Pharmaceuticals and Care Products in the Environment - American

Chapter 2

Pharmaceuticals and Metabolites as Contaminants of the Aquatic Environment

Downloaded by UNIV OF ARIZONA on July 28, 2012 | http://pubs.acs.org Publication Date: July 30, 2001 | doi: 10.1021/bk-2001-0791.ch002

Thomas Ternes ESWE-Institute for Water Research and Water Technology, Soehnleinstrasse 158, D-65201 Wiesbaden, Germany (Telephone: +49 611-7804343; Fax: +49 611-7804375; email: [email protected])

The occurrence of pharmaceuticals in the aquatic environment was mainly neglected in previous decades, although enormous quantities are used world-wide. In our laboratory, we therefore developed analytical procedures for a total of 84 drug-related analytes, enabling the simultaneous determination of polar drug residues belonging to different medicinal classes, such as lipid regulators, antibiotics, antiepileptics, anti-inflammatory agents, and estrogens in raw sewage, sewage treatment plant (STP) effluents, and river and drinking water. In a search for target analytes, 36 of 55 pharmaceuticals and 5 of 9 metabolites were quantified in at least one STP effluent. The highest concentration of drug residues were measured for the antiepileptic carbamazepine, with a maximum of 6.3μg/L;X -ray contrast media were found in concentrations as high as 15 μg/L (iopamidol). In 40 German rivers and streams, 31 pharmaceuticals and five metabolites were quantified in at least one sample. Highest median values were detected for bezafibrate (0.35 μg/L) and carbamazepine (0.25 μg/L). Frequently, in small rivers and streams, much higher concentrations were detected than in higher-order rivers such as the Rhine or Main. Even in groundwater samples taken close to stream banks, sometimes relatively high concentrations of pharmaceuticals (e.g., up to 1.1 μg/L for

© 2001 American Chemical Society

In Pharmaceuticals and Care Products in the Environment; Daughton, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

39

40 carbamazepine) were detected. Hence, in the aquatic environment, drug residues are ubiquitously distributed. In distinct contrast, for drinking water only 10 of 69 target pharmaceuticals were found - always in the lower ng/L-range and in very few samples.


Introduction While tons of individual pharmaceuticals have been used yearly in various countries over the last few decades, few investigations have been published about the assessment of their environmental relevance - a result of their inadvertent discharge from sewage treatment and land run-off. A survey of the current knowledge about exposure, effects, and environmental relevance is summarized in the reviews of Halling-S0rensen et al. (i), Daughton and Ternes (2), J0rgensen and Halling-Sorensen (3), and Ternes and Wilken (4). In Germany for instance, up to 1001 of individual drugs are prescribed every year. This amount underestimates the total usage of all drugs, many of which can be purchased without a pharmacy prescription and others procured illegally. Table I gives a rough estimate of the yearly tonnage of human medicines sold in Germany - both by prescription and "over the counter" (OTC). For example, approximately 100 t of ibuprofen was prescribed in 1997, with the remaining 80 t being OTC.

Table I: Estimation of quantities of selected pharmaceuticals sold for use in human medicine in Germany (5, 6)

Pharmaceuticals in human medicine Acetylsalicylic acid Ibuprofen Iopromide Carbamazepine Diclofenac Sulfamethoxazole Metoprolol Bezafibrate 17a-Ethinylestradiol

1997 in tons >500 180 130 80 75 60 52 45 0.050

Due to such high usage levels, detectable concentrations of drugs and their metabolites should not be unexpected in sewage. The overall concentrations, however, depend on each drug's pharmacokinetic behavior (half life, urinary/fecal excretion, metabolism, etc.). Many pharmaceuticals are excreted


41


mainly as metabolites, and thus in addition to the non-metabolized compounds, principal metabolites have to be analyzed. However, metabolites formed by conjugation (e.g., with glucuronic acid or sulfate) within phase II reactions are likely cleaved during sewage treatment to yield the non-metabolized (free) pharmaceuticals, and hence may increase the relevant environmental concentrations. In diluted aerobic batch experiments, this phenomenon can be illustrated by the cleavage of two glucuronides of 17P-estradiol (17P-estradiol17-glucuronide and 17 P-estradiol-3-glucuronide) and of 4-methyliumbelliferylβ-D-glucuronide (10). Figure 1 gives two examples for phase I and phase II metabolism. Clofibric acid, which is the principal and active metabolite of three lipid regulators (clofibrate, etofyllin clofibrate [theofibrate], and etofibrate) is conjugated to clofibric-O-P-acylglucuronide (11, 12). More than 90% of the clofibric acid, administered in form of the pro-drugs clofibrate, etofyllin clofibrate, and etofibrate, is excreted as glucuronide. Another example is ibuprofen, which is first hydroxylated and then conjugated to ibuprofen-O-P-hydroxyglucuronide. Approximately 9% of ibuprofen is excreted as hydroxy-ibuprofen, 17% as the glucuronide of hydroxy-ibuprofen, and the remaining percentage is allocated to further metabolites and the non-metabolized ibuprofen (11-13).

Ibuprofen

Ibuprofen-OH (9 %)

conjugate (17 %)

Figure 1. Metabolites of clofibric acid and ibuprofen excreted by humans A prerequisite for determining the contamination of environmental matrices with metabolites is the availability of reference compounds. The availability of metabolite reference standards, however, is often problematic because most are not commercially available. Drug manufacturer research divisions can be another source from which to request reference compounds. When all attempts fail to procure metabolites, they must be synthesized, often at great time and expense.


42 The fates of veterinary and human drugs after urinary or fecal excretion are quite different. In general, excreted human pharmaceuticals pass through STPs prior to entering rivers or streams, whereas veterinary drugs are more likely to directly contaminate soil and groundwater (without previous sewage treatment) when liquid manure is used for top soil dressing (Fig. 2). After rainfall, surface waters can be polluted with human or veterinary drugs by run-off from fields (esp. agricultural) treated with digested sludge or livestock slurries; groundwater can also be contaminated.


Veterinary Drugs Feed Additives I excretion 1 manure

Σ_

J

I landfill site]

(run-off)-j

S O

j i ~]

I fish farms ]

X [~

river, creek

drug manufacturer

—^

groundwater

C^Drinking water^)

Figure 2. Fate ofpharmaceuticals in the environment The use of pharmaceuticals, especially antibiotics, in fish farms is a unique source for contamination of rivers and lakes, which receive the effluents of those ponds. Discharge of sewage from pharmaceutical manufacturing into surface waters is another point source (8). Transport of drugs via bank filtration from highly contaminated surface water into groundwater is also a possibility, as is the infiltration of sewage directly from sewer drain leakages. Drugs disposed together with domestic refuse can reach landfill sites, which can lead to groundwater contamination by leaching (9). The use/disposal of raw sewage or STP effluents by spray and broad irrigation on agricultural areas is yet another route of introduction.


43 Analytical Methods In our laboratory, analytical procedures were elaborated for a total of 84 analytes, enabling the simultaneous determination of polar drug residues belonging to different medicinal classes such as lipid regulators, antibiotics, and estrogens, in sewage as well as drinking water (Fig. 3). Five methods were used for detenriining drugs and their metabolites in the lower ng/L-range. These incorporated solid phase extraction (SPE), derivatization, and detection by G C / M S and G C / M S / M S or LC-electrospray/MS/MS. The first multi-residue method determines betablockers and β sympathornimetics, as well as neutral drugs like diazepam or carbamazepine, at concentrations in the ng/L-range after solid phase extraction (14). A second multi-residue method allows the determination of acidic drugs possessing a carboxylic moiety, and additionally in some cases one or two hydroxy groups, down to the lower ng/L-range (75). Lipid regulators, antiphlogistics (anti inflammatory agents), and three metabolites with carboxylic and hydroxy groups are included. In the third method, 18 antibiotics can be analyzed simultaneously after lypholization and LC-electrospray tandem M S detection (16). With the fourth method, X-ray contrast media were determined after SPE and LC-electrospray tandem M S detection down to 10ng/L (17). Finally, contraceptives and natural estrogens were detected by G C / M S / M S after SPE extraction, silica gel clean-up, and silylation (18).


2

17 Betablockers Sympathomimetics Antiepileptics 11 Hormones

16 Antiphlogistics Lipid regulators Metabolites LC/MS/MS (64) GC/MS(/MSL/

26 Antibiotics X-ray contrast media

Cytostatic agents Psychiatric drugs Vasodilator

Figure 3. Number of analytes from different medicinal classes that can be determined in the lower ng/L-range with the analytical methods developed


44 E x p o s u r e of Sewage and the A q u a t i c E n v i r o n m e n t to D r u g


Residues The very first reports of drug residues in STP effluents and the environment were mainly focused on clofibric acid, the active metabolite of three lipid regulators. Garrison et al. (19) and Hignite and Azarnoff (20) detected clofibric acid in the lower μg/L-range in treated sewage in the United States. Waggott (21) determined clofibric acid in the river Lee (Great Britain) at concentrations below 0.01 μg/L, and in Spain clofibric acid was even detected in groundwater samples by Galceran et al. (22). Stan et al. (23) and Heberer and Stan (24) identified clofibric acid up to 0.27 μg/L in Berlin tap water. On Iona Island (Vancouver/Canada), the two antiphlogistics, ibuprofen and naproxen have been identified in sewage by Rogers et al. (25). More systematic studies on the fate of drugs after excretion were reported by Van der Heide and Van der Pals (26) and Richardson and Bowron (8). Recently, a multitude of pharmaceuticals and metabolites from distinctive medicinal classes such as anti-inflammatory agents, betablockers, β sympathomimetics, antiepileptics, antibiotics, X-ray contrast media, lipid regulators, contraceptives, and antineoplastics were identified in sewage and rivers. Two recent reviews, Halling-Sorensen et al. (/) and Daughton and Ternes (2) summarize the literature in this new emerging field about the environmental relevance of pharmaceuticals. 2

Contamination of sewage with pharmaceuticals Composite samples from a German municipal STP close to Frankfurt/Main were taken daily from the raw influent and the corresponding final effluent over a period of 6 days (7). In the influent, the concentration levels ranged up to 26 μg/L for acetaminophen and in the effluent up to 2 μg/L for carbamazepine and metoprolol. The elimination rates of the investigated drugs during passage through the STP ranged from negligible within in the standard deviation (appr. 7% for carbamazepine) to greater than 99% (salicylic acid) in the monitoring program presented in Table II. Generally more than 60% of the drug residues detected in the influent were removed. Only carbamazepine, clofibric acid, phenazone (antipyrine), and dimethylaminophenazone (aminopyrine) showed lower average removal rates. The antiepileptic carbamazepine was not significantly removed, thus nearly the same loads entering the STP were discharged into the receiving water. However, for other periods in the same STP much lower removal efficiencies for pharmaceuticals were observed, presumably influenced mainly by conditions of microbial activity in the


45

Table II. Mean concentration and loads of selected pharmaceuticals in the effluent of a municipal STP close to Frankfurt/Main (investigation of corresponding 6 daily composite raw influent and 6 daily composite final effluents) Substance

Mean cone. influent in g/L

Mean cone.

Load

Removal

Load

in %

effluent in μξ/L influent in g/d effluent in g/d

M

3


Meanflowrate from 28 Jan. -02 Feb. 1997: 55320 m /d 341 17

81112

102111

3212

6914

241 127

2517

9013

7318

2514

6617

14501320

99

0.09 10.02

1614

411

8317

0.9610.24

310188

53113

7519

0.9410.11

0.2910.06

5216

1613

6917

Fenofibric acid

1.1 10.2

0.3810.06

5919

21 13

6418

Clofibric acid

1.210.2

0.6010.09

691 10

3315

51 110

2.210.4

2.010.2

122120

114112

Non (7 1 9)

0.25 10.06

0.1710.03

1413

912

331 15

Acetylsalicylic acid Diclofenac

3.2±1.2

0.62 10.30

1.910.2

0.5810.03

Ibuprofen

4.410.5

0.4510.13

Naproxen

1.310.1

0.45 10.08

2616

90

Carazolol

0.19 ± 0 . 0 3

0.07 ± 0.02

10± 1

3± 1

66 ± 8

c

a

LOQ: Limit of quantification

b

has only been detected in two influent and two corresponding effluent samples

c

n.d.: not detectable


46


biological processes. For example, a major rainfall incident reduced the removal rates for some pharmaceuticals by more than 50% (7). In a search for target analyses, 36 of 55 pharmaceuticals and 5 of 9 metabolites were quantified in at least one STP effluent. The highest concentrations of drug residues were measured for the anti-epileptic carbamazepine, with a maximum of 6.3 μg/L. X-ray contrast media were found in concentrations as high as 15 μg/L (iopamidol) and 11 μg/L (iopromide) (27). In addition to X-ray contrast media and carbamazepine, the lipid regulators bezafibrate and gemfibrozil, the anti-inflammatory agents diclofenac, ibuprofen, indometacine, naproxen, ketoprofen, and phenazone, and the betablockers metoprolol, sotalol, and propranolol were present with elevated concentrations in the majority of the 49 German municipal STP effluents investigated.

Metabolites of ibuprofen Drug metabolites have high relevance as environmental contaminants since they are known to be the main excretion products. Figure 4 shows a G C / M S total ion chromatogram of a raw sewage sample that has been analyzed after SPE (RP-C ) followed by derivatization with diazomethane. Pharmaceutical residues are clearly the predominant peaks in the chromatogram, and certain metabolites are clearly of major significance. The determination of the two main metabolites of ibuprofen, 2-[4-(2-hydroxy-2-methylpropyl)phenyl]propionic acid (hydroxy-ibuprofen) and 2-[4-(2-carboxypropyl)phenyl]propionic acid (carboxy-ibuprofen) in municipal sewage, STP effluents, and even river water underlined the occurrence and relevance of metabolites in the environment (28). Since ibuprofen metabolites, just as with most of the other metabolites, could not be purchased commercially, they were extracted from the urine of a volunteer by solid phase extraction and semipreparative H P L C . Up to 6.7 μg/L of hydroxy-ibuprofen were quantified in raw sewage. While ibuprofen was eliminated nearly quantitatively during passage through a German STP, less than 20% of hydroxy-ibuprofen was removed. In 12 investigated German rivers, a median value of 0.34 μg/L hydroxy-ibuprofen was determined. The concentrations of the hydroxy metabolite were significantly higher in all water samples investigated than those of the parent drug ibuprofen. I8

Contamination of sewage with natural estrogens and contraceptives In the German municipal STP close to Frankfurt/Main, the raw sewage was contaminated by 17p-estradiol and estrone, with average concentrations of 0.015 μg/L and 0.027 μg/L, respectively, yielding loads up to 1 g/d (18). The calculated removal rates were much lower than those obtained in the Brazilian STP (18). For instance, the loads of estrone and 17a-ethinylestradiol were not


47

Abundance Clofibric acid Ibuprofen-OH


Ibuprofen

ibuprofen-COOH

Gemfibrozil -COOH

is (iMg) Gemfibrozil

my^0 — ι — ι — ι — r

15.00

20.00

25.00

I

I

I

»

»

30.00

I

1

ι

t

35.00

I

40.00

Retention time in min

Figure 4. G C / M S total ion chromatogram of raw sewage for acidic drugs

Figure 5. Removal of Ibuprofen metabolites in a municipal STP

American Chemical Society Library 1155 16th St., N-W. In Pharmaceuticals and Care Products in the Environment; Daughton, C., et al.; Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 2001.


48 appreciably reduced while passing through the German STP. Considering the standard deviation, no elimination rate could be observed. However, 16ahydroxy estrone and Πβ-estradiol were removed, with reduction in concentrations of 68% and about 64%, respectively. Because the efficiency of an STP for the elimination of drugs is influenced by several parameters such as microbial activity or rain events (7), only a long-term study can reveal whether the differences result from increased temperatures or whether other contributory factors are responsible. However, 17P-estradiol and 16a-hydroxyestrone were basically eliminated with a higher efficiency than 17a-ethinylestradiol and estrone. In STP effluents, primarily the natural estrogens estrone, 17P-estradiol, 16a-hydroxyestrone, as well as the synthetically altered contraceptive 17aethinylestradiol could be found - in the lower ng/L-range (18). Mestranol (17aethynylestradiol-3-methyl ether) was only present in three samples and 17βestradiol-17-valerate could not be detected at all (Table III). Estrone was determined with a maximum concentration of 0.070 μg/L and a median value of 0.001 μg/L. Additionally, median values could be calculated for 16ahydroxyestrone as 0.001 μg/L. The contamination of STP effluents are within the concentration ranges found by Aherne and Briggs (29), Wegener et al. (30), Belfroid et al. (31), Desbrow et al. (32), and Routledge et al. (33).

Contamination of rivers and streams Recent investigations reveal that more than 30 different pharmaceuticals belonging to nearly all important German medicinal classes could be found up to the μg/L-range in rivers and streams. In 40 German rivers and streams, a total of 31 pharmaceuticals and five metabolites were quantified. Highest median values were detected for bezafibrate (0.35 μg/L) and carbamazepine (0.25 μg/L). For instance, maximum concentrations were determined in German rivers up to 1.3 μg/L for carbamazepine (antiepileptic) and 1.2 μg/L for diclofenac (antiphlogistic). Frequently, in small rivers and streams, much higher concentrations were detected than in larger-order rivers like the Rhine or Main. Sometimes combined concentrations over 6 μg/L were quantified for the pharmaceuticals occurring in one sample (Fig. 6). These streams, located in the Hessian Ried, contain a high proportion of STP effluents, as indicated by their relatively high boron concentrations (ranging from 0.25 to 1.0mg/L). Hence, the higher the proportion of treated sewage in the receiving water, the greater the potential for contamination by pharmaceutical residues (7).

Contamination of groundwater In German groundwater samples taken close to stream banks in the Hessian Ried close to Frankfurt/Main, relatively high concentrations of pharmaceuticals


49 Table III: Concentrations of natural estrogens and contraceptives in German municipal S T P effluents.


Substance

LOQ in μg/L

number n>LOQ of STPs

median in μg/L

90maximum percentile in μg/L in μg/L

Estrone

0.001

38

20

0.001

0.021

0.070

17P-Estradiol

0.001

38

13

Pharmaceuticals and Care Products in the Environment - American

Recommend Documents