Environ. Sci. Technol. 2004, 38, 5577-5583
Aqueous Chlorination Kinetics of Some Endocrine Disruptors M A R I E D E B O R D E , * ,‡ S Y L V I E R A B O U A N , † H E R V EÄ G A L L A R D , ‡ A N D BERNARD LEGUBE‡ Laboratoire de Chimie de l’Eau et de l’Environnement, UMR CNRS 6008, Ecole Supe´rieure d’Inge´nieurs de Poitiers 40, avenue du Recteur Pineau - 86022 Poitiers Cedex, France, and Laboratoire de Chimie Analytique, Faculte´ de Me´decine et Pharmacie - 34, rue du Jardin des Plantes, BP 199-86005 Poitiers Cedex, France
The aqueous chlorination kinetics of six endocrine disruptors (EDs: 4-n-nonylphenol, β-estradiol, estrone, estriol, 17R-ethinylestradiol, progesterone) were studied in the 3.50-12.00 pH range, at 20 ( 2 °C, in the presence of an excess of total chlorine. Under these conditions, all molecules with a phenolic group in their structure were rapidly oxidized by chlorine, whereas progesterone remained unchanged. In the first step, apparent kinetic rate constants were determined at various pH levels. Then each elementary reaction kinetic rate constant, i.e., the reaction of hypochlorous acid (HOCl) with ionized EDs and neutral EDs and an acid-catalyzed reaction of HOCl with neutral EDs, was calculated in the second step. The results showed that chlorination exhibits a second-order reaction rate. The rate constants for the acid-catalyzed reaction ranged from 3.02 × 104 M-1 s-1 (for 4-n-nonylphenol) to 1.822.62 × 105 M-1 s-1 (for hormones). The rate constants of HOCl reactions with ionized EDs were found to be equal to 7.5 × 104 M-1 s-1 (for 4-n-nonylphenol) and between 3.52 and 4.15 × 105 M-1 s-1 (for hormones), while the rate contants of HOCl with neutral EDs were much lower, i.e., between 1.31 M-1 s-1 (for 4-n-nonylphenol) and 3.744.82 M-1 s-1 (for hormones). At pH 7, the apparent-secondorder rate constants were calculated to range from 12.6 to 131.1 M-1 s-1. For a total chlorine concentration of 1 mg/L, the corresponding half-life times at pH 7 were about 65 min for 4-n-nonylphenol and 6-8 min for hormones.
Introduction An endocrine disruptor was defined as “an exogeneous substance that causes adverse health effects in an intact organism, or its progeny, consequent to changes in endocrine function” (1, 2). Numerous compounds have been reported to have these effects (2). Among them, (i) hormones naturally secreted by humans and animals, (ii) synthetic hormones mainly used for contraception or management of menstrual and menopausal disorders, and (iii) various chemical products (pesticides, phthalate plasticizers, alkylphenols, bisphenol A) are found in the environment as a result of industrial, agricultural, and sewage runoff. In wastewater treatment, natural and synthetic endocrine disruptors are subjected to * Corresponding author phone: 335 49 45 44 74; fax: 335 49 45 37 68; e-mail:
[email protected]. † Faculte ´ de Me´decine et Pharmacie. ‡ Laboratoire de Chimie de l’Eau et de l’Environnement. 10.1021/es040006e CCC: $27.50 Published on Web 09/22/2004
2004 American Chemical Society
a variety of treatment processes but are only partially removed. In a sewage treatment plant, Baronti et al. (3) documented an average removal efficiency for some estrogens of between 61% (for estrone) and 95% (for estriol). Many compounds are thus ultimately released into effluents and surface waters (4). Recently, the presence of endocrine disruptors in natural waters was clearly demonstrated (2, 5), i.e., concentrations have been detected in the nanogram/L range for hormones (6-8) and in the microgram/L range for chemical products (9, 10). The lowest reported concentrations of endocrine disruptors required to induce intersex are 10 ng/L for estradiol or estrone, 0.1 ng/L for ethinylestradiol (11), and 50 µg/L for nonylphenol (12). Therefore, in some cases, surface water concentrations of these disruptors can be near or equal to the lowest concentrations which were found to induce reproductive disturbances in male fish in controlled laboratory studies, so they might disrupt reproduction in wildlife. Surface water is often used as a source of drinking water. Little is currently known about the ultimate fate of endocrine disruptors, particularly in the disinfection steps. However, the authors of some recent studies on the effects of chlorination and ozonation on some endocrine disruptors considered that degradation is possible by such oxidation processes (13-16). In their studies on oxidation of pharmaceuticals during ozonation, Huber et al. (13) reported that 17R-ethinylestradiol was rapidly removed by ozone with a second-order rate constant of 3 × 106 M-1 s-1 at pH 7 and 20 °C. On the basis of chemical characteristics, they also estimated that, at pH 7 and 20 °C, the following compounds (with their corresponding second-order rate constants) would likely be removed: estradiol (106 M-1 s-1), 4-nonylphenol (1-10 × 106 M-1 s-1), bisphenol A (1-10 × 106 M-1 s-1), and testosterone (105 M-1 s-1). Few kinetic studies concerning chlorination have been conducted. However, Hu et al. (1416) studied products of aqueous chlorination of several endocrine disruptors. In these studies, rapid elimination of bisphenol A, 4-nonylphenol, and 17β-estradiol was reported at pH 7.5 and in the presence of 1.30 to 1.46 mg/L of chlorine: 80% removal of bisphenol A, 84% of 4-nonylphenol, and 100% of 17β-estradiol were achieved after 10 min reaction time. They also noted the production of numerous biologically active byproducts. The results of these studies, in addition to the well-known reactivity of phenolic compounds with chlorine (17, 18), suggest that other endocrine disruptors could also be degraded in this way. This study was designed to assess the potential of chlorination for the oxidation of six endocrine disruptors. These compounds, reported in Table 1, were chosen because they are commonly found in the environment. They include compounds from the 3 endocrine disruptor groups described earlier, i.e. chemical products (4-n-nonylphenol (NP)), natural estrogens and progestogens (β-estradiol (E2), estrone (E1), estriol (E3), and progesterone (P)), and synthetic hormones (17R-ethinylestradiol (EE2)). Chemically, natural and synthetic hormones have a common cyclopentan-o-perhydrophenanthrene ring (6). All endocrine disruptors studied, except progesterone, have a phenolic ring. The major aim of this study was to determine, for each compound, the rate constant of each elementary reaction in order to calculate the apparent rate constant at a given pH. In the first part, the chlorination kinetics were assessed in pure aqueous solution, between pH 3.50 and 12.00 and at 20 ( 2 °C. The reaction rate order and apparent rate constants of chlorination were determined for each molecule. In the second part, the rate constants of each elementary reaction were determined on VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Selected Endocrine Disruptors
the basis of the pH dependence of the rate constants and the speciation of chlorine and endocrine disruptors.
Experimental Section Standards and Reagents. All endocrine disruptors (NP, E2, E1, E3, EE2, P) were supplied by Aldrich (purity g 97%). Sodium hypochlorite solution was purchased from VWR International and was controlled to ensure equimolar concentrations of hypochlorite (ClO-) and chloride (Cl-) ions, with 14.0% (m/V) of active chlorine. All other reagents (Na2S2O3, NaOH, H2SO4, phosphate, etc.) were analytical grade or better and used without further purification. Solvents were HPLC grade. Ultrapurified water (18 MΩ cm) was obtained from Milli RO-Milli Q Millipore system. Analytical Methods. Each ED was analyzed by highperformance liquid chromatography (HPLC) using an automatic Waters 717 plus autosampler injector and a Waters 600E pump. Isocratic reversed-phase chromatography was 5578
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optimized for each ED using a C18 packed column (Hichrom spherisorb S50DS2, C.I.L. Cluzeau, 5 µm, 4.6 mm i.d., 25 cm long). A water-methanol mixture (55-90% of methanol, according to the studied ED) was used as the mobile phase, with a flow-rate of 0.8 mL/min. The compounds were detected either with a Waters 484 Tunable absorbance detector set at 217 nm for E1 or 250 nm for P or a Waters 474 scanning fluorescence detector with an excitation wavelength of λex ) 217 nm and emission wavelength of λem ) 300 nm for EE2, E2, E1 and λex ) 222 nm and λem ) 307 nm for NP. Commercial solution and 19 mM stock solution of chlorine were standardized by iodometry (19). In kinetic experiments, chlorine was analyzed by the N, N-diethyl-p-phenylenediamine (DPD) colorimetric method (20). pH was measured with a Tacussel LPH330T pH-meter equipped with a Radiometer Analytical combined electrode and previously calibrated with standard pH 4, 7, and 10 buffers.
FIGURE 1. Pseudo-first-order kinetic plot of estriol chlorination at 20 ( 2 °C, [HOCl]T ) 37.3 ( 0.4 µM and three pH levels. Symbols represent measured data, and the straight line is the linear regression. (Insert: estriol chlorination at pH 5.94 ( 0.02, 20 ( 2 °C, and various [HOCl]T). Kinetic Experiments. All experiments were performed in a batch reactor thermostated at 20 ( 2 °C, under pseudofirst-order kinetics conditions ([HOCl]T . 10 × [ED]T). Tested aqueous solutions (800 mL) were buffered using phosphate salts (10 mM) for a 6-9 pH range. For pH < 6 and pH > 9, pH values were preadjusted with H2SO4 and NaOH, respectively. The initial ED concentration was 1 µM. At least 35 µM of total chlorine was added. Chlorine variations were less than 5% under these conditions. The chlorine concentration was thus assumed to be constant during the kinetic experiments. Kinetic runs were initiated by injecting, under rapid mixing, an aliquot of a 19 mM chlorine solution. At constant time intervals, 3 mL of solution was withdrawn with a glass syringe and added to 4 mL HPLC vials containing 100 µL of sodium thiosulfate solution (100 g L-1) to quench the residual chlorine and stop the oxidation reaction. Samples were then analyzed using HPLC to determine the remaining ED concentration. When the EDs disappeared, the kinetic experiments were pursued until at least 50% ED consumption was achieved.
Results and Discussion Reaction Rate Order and Apparent Rate Constants of Chlorination. Among the studied EDs, only progesterone did not react with chlorine under our experimental conditions (i.e. 1 µM ED with 35 µM or 190 µM total chlorine for 30 min, at pH 3.80, 6.90, 7.00, and 8.25). For the other molecules, the chlorination experiments exhibited a pseudo-first-order dependence on the ED concentration, as demonstrated by the linear time-course plot of Ln([ED]T.t/[ED]T.0). Figure 1 presents an example of the pseudo-first-order kinetic plot of estriol chlorination at pH 3.50, 9.61, 10.53 and with a total chlorine concentration of 37.3 ( 0.4 µM. These plots were linear (r2 > 0.99) for > 85% reactions. The correlation coefficients were always superior to 0.96 for all reaction compounds. Therefore, EDs disappeared at a pseudo-firstorder rate with respect to the total ED concentration
d[ED]T ) kobs[ED]T v)dt
(1)
with kobs ) pseudo-first-order kinetic constant. The reaction order relative to chlorine was then examined for each ED by varying the chlorine concentration and monitoring the disappearance of EDs. The insert in Figure
1 presents the case of estriol at pH 5.94 ( 0.02, for a total chlorine range of 38.1-96.6 µM. This figure illustrates that the pseudo-first-order kinetic constant was proportional to the total chlorine concentration (r2 > 0.99). Similar results were obtained for the other EDs (r2 > 0.93). Therefore, the chlorination kinetics of NP, E3, E2, EE2, E1 is a second-order reaction, first-order relative to the free active chlorine concentration ([HOCl]T) and to the total ED concentration ([ED]T)
v)-
d[ED]T ) kapp[HOCl]T[ED]T dt
(2)
with
kapp ) kobs/[HOCl]T, second-order kinetic constant (3) The kapp values were determined for about 20 pH values for each ED. pH Dependence Profile and Rate Constants of Elementary Reactions. Figure 2 presents the pH profile of the apparent second-order rate constants for NP, EE2, and E1. This figure also gives the standard deviations for each rate constant. Similar profiles are obtained for E3 and E2. The results show that the second-order apparent rate constant was minimal at around pH 5 and maximal between pH 8 and 10, irrespective of the compound considered. Early kinetic studies of chlorination of phenols (17) and resorcinols (18) showed similar pH dependence profiles for second-order apparent rate constants, thus strongly suggesting that the phenolic ring in the estrogenic structure would control the reactivity of chlorine. Based on these earlier works, the pH profiles could be explained by taking the ionized (ED-) and neutral (ED) species of EDs (reaction 4) into account
ED / ED- + H+ KaED
(4)
[ED]T ) [ED] + [ED-]
(5)
with
and, the different species of chlorine (reactions 6 and 7):
Cl2 + H2O / HOCl + Cl- + H+ HOCl / ClO- + H+
KCl2
(6)
KaCl
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FIGURE 2. pH-dependence of the apparent-second-order rate constants of nonylphenol, ethinylestradiol, and estrone chlorination at 20 ( 2 °C. Symbols represent measured data and lines represent the model calculations. Under our experimental conditions, the sodium hypochlorite solution used was controlled to ensure that it was equimolar in hypochlorite and chloride. Based on reported equilibrium constant values for the hydrolysis of Cl2 (reaction 6) (21) and for [Cl-] ) [HOCl]T ) 3.5 × 10-5 M, the maximum Cl2 concentration was calculated to be very low at pH 3.5 (ca. 1 × 10-9 M). Further experiments showed that kapp increased linearly when the chloride concentration increased (results not presented), which could be explained by the reactivity of Cl2 with phenolic compounds. The results also showed that at low chloride concentrations (≈35 µM) kapp levels were not significantly different from kapp levels extrapolated for [Cl-] ) 0. Consequently, Cl2 species was considered to be negligible in our conditions, so only ClO- and HOCl species were taken into consideration (reaction 7). The concentration of free active chlorine ([HOCl]T) was as follows:
[HOCl]T ) [HOCl] + [ClO-]
(8)
The lower reactivity of EDs with chlorine at pH 5 could be explained by the slow reaction between HOCl and neutral ED species. However, the increase at more acidic values (pH < 4) could be explained by the catalytic effect of protons on the reaction between HOCl and neutral ED species (reactions 9 and 10).
ED + H2OCl+ f byproducts
k′1
(12)
with
k1 ) k′1/K1
(13)
Moreover, in the 5-10 pH range, the increased reactivity could be explained by the reaction between HOCl and ionized ED species (reaction 14).
ED- + HOCl f byproducts
k3
(14)
The maxima in the rate constant profiles might be explained if HOCl is the only active oxidant and the ClOreactivity is negligible. Considering that chlorination involves reactions 9, 10, and 14, the ED chlorination kinetics can be expressed as follows:
v)-
d[ED]T ) [HOCl](k1[ED][H+] + k2[ED] + k3[ED-]) dt (15)
Replacing [HOCl], [ED], and [ED-] by their expressions as ratios of [HOCl]T or [ED]T, the rate of EDs disappearance is
d[ED]T ) dt + (k1RED[H ] + k2RED + k3R ED-)RCl[HOCl]T[ED]T (16)
v)ED + HOCl + H+ f byproducts ED + HOCl f byproducts
k2
k1
(9) (10)
Following Rebenne et al. (18) who studied the chlorination of resorcinols, an alternative pathway involving H2OCl+ species could be proposed to explain reaction 9. If this mechanism applies in the case of EDs, reactions 11 and 12 would explain the increase in kapp at pH < 4
HOCl + H+ / H2OCl+ K1 5580
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with RCl ) hypochlorous acid fraction of total chlorine, with RED ) neutral ED fraction, and with RED- ) ionized ED fraction. Combining eqs 2 and 16, kapp is a function of pH
kapp )
k1[H+]3 + k2[H+]2 + k3[H+]KaED [H+]2 + [H+]KaED + [H+]KaCl + KaClKaED
(17)
The intrinsic constants k1, k2, and k3 were calculated by
TABLE 2. Second-Order Rate Constants Calculated for the ED Chlorination Mechanism (20 ( 2 °C, 3.5-12.0 pH Range) compounds
k′1 ((σ) (M-1 s-1)
k1 ((σ) (M-2 s-1)
k2 ((σ) (M-1 s-1)
k3 ((σ) (M-1 s-1)
4-n-nonylphenol 17R-ethinylestradiol β-estradiol estrone estriol
3.02 (0.34) ×107 2.04 (0.16) × 108 2.24 (0.17) × 108 2.62 (0.18) × 108 1.82 (0.15) × 108
3.02 (0.34) × 104 2.04 (0.16) × 105 2.24 (0.17) × 105 2.62 (0.18) × 105 1.82 (0.15) × 105
1.31 (0.13) 4.33 (0.53) 3.78 (0.42) 3.74 (0.57) 4.82 (0.50)
7.5 (0.27) × 104 3.52 (0.10) × 105 3.64 (0.11) × 105 4.15 (0.17) × 105 3.56 (0.12) × 105
FIGURE 3. Linear correlation between the log(k3) and molecule pKa for phenolic compounds monitored: (b) in this study, (O) by Gallard and Von Gunten (17). multiple regression of the experimental kapp data. Values of k1, k2, and k3 were obtained for the lowest quadratic mean deviation, defined as Σ((kapp exp - kapp theo)2/(kapp exp)2), where kapp exp and kapp theo represent the experimental and theoretical apparent second-order rate constants, respectively. The chlorine acidity constant used for the calculation was pKaCl ) 7.54 (22). ED pKa values were derived from the literature and are reported in Table 1. For E2 and E1, the pKa values were estimated according to Perrin (25) from the analogy of their structure with EE2 and E3. Figure 2 represents the experimental and the modeled pH profiles for NP, EE2, and E1. Similar correlations were obtained for E3 and E2. A good correlation between the experimental and modeled values was obtained. In the case of 4-n-nonylphenol, the theoretical curve seems to be slightly shifted toward the right as compared to the experimental values at pH > 7. For these pH values, the total reaction was mainly controlled by the reaction between HOCl and the ionized ED. This shift could thus be explained by uncertainty concerning the pKa value which would underestimate the ionized ED concentration. The best fit could be obtained for a pKa value of 10.4, whereas a value of 10.7 was given by Maguire and used in this study (23) and the theoretical value of 10.25 was calculated by Lipnick et al. (26). Table 2 shows, for each studied compound, the values of k1 (HOCl-acid-catalyzed reaction), k2 (HOCl reaction with ED), and k3 (HOCl reaction with ED-) as well as the k′1 value (H2OCl+ reaction with ED) from eq 13 with K1 ) 10-3, according to Rebenne et al. (18). As expected from their molecular structure, the 4 hormones that have a phenolic ring showed similar rate constants. The rate constants of chlorine with dissociated forms range from 3.52 × 105 to 4.15 × 105 M-1 s-1, which is about 10-fold higher than the corresponding rate constants of phenol (17) and 4-nnonylphenol. Therefore the chemical structure of these hormones molecules seems to enhance the chlorine reactiv-
ity. This suggested that there could be a similar reactivity for some other hormones that have a phenolic ring (e.g. equilin, equilenin, diethylstibestrol, etc.). As for other phenolic compounds (17, 18), the reactivity of chlorine with neutral ED species was 104 to 105 times weaker than with the ionized form. Attenuation of the electron-donor character of the hydroxyl group, i.e., change of the O- group in OH, thus decreases the reaction rates. For pH values between 4 and 6, ED chlorination is mainly controlled by the reaction between HOCl and undissociated species. At higher pH, the reaction between HOCl and the dissociated species is prevalent. For aromatic compounds, the substituent effect on the second-order rate constants with oxidants can be illustrated by linear free energy relationships using the Hammett-type correlation for example. Good correlations were also obtained for phenol chlorination using pKa values (17, 18). Figure 3 shows that in the case of NP, the plot of k3 vs pKa was in line with the correlation obtained with data from Gallard and Von Gunten (17) for phenolate species substituted in the ortho and para positions. Even though the 3 estrogens have a phenolic ring, they did not fit with the above correlation because they also have a much more complex structure (steroid moiety) than phenols, which probably affects the overall charge density and thus the reactivity with chlorine. To assess the effect of chlorine disinfection on EDs in drinking water treatment conditions, the half-life times of each compound were calculated for 3 chlorine concentrations (0.1, 0.5, and 1 mg/L) at pH 7. Table 3 gives the apparent second-order rate constant values and the corresponding half-life times for each studied ED at these chlorine concentrations. These results show that chlorination of drinking water prompted rapid elimination of the studied estrogens at a chlorine concentration of 1 mg/L (t1/2 ) 6-8 min). The reaction with nonylphenol was much slower, with a half-life time of 65 min at a 1 mg/L chlorine concentration. VOL. 38, NO. 21, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Apparent-Second-Order Rate Constants and Half-Life Times Calculated at pH 7, 20 °C for Total Chlorine Doses Ranging from 0.1 to 1 mg/L t1/2 (min)
compounds
kapp (M-1 s-1)
chlorine concn 0.1 mg/L
chlorine concn 0.5 mg/L
chlorine concn 1 mg/L
4-n-nonylphenol 17R-ethinylestradiol β-estradiol estrone estriol
12.6 112.1 115.2 131.1 113.6
651 73.2 71.2 62.6 72.2
130 14.6 14.2 12.5 14.4
65.1 7.3 7.1 6.3 7.2
In the presence of ozone, and under the same conditions, i.e., pH 7 at 20 °C, the second-order rate constants estimated for E2, EE2, and NP are about 106 M-1 s-1 (13), which is significantly higher than for chlorine. For an ozone concentration of 0.4-1 mg/L, the half-life times are < 0.1 s. Ozonation would therefore be much more efficient for removing the selected EDs. Even though high quantities of estrogens can be removed by biological treatments (3), municipal wastewaters are known to be an important source of endocrine disruptors in the environment. Typical wastewater chlorination dosages of 1 to 4 mg/L are used with a minimum contact time of 15 min. Assuming a theoretical constant chlorine concentration of 2 mg/L and a minimum 15 min contact time, the calculated estrogen removal efficiency is about 95% for a 7-8 pH range. For NP under the same conditions, the removal efficiency is only 27%, so a contact time of 140 min is necessary to achieve 95% removal efficiency. Chlorination of municipal wastewater thus not only helps to further remove such compounds but also participates in the discharge of chlorinated estrogens. To assess the overall efficiency of oxidation treatments, byproducts of parent EDs should be identified, and their reactivity with oxidants and their biological activity should be evaluated. The structure of chlorinated ED byproducts and their reactivity with chlorine can be estimated from the known reactivity of structurally related compounds. Considering the nonreactivity of progesterone and the pH profile of rate constants of other phenolic-like EDs, the phenol moiety is probably the chlorine reaction site. According to Burttschell et al. (27), phenol chlorination proceeds by the stepwise substitution of 2, 4, and 6 positions of the aromatic ring. In this case, two primary byproducts could be expected from the chlorination of the studied EDs: 2 monochloro-ED and 2,6 dichloro-ED (where OH is in position 1). For NP and E2, these two byproducts have been analyzed or suspected (15, 16). For phenol, each chlorine substitution causes a decrease of 1 order of magnitude for the elementary rate constant of phenolate with chlorine (17). Moreover, chlorine substitution makes the substituted phenol more acidic (i.e. decrease in pKa values with increasing chlorine substitution). The lower reactivity of chlorinated phenolates is thus compensated by their higher concentration at pH 6-8. Then the magnitudes of the apparent chlorination rates of phenols in the neutral pH range are similar to those of chlorophenols (28). If chlorinated ED byproducts have a similar behavior, at neutral pH, chlorinated EDs would likely be degraded with apparent rates similar to those of the parent EDs. For NP and E2, mono and dichloro byproducts were monitored using HPLC (15, 16). The results showed that these compounds are effectively removed by chlorine but can remain for more than 1-2 h in the presence of low residual chlorine concentrations. Finally, some recent studies showed that estrogenic activity is usually reduced as a result of chlorination (15, 16, 29). However, chlorination of a solution of E2 (10-7 M, pH 7) 5582
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with 1.5 mg/L of chlorine for 10 min contact time did not show a significant decrease in estrogenic activity (29). Under these chlorination conditions, residual E2 was calculated to be only 23% using the rate constants determined in our study. As shown by Hu et al. (15), these results confirm that the initial chlorinated byproducts of E2 exhibit estrogenic activity similar to that of their parent compounds. Further chlorination led to more drastic transformation of the molecule (ring opening), with a loss of estrogenic activity. Further identification, kinetic studies, and evaluation of the biological activity of chlorinated byproducts are thus required to model the fate of these compounds in the environment and assess the health impact of drinking tap water.
Acknowledgments The authors thank Prof. Marc Arnaudon, Mathematics section, University of Poitiers, for helpful discussions and advice for the statistical calculations.
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Received for review January 12, 2004. Revised manuscript received July 26, 2004. Accepted August 2, 2004. ES040006E
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