2 x IC-6* 9 PenV 350.38'' i 2.09^ 588' 8 x IC- * CP 261.V 41-53^ (50) 0.63° 40,000* 0.15'

Note. Parentheses indicate figures used in fate modeling in the case of intervals or differences between references. ° Octanol-water partitioning coefficient. ' Verschueren (1996X e EpiWin database (EpiWin, 1995). d Various values have been reported: 4.745 (Rathner and Sonnenbom, 1979), 4.75 (Norporth el al, 1973), 4.83 (Tabak et al, 1981), 5681 (Rurainski et al., 1977), and 5681-5944 (Verschueren, 1996). There seems to be a factor 103 error in some of the references. 4.75 mg/liter is conservatively used and seems to be the most likely value. • Estimated with QSAR (EpiWin, 1995). ^Kieth and Walters (1992). * HSDB. * The Merck Index (1989). • Decomposes at 120-128''C (HSDB). •'Reynolds (1982).

listed in Table 1 are excreted to the regional environ- number of micro-organisms, including Escherichia coli ment via the sewer systems. and Psudomonas aeruginosa, which are found in wastewater treatment plants as well as the environ- Biodegradation. Biodegradation is to be indicated ment. Once the ring has been opened, the resulting according to OECD Test Guidelines 301 and 302. In penicilloic acid would be readily mineralized by envi- BUSES, biodegradation is by default set to "not biode- ronmental micro-organisms. Additionally, hydrolysis gradable." This seems to be a proper value for CP, of penicillins by microbial amidases produces 6-amin- which has reached this ranking in a closed-bottle test openicillanic acid and the corresponding acid (OECD 301D) (Kummerer et al.. 1996) and in a labo- group..." and "Amoxicilliri will also undergo pH de- ratory wastewater treatment plant experiment (Ste- pendent hydrolysis at ambient environmental condi- ger-Hartmann et al., 1997). tions." A biodegradation experiment carried out using EE2 also cannot be assumed to biodegrade signifi- a modified version of the Sturm test showed a 50% cantly. This was concluded based on laboratory exper- mineralization in 216 days. The 50% plateau was iments carried out in connection with an environmen- reached in approximately 140 days after a 20-day lag- tal assessment conducted as support to an FDA phase. d-Glucose was used as a reference and rapidly approval application for a new EE2-containing drug reached a plateau of approximately 75% mineraliza- (FDA, 1996a). Norpoth et al. (1973) report from a study tion. It is speculated that the degradation probably where 100% of the EE2 test substance remained after would have proceeded at a faster rate in the presence of a 5-day aerobic sludge experiment. Tabak and Bunch biomass concentrations used in aerobic wastewater (1970) report that EE2 was less biodegradable than treatment plants (approximately 2500 mg/liter TSS, estrone, estradiol, and estriol in a laboratory experi- while 25 mg/liter was used in the experiment). It is ment, where the degradation was investigated in concluded that amoxicillin may reasonably be expected sludge supplied with nutrient and an optimum content not to persist or accumulate in the environment (FDA, of microorganisms. Five percent of EE2 remained after 1996b). Based on this. Pen V is expected to be "readily 2 weeks. The latter study indicates some biodegrada- biodegradable." However, a cross-check calculation rat- tion and the EUSES calculations are therefore run ing Pen V as "not biodegradable" will be carried out. twice assuming "inherently biodegradable, not fulfill- ing criteria" and "not biodegradable," respectively. No data have been found on the biodegradability of RESULTS Pen V. However, an environmental assessment of the structurally related amoxidllin has been carried out in The calculated daily human intakes are presented in connection with the registration of a drug containing Table 3. the substance (FDA, 1996b) from where the following It can be seen that the assumptions on biodegrad- is cited: "Amoxicillin trihydrate is an aminopenicillin ability of Pen V and EE2 do not influence the results as and as such unstable to a wide range of bacterial beta- dramatically as could be expected. For Pen V there is lactamates. In general, penicillins are susceptible to only a factor 19 for the regional and a factor 13 for the Q enzymatic opening of the beta-lactam ring found in a local scenario between the estimated daily human in- PHARMACEUTICALS IN THE ENVIRONMENT—A HUMAN RISK? 217

TABLE 3 concentrations. No data have been found on EE2 mea- o Estimated Daily Human Doses Based surements in the foodstuffs included in EUSES. on EUSES Calculations As indicated in Table 4, there are big differences between the regional and local scenarios in terms of Daily human dose, regional Daily human dose, local exposure routes for Pen V. Fifty-seven percent comes mg/(kg body wt x day) Substance mg/(kg body wt x day) from drinking water, 39% from fish, and 4% from leaf 7 6.32 x 10-7 crops in the regional scenario, whereas the distribution EE2 1.37 x KT 6 (2.02 x io-7r (1.22 x io- y for the local scenario is 23% from drinking water, 16% 6.06 x IQ-6 9.54 X lO-5 PenV -3 0 from fish , 58% from leaf crops, and 3% from root crops. (1.16 X lo-'r (1.23 X lO ) Compared to EE2, these figures indicate a lower ten- 8 2.57 X lO-7 CP 7.59 x 10" dency of bioaccumulation in fish as a result of the lower ' Figures in parentheses refer to calculations carried out assuming logPow. No data have been found on Pen V in drinking "not biodegradability." CP is beforehand assumed 'not biodegrad- water or foodstuffs. able.' CP has a low Pow and a high water solubility, and about 90% of the dose is estimated to come from drink- ing water, a minor part from fish (7-8%), and for the takes based on "not" and "readily" biodegradability, local scenario about 4% from leaf crops (see Table 4). •jtoectively. The factors for EE2 between "inherently No data have been found on CP in drinking water or ^Begradable, not fulfilling criteria" and "not biode- foodstuffs. However, German measurements at a sew- gradable" are below two. age treatment plant which among others receives dis- For EE2 the main exposure is estimated to originate charges from hospitals using antineoplastic drugs from fish (80% for local scenario and 93% for regional showed values—though uncertain as they were dose to scenario), which is not surprising considering the rel- the detection limit—in the range of 7-17 ng/liter in the atively high octanol-water partition coefficient (Table effluent (Steger-Hartmann, 1995). The estimated 2). The remaining dose is estimated to originate from drinking water concentrations of CP using EUSES are drinking water (5-6%) and for the local scenario from about 2.5-8 ng/liter, which therefore seems a worst root crops (12%) and leaf crops (3%) (see also Table 4). case. -s The estimated EE2 concentrations in drinking water * Xre 0.30 and 1.20 ng/liter for the regional and local DISCUSSION • jcenarios, respectively. In the literature, several drink- ing-water measurements have been presented. Rath- ner and Sonnenbom (1979) refer to a Dutch investiga- EE2 tion where 0.6 ng/liter was found. Rurainski et al. (1977) report average values of 0.83-6.4 ng/liter (indi- The local scenario gives a human daily intake value 7 -1 -1 vidual measurements ranging from 0 to 22.5 ng/Uter) of 6.32 X 10~ mg EE2 X kg body wt X day in drinking water from wells in south and southeast corresponding to 44 ng/day (0.044 /xg/day) for a 70-kg ^^rmany. Recent results from the Bavarian area person or 85 ng/day (0.085 /xg/day) when assuming • Bwed EE2 concentrations in river water below the EE2 to be not biodegradable. These values refer to an ^Stection limit of 0.2 ng/liter, and only a few wastewa- oral intake through drinking water and food. ter treatment plant effluent samples showed values of It is difficult to define a human no-effect level for 0.3-0.5 ng/liter (Kalbfus, 1997). Results from Israel . endocrine-acting substances. However, in relation to showed 12-20 ng/liter during drought periods (Shore et effects on wildlife, extra attention has lately been paid al, 1993). Finally, in British measurements, Aheme et to estrogens as British investigations have made prob- al. (1985) did not find any EE2 in drinking water able that the elevated occurrence of hermaphrodite fish samples with an EE2 detection limit of 5 ng/liter and in English rivers is the result of estrogens in wastewa- Aheme and Briggs (1989) report EE2 measurements ter treatment plant effluent (Purdom et al, 1994; from 1987 of

TABLE 4 n Distribution Expressed in Percentages between the Human Exposure Routes

Drink water Leaf crops Root crops Fishes Dairy prod Meat Air

EE2, reg 6 0.1 0.4 93 4 X lO"3 6 x lO"3 9 x lO-6 EE2, local 5 3 12 80 4 X lO"3 7 x lO-3 6 X lO"4 3 Pen V, reg 57 4 0.2 39 8 X ur 2 x lO"3 3 x lO"9 Pen V, local 23 58 3 16 8 X ur3 2 x lO"3 2 x lO"8 CP, reg 92 0.1 1 X lO"2 8 1 X lO"2 6 x lO"4 7 x lO-5 CP, local 88 4 0.7 7 1 X lO"2 6 x lO-" 7 x lO-5

cally as having the same mode of action as E2. EE2 has action of which may trigger effects at very low concen- approximately the same affinity to the estrogen recep- trations, and therefore it is difficult to establish a tol- tor as E2 (Bergink et al., 1983; Kaspar and Witzel, erable/acceptable daily intake (TDI/ADI). However, the 1985; Cheskis, 1997), but at the same time EE2 is Committee for Veterinary Medicinal Products (CVMP) believed to be slightly more efficacious than E2 once at has defined maximum tolerable residue levels of peni- the receptor (Cheskis, 1997). Taken together, it seems cillins in food. Based on relatively few reported inci- justifiable to compare the oral EE2, dose with endog- dents of allergic reactions traceable to penicillin resi- enously produced E2 in an assessment of the risk. In dues in foods, it seems that at least 10 IU prepubescent boys, the endogenous E2 production (international units) penicillin is necessary to provoke amounts to 6 Mg/day (Farber and Arcos, 1983; CVMP, an allergic reaction (CVMP, 1997b). Ten international 1997a) and in adult men to 45-48 p.g/day (Farber and units corresponds to 5.9 ng as 1 IU equals 0.59 /xg Arcos, 1983; Griffin, 1996) in the testis (10-15%) and (Reynolds, 1982). The estimated daily Pen V intake is in peripheral tissue (85-90%). The absolutely worst- at this level when assessing "readily biodegradability," case estimated human daily EE2 intake (0.085 (xg/day) but is exceeding this "limit value" when assuming a can be seen to be much lower. When considering that poor biodegradability. there has been assumed a 100% discharge to the envi- However, especially for Pen V the estimated daily ronment of all prescribed EE2, a worst-case FUSES intakes must be assumed to be very conservative as all simulation of the environmental fate and human expo- of the substance was assumed to be in the acidic form sure, and a 100% bioavailability of the EE2 obtained to justify the fugacity calculations in FUSES. Pen V through drinking water and foodstuffs, it seems un- will almost exclusively be in the ionic form (pKa = likely that the human EE2 exposure via the environ- 2.73). Furthermore, as previously described, penicillins ment contributes a significant risk. will undergo extensive enzymatic hydrolysis in waste- Though a direct comparison is difficult due to the water treatment plants and in the ambient environ- great difference in oral potency, this conclusion can be ment, and Pen V is approximately 75% metabolized in supported by considering that the excretion of "natu- humans. Thus, realistic human intakes must be as- ral" estrogens—more than 1 ton, as calculated earli- sumed to be well below the indicated "limit value," er—is a factor 300 above the emission of EE2 and EE2 though a better fate modeling would be desirable. metabolites. Resistance is another impact of concern in relation to On the other hand, it should be remembered that utilization of antibiotics. This end point, though very EE2 is a synthetic estrogen and that caution should be important, has not been dealt with in this study as it taken when comparing oral EE2 exposure with endog- seems more relevant in relation to the direct clinical/ enous E2 produced in the testis and in peripheral tis- therapeutical application of the antibiotics. More infor- sue. Additionally, the referred results on the probable mation on this point can be found in Eager (1997) for toxicity of EE2 on fish at extremely low concentrations Danish conditions. should call for caution. Overall, it can be concluded that Pen V and other However, based on the present knowledge, human penicillins cannot be assumed to pose a significant risk caused by EE2 in the environment may be rated as human risk through indirect environmental exposure, insignificant. though very sensible persons which may react on a few molecules cannot be absolutely safe as long as penicil- PenV lins are used as anti-infectives. For Pen V, the human daily intake for the local CP scenario was estimated to 6.06 X 10-5 mg X kg body wt-1 X day-1 corresponding to 4.2 fig/day for a 70-kg The worst-case scenario for CP gives a human daily person or 86 /tg/day when assuming Pen V to be not intake value of 2.57 x 10~7 mg x kg body wt-1 X day-1 Q biodegradable. corresponding to 0.018 /xg/day (18 ng/day) for a 70-kg Again we are dealing with a substance, the mode of person. PHARMACEUTICALS IN THE ENVIRONMENT—A HUMAN RISK? 219

As for EE2 and Pen V it is difficult to put up a human In summary, it seems unlikely that human CP expo- ) no-effect level for CP as one of its toxic actions is sure through the environment will contribute a signif- genotoxicity. However, quite a few human data are icant risk. However, it should be remembered that CP available from the cancer therapy treatment. Steger- acts along with a multitude of other pharmaceuticals Hartmann (1995) has in his dissertation—based on and environmental pollutants with carcinogenic poten- human data—estimated a connection between a life- tial and therefore may contribute to the overall toxic time relative risk as a function of the cumulated CP load toward the environment and therefore indirectly dose. The relative risk reflects the probability of get- toward humans. ting more cancer cases than expected from the back- ground level. It is estimated that the relative risk will CONCLUSION not exceed 1 until the cumulative dose reaches 14 g CP in a lifetime (Steger-Hartmann, 1995). This value cor- The human risk connected to environmental expo- responds to approximately 480 /xg/day when assuming sure for the pharmaceuticals EE2, Pen V, and CP for 365 days per year in 80 years and can be seen to be Danish conditions was assessed based on the diffuse more than a factor 25,000 above the worst-case esti- emissions from the use phase of the drugs. Overall, mated daily human intake. Steger-Hartmann (1995) there seems to be a negligible human risk connected to supports this finding by referring to the assumed the present consumption volumes of these drugs. .orldwide number of cancer cases resulting from ther- The study has been carried out reflecting Danish utically used antineoplastics, estimated to be about conditions, but the results are believed to be valid for ^800ibo (Schmall, 1981), which must be rated as a relative regions with similar drug consumption profiles. low number taking into account the widespread use of This should not, however, lead to an uncontrolled these substances. However, the connection between usage of the drugs as there are uncertainties in the relative risk and cumulative CP dose has been carried assessment, as the drugs and their metabolites, al- out assuming a threshold mechanism. This procedure though appearing insignificant independently, contrib- is questionable when dealing with risk assessments of ute to the overall toxic load of the environment and as genotoxic carcinogens though the thorough examina- there are indications that drugs may pose ecotoxico- tion of the CP literature in Steger-Hartmann (1995) logical hazards. The latter especially addressed in re- could indicate a threshold. Furthermore, using the rel- lation to estrogen (including EE2) emissions from sew- 0 ative risk approach it can be statistically difficult to age treatment plants, for which British researchers extract an extra risk caused by the use of CP as it is have indirect evidence that these emissions could be compared to a relative high background level. the cause of an elevated number of hermaphrodite fish Another approach for carrying out risk assessments in English rivers. Caution should also be taken with of (genotoxic) carcinogens is to calculate a TD1/ADI Pen V, where very sensitive individuals reacting on from a low-dose extrapolation allowing an extra life- even a few molecules may have a reaction triggered at time risk of typically 10_6 or 10_6. Several mathemat- extremely low exposure levels. Further, in relation to ical models for extrapolating to low-dose risks have Pen V and other antibiotics, the resistance problem is m developed; see for instance Zeise et al. (1987) for a important. imprehensive review. One of the most conservative The three drugs studied were chosen as being some ^^mo

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