ORIGINAL PAPER

Persistence of gemfibrozil, naproxen and mefenamicacid in natural waters

Lilia Araujo • Noreiva Villa • Nuris Camargo •

Maikellys Bustos • Theobaldo Garcıa •

Avismelsi de Jesus Prieto

Received: 16 November 2008 / Accepted: 21 April 2009 / Published online: 20 November 2009

� Springer-Verlag 2009

Abstract The occurrence of pharmaceuticals in natural

waters is a potential threat to human nutrition and eco-

system quality. The persistence of the acidic pharmaceu-

ticals gemfibrozil, naproxen and mefenamic acid was

studied in surface waters of Maracaibo Lake and Tule

reservoir (Venezuela) under laboratory conditions. A quick

and easy analytical method was developed for the deter-

mination of the acidic drugs at microgram per liter levels

using aqueous derivatization, liquid–liquid extraction and

gas chromatography–mass spectrometry. Pharmaceuticals

degradation followed a pseudo first-order kinetic and their

half-lives were calculated for every experimental condi-

tion. Under sunlight, naproxen and mefenamic acid were

degraded at moderate rates with half-lives from 9.6 ± 0.5

to 27.0 ± 6.6 days, while gemfibrozil had a higher per-

sistence (t1/2 = 119.5 ± 15.6 - 288.8 ± 61.3 days).

Keywords Pharmaceuticals � Surface waters � Kinetics �Persistence � Analytical method

Introduction

In recent years, residues of pharmaceuticals have been

detected in various aquatic environments (Nikolau et al.

2007; Zuccato et al. 2006). Pharmaceuticals have been

designed for their biological activity. With respect to their

purpose, they should be considered as potentially signifi-

cant environmental contaminants. These compounds that

are widely used in human and veterinary medicine are

excreted unchanged or as active metabolites and continu-

ously discharged into municipal wastewaters. Incomplete

removal during wastewater biological treatments results in

their presence in effluents and finally in surface waters

(Tauxe-Wuersch et al. 2005). Among the detected sub-

stances, acidic drugs belong to one of the most important

groups of pharmaceuticals.

Gemfibrozil (Fig. 1) is an acidic drug, prescribed as lipid

regulator to lower plasma triglycerides, but it is also

reported to lower very low-density lipoproteins and total

cholesterol and to increase high-density lipoproteins.

Naproxen and mefenamic acid (Fig. 1) are acidic drugs that

are commonly used as anti-inflammatory, analgesic and

antipyretic. Mefenamic acid is indicated for relief of mild to

moderate pain, and for the treatment of primary dysmen-

orrhea. Naproxen has been detected in wastewater treatment

effluents at concentrations up to 5.22 lg/L (Andreozzi et al.

2003). Nakada et al. (2006) investigated the presence of

naproxen and mefenamic acid and found maximum effluent

concentrations of 0.139 and 0.396 lg/L, respectively.

Gemfibrozil was found in effluents at 0.18 lg/L (Bendz

et al. 2005). In the aquatic environment, naproxen was

detected in surface waters at concentrations of up to

0.4 lg/L (Ollers et al. 2001) and gemfibrozil at about 0.75–

1.5 lg/L (Sanderson et al. 2003). Hilton and Thomas (2003)

found 0.065 lg/L of mefenamic acid in surface waters.

Nowadays, environmental risks associated with the

presence of such compounds in water samples are still

unknown. To date, there is little available information

about the adverse effects of gemfibrozil and naproxen in

aquatic organisms.

The toxic effects of gemfibrozil were investigated by

Zurita et al. (2007) using three bioassays. The most sensi-

tive system was the immobilization of the cladoceran

L. Araujo � N. Villa � N. Camargo � M. Bustos � T. Garcıa �A. Prieto (&)

Laboratory of Analytical Chemistry and Electrochemistry,

Faculty of Engineering, University of Zulia,

PO Box 4011-A-526, Maracaibo, Venezuela

e-mail: aprieto@luz.edu.ve

123

Environ Chem Lett (2011) 9:13–18

DOI 10.1007/s10311-009-0239-5

Daphnia magna, followed by the inhibition of biolumi-

nescence of the bacterium Vibrio fischeri and the inhibition

of the growth of the alga Chlorella vulgaris. The crustacean

D. magna was the most sensitive system to gemfibrozil with

a mean effective concentration (EC50) of 120 lM after 72 h

of exposure. According to the results, gemfibrozil should be

classified as harmful to aquatic organisms. However,

comparing the concentrations in water and the toxicity

quantified in the assayed systems, gemfibrozil is not

expected to represented acute risk to the aquatic biota.

On the other hand, Mimeault et al. (2005) report that

gemfibrozil has the potential to be taken up from water and

concentrated in goldfish blood. In experiments with aque-

ous gemfibrozil exposure, bioconcentration factors in

plasma relative to the nominal concentrations in water were

between 16 and 89. These results imply that uptake through

the gills is an important route for bioconcentration of this

pharmaceutical in fish blood. The authors reported that

plasma testosterone levels were reduced by 49 and 72%

compared with controls when goldfish were exposed to 1.5

and 1,500 lg/L of gemfibrozil in water, respectively.

Testosterone is essential for the endocrine control of

reproduction and spermatogenesis. The fact that environ-

mental level of gemfibrozil decreased testosterone levels

by 49% after 14 days provides strong evidence that this

compound may be acting as an endocrine disruptor in this

species of fish.

Ecotoxicity tests of naproxen and its photo transforma-

tion products in the aquatic environment were performed

on algae Pseudokirchneriella subcapitata, rotifers Brachi-

onus calyciflorus and crustaceous Thamnocephalus

platyurus and Ceriodaphnia dubia, to evaluate the acute as

well as chronic effects. Chronic tests showed higher tox-

icity than acute tests. The photoproducts of naproxen,

obtained by solar simulator irradiation, were significantly

more toxic than the parent compound (Isidori et al. 2005).

Due to the biological activity of these emergent con-

taminants, the evaluations of environmental impact in the

aquatic environments require that their persistence in the

environment must be understood.

The present study was conducted to examine the persis-

tence of gemfibrozil, naproxen and mefenamic acid in water

samples of Tule reservoir and Maracaibo Lake (Venezuela)

under laboratory conditions. The persistence was followed

used a new analytical method by means of aqueous meth-

ylation, liquid–liquid extraction and gas chromatography–

mass spectrometry. The influence of solar light, adsorption

on particulate and volatilization were examined.

Experimental

Chemicals

All reagents were of analytical reagent grade unless stated

otherwise. Water was purified with a Nanopure system

(Barnstead, USA). Gemfibrozil, naproxen and mefenamic

acid were supplied by Sigma (St Louis, MO, USA). A

stock standard solution of 1,000 lg/mL of each compound

was prepared in basic deionized water. Working solutions

were obtained by appropriated dilutions with deionized

water. The derivatization reagent dimethyl sulfate (DMS)

was purchased from Riedel-de Haen. Tetrabutylammonium

hydrogen sulfate (TBA-HSO4) was obtained from Fluka.

Analytical method

As much as 25.0 mL of standard solution or water sample

was placed in a separator funnel of 50 mL. Phosphate

buffer solution (pH 6.0, 3.0 mol/L, 3.0 mL) and Na2SO4

(10.0 g) were then added and the sample was agitated.

After addition of the ion-pairing reagent (TBA-HSO4,

OCOOH

CH3

CH3 CH3

CH3

(a)O

COOH

CH3

H3C

(b)

HN

COOH CH3

CH3

(c)

Fig. 1 Chemical structure of

a gemfibrozil, b naproxen,

c mefenamic acid

14 Environ Chem Lett (2011) 9:13–18

123

0.1 M, 0.25 mL), 100 lL of derivatization reagent (DMS)

was added and agitated. After 5 min, water samples were

extracted by shaking for 3 min with 1.0 mL n-hexane. The

extract was dried by passage through anhydrous sodium

sulfate. Finally, 1 lL of extract was injected into the gas

chromatography–mass spectrometry system.

Concentration values for persistence experiments were

performed using a 6890N series gas chromatograph

equipped with a split–splitless injector for the HP-5MS fused

silica capillary column (30 m 9 0.25 mm i.d., 0.25-lm

film thickness), and 5973 quadrupole mass selective

detector (Agilent Technologies, USA). The injector tem-

perature was set at 250�C and the transfer line temperature

was 260�C. The oven temperature was held at 50�C for

3 min and then heated to 250�C at a heating rate of 30�C/

min. The temperature was held at 250�C for 4.5 min. The

carrier gas was helium (purity 99.999%) at a flow rate of

1 mL/min. The samples were automatically injected using

the splitless mode. The mass spectrometer detector was

tuned by maximum sensitivity autotune. The following

mass to charge (m/z) values were acquired in the electron

impact ionization mode by single ion monitoring and used

for quantification of the analytes: 143–264 for gemfibrozil,

185–244 for naproxen and 223–255 for mefenamic acid.

Triphenyl phosphate was used as internal standard.

Persistence study

The persistence study for gemfibrozil, naproxen and mefe-

namic acid was carried out during the period September

2007 to February 2008 in water samples of Tule reservoir

and Maracaibo Lake, located in Zulia State, Venezuela. The

mean temperature oscillated between 26 and 37�C. The

physical–chemistry parameters for water samples of reser-

voir were as follows: pH 7.48, and the hardness was 71.0 mg

CaCO3/L. The conductivity at 25�C was 0.19 mS/cm and the

alkalinity 70.0 mg CaCO3/L. The chemistry oxygen demand

(COD) was 11.6 mg/L. Total solids and suspended solids

were 132.0 and 6.0 mg/L respectively. For water samples of

the Lake, the pH was measured as 7.3. The hardness was

762.5 mg CaCO3/L, conductivity at 25�C was measured as

750 mS/cm and alkalinity as 45.0 mg CaCO3/L. Total solids

and suspended solids were 4918.0 and 33.0 mg/L, respec-

tively. The COD was 124.8 mg/L. Four experimental

conditions with two repetitions in each condition were used

in the evaluation of the effects. Table 1 describes the

experimental conditions used in the study of persistence. The

pharmaceuticals were spiked in 2-L samples of surface

waters at an initial concentration of 75.0 lg/L and placed in

4-L bottles. The concentration of the acidic drugs was

monitored over time in each repetition, beginning day 1, and

then during the following days: 3, 5, 7, 9, 11, 15, 21, 30, 42,

59, 72, 94, 100, 135 and 150.

Results and discussion

Preliminary experiments were carried out to optimize the

main parameters affecting the aqueous methylation, liquid–

liquid extraction and gas chromatography–mass spec-

trometry of gemfibrozil, naproxen and mefenamic acid.

The liquid–liquid extraction technique was chosen, as it is

a simple and reliable technique for extraction of methyl

esters of acidic drugs in water. In these studies, deionized

water samples spiked with the appropriate amount of the

standard solution were used.

For 25 mL of samples, sensitive responses were

obtained using 100 lL of DMS, 0.25 mL of 0.1 M TBA-

HSO4, 10.0 g Na2SO4, pH 6.0 and 5 min derivatization

time in combination. Liquid–liquid extraction was per-

formed using 1 mL of n-hexane.

Detectable yields of methyl esters were achieved for the

analytes and identified on the basis of their mass spectra.

Calibration graphs for deionized water samples, monitored

using SIM mode were linear for the concentration range

2–100 lg/L. The detection limits were 0.18 lg/L for

gemfibrozil, 0.11 lg/L for naproxen and 0.13 lg/L for

mefenamic acid. The relative standard deviation at 25 lg/L

was between 1.64 and 7.65% (eight determinations). The

study of recovery for levels of 10.0 and 50.0 lg/L showed

recoveries between 85.4 and 106%. These values indicate

an adequate level of accuracy and precision for the new,

quick and easy methodology proposed utilizing aqueous

derivatization, liquid–liquid extraction and gas chroma-

tography–mass spectrometry.

To determine the kinetics of the degradation, plots of

concentration against time, starting day 1, were made and

an exponential regression analysis was then performed on

each data set in which the pharmaceuticals were degraded.

The rate constant, k, was calculated from the first-order rate

equation:

Ct ¼ C0e�kt

where Ct represents the concentration of pharmaceutical at

time t, C0 represents the initial concentration (both concen-

trations expressed in lg/L) and k is the rate constant in days-1.

Table 1 Experimental conditions in the study of persistence

Condition Water (2 l sample) Temperature

(�C)

Light Recipient

(glass)

1 Filtered 26–37 Sun Transparent

(closed)

2 Filtered 26–37 Darkness Amber

(closed)

3 Non-filtered 26–37 Sun Transparent

(closed)

4 Non-filtered 26–37 Sun Transparent

(open)

Environ Chem Lett (2011) 9:13–18 15

123

The confirmation of the order rate kinetics was derived from

the linearity of the plots of ln Ct against time. The regression

coefficients varied from 0.66 to 0.97, demonstrating good

correlation of the data and the subsequent establishment of a

pseudo first-order degradation kinetics (Figs. 2, 3).

The half-life (t1/2) was determined from the following

equation:

t1=2 ¼ ln 2=k:

The half-lives were calculated for the three acidic drugs

under the four experimental conditions in water samples of

Maracaibo Lake and Tule reservoir. The values of standard

deviations for rate constants were used to estimate

confidence intervals for half-lives at 95% confidence

level. The results are shown in Table 2.

The most rapid degradation rate was observed in non-

filtered open recipient samples exposed to sunlight, where

environmental factors such as sunlight, adsorption on par-

ticulates and biodegradation were present.

On the other hand, the half-lives for the pharmaceuticals

increased using filtered water in darkness where the effects

of solar photo degradation, adsorption on particulates and

volatilization of pharmaceuticals were not present, and

where only the activity of micro-organisms present in the

0

1

2

3

4

5

ln C

Time (days)

0

1

2

3

4

5

ln C

Time (days)

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160

0 20 40 60 80 100 120 140 160

0 20 40 60 80 100 120 140 160

ln C

Time (days)

(a)

(b)

(c)

Fig. 2 Pseudo first-order kinetic plot for a gemfibrozil, b naproxen

and c mefenamic acid in Tule reservoir water samples at four

experimental conditions: filtered/closed/sunlight (open circle), fil-

tered/closed/dark (filled circle), non-filtered/closed/sunlight (filledsquare) and non-filtered/open/sunlight (filled triangle)

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160

ln C

Time (days)

0

1

2

3

4

5

ln C

Time (days)

0

1

2

3

4

5

0 20 40 60 80 100 120 140 160

ln C

Time (days)

(a)

(b)

(c)

0 20 40 60 80 100 120 140 160

Fig. 3 Pseudo first-order kinetic plot for a gemfibrozil, b naproxen

and c mefenamic acid in Maracaibo Lake water samples at four

experimental conditions: filtered/closed/sunlight (open circle), fil-

tered/closed/dark (filled circle), non-filtered/closed/sunlight (filledsquare) and non-filtered/open/sunlight (filled triangle)

16 Environ Chem Lett (2011) 9:13–18

123

Lake and reservoir water and chemical degradation

intervened.

In the experiment using non-filtered water and open

recipients, the most rapidly degrading acidic drug was

naproxen (t1/2 = 10.2 ± 0.5 days for Maracaibo Lake and

14.6 ± 1.1 days for Tule reservoir), followed by mefe-

namic acid (t1/2 = 15.5 ± 2.9 days for Maracaibo Lake

and 17.5 ± 1.7 days for Tule reservoir), while the most

persistent pharmaceutical was gemfibrozil (t1/2 = 119.5 ±

15.2 days for Maracaibo Lake and 288.8 ± 61.3 days for

Tule reservoir).

The evaluation of the effect of photodegradation on the

persistence of acidic drugs was carried out comparing the

results of the experiments using filtered water in darkness

with filtered water exposed to sunlight. For the case of

Maracaibo Lake, the half-life times of gemfibrozil,

naproxen and mefenamic acid were significantly lower at

95% confidence level in the filtered water exposed to

sunlight with respect to filtered water in the darkness. This

is shown in Table 2, where for the condition of filtered

water exposed to sunlight, gemfibrozil, naproxen and

mefenamic acid had a t1/2 of 182.4 ± 27.6, 10.7 ± 0.7 and

27.0 ± 6.6 days, respectively, while for filtered water in

darkness the t1/2 were 277.3 ± 60.8, 385.0 ± 90.2 and

66.6 ± 13.9 days, respectively. Similarly, naproxen

showed a significant effect of photodegradation in the

water samples of Tule reservoir. This demonstrates that

sunlight plays an important paper in the degradation of the

studied acidic drugs.

To analyze the effect of adsorption on particulates on

degradation of the pharmaceuticals studied, the results of

the experiments of filtered and non-filtered water, exposed

to sunlight in both cases, were compared. It was observed

that in the majority of the cases, the t1/2 acidic pharma-

ceuticals of the filtered samples did not differ significantly

from that of the non-filtered samples (Table 2). This

demonstrates a low effect of particulate material present in

the non-filtered samples on the degradation of acidic drugs,

though the compounds provide potential for adsorption to

organic material as can be inferred from their log Kow

(4.77, 3.26 and 4.29 for gemfibrozil, naproxen and mefe-

namic acid, respectively). These results could be explained

on the basis that gemfibrozil, naproxen and mefenamic acid

with pKa values from 4.9 to 3.9 occur as ions at basic pH

and are, therefore, not readily adsorbed by particulate

material and remain in the aqueous phase.

In evaluating the effect of volatilization on persistence,

in general, similar t1/2 values were observed for acid

pharmaceuticals in the non-filtered water samples in closed

recipients as opposed to open recipients (both experimental

conditions were exposed to sunlight), which evidences

invaluable degradation effect by the volatilization mecha-

nism. These results agree with the physical–chemical

characteristics of the evaluated substances.

On the other hand, it must be remarked that the type of

water does not seem to be a significant factor in the per-

sistence of acidic drugs studied.

Conclusions

During 150 days, degradation kinetics of gemfibrozil,

naproxen and mefenamic acid in water samples of Mara-

caibo Lake and Tule reservoir were studied experimentally.

The degradation kinetic of acidic drugs follows a pseudo

first-order reaction. The results demonstrate that naproxen

is the most photolabile among the three acidic drugs

studied with half-lives from 10.2 ± 0.5 to 14.6 ± 1.3 days.

Mefenamic acid presented half-lives ranging from

15.5 ± 2.9 to 27.0 ± 6.6 days, while the most persistent

pharmaceutical was gemfibrozil (t1/2 = 119.5 ± 15.6 to

288.8 ± 61.3 days). These will aid in the understanding of

the fate of gemfibrozil, naproxen and mefenamic acid in

the aquatic environment.

Acknowledgments We thank ONCTI for financing this study.

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Condition Gemfibrozila Naproxena Mefenamic acida

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Reservoir water 239.0 ± 45.5 13.2 ± 0.9 23.0 ± 6.2

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Environ Chem Lett (2011) 9:13–18 17

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