water partitioning of

Apr 13, 1987 - enough for implementation on a programmable calculator or microcomputer. The data and methods reported should be useful for fate and ...
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Environ. Sci. Technol. 1988, 22, 286-292

enough for implementation on a programmable calculator or microcomputer. The data and methods reported should be useful for fate and transport calculation for gasolinerange hydrocarbons. Registry No. MTBE, 1634-04-4;hexane, 110-54-3; benzene, 71-43-2; methanol, 67-56-1; ethanol, 64-17-5; water, 7732-18-5.

Literature Cited (1) Triday, J. J. Chem. Eng. Data 1984, 29, 321-324. (2) Brandani, V.; Chianese, A.; Rossi, M. J. Chem. Eng. Data 1985,30, 27-29. (3) ROSS,S.; Patterson,R. E. J. Chem. Eng. Data 1979,24,111. (4) Leinonen, P. J.; Mackay, D. Can. J . Chem. Eng. 1973,51, 230-233. (5) Bannerjee, S. Environ. Sci. Technol. 1984, 18, 587-591. (6) Munz, C.; Roberts, P. V. Environ. Sci. Technol. 1986,20, 830-836.

(7) Sutton, C.; Calder, J. A. J . Chem. Eng. Data 1975, 20, 320-322. ( 8 ) Polak, J.; Liu, B. C. Y. Can. J . Chem. 1973,51,4018-4023. (9) Holmes, M. J.; Van Winkle, M. Ind. Eng. Chem. 1970,62(1), e .

21.

(10) Anderson, T. F.; Prausnitz, J. M. Znd. Eng. Chem. Process Des. Dev. 1978, 17, 552-561. (11) Prausnitz, J. M.; Anderson, T.; Grens, E.; Eckert, C.; Hsieh,

R.; O'Connell, J. Computer Calculations for Multicomponent Vapor-Liquid and Liquid-Liquid Equilibria; Prentice-Hall: Englewood Cliffs, NJ, 1980; p 334. Received for review April 13,1987. Accepted July 16,1987. This work was supported by the Louisiana State University Hazardous Waste Research Center via a grant from the US.Environmental Protection Agency. However, it does not necessarily reflect the views of the Agency, and no official endorsement should be inferred.

Thermodynamics of FishIWater and Octan-I-ol/Water Partitioning of Some Chlorinated Benzenes Antoon Opperhulzen," Peter Sern8, and Jan M. D. Van der Steen

Laboratory of Environmental and Toxicological Chemistry, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands The thermodynamic properties of the partitioning of chlorobenzenes between fish and water have been investigated. It is shown that bioconcentration by fish of polychlorobenzenes is accompanied by positive enthalpy and entropy changes. The free energy of this transfer process at room temperature is dominated strongly by the favorable entropy contribution. In contrast, the partitioning of these compounds between octan-1-01and water is accompanied by negative enthalpy and by small negative or positive entropy changes. These results demonstrate that octan-1-01is a poor model of the fish lipids and that generally octan-1-ol/water partition coefficients will not give reliable predictions of bioconcentration factors. In particular, the slopes of plots of octan-1-ol/water partition coefficients against bioconcentration factors will not be the same for different compounds. In addition, preliminary results are presented on the extrathermodynamic relationships between molecular structure and enthalpy, entropy, and free energy changes during both accumulation in fish and octan-1-ol/water partitioning of chlorobenzenes.

Introduction Although there are many concerned with the nature of the accumulation of hydrophobic chemicals in fish or other aquatic species, very few data have been reported considering the thermodynamic background of this partition process (1, 2). Generally only relationships between bioconcentration factors (K,) or uptake and elimination rate constants ( k l and kp, respectively) and physicochemical data are presented (3-5). For instance, the relationship between K , and the octan-1-ol/water partition coefficient (Kd,,J has been reported many times (3-7). Use of such relationships presupposes that the thermodynamics of the various partition processes are proportional (8). Equilibrium partitioning of a chemical between two physicochemical phases can be expressed by Kd = e-AGO/RT

(1)

in which R denotes the gas constant, T denotes the abso286

Environ. Sci. Technol., Vol. 22, No. 3, 1988

lute temperature, and AGO denotes the Gibbs free energy of transfer between the phases. Hence, the relationship between log K,and log Kd,octcan be considered as a relationship between the Gibbs free energies of the two processes, i.e., an example of a linear free energy relationship (LFER) The concept of LFER was initially proposed by Collander (9,IO). Subsequently, such relationships have been reported between various types of liquid-liquid distributions for many types of chemicals. Satisfactory relationships are usually obtained for structurally related compounds. In addition, application of the LFER concepts to other types of distribution processes such as adsorption or solubility also provided satisfactory results (11,12). Relationships between Kd,octand reversed-phase high-performance liquid chromatography (HPLC) retention (13) or Kd,oct and fish bioconcentration factors, for instance, are usually linear, but sometimes significant deviations from linearity have been found (3, 4, 7, 14). Although the concept of LFER is often employed, few studies report on the full thermodynamics of the distribution processes involved. Recently, it has been shown that, even if the free energies of two processes appear to be linearly related, this does not necessarily indicate that the thermodynamic background of partitioning in the two distribution processes is similar (15). In this study, preliminary results are reported for the elucidation of the full thermodynamics of the exchange of hydrophobic chemicals between water and fish and the thermodynamic background of the generally accepted relationship between K , and Kd,oct.In addition, a first attempt is made to find extrathermodynamic relationships between a solute's structure parameters and the partial thermodynamic parameters of the fish/water and octan1-ol/water partitioning, Le., AAG, AAH, and AAS.

.

Materials and Methods Chemicals. 173-Di-,1,3,5-tri-,1,2,3,4-tetra-,penta-, and hexachlorobenzene were obtained from Analabs. After

0013-936X/88/0922-0286$01,50/0

0 1988 American Chemical Society

Table I. Logarithms of Bioconcentration Factors (log Kc,fat)of Selected Chlorobenzenes in Guppy (Poecilia reticdata )

a

experimental temperature, K 301 306

chlorobenzene

286

292

1,3-di1,3,5-tri1,2,3,4-tetrapenta-

3.77 f 0.04 4.32 f 0.05 4.70 f 0.04 5.11 & 0.04

3.78 h 0.03 4.35 f 0.02 4.74 f 0.03 5.18 f 0.04

3.82 f 0.06 4.38 f 0.04 4.75 f 0.04 5.20 f 0.06

3.84 f 0.04 4.43 f 0.08 4.84 f 0.04 5.28 f 0.08

hexa-

5.57 f 0.04

5.62 f 0.06

5.66 f 0.07

5.76 f 0.06

293 (4) 4.15 5.42 4.41 (3) 5.41 (16) 5.46

288 (17)a 3.70-3.96 4.34-4.67 4.80-5.13 5.19-5.36 5.34-5.57 (16) 5.16-5.37

Measured in rainbow trout.

recrystallization albchemicals were >97 % purity as confirmed by GC-ECD. Analytical-grade pentane (Janssen) was used for extraction. Fish. Two-year-old female guppies (Poecilia reticulata) with lengths between 15 and 20 mm, with weights between 206 and 283 mg, and with a mean fat weight of 5 f 2% were used. In each experiment, six fishes were exposed to 10 L of water that contained the test compounds. Throughout the experiments pure oxygen was added by a capillary glass tube, in order to prevent excessive evaporation of the test chemicals. Aquarium water was 50% Amsterdam tap and 50% demineralized water and contained no detectable background concentrations of chlorobenzenes. Analysis of the fish that were not exposed showed no background concentrations. Chemical Analysis. Water (100-200 mL), sampled at the start and end of the experiments, was extracted twice with n-pentane (25-50 mL). Of the pentane solution, 1 p L was injected in a Tracor 300 GC gas chromatograph, which was equipped with a linearized electron capture detector (ECD). Water (20 mL) sampled during the experiment was extracted twice with 10 mL of pentane before analysis in a gas chromatograph. After being sampled (six fishes each sample), the fish were killed by immersion in liquid nitrogen, and the composite sample was homogenized in a mortar. The homogenate was heated under reflux in a 1:l water-pentane mixture for 90 min. Further preparation of the samples was similar to the method that has been reported elsewhere (3, 7). Spiking of clean water (100-200 mL) and fish (six fishes) with a 1-mL solution of all chlorobenzenes in pentane yielded recoveries between 94 and loo%, and between 71 and 100% respectively, when the above analytical method was applied. Octan-1-ol/Water Partition Coefficient. The Kd,oct values were determined after the addition of 1 mL of a solution of octan-1-01with each of the test compounds to 1L of distilled water. The two phases were shaken vigorously for 1 h and allowed to equilibrate for 3 days. Samples were then taken from both the organic and the aqueous layers and were analyzed (after extraction) with a gas chromatograph. All Kd,oct values are means of three replicate measurements. Exposure. Bioconcentration factors were measured in accelerated static tests and were calculated from the ratio between the steady-state concentrations at the end of the exposure period. To check whether or not steady state was achieved, water samples were taken at regular intervals during the exposure, as was proposed by Banerjee et al. (16). Since this method is only successful if there are no significant losses of test compound from the aquaria, the mass balance of each of the compounds was checked. For all test compounds, the loss during the experiments was less than 4%. In a preliminary study the time required to reach equilibrium was determined. On the basis of this

test, the applied exposure period was 48 h for all tests. The initial aqueous exposure concentrations were obtained by diluting water that was saturated with chlorobenzenes. The saturated aqueous solutions were obtained by a water-saturating method that has been described elsewhere (7). Bioconcentration factors were measured in duplicate at 13,19,28, and 33 OC. This temperature range was chosen after control of the short-term survival of guppies, which were bred at 22 "C. Since no acclimation period was used in the test, temperatures below 13 OC and above 33 "C resulted in high mortality of the fish. Results and Discussion It has been shown previously that uptake rate constants of chlorobenzene congeners by fish are very high (4). Since the elimination rate constants are much lower, significant accumulation of all congeners is found (4, 17). Equilibrium between increasing concentrations in fish and decreasing concentrationsin water was achieved within 2 days in all experiments at the various experimental temperatures. Measured bioconcentration factors, which are based on the lipid content of the fish (Kcfat),are listed in Table I, together with data obtained from the literature. As is shown, the measured Kc,fatvalues of this study are in good agreement with data reported previously. The differences between the Kc,fat values determined at different temperatures are not due to the different oxygen saturation levels, which ranged from 9.8 mg/L at 13 "C to 6.8 mg/L at 33 OC. It has been shown elsewhere that variation of oxygen concentration between 2.5 and 9.0 ppm at 22 "C does not influence the bioconcentration of hydrophobic chemicals (28). In addition, the difference between the bioconcentration factors cannot be due to a temperature gradient between the water and the fish bred at 22 "C, because temperature acclimation of small fish such as guppies is achieved very rapidly, due to their low heat capacity. Only in large fish with very high metabolic rates such as tunas may small temperature differences exist between body temperature and water temperature for some time during acclimation (19). Although the bioconcentration factor of a hydrophobic chemical is often considered as being a pure partition coefficient, comparable to liquid-liquid partition coefficients (649, this consideration is rather doubtful since neither fish nor fish lipids are homogeneous phases. This means that elucidation of the thermodynamics of the waterlfish exchange process is not straightforward. To simplify analysis, three assumptions can be made: (i) Partitioning between fish lipids and water is essentially regulated by passive transport processes. (ii) Lipids represent a homogeneous physicochemical phase. (iii) The fish lipid's mean molar volume can be represented by the molar volume of octan-1-01or another homogeneous phase. Results have often been reported that support the first Environ. Sci. Technol., Vol. 22, No. 3, 1988 287

Table 11. Logarithms of Octan-1-ol/Water Partition Coefficients (log Kd,oct)of Selected Chlorobenzenes

chlorobenzene

286

1,3-di1,3,5-tri1,2,3,4-tetrapentahexa-

3.72 f 0.02 4.40 f 0.07 4.83 f 0.04 5.20 f 0.04 5.68 f 0.08

exuerimental temuerature. K 292 301 3.55 f 0.04 4.32 f 0.04 4.61 f 0.03 5.05 f 0.07 5.70 f 0.08

3.48 f 0.04 4.04 f 0.40 4.37 f 0.06 4.70 f 0.06 5.58 f 0.04

306

ref 21

ref 22

ref 23"

3.42 f 0.04 3.93 f 0.03 4.25 f 0.04 4.66 f 0.02 5.17 f 0.05

3.38 4.31 4.60 5.20 5.50

3.38 4.02 4.55 5.03 5.47

3.58 4.27 5.05 5.79 6.53

Calculated value. Table 111. Free Energy, Enthalpy, and Entrogy Changes in kJ mol-', during Transfer of Some Selected Chlorobenzenes from Water to Fish and to Octan-1-01 at 292 K

chlorobenzene 1,3-di1,3,5-tri1,2,3,4-tetrapentahexa-

water to fish

water to octan-1-01 AHo,,O TASmtQ

AGfa,O AHfat" TASfato AGdo

-26.3 -29.5 -31.7 -34.2 -36.6

6.0 10.5 13.5 16.0 17.9

32.3 40.0 45.2 50.2 54.3

-25.0 -29.4 -31.0 -33.6

-15.0 -21.7 -26.3 -30.8

assumption (7,20). Although it is obvious that the latter two assumptions do not hold true, they nevertheless may give satisfactory estimates of the actual thermodynamics. To enable comparison of the thermodynamics of fish/ water and octan-1-ol/water partitioning, partition coefficients of selected chlorobenzenes were measured at different experimental temperatures. Experimental octan1-ol/water partition coefficients of the test compounds measured at different temperatures together with data obtained from the literature (21-23) are listed in Table 11. Liquid-liquid partition coefficients (&*) are usually expressed by log &* = log (xb/x,) -AGob,,/2.303RT (2) in which X, and Xb denote the solute's mole fraction solubility in solvents a and b and AGOb,, denotes the Gibbs free energy of the solute's transfer from solvent a to solvent b (J mol-l). The Gibbs free energy can be expressed by AGO = AH" - TAS" (3) in which AH" denotes the enthalpy change (J mol-l) and AS" (J mol-l K-l) denotes the entropy change accompanying the transfer process. Accepting assumptions i-iii and combining eq 2 and 3, the partial molar thermodynamic parameters of water to fish transfer can be calculated from log K,.fa, = -AG0fa,/2.303RT - log (55.5/6.4) (4) The factor 55.516.4, which is the ratio of molar densities of water and octan-1-01, is added to enable thermodynamic analysis with molar concentrations rather than with mole fractions. From eq 4 and the van't Hoff plot of log K,,fat versus T1(Figure l), AHfat",AGfat0,and Asfato can be calculated. These values are listed in Table 111. It should be noted that the calculations are dependent on the reliability of experimental bioconcentration factors. The data show that the negative free energy of transfer from water to fish is dominated strongly by a gain in entropy, which is favorable for the bioconcentration process. Such large positive entropy changes have been also reported for the transfer of hydrophobic chemicals from water to other types of nonaqueous phases such as organic solvents (15, 24) or liposomes (23). Hence, it has sometimes been proposed that for these chemicals this entropy 288

Environ. Sci. Technol., Vol. 22, No. 3, 1988

3,6&-T3r

4

1

3 w T-l 1000

T 1 1000

10.0 7.7 4.7 2.8

Flgure 1. van't Hoff plots of bioconcentration factors.

T1lOOO

4 3.

.

0 T - l 1000

i

Figure 2. van't Hoff plots of octan-1-ol/water partition coefflcients.

change is in fact the most important factor determining their hydrophobicity (26). The gain in entropy during water fish transfer can be explained by a loss of strueturing of the aqueous phase when the chlorobenzenes are removed from it. For the bioconcentration of Aroclor 1254, a similar large gain in entropy was found previously ( 1 ) . Positive entropy changes were also reported for the bioconcentration of 4-aminoantipyrine and ethanol by fish (2). For these compounds however, it was found that the transfer free energy was not dominated by the entropy but by the enthalpy change at room temperature. As is also shown in Table 11,the bioconcentration of all chlorobenzenes is accompanied by an increasing positive enthalpy change, i.e., unfavorable for the transfer process, with an increasing number of chlorines. These endothermic enthalpies are in agreement with the positive enthalpy found for the bioconcentration of Aroclor 1254 by fish (I). Such unfavorable enthalpy changes are also reported for the transfer of weakly hydrophobic chemicals from water to 2,2,4-trimethylpentane (24). It is remarkable that, in contrast to the water to fish transfer, the transfer from water to octan-1-01of chlorobenzenes is accompanied by exothermic enthalpy changes -+

and by small changes of the entropy (Figure 2 and Table 111). However, this is consistent with thermodynamic data for the octan-1-ol/water partitioning of other classes of hydrocarbons (15, 27, 28). The contrast in endothermic AHO(fish-w) and exothermic AHo(octcw) can, according to Krishnan and Friedman (29),be explained by the differences between the solvation enthalpy of solutes in fish lipid and those in octan-1-01. By employing the concept of solvation of solutes as initially proposed by Eley (30),two steps can be distinguished during the solvation of a compound. First, cavities have to be formed in the solvent to enable the solute to be placed there. This cavitation will give an endothermic contribution to the solvation enthalpy. Secondly, solvent-solute interactions will give an exothermic contribution to the solvation enthalpy after the placement of the solute into the cavity. Therefore, the difference between the solvation enthalpies of chlorobenzenes in octan-1-01and those in water may be explained as being mainly due to the difference in the endothermic enthalpy contribution from cavitation. This explanation was proposed by Spencer et al. (31),who showed that the endothermic enthalpy of cavitation increases with increasing polarity of the solvent. The relative importance of the cavitation and the interaction contributions to the enthalpies of solvation are highly dependent on the natures of both the solute and the solvent. For instance, the presence of the hydroxy group in alcohols may have a significant influence on the solvation enthalpy of aromatic hydrocarbons, due to interactions of this group with the ?r-system of the solutes. The positive enthalpy changes accompanying the transfer of the hydrophobic chlorobenzenes from water to fish lipids appears a little surprising, since these changes are unfavorable for the transfer process. This may be explained, however, as being due to very large enthalpies of cavitation in the fish lipids. In structured phases, such as lipid membranes that are only 5.0-7.0 nm thick, this cavitation enthalpy may be much larger than in bulk phases such as octan-1-01. Since the disturbance of lipid membranes due to cavity formation will be dependent on the size of the solutes, it is clear that the positive enthalpy of transfer increases with increasing humber of chlorine atoms on the phenyl ring. The contrast in the partial thermodynamic properties of partitioning between fish/water and octan-1-ol/water is comparable to that found for halogenated phenols for dimyristoylphosphatidylchloline liposomes/water and octan-1-ol/water partitioning (32,33). Rogers and Wong (32) found exothermic enthalpy change and only small (negative) entropy changes for the transfer of halogenated phenols from water to octan-1-01, which is consistent with the data of chlorobenzenes presented here (Table 111). However, for the transfer of the halogenated phenols from water to dimyristoylphosphatidylcholine liposomes below the transition temperature of the vesicles, Rogers and Stanley (33)found endothermic enthalpy and large positive entropy changes. The entropy contribution dominated the partitioning process at 288 K. In addition, it was found by Rogers and Stanley that, above the transition temperature where the lipid vesicles have a less structured (molten) phase, the enthalpy contribution switches from endothermic to exothermic and the entropy contribution (TAS) switches from positive to negative, the free energy being almost uninfluenced. These observations are fairly consistent with those found by Diamond and Katz (34)for the partitioning of weakly hydrophobic chemicals between water and dimyristoyllecithin. I t was suggested by the

latter authors that below the transition temperature the hydrophobic region of the “frozen”lipids behave like pure nonpolar organic solvents, while above the transition temperature the “molten”lipids behave more like alcohols. From these results, and from the thermodynamic analysis of the fish/water bioconcentration process, we suggest that frozen lipid vesicles resemble natural membranes more than molten lipids do. On the other hand, octan-1-ol/water partitioning seems to better resemble the partitioning between molten lipid and water phases. Although the enthalpy contribution is unfavorable for the transfer from water to fish, the fish/water partition coefficients are very high. This is due to the large positive entropy contribution to the free energy of transfer. This is not surprising since it has been shown elsewhere that the low aqueous solubilities of hydrophobic chemicals such as polychlorinated benzenes and biphenyls (35, 36) are mainly expressions of unfavorable entropy contributions to the free energies of solution. Hence removal of the hydrophobic solutes from the aqueous phases will be associated with a large gain in entropy. That on the other hand the entropy contribution to the free energy of transfer from water to octan-1-01is low shows that in both in water and octan-1-01an increase in structuring of the solvent is achieved after solvation. Partition coefficients or Gibbs free energies of specific distribution processes such as octan-1-ol/water partitioning are usually employed to express the nature of functional groups in the solute’s structure. For instance, the substituent parameter ( T ) as was proposed by Hansch and co-workers (37) for the octan-1-ol/water system is often considered to be the hydrophobic parameter of the functional substituent. This parameter is generally expressed by (5) log Kd,oct,X - log Kd,oct,H = “X in which X represents a derivative of an unsubstituted reference chemical H and ?rx represents the influence of the substituent in this derivate on the octan-1-ol/water partition coefficient relative to that of the reference chemical. The extrathermodynamic contribution of a substituent to the free energy of the octan-1-ol/water partition coefficient (AAGo,c,,x) can be expressed by AAGooct,X

=

AAGooct,X

- AAGooct,H

(6)

As has been shown in various studies, chlorine substitution at different places in an aromatic system does not usually provide an unique ?rcl value (38-40). For instance the ?r values of chlorine atoms substituted at the 2 or 6 position in polychlorobiphenyls are much lower than those of chlorines at the 3,4, or 5 position (38,40). Thus after combining 2 and 5, no unique extrathermodynamic free energy group contribution (AAGooc,,cl) will be found for chlorine atoms. This is supported by the data listed in Tables 111and IV. It has been proposed, however, that the hydrophobicity of nonelectrolyte solutes is related to the solute’s total surface area (TSA) or to its total molecular volume (TMV) ( 3 5 , 4 1 ) . Since TSA and TMV of structurally related compounds are almost proportional (35))only the former parameter will be considered here. As is shown in Table V, normalization of the contributions of chlorines to the octan-1-ol/water partition coefficients of chlorobenzenes, on the basis of the substituents contribution to the solute’s total surface area, yields an extrathermodynamic free energy contribution (AAGo/A2) of approximately 0.31 kJ/A2 molb1. The extrathermodynamic contributions to the entropy and enthalpy changes for one single chlorine atom in a phenyl ring, with no adjacen substituents other than hydrogen, are listed in Table VI. Environ. Scl. Technol., Vol. 22, No. 3, 1988 280

Table IV. Differences between the Free Energy, Enthalpy, and Entropy Changes of Selected Chlorobenzenes and 1,3-Dichlorobenzene for the Water to Fish and Water to Octan-1-01 Transfers at 292 K and the Differences in Total Surfaces Areas

fish/water partitioning

octan-1-ol/water partitioning

chlorobenzene

AAGfnt

Amfnt

AASfnt

AAGoct

1,3-di1,3,5-tri1,2,3,4-tetrapentahexa-

-3.2 -5.4 -7.9 -10.3

4.5 7.5 10.0 11.9

24.4 44.2 61.3 75.3

-4.4 -6.0 -8.6

A-wt

-6.7 -11.3

-15.8

AASOC,

6 TSA

-7.9 -18.2 -24.7

12.9 20.8 28.7 34.1

Table V. Contribution of Chlorine Substitution in Benzene to the Free Energy, Enthalpy, and Entropy of Water to Fish and Octan-1-01 Transfer at 292 K per Unit of Total Surface Area, As Expressed per Square Angstrom

chlorobenzene

octan-1-ol/water partitioning

fish/water partitioning

1,3,5-tri1,2,3,4-tetrapentahexa-

-1-50

AAGfnt A M f n t AAS,,

AAGmt A m o ,

AAS,

-0.25 -0.26 -0.28 -0.30

-0.34 -0.29 -0.30

-0.61 -0.88 -0.86

0.35 0.36 0.35 0.35

2.05 2.13 2.14 2.21

-0.52 -0.54 -0.55

Table VI. Mean Contribution to the Free Energy, Enthalpy, and Entropy of Transfer from Water to Fish and Octan-1-01 for Substitution of One Single Chlorine Atom in Benzene (=12.9 A*) at 292 K

fish/water partitioning AAGfnto AMfn: AASfBto

-3.51 kJ mol-' 4.55 kJ mol-' 27.51 J mol-' K-'

octan-1-ol/water partitioning AAGwto AA",cto AAS,+O

-4.00 kJ mol-l -6.92 kJ mol-' -10.11 J mol-' K-'

responsible for the partitioning of chemicals between two phases, with AHo and ASo of the process being approximately constant, if AMo is proportional with AASO, i.e., A A H O = OAAS'. The proportionality factor /3 is termed compensation temperature, due to its dimension. Observed proportionality is called enthalpy-entropy compensation (42,43). Since within the small experimental temperature range (13-33 "C)enthalpy-entropy compensation was observed for the partitioning of chlorobenzenes with two to five chlorine atoms between water and octan-1-01, a general expression of the extrathermodynamic free energy contribution may be given by (Figure 3a) (7)

in which Pact is 714 K. It must be noted that compensation temperatures were calculated from eq 7 (Figure 3a) rather than from A S - AH values, in order to reduce the experimental artifacts (42, 43)* The results of a thermodynamic analysis of the water fish transfer are shown in Figure 3b. The extrathermodynamic group contribution from chlorines t o the transfer of chlorobenzenes from water to fish can be expressed by

-

AAG'fat/A2 = AAH',t/A2(l

-

T/PfaJ

Environ. Sci. Technol., Vol. 22, No. 3, 1988

'

$0

'

AH

AH

Flgure 3. Relationship between free energy (AGO) and enthalpy (AH') changes of octan-1-ol/water partitioning (a) and bioconcentration by fish (b) of selected chlorobenzenes.

3u

'23

5

6

7

8

'OQKdoct

van't Hoff plots but from direct microcalorimetric measurements. The negative extrathermodynamic entropy contribution of chlorine substitution found for the chlorobenzenes in this study (Table VI) is also in agreement with the phenol and aniline data. As is shown in Figure 2e, the experimental data for hexachlorobenzene did not allow estimation of the enthalpy of transfer in the octan-1-ol/water partition process by employment of a van't Hoff plot. For water to fish transfer, substitution of a chlorine on benzene showed a positive extrathermodynamic entropy and enthalpy contribution, in contrast to that for the water to octan-1-01transfer. If the addition of one single chlorine atom to benzene is considered (6 TSA = 12.9 A2), it can be calculated by combining eq 2, 4, 5, 7, and 8 that the log Kd,octof chlorobenzenes will increase by 0.60 at an experimental temperature of 298 K, while log Kcfatwill increase by 0.57. The value of rC1 = 0.60 is close to values reported in the literature (38, 39). Addition of one to five chlorines to benzene for all possible configurations will give rise to increases of both AGf+, and AGO-,, and thus in log Kd,octand log Kc,fat, which will be proportional. By combining eq 6 and 8, the slope a of a plot of log against log Kc,fatcan be calculated from

(8)

from which Pet = 160 K was calculated. As is shown in Table VI, addition of one single chlorine atom yields a contribution of -6.92 kJ mol-' to the free energy of transfer from water to octan-1-01, which is close to that reported for chlorine substitution in phenol and aniline (28). The latter values were not obtained from 290

-30

Flgure 4. Relationship between log K,,f,t and log Kd,octat T = 33 ' C (top line) and T = 13 O C (bottom line).

It had been argued that one single mechanigm may be

AAGooct/A2 = AM'oct/A2(1 - T/Poct)

'

A t T = 301 K, a slope of 1.00 is calculated, which is in agreement with the experimental slope of this relationship between log Kc,f,, and log Kd,octand also with the data reported in the literature (4). For a series of congeners for which enthalpy-entropy compensation is found in both processes, the slope a of eq 9 will be a variable depending

only on experimental temperature T. For the various experimental temperatures used here, the slopes are calculated and listed in Table V. The influence of the experimental temperature on the log K,,fat- log Kd,, curves is illustrated by the experimental plots obtained at T = 13 and 33 OC,shown in Figure 4. That the slopes of the log K,,,, - log Kd,&plots at room temperature are close to unity is coincidental and does not indicate similarity between the thermodynamics of water to octan-1-01and water to fish transfer processes of chlorobenzenes as is shown in Tables I11 and IV. This may also hold true for the slopes of log K, - log Kd,octcurves of other hydrophobic chemicals (44,45). Thus, although the thermodynamics of transfer of hydrophobic chemicals from water to fish and from water to octan-1-01 are completely different as far as the enthalpy changes are considered, some proportionality between log K,,fatand log Kd,&may be expected for structurally related compounds. This only holds true if in both processes enthalpy-entropy compensation is found. In general, however, for the octan-1-ol/water system such compensation is not found for various functional groups (27,46). Therefore, no unique relationship between log Kc,fatand log Kd,octcan be expected for different types of hydrophobic chemicals. Loss of compensation in one or both processes will usually result in loss of proportionality between the bioconcentration factor and the octan-1-01water partition coefficient. This phenomenon, which was found in this study for hexachlorobenzene, may help to explain the often observed loss of linearity of the distribution coefficientsof chemicals with estimated log &,oct values larger than 5.

Conclusions Analysis of the thermodynamic parameters (AGO, AHo, and ASo) for the accumulation of chlorobenzenes by fish indicates that this process is associated with an endothermic enthalpy change, which is unfavorable for this transfer process but is dominated by a favorable increase in entropy. In contrast, octan-1-ol/water partitioning of these compounds is accompanied by small changes in entropy and is dominated by exothermic enthalpy contributions. The differences in the therodynamic properties of these processes, especially those in the enthalpy changes, arise from the different structures of fish lipids and octan-1-01 phases. It must therefore be concluded that octan-1-01 is not a good representative of the physicochemical properties of fish lipids. It can be shown that only under specific conditions linear relationships between log K,,fatand log Kd,octwill be expected for structurally related chemicals. The slopes of such relationships may usually deviate significantly from unity and will vary with experimental temperature. In addition, these relationships will not be the same for all types of unmetabolizable hydrophobic chemicals. This is because in general the thermodynamics of partitioning between fish/water and octan-1-ol/water will show no similarity for different classes of chemicals and no enthalpy-entropy compensation will usually be found in both octan-1-ol/water and fishlwater partitioning. Registry No. Chlorobenzene, 108-90-7;1,3,5-trichlorobenzene, 108-70-3;1,2,3,4tetrachlorobenzene,634-66-2;pentachlorobenzene, 608-93-5;hexachlorobenzene, 118-74-1;water, 7732-18-5.

Literature Cited (1) Matsuo, M. Chemosphere 1980,9,671-675.

(2) Matsuo, M. Chemosphere 1981,10,491-494. (3) Bruggeman, W. A.; Opperhuizen, A,; Wijbenga: A.; Hutzinger, 0. Toxicol. Environ. Chem. 1984, 7, 173-189. (4) Konemann, H.; Van Leeuwen, K. Chemosphere 1980,9, 3-19. (5) Watarnai, H.; Tanaka, M.; Suzuki, N. Anal. Chem., 1982, 54, 702-705. (6) Neely, W. B.; Branson, D. R.; Blau, G. E. Enuiron. Sci. Technol. 1974,13,1113-1115. (7) Opperhuizen, A.; Van der Velde, E. W.; Gobas, F. A. P. C.; Liem, A. K. D.; Van der Steen, J. M. D.; Hutzinger, 0. Chemosphere 1985,14,1871-1896. (8) MacKay, D. Enuiron. Sci. Technol. 1982,16,274-278. (9) Collander, R. Acta Chem. Scand. 1949,3,717-747. (10) Collander, R. Acta Chem. Scand. 1951,5, 774-780. (11) Coates, M.; Connell, D. W.; Barron, D. M. Environ. Sci. Technol. 1985,19,628-632. (12) Kenega, E. E.; Gohring, C. A. I. ASTM Spec. Tech. Publ. 1980,NO.707,78-115. (13) Hafkenscheid, T. L.;Tomlinson, E. Znt. J. Pharm. 1983, 16,225-239. Segiura, K.; Ito, No;Matsumoto, N.; Mihara, Y.; Murata, K.; Tsukakoshi, Y.; Goto, M. Chemosphere, 1978, 7, 731-736. Riebesehl, W. Ph.D. Thesis, University of Amsterdam, 1984. Banerjee, S.; Sugatt, R. H.; O’Grady, D. P. Environ. Sci. Technol. 1984,18,79-81. Oliver, B. G.;Niimi, A. J. Enuiron. Sci. Technol. 1983,17, 287-291. Opperhuizen, A.; Schrap, S. M. Environ. Toxicol. Chem. 1987,6,335-342. Sharp, G. D. In The Physiological Ecology of Tuna’s;Sharp, G. D., Dizon, A. E.; Eds.; Academic: New York, 1978;pp 213-232. Boese, B. L. Can. J. Fish. Aquat. Sci. 1984,41,1713-1718. Chiou, C. T. Enuiron. Sci. Technol. 1985,19, 57-62. Miller, M.M.; Ghodbane, S.; Wasik, S. P.; Tewari, Y. B.; Martire, D. E. J. Chem. Eng. Data 1984,29,184-190. Yalkowsky, S.H.; Valvani, S. C. J. Pharm. Sci. 1980,69, 912-922. Riebesehl, W.; Tomlinson, E.; Grunbauer, H. J. M. J. Phys. Chem. 1984,88,4775-4779. Ahmed, M.; Burton, J. S.; Hadgraft, J.; Kellaway, I. W. J. Membr. Biol. 1981,58, 181-189. Janin, J.; Chothia, C. J. Mol. Biol. 1976, 100, 197-211. Berti, P.; Cabani, S.; Conti, G.; Mollica, V. J. Chem. SOC., Faraday Trans. 1 1986,82,2547-2556. Riebesehl, W.; Tomlison, E. J. Solution Chem. 1986,15, 141-150. Krishnan, C. V.; Friedman, H. L. In Solute-Solvent Znteractions; Coetzee, J. F., Ritchie, C. D., Eds.; Marcel Dekker: New York, 1969;pp 2-97. Eley, D. D. J . Chem. Soc., Faraday Trans. 1 1939, 35, 1249-1255. Spencer, J. N.; Gleim, J. E.; Blevins, C. H.; Garrett, R. C.; Meyer, F. J. J. Phys. Chem. 1979,83,1249-1255. Rogers, J. A.; Wong, A. Int. J. Pharm. 1980,6, 339-348. Rogers, J. A.; Stanley, S. S. Biochim. Biophys. Acta 1980, 598,392-404. Diamond, J. M.; Katz, Y. J. Membr. Biol.1974,17,121-154. Opperhuizen, A.; Gobas, F. A. P. C.; Van der Steen, J. M. D.; Hutzinger, 0. Environ. Sci. Technol., in press. Opperhuizen, A.; Benecke, J. I.; Parsons, J. R. Environ. Sci. Technol. 1987,21,925-926. Hansch, C.; Maloney, D.; Fujita, T.; Muir, R. Nature (London)1962,194,178-180. Bruggeman, W.A.; Van der Steen, J.; Hutzinger, 0. J. Chromatogr. 1982,238,335-346. Konemann, H.; Zelle, R.; Busser, F.; Hammers, W. E. J . Chromatogr. 1979,178, 559-565. Opperhuizen, A. Toxicol. Environ. Chem. 1987,15,249-264. Pearlman, R. S.;Yalkowsky, S. H.; Banerjee, S. J. Phys. Chem. Ref. Data 1984.13. 555-562. Krug, R. R.; Hunter, W: G.iGrieger,R. A. Nature (London) 1976,261,566-567. Environ. Sci. Technol., Vol. 22, No. 3, 1988

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Tomlinson, E. Int. J. Pharm. 1983, 13, 115-144. Oliver, B. G.; Niimi, A. J. Environ. Toxicol. Chem. 1984, 3,271-277. McKim, J.; Schmieder, P.; Veith, G. Toxicol. Appl. Pharmacol. 1985, 77, 1-10.

(46) Kinkel, J. F. M.; Tomlinson, E.; Smit, P. Int. J. Pharm. 1981,9, 121-138. Received for review March 21, 1987. Accepted September 16, 1987.

Ozone Fading of Natural Organic Colorants: Mechanisms and Products of the Reaction of Ozone with Indigos Daniel GrosJean,+Paul M. Whitmore,$ and Glen R. Cass” Environmental Engineering Science Department and Environmental Quality Laboratory, California Institute of Technology, Pasadena, California 91 125

James R. Druzik The Getty Conservation Institute, 4503-6 Glencoe Avenue, Marina del Rey, California 90292-6537

Indigo, dibromoindigo, and colorants containing thioindigo and tetrachlorothioindigo were exposed in the dark to dry, purified air containing ozone (10 ppm) for 4 days, and the exposed samples were analyzed by mass spectrometry. Under the conditions employed, indigo and dibromoindigo were entirely consumed, and the major reaction products were isatin and isatoic anhydride from indigo and bromoisatin and bromoisatoic anhydride from dibromoindigo. Thioindigo and its chloro derivative also reacted with ozone, though at a slower rate; the corresponding substituted isatins and anhydrides were tentatively identified as reaction products. These results can be rationalized in terms of‘ a mechanism involving electrophilic addition of ozone onto the unsaturated carboncarbon bond. This mechanism adequately describes the observed loss of chromophore (fading) for all indigos studied and presumably applies to other indigo compounds as well. The reaction products of indigo, isatin and isatoic anhydride, were not ozone fugitive under our conditions. Introduction

Studies carried out in this laboratory have shown that many artists’ organic colorants fade substantially when exposed to ozone in the dark (1-3). These studies involved 12-week exposures to air containing 0.3-0.4 parts per million (ppm) of ozone at ambient temperature (21-25 “C) and humidity (RH = 46-50%). Examination of data for ambient levels of ozone in urban air, together with investigations of ozone levels inside museums ( 2 , 4 )and of the corresponding indoor-outdoor relationships ( 2 , 5 ) ,indicate that the total ozone dose (concentration X duration of exposure) to which the colorants were subjected in those laboratory studies is equivalent to about 6 years inside a typical air-conditioned building in Los Angeles. Organic compounds that were identified as ozone fugitive in our experiments included modern (synthetic) as well as natural colorants. Many alizarin lakes, including a synthetic indigo substitute, formulated from lamp black, copper phthalocyanine, and an alizarin lake, faded noticeably upon exposure to ozone (2). The products and mechanisms of the reaction of ozone with alizarin lakes and ‘Also with DGA, Inc., 4526 Telephone Rd., Suite 205, Ventura, CA 93003. *Present address: Center for Conservationand Technical Studies, Harvard University Art Museums, 32 Quincy St., Cambridge, MA 02138. 292

Environ. Sci. Technol., Vol. 22, No. 3, 1988

related colorants have been investigated recently (6). Natural indigo was found to be even more ozone fugitive than its modern substitute (3). Although ancient works of art created with natural colorants such as indigo may have “survived” well before the recent introduction of ozone and other anthropogenic pollutants into urban air, the demonstrated ozone fading of these natural colorants has direct implications for current practices in the conservation of museum collections. This paper describes the methods and results of a study focusing on the confirmation that ozone is indeed responsible for the fading observed in the above studies, on the identification of the products of the ozone-indigo reaction, and on the discussion of the corresponding reaction mechanisms that account for the loss of the chromophore, i.e., ozone fading. To test the general applicability of our findings, several indigo derivatives have been investigated as well. These include thioindigo, 6,6’-dibromoindigo, and several colorants containing chlorinated thioindigo. The chemical structures of these colorants are given in Figure 1. Thioindigo was included as a simple structural homologue of indigo. Dibromoindigo, the Royal Purple dye of fabrics widely traded by the Phoenicians in the first millenium B.C., has been used as a natural colorant since at least the 13th century B.C. (7, 8). The chlorinated thioindigos have found applications as artists’ pigments and in the printing ink and paper industries (9). Also included in this study are the results of experiments involving the reaction of ozone with the major products of the ozone-indigo reaction. Experimental Section

Colorants Studied. Indigo (MI262, CAS Registry No. 482-89-3, CI no. 73000) was obtained from Aldrich Chemical Co, Milwaukee, WI, and was used without further purification following verification of its structure by mass spectrometry (see Mass Spectrometry Analysis). A second indigo sample was obtained from Fezandie and Sperrle (F&S). Dibromoindigo (MI 420, CI no. 75800) was obtained from Dr. F. D. Preusser of The Getty Conservation Institute, Marina del Rey, CA, and from the Forbes Collection (sample 7.01.8, Tyrian purple, murex) maintained at the Fogg Museum, Harvard University, Cambridge, MA. Thioindigo (M, 296), Vat Red 41, was obtained as a liquid emulsion and as a “press cake” from Hoechst. Thioindigoid Violet (Binney and Smith PR 88 MRS, CI no. 73312) and Thioindigo Red (BASF PR 88, CI no. 73312)

0013-936X/88/0922-0292$01.50/0

0 1988 American Chemical Society