Baseline Toxicity (Narcosis) of Organic Chemicals ... - ACS Publications

Mar 30, 2002 - Baseline toxicity of a selection of industrial chemicals and pharmaceuticals is determined experimentally with a new in vitro test syst...
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Environ. Sci. Technol. 2002, 36, 1971-1979

Baseline Toxicity (Narcosis) of Organic Chemicals Determined by In Vitro Membrane Potential Measurements in Energy-Transducing Membranes B E A T E I . E S C H E R , * ,† R I K I . L . E G G E N , † ULRICH SCHREIBER,§ ZACHARIAH SCHREIBER,† ERIKA VYE,† BIANCA WISNER,† AND R E N EÄ P . S C H W A R Z E N B A C H † Swiss Federal Institute for Environmental Science and Technology (EAWAG), and Swiss Federal Institute of Technology (ETH), CH-8600 Du ¨ bendorf, Switzerland, Julius-von-Sachs Institute for Biosciences, University of Wu ¨ rzburg, D-97082 Wu ¨ rzburg, Germany

Baseline toxicity of a selection of industrial chemicals and pharmaceuticals is determined experimentally with a new in vitro test system (Kinspec) using membrane vesicles isolated from a photosynthetic bacterium, Rhodobacter sphaeroides. This test system is selective and more sensitive than other mechanistic test systems for baseline toxicity. The only concomitantly determined mechanism is uncoupling, which can be distinguished from baseline toxicity by pH-dependent measurements. Because the tests system contains only the target site for baseline toxicants, the biological membrane, effective target site concentrations can be directly related to observed effects by combining the in vitro test with membranewater partition experiments. No differences were found between the effective membrane concentrations of nonpolar and polar compounds, confirming the earlier hypothesis that differences in lethal body burdens are primarily caused by unequal distribution of the compounds between target and nontarget lipids and not by different mechanisms. A selection of pharmaceuticals with various specific modes of toxic action exhibited the same constant effective membrane concentrations as found for pure baseline toxicants. In mixtures of four to six components, the pharmaceuticals were concentration-additive with each other and with the pure baseline toxicants. A potential application of the proposed test system lies, therefore, in assessing the cumulative baseline toxicity in complex environmental mixtures.

Introduction Approximately 60% of all industrial chemicals exhibit no specific toxicity (1). Nevertheless, if such chemicals are persistent and bioaccumulating, they may pose a hazard to the environment by acting as baseline toxicants (2-6). * Corresponding author phone: 0041-1-823 5068; fax: 0041-1823 5471; e-mail: [email protected]. † Swiss Federal Institute for Environmental Science and Technology and Swiss Federal Institute of Technology. § University of Wu ¨ rzburg. 10.1021/es015844c CCC: $22.00 Published on Web 03/30/2002

 2002 American Chemical Society

Although baseline toxicity, which is also often referred to as narcosis, is the minimal toxicity that a compound may elicit, it is relevant for assessing the risk of complex mixtures in the environment. In particular, for mixtures of large numbers of compounds, all of which are present below the threshold level of specific toxicity, the underlying cumulative baseline toxicity might be determining the overall toxic effect (7-9). A method that is able to specifically measure baseline toxicity is therefore an important tool for the characterization of effects of single compounds and of complex environmental mixtures, in particular, within mechanism-based test batteries (10). In this paper, a novel in vitro method for the determination of baseline toxicity is presented that uses energy-transducing membrane vesicles (chromatophores) isolated from a photosynthetic bacterium, Rhodobacter sphaeroides. The method described is one application of a modular test system that was developed to quantitatively evaluate different toxic mechanisms on energy-transduction, including inhibition of the electron-transfer chain (11), uncoupling (11, 12), inhibition of adenosine triphosphate (ATP) synthesis (13), and nonspecific membrane toxicity of surfactants (14). The partitioning of baseline toxicants into the energy-transducing membrane causes the membrane to lose its insulating capacity and to become permeable to small ions. This disturbance of the membrane integrity can be determined quantitatively by following the membrane potential through measurement of the absorbance change of the carotenoids in the chromatophores, which is directly proportional to the membrane potential (15). The membrane potential can also be destroyed through uncoupling (11, 12). However, the uncoupling effect is strongly pH-dependent (16), while baseline toxicity is independent of pH. pHdependence is the selectivity criterion to distinguish between uncoupling and baseline toxicity (17). The target sites of hydrophobic toxicants are biological membranes, where nonspecific disturbance of membrane integrity and functioning occur (3). Because the test system contains only biological membranes and no nontarget tissue like storage lipids, DNA, or soluble proteins, it is also suitable to investigate the question of whether there is a difference in effective membrane concentrations of nonpolar and polar toxicants. For a long time, it was commonly accepted that nonpolar and polar chemicals should be classified in two different groups of mode of action, nonpolar and polar narcosis (18, 19), but more recent work suggests that such a distinction is not justified (3, 20-22). The earlier distinction as two modes of action was based on several observations. (i) Quantitative structure-activity relationships (QSAR) of baseline toxicants with the octanol-water partition coefficient (log Kow) as a physicochemical descriptor yielded two separate lines for nonpolar and polar compounds (18). However, octanol is not an optimal surrogate to model biological membranes. Better surrogates are membrane vesicles made up of phospholipid bilayers, so-called liposomes (23-27). When the liposome-water partition coefficient (log Klipw) is used as descriptor in QSARs of toxicity, nonpolar and polar compounds fall on one regression line (21). (ii) Total concentrations in fish at the time of death, socalled lethal body burdens, were smaller for polar compounds than for nonpolar compounds (3, 28-30). However, modeling of the internal distribution of the chemicals within a fish or other aquatic organisms indicated that the concentration in the membrane (target lipid) is uniform for nonpolar and polar compounds while the concentration in storage lipids VOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(nontarget lipids) is much smaller for polar than for nonpolar compounds (30). The proposed test system offers the opportunity to measure directly the effects in the target site and can therefore be used to experimentally verify these distribution models. (iii) Mixture toxicity studies (31) as well as behavioral and physiological studies (32-34) also gave evidence of a distinction of nonpolar and polar narcotic mechanisms. The results of the present study will also be discussed in the light of these earlier findings. In the work presented in this paper, the baseline toxicity of eight polar and eight nonpolar compounds has been evaluated. This test set has been selected as a sample representing wide diversity from a large database using principle component analysis (35) and has already been used in detailed investigations of membrane-water partitioning (23, 24), aquatic toxicity (36, 37), and QSAR studies ( 21, 38). In addition, we evaluated the baseline effects of 10 pharmaceuticals, most of which have been detected in the aquatic environment (39, 40). Each of these pharmaceuticals acts according to one or more specific modes of toxic action. In the environment, these chemicals are most likely present below their threshold for specific effects, but they may contribute to the cumulative baseline toxicity (8, 41). A series of mixture experiments was, therefore, also conducted with the goal to test the hypothesis of additivity of membrane concentrations of baseline toxicants.

Material and Methods Chemicals. The compounds were purchased from the following companies (purity in parentheses): p-xylene (g99.9%), nitrobenzene (g99.9%), 3-nitroaniline (g99%), 2,4,5-trichloroaniline (g98%), and 2-phenylphenol (g99%) from Riedel-de Ha¨en (Seelze, Germany); 1,3,5-trichlorobenzene (g98%), 2-butoxyethanol (g99.8%), 3-pentanol (g99.5%), chlorobenzene (g99.5%), 1-butanol (UV-spectroscopy quality), 1-hexanol (g99.5%), and 4-n-pentylphenol (g99%) from Fluka (Buchs, Switzerland); 2-nitrotoluene (g99%), 2-allylphenol (g98%), and 4-chloro-3-methylphenol (g99%) from Aldrich (Buchs, Switzerland); acetaminophen (g99.7%), clofibrate (100%), diclofenac (100%), ethinylestradiol (g98%), ibuprofen (100%), and propranolol (100%) from Sigma (Buchs, Switzerland); 2,4,5-trichlorotoluene (neat) from Supelco (Bellefonte, PA); and diazepam (100%) from Hoffmann-La Roche (Basel, Switzerland). 3-(N-Morpholino)propanesulfonic acid buffer (MOPS; pKa ) 7.2) was obtained (Fluka). The inhibitors antimycin and myxothiazol were purchased from Sigma. Time-Resolved Photometry. The kinetic single-beam spectrophotometer (Kinspec I) developed for the concomitant measurement of uncoupling and the inhibitory effect on the electron-transfer chain (11) can also be used for the determination of baseline toxicity. Additionally, a second, newly developed photometer (Kinspec II) exclusively measures the membrane potential (i.e., uncoupling and baseline toxicity). Major advantages of this new instrument in comparison to the original Kinspec I are a higher signal/noise ratio, a more compact and robust design, as well as lower costs. Kinspec II was developed on the basis of a pulse-amplitudemodulated chlorophyll fluorometer PAM-101 (Heinz Walz GmbH, Effeltrich, Germany) in conjunction with an emitterdetector unit ED-101US (Heinz Walz GmbH), which was modified for the purpose of split-beam 505 nm absorbance measurements. The new system displays similar properties as previously described single-beam photometers (42, 43), which were developed for measurements of P700 and P515 absorbance changes in isolated chloroplasts. In the new system, a light-emitting diode (LED) with peak emission at 505 nm (type NSPE 590S; Nichia, Tokyo, Japan) serves as source of high-frequency pulse-modulated measuring light 1972

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(100 kHz). The bandwidth of the LED light (30 nm halfbandwidth) is further narrowed by an optical interference filter with peak transmission at 505 nm (half-bandwidth 8 nm; Omega Optical Inc., Brattleborro, VT) before it is divided into two beams by a beam splitter. Part of the measuring light is guided via a 10 × 10 × 100 mm quartz rod to the 10 × 10 mm cuvette containing the sample, while another part is directed via a flexible light guide to a reference detector. The measuring light transmitted by the sample is guided via a second 10 × 10 × 100 mm quartz rod to the detector unit of the ED-101US. This unit was modified for split-beam measurements; the reference detector, which consists of the same type of 10 × 10 mm PIN photodiode as the transmittance detector (type S 1723; Hamamatsu, Japan), is mounted within the same detector unit. Both detectors are protected against the long wavelength “actinic light” (see the following discussion) with the help of short-pass filters (DT Cyan, Balzers, Liechtenstein). A conical metal pin can be moved in and out of the reference beam such that transmittance and reference signals are equal. In this way, the reference signal is used for compensation of the transmittance signal. Hence, unavoidable fluctuations and drifts of the measuring light LED, which are common to both signals, are effectively eliminated in the difference signal. This signal is fed into the detector input of the PAM-101, where a high gain setting can be applied in order to obtain high level difference signals compatible with the A/D converter. “Actinic light”, for driving photosynthetic electron transport in the chromatophores, was applied either with a xenon discharge lamp (type XE-ST, Heinz Walz GmbH) or a high power LED lamp (in conjunction with a HPL-L875 LED-array-cone; Heinz Walz GmbH). The XE-ST provides flashes with 1 µs half-peak width. The flashes were filtered via a near-infrared color glass filter (3 mm RG 780, Schott Glaswerke, Mainz, Germany). Flash intensity incident on the sample amounted to ca. 1016 quanta cm-2 flash-1, which was saturating. The HPL-L875 provided saturating light pulses (peak wavelength 875 nm, 35 nm halfbandwidth) of 1 ms duration, with the intensity amounting to ca. 2 µmol quanta cm-2 s-1. Control of the instrument, data acquisition, and data analysis were performed with Labview, version 6.0. (National Instruments, Austin, TX). Determination of Baseline Toxicity. The majority of the baseline toxicants were measured with the Kinspec I (11); all measurements with pharmaceuticals and some baseline toxicants as well as the mixture experiments were performed with Kinspec II. Although the extent and the kinetics of the membrane potential differed between the two systems, the resulting toxicant’s effect was equivalent, as a comparative validation of the new test system for several baseline toxicants and uncouplers in both test systems showed (data not shown). All measurements were performed in the presence of a redox buffer. In earlier studies, the experiments had been performed with redox control under an argon stream (11, 14). This adaptation of the method became necessary because of the higher volatility of the test chemicals as compared to uncouplers, such as substituted phenols, and surfactants. Consequently, the system had to be closed and oxygen had to be allowed to be present. Although this adaptation made the performance of an experiment much easier and faster, it also has disadvantages because of the limited stability of the redox buffer, which becomes reductively depleted causing the ambient redox potential to decrease approximately 2 h after the beginning of the measurements. Membrane vesicles (chromatophores) were prepared and characterized as described previously (11). For a given assay, frozen chromatophores were thawed and diluted to a concentration of approximately 150 nM of reaction center in 2-5 mL of buffer containing 50 mM MOPS, 100 mM KCl, and 1 mM each of succinate and fumarate at pH 7 and an ambient redox potential of approximately 30 mV in a

precision cell (1 × 1 × 6 cm) made of special optical glass closed tightly by a screw cap with septum. Hence, headspace varied from 1 mL (for the more volatile compounds) to 4 mL, to keep loss to the headspace below 5%. For example, up to 3% of 1,3,5-trichlorobenzene, the most volatile compound in the test set, and 5% of p-xylene, a less volatile but also less hydrophobic compound, were present in the headspace according to three-phase equilibrium partitioning calculations (44). All other headspace concentrations were calculated to be lower. All additions were made by injection through the septum. The chromatophore suspension was equilibrated in the darkness with stirring for 30 min. Then 10 µM of antimycin and 4 µM of myxothiazol (final concentrations, dissolved in a total of 10 µL of ethanol) were injected through the septum and equilibration was continued for another 30 min. Antimycin and myxothiazol inhibit the cytochrome bc1 complex of the electron-transfer chain. They were added to avoid any interference due to possible inhibitory activity of the cyt bc1 complex and to exclude the slow phase of build-up of the membrane potential that could interfere with the decay kinetics (11). Then, a control measurement of the absorbance change at 505 nm (∆A505) was performed followed by incremental addition of toxicant dissolved in buffer, ethanol, or DMSO (only for the four most hydrophobic compounds) and repetitive measurements of ∆A505. A maximum of 50 µL of ethanol or 35 µL of DMSO were added, which by themselves at these concentrations did not exhibit any effect. The suspension was equilibrated after each addition for 10 min. A measurement consisted of the following routine: (i) stirrer off and measuring light on, (ii) 5 s pause, (iii) data read for 20 ms before flash, (iv) flash, (v) data read for 150 ms after flash, (vi) measuring light off and stirrer on, and (vii) 1 min dark equilibration. Each measurement was repeated 4 times. For two compounds, 2,4,5-trichlorotoluene and chlorobenzene, we did not obtain an analyzable concentrationeffect curve. For these two compounds and also for 1,3,5trichlorobenzene and p-xylene (i.e., for the four most hydrophobic and most volatile compounds studied), a different experimental design was used. The approximate effective concentration was predicted from a QSAR of the already obtained data. Consequently, an experiment with this single concentration, but with an hour of equilibration after addition of the compound, was performed. The results did not differ between the two experimental designs. This comparison assured that equilibration time did not produce any artifacts, even for the most hydrophobic compounds. For the mixture experiments, five separate 5-mL vials were prepared as controls and five vials, each with the mixture components in a total of 30 µL of DMSO, were added 30 min after the initial equilibration started. After another 30 min of equilibration, the measurement was performed. The n components of a given mixture were present at 1/n of the effective concentrations (EC) (for definition, see the following section). Liposome-Water Distribution Ratios. Liposome-water distribution ratios at pH 7, log Dlipw (pH 7) were determined for clofibrate, diazepam, and ethinylestradiol with the Transil method (45). Lipid bilayers consisted of egg yolk phosphatidyl choline. Experimental details are described in ref 45.

Results and Discussion Data Analysis. Baseline toxicants exhibit two types of effects on the membrane potential (Figure 1A); first, the build-up of the membrane potential following a single turnover flash is lowered (i.e., the maximum absorbance change at 505 nm is inhibited), and second, the relaxation of the membrane potential after it has reached its maximum is accelerated. The effect on the build-up of the membrane potential is less sensitive to toxicants and less reproducible than the effect

FIGURE 1. (A) Typical raw data of experiments with Kinspec II shown for one control absorption trace and one absorption trace after addition of 7.8 mM nitrobenzene. The two effects of baseline toxicants are indicated with arrows in the figure. In the inset, the derivation of the first-order decay rate constant kobs (in the example kobs ) 2.9 s-1) is illustrated. (B) Concentration-effect curve for butoxyethanol. The symbols represent the experimental data points; error bars are standard deviations of the measurements. The line is the fit of the data to the log-logistic model (eq 1). on the decay kinetics (14). Therefore, the rate of decay of the membrane potential is used as an indicator of the nonspecific disturbance of the membrane integrity. The decay of the membrane potential is accelerated in the presence of baseline toxicants, and this acceleration of decay can be quantified by a first-order decay rate constant kobs (Figure 1A, inset), whose derivation is presented in ref 11. The concentration-effect curves of the baseline toxicants of the present study appeared like the lower part of a sigmoidal curve as is depicted for butoxyethanol in Figure 1B. Typical experiments were conducted up to a kobs ≈ 5 s-1, but the less hydrophobic compounds often reached only kobs ≈ 1 s-1. The more hydrophilic compounds required very high aqueous concentrations, and sometimes precipitation occurred. Unlike for uncouplers, for which a mechanistic model determines the mathematical function of the concentrationeffect curve, there is no evident model to describe the concentration-effect curves for baseline toxicants in the VOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Membrane-Water Distribution Ratios and Effect Concentrations for the Test Set of Baseline Toxicants

1,3,5-trichlorobenzene 2-butoxyethanol 2,4,5-trichlorotoluene 3-pentanol chlorobenzene p-xylene 1-butanol 1-hexanol 2-nitrotoluene nitrobenzene 3-nitroaniline 2,4,5-trichloroaniline 4-n-pentylphenol 2-allylphenol 4-chloro-3-methylphenol 2-phenylphenol

classa

log Dlipwb

w log (1/EC0.5 (M))

lower 95% CIc

upper 95% CIc

lip EC0.5 (mmol/kglip)

lower 95% CLc

upper 95% CLc

dfd

sre

np np np np np np np np p p p p p p p p

3.95 0.60 4.77 1.00 2.81 2.98 0.45 1.91 2.41 2.01 2.17 4.16 4.31 3.06 3.34 3.46

4.06 1.40 5.21 1.59 2.93 3.08 1.21 2.40 3.22 2.48 2.56 4.57 5.30 3.68 4.15 4.37

4.32 1.46 nd 1.68 nd 3.42 1.26 2.47 3.33 2.55 2.64 4.62 5.43 3.78 4.25 4.43

3.84 1.33

770 158 361 251 756 794 172 320 154 340 409 387 102 237 153 123

429 136

1295 183

9 24

0.179 0.405

204

309

17

0.532

364 154 278 121 291 339 345 75 190 124 108

1704 190 366 192 393 493 438 137 301 187 139

4 14 16 20 22 21 28 15 17 17 17

1.522 0.265 0.607 0.682 0.556 0.567 0.311 0.667 0.464 0.408 0.323

1.50 2.75 1.17 2.35 3.13 2.42 2.48 4.52 5.17 3.58 4.07 4.32

a Former classification: np ) nonpolar, p ) polar. b From ref 23. c CL ) confidence limit; nd ) not determined. d Degrees of freedom, here corresponding to number of experimental data points in one concentration-effect curve minus one. e Standard deviation of the residuals of the fit of the concentration-effect curve (eq 1) sr ) x(sum of squares/ degrees of freedom).

Kinspec system. For surfactants, which also act as baseline toxicants, the concentration-effect curves were biphasic, most likely due to the presence of micelles at higher concentrations, and the EC was not deduced from an analytical form of the concentration-effect curve but from a linear interpolation between the nearest measuring points in the curve (14). Despite its simplicity, the disadvantages of this approach are very large confidence intervals of the interpolated concentrations. Effects that are influenced by many independent factors, each of which is ideally normally distributed, often show a log-normal and log-logistic distribution of responses. Such a behavior results in a sigmoidal concentration-effect curve. Equation 1 represents the log-logistic concentration-effect curve

kobs )

kmax obs 1 + e(-b1-b2 log C)

(1)

where log C refers to the decadic logarithm of the concentration (Cw for aqueous concentration in units of M and Clip for lipid-based membrane concentration in units of mol‚kglip-1), kmax obs is the upper limit of effect, and b1 and b2 are fitting parameters for the slope and location of the concentration-effect curve. However, due to the experimental limits of the Kinspec system, we cannot reach high enough effects to see saturation, even though in principle the effect cannot rise to indefinite but has to have a certain limit. It was observed that activity decreased or leveled off after approximately 2 h of measurement, but this is an artifact of the test system, presumably due to depletion of the redox buffer or other unstable components of the redox system. However, for uncouplers that are effective at much lower concentrations in the membranes, a saturation of the concentration-effect curve was observed at kobs values of approximately 20 s-1 (unpublished data from our lab). We -1 therefore used a kmax obs ) 20 s . A sensitivity analysis showed -1 but that results were equal between kmax obs ) 20 and 50 s quality of the fit decreased at kmax values below 15 s-1. obs Alternatively, a probit model, which is based on the lognormal distribution and differs from the logistic distribution by slightly narrower tails, yielded slightly worse quality of fits (data not shown). Because there is no upper limit of effect, an endpoint effective concentration cannot simply be defined as 50% of a maximum effect, as it is usually done in ecotoxicology. The 1974

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effective concentration EC was derived from the toxic endpoint kobs ) 0.5 s-1. In earlier studies, a toxic endpoint representing a half-time of decay of 500 ms (i.e., kobs ) 1.38 s-1) was chosen (12, 14). In this study, a lower endpoint was chosen for two reasons. First, the aqueous concentrations required to obtain the higher endpoint are very large for the less hydrophobic compounds. Second, the membrane concentrations for the new endpoint of kobs ) 0.5 s-1 are the same magnitude as toxic membrane concentrations in aquatic organisms (see the following discussion). The effective concentration of the compounds in the membrane lipids, EClip 0.5, and the free aqueous effective concentration, ECw , 0.5 were calculated from the nominal effective concentration, ECtot 0.5, which is defined as the total amount of compound per total volume, and the liposomewater distribution ratio at pH 7, Dlipw with eqs 2 and 3 (Table 1). lip ) EC0.5

tot EC0.5

1 Dlipw

w EC0.5 )

(2)

+ [lip]

lip EC0.5 Dlipw

(3)

where [lip] is the mass concentration of the membrane lipids in the assay in units of kglip‚L-1. Dlipw has the units of L‚kglip-1 and corresponds to the liposome-water partition coefficients Klipw for the neutral compounds. For the weak acids and bases, Dlipw was calculated from the liposome-water partition coefficients of neutral and charged species and the acidity constants with the equations derived in ref 46 or directly measured at pH 7 (Table 1). For the conversion from nominal concentrations to concentrations in the membrane, it was assumed that sorption to the proteins of the chromatophore membranes and to the soluble cytochromes was negligible. This assumption had been confirmed earlier for a series chlorophenols with a log Dlipw range of 2.5-5 at pH 7 (46) and should also hold for the compounds investigated here. Loss due to sorption to the walls of the cuvette was minimized because only glass was in contact with the solution, and the surfaceto-volume ratio was kept small. The headspace was kept to a minimum for the more volatile compounds (see Materials and Methods section).

TABLE 2. Effective Concentrations in the Membrane, EClip 0.5 (mmol/kglip)

all baseline toxicants nonpolar chemicals polar chemicals pharmaceuticals

lip FIGURE 2. QSAR of (A) ECw 0.5 and (B) EC0.5 as a function of Dlipw. The line represents (A) a linear regression through all data of classical baseline toxicants (eq 5) or (B) the constant EClip 0.5 of 286 mmol/kglip; (2) nonpolar compounds, (1) polar compounds, (]) pharmaceuticals.

Comparison of the Kinspec Results with Acute Toxicity lip Data. The effective concentrations ECw 0.5 and EC0.5 of the 16 selected nonpolar and polar compounds are listed in Table 1. The results obtained in the Kinspec system correspond well to acute toxicity data. For instance, the 96-h LC50 values (lethal concentration for 50% of the organisms) for Poecilia reticulata are linearly correlated to ECw 0.5 through eq 4. w log (1/EC0.5 ) ) (1.12 ( 0.06) log (1/LC50) - (0.83 ( 0.23)

r2 ) 0.963,

n ) 16, F ) 361,

s ) 24

(4)

The slope of this regression is almost 1. The fish are up to 5 times more sensitive to the less hydrophobic compounds than the Kinspec system, while the sensitivity is approximately equal in both test systems for the more hydrophobic compounds. Overall, the sensitivity of the Kinspec system is in the same concentration range as for acute toxicity studies on aquatic organisms. Nonpolar versus Polar Narcosis. A single regression line (eq 5) is obtained when plotting the log ECw 0.5 values against log Dlipw (Figure 2A). w log (1/EC0.5 ) ) (0.93 ( 0.06) log Dlipw + (0.66 ( 0.17)

r2 ) 0.952,

n ) 16, F ) 280,

s ) 24

median

mean

lower 95% CL

upper 95% CL

286 341 196 189

343 448 238 192

218 215 135 126

468 680 351 259

distinction between nonpolar and polar narcosis is usually not observed (18). Despite the precaution to avoid losses to headspace and other components of the test system, it is possible that the variability of the calculated EClip 0.5 is partially due to experimental artifacts. If the two groups of polar and nonpolar narcotics are analyzed independently, the EClip 0.5 values of the nonpolar compounds are slightly higher than those of polar compounds, but the difference is not statistically significant. An unpaired t test showed that both polar and nonpolar test sets of compounds were not significantly different (P ) 0.07). Our results, determined with a completely independent method, further validate the hypothesis of Vaes et al. that the difference between nonpolar and polar narcotics is an artifact of the inappropriate choice of the QSAR descriptor (21). This is also corroborated by an analysis of the data using the solvation parameter model (22, 47). This model describes the partitioning between two phases (in our case, between water and octanol, liposomes, or biological organisms) with a cavity model of solvation. This model has already been applied to describe toxicity data (22, 48). A linear solvation energy relationship (LSER) links solute properties to system properties characteristic for the partitioning process. Solute properties relevant for the partitioning process include the molar volume of the compound, Vx (cm3‚mol-1), which is important for the size of the cavity to be created in the (bio)partitioning medium. Parameters that describe the solute-solvent interaction include the air/hexadecane partition constant Kair/hexadecane (m3/m3), which describes the van der Waals interactions, the solute’s dipolarity/polarizability π2H, the solute’s effective hydrogen-bond acidity ∑R2H, and the solute’s effective hydrogen-bond basicity ∑β2o. Analysis of the solute parameters of our test set (data from refs 22, 49, 50, and 51, listed in Table 1, Supporting Information) revealed that the H-bond acidity and basicity are not suitable discriminators for nonpolar and polar compound, although several studies have used such parameters to bring QSARs of polar and nonpolar narcotics together (52). The only discriminator between the two sets is the π2H descriptor. The polar narcotics have significantly higher π2H values than the nonpolar narcotics. Interestingly, the dipolarity/polarizability parameter π2H has a significant coefficient only in LSERs for Kow (50) but not in the equations for Dlipw and the aquatic toxicity (22). Hence, Kow is not representative for biopartitioning and, by deduction, neither is π2H. The LSER for the Kinspec data and for Dlipw and Kow using all five descriptors are given in the Supporting Information. After omission of the insignificant descriptors dipolarity/ polarizability π2H and Kair/hexadecane, the following threeparameter LSER results (eq 6).

(5)

The effective membrane concentration, EClip 0.5, is fairly constant (Figure 2B) and amounts to approximately 300 mmol‚kglip-1 (for statistics, see Table 2). Three values of the nonpolar narcotics are somewhat higher than all other values, but these values also have very wide confidence intervals. In addition, the outliers of the regression appear for more hydrophobic compounds (log Kow g 2.7), for which a

w log (1/EC0.5 ) ) (4.75 ( 0.38)Vx + (1.04 ( 0.36)

(-3.89 ( 0.37) 2

r ) 0.969,



o

∑R

H 2

+

β2 + (-0.80 ( 0.41)

n ) 16, F ) 125,

s ) 24

(6)

This equation has solvent parameters (regression coefficients) very similar to the LSER for Dlipw (eq 7), confirming that the VOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Effective membrane concentrations of polar (0) and nonpolar (]) compounds in algae, daphnia, fish (data and model from ref 17), and in the Kinspec system. target site of effect in chromatophores has very similar properties to the lipid bilayer in liposomes.

log Dlipw ) (4.33 ( 0.38)Vx + (0.68 ( 0.36) (-4.50 ( 0.37) r2 ) 0.970,

∑β

o 2

n ) 16, F ) 128,

∑R

H 2

+

+ (-0.61 ( 0.41) s ) 25

(7)

H-donor functions slightly increase the toxicity, while Hacceptor functions strongly decrease the toxicity. Overall, the cavity term dominates the toxicity (i.e., the transfer from the aqueous phase to the target site is the dominant process responsible for baseline toxicity). These results are in agreement with LSER for acute toxicity toward fish, daphnids, Vibrio fischeri, and other short-term bioassays (22, 48). The relatively constant effective membrane concentrations in the Kinspec system are also consistent with the hypothesis of van Wezel et al. that the difference in internal effect concentrations between nonpolar and polar compounds is due to uneven distribution into target and nontarget lipid tissue (30). We have reevaluated the threecompartment equilibrium-partitioning model proposed by van Wezel et al. using liposomes as surrogates for the target lipids (i.e., the biological membrane) and hexane as surrogate for neutral storage lipids (17). For fish, 95% of the wet body weight was assumed to be aqueous phase and the remaining 5% lipids. The lipid compartment was split into 3.75% storage lipids and 1.25% membrane lipids (30). Partitioning to other material (e.g., proteins) was neglected. For daphnia, lipid contents of 0.69% membrane lipids and 0.89% neutral lipids were assumed (53). For algae, no storage lipids were included in the model. All data and the mass balance equations are reported in ref 17. The modeled storage lipid concentrations varied over several orders of magnitude. The polar narcotics had significantly smaller storage lipid concentrations than the nonpolar narcotics. In contrast, the effective membrane concentrations were indistinguishable between nonpolar and polar compounds and did not vary much between the Kinspec system and the three aquatic organisms, algae, daphnia, and fish (Figure 3) (17). Note, however, that the endpoints in the four tests systems are not equivalent and therefore not directly comparable. One remaining inconsistency between results from the literature and the present study is the behavioral and physiological assessment of the responses of fish to baseline toxicants (32-34, 54-56). A set of respiratory-cardiovascular responses was used to define so-called “fish acute toxicity syndromes”. The response set determined with rainbow trout, which were analyzed with principal component analysis, identified clear differences between the two sets of compounds. While nonpolar narcotics lead to hypoactivity, the 1976

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fish reacted to polar narcotics with hyperactivity. It can be speculated that mechanisms and effects other than baseline toxicity or possibly even differences in the toxicokinetic phase are responsible for the discrepancy between these studies on fish behavior and physiology and our in vitro studies on isolated membranes. Comparison of the Kinspec Method with Other In Vitro Tests for Determining Baseline Toxicity. A widely applied test for membrane damage is the neutral red assay, which has already been used in mode-of-action-based ecotoxicological test batteries (1, 10). This test was initially developed for mammalian cells but has been applied also to fish cell lines (57). The principle underlying the test is that dead or damaged cells cannot retain the cationic dye, neutral red, which was accumulated passively in the lysosomes prior to the toxicity experiment. The measure of cytotoxicity, the NR50-value, is defined as the concentration of toxicant that induced a 50% decrease of absorbance of the retained dye, which is measured photometrically after digestion of the cells. The ratio of 24-h NR50 in mouse fibroblasts cells to the ECw 0.5 values from the Kinspec system is 7 for 1,3,5-trichlorobenzene, 5 for chlorobenzene, and 19 for 2,4,5-trichloroaniline (57). NR50 data determined with goldfish scale cells for a series of chlorophenols, which act as baseline toxicants or uncouplers, was 3-60 times less sensitive than the corresponding ECw 0.5 from the Kinspec system (58). Only two pharmaceuticals, propranolol and acetaminophen, had approximately equal effective concentrations in both test systems (59). Overall, it can be concluded that the neutral red assay is less sensitive than the Kinspec test. Baseline toxicity might also be caused by disturbance of membrane-bound proteins (2). However, the activity of the Na+/K+ ATPase in erythrocyte ghosts (membrane of blood cells) was not affected up to membrane concentrations of 110 to 1100 mmol/kglip (60). Therefore, measurement of the activity of this membrane-bound enzyme is not a good tool for the assessment of baseline membrane toxicity. Ligandgated ion channels are the most important target sites for narcotics in the mammalian nervous system (4), but none of the wide range of available experimental techniques in this field has been systematically evaluated for its application in effect assessment of environmental pollutants. The mitochondrial membrane potential has also been used as indicator of cytotoxicity (61). Measurements were performed with fluorescent dyes, whose fluorescence is influenced by the membrane potential. This method is conceptually equivalent to the Kinspec method, but results are qualitative only and the test is quite insensitive, thus requiring high concentrations. Baseline Toxicity of the Pharmaceuticals. Out of the test set of 10 pharmaceuticals (for names and physicochemical descriptor, see Table 3), three of the less hydrophobic compounds, caffeine (log Kow ) -0.07 (62)), sulfamethoxazole (log Kow ) 0.89 (62)), and amoxicillin (log Kow ) 0.87 (63)), did not show an effect in the Kinspec system up to nominal concentrations of 70, 0.5, and 3 mM, respectively. All other pharmaceuticals clearly exhibited baseline toxicity (Table 3). Their ECw 0.5 values fit perfectly into the QSAR equation of baseline toxicity (eq 5 and Figure 2A), and their EClip 0.5 values have a median of approximately 200 mmol/kglip (for statistics, see Table 2) and do not differ statistically from the membrane concentrations of the other test set of baseline toxicants (unpaired t test; p ) 0.11). The test set of pharmaceuticals included weak organic acids as well as one weak base, which were partially deprotonated/protonated to their charged conjugate species at pH 7. The base diazepam is fully neutral at pH 7. In principle, weak organic acids and bases can also act as uncouplers in energy-transducing membranes (64). It was therefore necessary to check if these acidic and basic

TABLE 3. Membrane-Water Distribution Ratios and Effective Concentrations for the Test Set of Pharmaceuticals

acetaminophen clofibrate diazepam diclofenac ethinylestradiol ibuprofen propranolol

pKa

log Dlipw

w log (1/EC0.5 (M))

lower 95% CIi

upper 95% CI

lip EC0.5 (mmol/kg)

lower 95% CI

upper 95% CI

dfj

srk

10.1a

0.51e 3.53f 2.79f 2.67g 3.81f 1.91g 2.77h

1.23 4.22 3.52 3.17 4.46 3.03 3.58

1.29 4.36 3.74 3.21 4.50 3.07 3.71

1.18 4.08 3.30 3.13 4.42 2.99 3.45

189 204 186 314 224 77 153

167 150 112 284 204 70 113

213 279 308 346 246 85 207

21 15 9 28 12 18 26

0.298 0.520 0.457 0.191 0.205 0.161 0.320

3.31b 3.99c 4.45c 9.24d

a Reference 31. b Reference 74. c Reference 75. d Reference 76. e Corresponds to the K f ow value (77). This work, determined at pH 7 with egg yolk liposomes using the Transil method (see Materials and Methods section). g Calculated for pH 7 from the partition coefficients of the neutral and charged species, log Klipw,HA ) 4.45 and log Klipw,A ) 2.64 for diclofenac and log Klipw,HA ) 3.80 and log Klipw,A ) 1.81 for ibuprofen (75). h Calculated for pH 7 form the partition coefficients of the neutral and charged species, log Klipw,A ) 3.24 and log Klipw,HA ) 2.76 (76). i CL ) confidence limit. j Degrees of freedom, here corresponding to number of experimental data points minus one. k Standard deviation of the residuals of the fit of the concentration-effect curve (eq 1) sr ) x(sum of squares/ degrees of freedom).

TABLE 4. Mixture Experiments mixture

componentsa

I

2-butoxyethanol 1,3,5-trichlorobenzene 1-hexanol 2-nitrotoluene 2,4,5-trichloroaniline 4-chloro,3-methylphenol diazepam diclofenac ethinylestradiol ibuprofen 2-butoxyethanol 1-hexanol 2,4,5-trichloroaniline diazepam diclofenac ethinylestradiol

II

III

standard kobs (s-1) deviation p valueb 0.467

0.144

0.600

the components of the mixtures acted concentration-additive (i.e., obeyed eq 8), then the observed effect should equal to the endpoint effect kobs ) 0.5 s-1. n

i)1

0.525

0.246

0.814

0.438

0.231

0.537

a Each of the n components is present in the mixture at 1/nth of its effective concentration EC. b One sample t test; theoretical mean 0.5 s-1. All p values indicate that the experimental kobs are not different from the value predicted from concentration-addition.

pharmaceuticals act as baseline toxicants or if they have an additional nontarget, but specific mechanism, in energytransducing membranes (i.e., if they can act as uncouplers). Ibuprofen was slightly more toxic than expected from baseline toxicity with an excess toxicity of a factor 3.1, but this value is barely outside the 95% confidence interval of effective membrane concentrations for baseline toxicants. Diclofenac showed no excess toxicity. The acidic groups of these two pharmaceuticals are carboxylic acids, for which the charge on the deprotonated conjugate base is not well-delocalized in the aromatic ring system but is rather isolated. Only a well-delocalized anion is membrane-permeable and can thus contribute to uncoupling (64). Aromatic bases are known to be weaker uncouplers than the corresponding acids because of the positive potential in the interior of the lipid bilayer (65). Aliphatic amines have no opportunity to delocalize the positive charge. In consistency with this expectation, the tertiary aliphatic amine propranolol did not show any excess membrane toxicity. Mixture Experiments. Three mixture experiments were performed with n components (n ) 4-6). Each component i was present at concentration Ci, at 1/nth of its EC0.5i. The compositions of the mixtures are listed in Table 4. Mixture I was a selection of three nonpolar and three polar compounds from the test set of classical baseline toxicants, mixture II consisted of four pharmaceuticals, and mixture III consisted of three classical baseline toxicants and three pharmaceuticals. The following assumption was tested: if

C0.5i

∑EC

)1

(8)

0.5i

All mixture combinations exhibited the expected concentration additivity (Table 4). The results obtained with mixture I are inconsistent with results from the literature (18, 31) but confirm that nonpolar and polar narcosis are not different mechanisms. In the mixture studies performed by Broderius and co-workers, concentration additivity was found in binary mixtures of nonpolar narcotics with octanol and of polar narcotics with phenol (18, 31). In that study, additive and nonadditive effects were actually used as discriminator between nonpolar and polar narcosis. These results are contradictory to what is now expected from theory, while the present results confirm the predictions. Another goal was to investigate if baseline toxicants were additive with pharmaceuticals with respect to their nontarget effect, baseline toxicity. The results of mixture II and III clearly show that pharmaceuticals and classical baseline toxicants act additive in mixtures. This finding is particularly relevant for mixtures where the single compounds are present at concentrations below the effect threshold of their specific effect(s) but contribute to the cumulative baseline toxicity. Such conditions are likely to be encountered in effluents of wastewater treatment plants and in the aquatic environment, whereas in industrial effluents as well as in urine and manure, a dominance of specific effects could be expected. Resulting consequences for the risk assessment of pharmaceuticals imply that not only the specific effects (e.g., endocrine disruption) have to be considered but that, additionally, a cumulative risk assessment of the nonspecific, but additive, baseline toxicity has to be performed. Because, at least in our study, the slopes of the concentration-effect curves are similar for the different baseline toxicants (data not shown), the concept of toxic equivalence factors can be applied to estimate the risk posed by a mixture of baseline toxicants (66). Then, the risk quotients of the single components of a given mixture can be added up (67). When assessing the risk of a complex environmental mixture, both a cumulative assessment of the baseline toxicity and a common mechanism specific risk assessment should be carried out to assess the multiple role of potential cumulative effects. Potential Applications of the Kinspec Test System. In comparison with other in vitro test systems specific for membrane toxicity, the Kinspec system seems to be advanVOL. 36, NO. 9, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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tageous regarding selectivity, reproducibility, sensitivity, and ease of operation. Selectivity is assured in an in vitro system that contains only the target sites and where the only simultaneously detected mechanism, uncoupling, can be clearly distinguished from baseline toxicity (17). Reproducibility has been shown to be adequate over several years and different batches on membrane vesicles (16). The test is more sensitive than other in vitro tests and exhibits similar sensitivity as acute toxicity tests. While the earlier version of the Kinspec system required continued maintenance of system conditions, the new system is more automated. It may therefore serve as important component in any mechanism-based test battery, both for the effect assessment of single compounds and for mixtures. For single compounds, the baseline toxicity test is relevant because the majority of industrial chemicals act as baseline toxicants. In addition, it may serve as a control to ensure that the response in specific test systems is not caused by unspecific cytotoxicity. For the assessment of complex environmental samples, this method is additionally an indirect measure of the bioavailable total molar concentration of pollutants and therefore may complement other methods for biomimetic extractions (68-73). It has also the potential to deal adequately with hydrophobic chemicals in environmental mixtures, which often pose bioavailability problems in conventional test systems.

Acknowledgments We thank Thomas Fleischmann for experimental assistance with the liposome-water partitioning experiments. Helpful discussion with Gert-Jan de Maagd, Joop Hermens, Kai-Uwe Goss, and Mario Snozzi are gratefully acknowledged. We thank Martin Scheringer and Zach Schreiber for reviewing the manuscript.

Supporting Information Available LSER data and solute parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review December 17, 2001. Revised manuscript received March 1, 2002. Accepted March 4, 2002. ES015844C

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