of Hydrophobic Organic Chemicals in-Fish Based on Diffusive Mass

In a series of size classes of fathead minnow, bio- concentration kinetics and bioconcentration factors of five polychlorinated benzenes and biphenyls...
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Environ. Sci. Techno/. 1995, 29, 2769-2777

Kinetic; of Hydrophobic Organic Chemicals in-Fish Based on Diffusive Mass Transfer and Allometric Relationships D I C K T . H . M. S I J M * A N D A L E X VAN D E R L I N D E Enuironmental Chemistry Group, Research Institute of Toxicology, P.O. Box 80.058, NL-3508 TB Utrecht, The Netherlands

In a series of size classes of fathead minnow, bioconcentration kinetics and bioconcentration factors of five polychlorinated benzenes and biphenyls are weight related. A simple bioaccumulation model for hydrophobic chemicals in fish is presented that is based on diffusive mass transfer. The model contains physical-chemical and size-related parameters. The model calculations are in good agreement with experimental values from the literature for uptake and elimination rate constants. The model, however, partly explains the observed size and octanol/water partition coefficient (Kow) related uptake and elimination rate constants in the present study with juvenile fathead minnow. Physiological factors other than sizerelated parameters, such as uptake by skin, are suggested to be important as well. The main fishrelated parameters are (gill) exchange surface area and lipid content; the main physical-chemical parameters are KO, and molecular weight.

Introduction Aquatic organisms are exposed to a variety of xenobiotics. Bioconcentration plays an important role in hazard assessment procedures. Bioconcentration is the result of uptake by organisms from and elimination to the ambient environment of chemicals. The equilibrium bioconcentration factor for organic chemicals increases with increasing hydrophobicity, usually expressed as the octanollwater partition coefficient (1,2). Hitherto, many relationships have been derived that relate bioconcentration factors of hydrophobic chemicals for fish and physical-chemical properties, such as the octanol/water partition coefficient (&). While bioconcentration factors appear to be less dependent on species or size of the organism,particularly when lipid-normalized (31, bioconcentration kinetics appear to be highly dependent on species, size, and age of the organism (4-10). * Corresponding author fax: **31/30-532837;e-mail address: : D. [email protected].

0013-936)(/95/0929-2769$09.00/0

D 1995 American Chemical Society

Bioconcentration factors represent steady-state situations where concentrationsin fish and water do not change and may predict the maximum concentrations that can be reached in fish following aqueous exposure. Bioconcentr$on models are based on diffusion processes through series of aqueous and lipid layers (9-16). The rate at which chemicals pass aqueous and lipid layers is determined by the resistance the chemicals experience in each phase. Earlier studies showed that the uptake rate constant (kl)increased with &, for organic chemicals having a log &, < 3, that kl was independent of &, for chemicals with log &, between 3 and 6, and decreased with KO, for chemicals with a log %, > 6 (2,17), which is corroboratedby invitro studies using artificialmembranes (18). Also it was found was that the elimination rate f constant (k2)was independent of &, for chemicals with log &, < 3 and decreased with increasing KO, (2). The explanationfor the aforementioneddependency of kl and k2 on &, was that uptake and elimination of the organic chemicals are mass transfer processes through series of aqueous and lipid layers within the organism and the ambient water. The experimentally observed breaking point at log &, x 3 is a result of the assumption that the dominant resistance changes from transport through the lipid layer to transport through an aqueous layer, with increasing hydrophobicity. Bioconcentration kinetics, however, may also be very importantfor hazard assessment. The uptake rate constant (kJis a measure of how fast concentrationsin an organism may increase. The elimination rate constant (k2) is a measure of how fast the concentration in an organism decreases and how fast steady-state is reached between concentrations in the organism and the ambient environment. Uptake kinetics give informationon how fast chemicals are taken up. This is important (i)for short-term exposure, e.g., during accidental contamination, when steady-state may not occur and the rate of increase of the chemical concentration in fish is important and (ii) for long-term exposure of chemicals that have either extremely low solubilities or reach steady state after long period of times. Organisms that have high uptake rates will attain initially higher concentrations than those that have lower uptake rates when exposed to the same aqueous concentration. Elimination kinetics give information on how fast chemicals are depurated from organisms and thus on the biological half-life of a xenobiotic. In addition, the elimination rate influences the time needed for steady state. Organisms that have low elimination rate constants retain chemicals longer than organisms that have higher elimination rate constants. Biotransformation may increase the physical-chemical elimination rate, thereby accelerating depuration of the parent compound (6). In the present study, however, the model assumes that the organic chemicals are not biotransformed. It is very interesting if size-related bioconcentration kinetics can be understood, and how a diffusive based bioconcentration model can include size-related parameters. In the present study, size-related bioconcentration kinetics of a series of polychlorinated benzenes and biphenyls are studied in fathead minnow (Pimephales

VOL. 29, NO. 11, 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY

2769

TABLE 1

Fish Age, Size (F), and Lipid Content (a) age (months) 1 2 4 7 17 20 a

kg)

Fa

a (kghJkg)

no. of fish

0.238 0.0996 0.0916 0.0741 0.0147 0.0358

25 24 25 25 30 41

*

0.045 0.036 0.068 f 0.048 0.22 i 0.09 0.41 i 0.13 1.17 f 0.25 0.67 f 0.21

n = 11 (fish from uptake period only).

TABLE 2

Physical-Chemical Preperties and Initial Aqueous Concentration (ugll) of Polychlorinated Benzenes and Biphenyls TrCBz

TeCBz

pCBz

TCB

HCB

292 6.18 30

360.9 6.g8 1

0.75 0.32 0.33 0.62 3.5 3.0

0.060 0.048 0.046 0.087 0.14 0.14

Physical-Chemical Properties Ma log Kow sa(pg/L)

181.45 4.13gb 21 103

215.9 4.635b 7.8 103

250.3 5.183b 650

Initial Aqueous Concentration(pg/L) age (months) 1 2 4 7 17 20 a

21

7.8 9.0

18 8.7 7.1

3.9 1.9 2.2 4.2 2.6 1.9

7.2 3.0 3.6 6.3 27 20

Water temperature was 20 i 1 "C; oxygen concentrations was always greater than 80% of saturation. Extraction and Cleanup. Water samples were extracted with hexane. Fish were killed by immersion into liquid nitrogen and stored in the refrigerator until extraction.They were homogenized in a mortar and heated under reflux with 50 mL of hexane and 50 mL of water during 1.5h. After centrifugation (20 min 9OOg),one part of the hexane layer was completely evaporated for determination of the lipid content by gravimetry. The other part of the hexane was concentrated under a gentle stream of nitrogen and eluted over silica-HzS04 and silica-NaOH, respectively, to remove lipids and other interfering agents (6).Both water and fish samples were concentrated to appropriate volumes for analysis. Detection limits for allchlorinatedchemicals used in the present study in fish samples were approximately 0.1 pglkg. Analysis. An HP-5880A GC, which was equipped with a "Ni-ECD and a DB-5 column (15 m, dl = 0.23 pm, i.d. = 0.35 mrn), was used for analysis. Carrier gas was helium makeup gas was Ar/CH4 (90/10). A splitless injector (250 "C) was used. The column temperature program was 60 "C for 1 min, followed by a temperature increase of 20 "C/ min to 250 "C. The detector temperature was 325'C. Bioconcentration Model. Experimental. Bioconcentration is assumed to follow a first-orderone-compartment model according to Branson et al. (21):

Data from ref 26. Data from ref 25.

promelas). A bioconcentration model is presented that includes fish size and physical-chemical related parameters, such as the influence of aqueous and lipid diffusion path lengths, diffusion coefficientsin water and lipid, lipid content, surface exchange area, molecular weight, and lipid/ water partition coefficient. In addition, the implications of bioconcentration kinetic parameters for biological halflives and uptake rates are discussed.

Experimental Section Fish. Fathead minnow (P.promelas)were bred and reared in our laboratory. Six age classes were used of which age, weight, and hexane-extractable lipids are summarized in Table 1. Chemicals. 1,2,3-trichlorobenzene (TrCBz), 1,2,3,4Tetrachlorobenzene (TeCBz),pentachlorobenzene (pCBz), 2,2',5,5'-tetrachlorobiphenyl (TCB), and 2,2',4,4',5,5'hexachlorobiphenyl (HCB) were used as hydrophobic organic test chemicals (Table 2). Hexane was distilled before use. Experiments. Between 24 and 41 fish of each age class were exposed to the test chemicals in water during 5 d in an aquarium of 10 or 20 L in a static system (Table 1).One fish was sampled after 2, 4, 6, 8, 24, 48, 72, and 96 h, and three fish were sampled after 120 h of exposure. The test chemicals were dissolved in water using a generator column according to Opperhuizen (19). Following exposure, fish were transferred to a clean aquarium that was equipped with a carbon aeration filter to study elimination during 6 months. Fishwere sampledafter 1,2,3,4,8,16,31,64,106, and 202 d during the elimination period. Aqueous concentrations of the polychlorinated benzenes and biphenyls (Table 2) did not exceed their aqueous solubilities (20). 2770 1 ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 11,1995

In each size class, bioconcentration parameters were determined for all five hydrophobic test chemicals. The uptake rate constant klwas determined from the increase of the concentration in fish during the first hours of exposure, when elimination is assumed to be neghgible. The elimination rate constant k2 was determined from the log Cf versus time plot, the slope being equal to -k2/2.303 (22). The bioconcentration constant Kc was expressed as the ratio of kl/k2. The lipid-normalized bioconcentration factorKLwas determined by dividing & by the lipid content a. Since experiments were performed in static systems, evaporation losses were observed, which were modeled using a diffusive waterlair model as described by Schwarzenbach et al. (231,assuming a stagnant air layer of 0.3 cm and a stagnant aqueous layer of 0.03 cm, which were determined from a control aquarium without fish. Theory. The diffusive mass transfer model that has been proposed by Gobas et al. (2)was used as a kinetic model to describe uptake and elimination of organic chemicals. A short description of the model and the most important equations will be repeated here. Bioconcentration in fish is the result of uptake from water and elimination from fish. Uptake as well as eliminationof organic chemicals are diffusive mass transfer processes: chemicals are transported through aqueous and lipid layers in series. For movement through lipid layer, both size and permeability of the chemicals are important (24),which are included in diffusion coefficients and lipid/ water partition coefficients.

Diffusion Coefficient (0).Diffusion coefficients describe the rate at which chemicalsdiffuse througha medium and are dependent on physical-chemical properties of the compound, temperature, and characteristics of a liquid medium, such as described in the Sutherland-Einstein relationship:

The uptake rate constant kl is described as kl

=

A

1A

T

4:

The elimination rate constant k2 is described as

-(-)

D = RT - 1 4nN1'3 N 6 q 3Mv

The bioconcentration factor is

K, = k l / k 2

(4)

where Mis molecular weight (g/mol),R is the gas constant (=8.314 J/mol.K), T is temperature (K), N is Avogrado's constant (6.0220 x IOz3mol-'), 7 is viscosity (Pas), and Y is molecular specific volume (m3/mol). The diffusion coefficient in pure water at 25 "C, Dw (m2/ s), can be estimated from (23)

The lipid-normalized bioconcentration factor KL is

4 = kllak2

Parametrization of Theoretical Model. A number of parameters are important for the rate of transfer from water to fish or vice versa. The role of each individual parameter on uptake and elimination rate constants for hydrophobic chemicals will be investigated. Lipidwater Partition Coefecient (Kid. Partition coefficients describe the tendency of chemicals to reside in one compartment over another and are used to normalize the chemical potentials in the lipid phase. The lipid/water partition coefficient is often related to the distribution coefficient between octanol and water (1). In the present study, &, is used as a surrogate for K,. KO, values for the polychlorinated benzenesand biphenylswere derived either from De Bruijn et al. (25) or from Mackay et al. (26'). Diffusion Path Length (&I. The distance the chemicals must travel from water to the organism or vice versa is important for the total resistance. When it is assumed that uptake of the chemicalspredominantly occurs by the gills, diffusion path lengths are restricted by anatomical configurations. The aqueous diffusion layer (d,, m) will be smaller than the distance between two gill lamellae. An estimate of the distance between gill lamellae depends on the size of a fish and is given by (27)

d = 20.5 x 1 0 - 6 ~ 1 0 0 0 ~ 0 ~ 1 1 4

(6)

where d is the distance between two gill lamellae (m) and F is fish weight (kg). Randall et al. (28) estimated that the mucous layer can be assumed to be the aqueous diffusion layer, which is at least 1pm and perhaps about 3 pm thick. We estimated the aqueous diffusion path length as 10%of the interlamellar distance d:

6, = 2.05

x 10-6~1000~0~114

(71

For 0.1 g of guppy, 6, thus results in 1.6 pm, and for 750 g of rainbow trout, 6, would be 4.4 pm. SurfaceArea(A) and Weight (l?. Transport ofchemicals is linearly related to the exchange area. When it is assumed that fish take up chemicals from water by the a s , the gill surface area is used as an exchange surface. An allometric relationship (29)is used to relate gdl surface area to weight according to (8) A = (5.59 f 3.16) x 10-4(1000~0~77*0~15 (8) where A is the gill surface (m2).

2.7 x lo-'

(5)

e 7 1

where M is molecular mass (glmol). However, since diffusion occurs in the aqueous diffusion layer, which contains mucus (which is 95%water and glycoproteinswith a high sialic acid content (241,and since in the close vicinity of the lipid membrane water may be more structured causing a higher resistance to pass (301,actual diffusion coefficientswillprobably be lower than those in pure water. The diffusion coefficient in the aqueous diffusion layer can be estimated when using the following assumptions. The uptake rate constant is independent of &, for chemicals having a log %, > 3, and is approximately 1000 Llkgd (or 1.16 x m3/kg.s)for 0.1 g guppy. Using eqs 2 and 8, and estimating 6, to be 1.6 pm (eq 71,D, will be m2/s. A very hydrophobic chemical, such as 2.0 x hexachlorobenzene (M = 285 glmol), would have an aqueous diffusion coefficient in pure water of 4.9 x mz/saccordingto eq 10,which is close to estimated diffusion coefficients in water of 6 x 10-lo m2/sof a series of alkyl p-aminobenzoate esters (18). The estimated aqueous diffusion coefficient in the aqueous diffusion layer is thus 0.04 times of that in pure water, which indicates that the viscosity of the aqueous diffusion layer would be 25 times that of pure water (eq 9). In the present model, an aqueous diffusion coefficient of the chemicals 1/25th that of in pure water is used: D, =

1.08

10-~ 71

Diffusion coefficientsin lipid (D,)can also be estimated by the Sutherland-Einstein relationship and differ from Dw in viscosity only, which is inversely related to the diffusion coefficient (eq 9). An estimate for the viscosity of lipids is that of olive oil, which is 0.084 Pas at 25 "C (31). For comparison,that ofwater at 25 "Cis 0.001Pas (31).The viscosity of the aqueous diffusion layer will probably have a viscosity 25 times that of pure water or 0.025 Pas. A first estimation of the diffusion coefficientsof chemicals in lipid is thus approximately 1/84th of those in water or 0.3 times that of the diffusion coefficient in the aqueous diffusion layer: D, = 0.30,

(12)

This approximationagrees well with other estimates, such as the diffusion coefficient in skin being 0.5 of that in the VOL. 29, NO. 11. 1995 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 12771

aqueous diffusion layer (32) and the experimentally determined diffusion coefficientsof antipyrine and iodoantipyrine in erythrocytes,which are 3.4 x 10-lom2/sand 5.8 x m2/s at 20 "C, respectively (33). Diffusion Path Length (6A. The distance, which chemicals must travel through lipids, is important for hydrophillic chemicals for which transport is lipid layer controlled. The lipid diffusion path length (d,, m) is estimated as follows. For guppy, the change between lipid and aqueous diffusion layer controlled uptake is for chemicals with a log KO, of about 3. This means that the resistances between the lipid compartment and the aqueous diffusion layer are approximately equal (from eq 2): S,/D, = dm/DmKm.For guppy, 6, = 1.6 x m, Kmis equal to = lo3, and for a chemical with log &, = 3, 0, is 3.5 x lo-" m2/s. The result is that 6, = 0.47 x m. This value is much higher than the thickness of a lipid membrane, which is in the order of 5 nm (34);however, it may reflect the total lipid diffusion path length, which the chemical must pass being transported through the lipid compartments of a fish (9). Lipid Content (a). Lipid content is important only for the elimination rate constant, but not for the uptake rate constant. Lipid content is not size-related and may vary significantly between and within species. Analysis of Parameters. Each parameter that affects bioconcentration kinetics is examined separately. A hypothetical fish is taken, which has the following charackg and a = 0.05 kgdkg. From eqs teristics: F = 0.1 x 2, 7, and 8, it follows that 6, = 1.6 x m, 6, = 0.47 x m andA = 9.49 x low5m2. The diffusion coefficients in water and lipid depend on molecular weight (eqs 11 and 12). The lipidlwater partition coefficient is set equal to Koxv. For a few parameters, uptake and elimination rate constants are plotted as a function of KO, to include hydrophobicity-related bioconcentration kinetics (Figures 1 and 2). Figure 1A,B shows the influence 6, has on uptake and elimination rate constant. Both kl and k2 decrease, and the maxima of the uptake and elimination rate constant shift toward lower KO,,with increasing aqueous diffusion path length for chemicals with a high &., Chemicals with a low KO, are not affected by 6,. The diffusion coefficient (0,) has the opposite effect as the aqueous path length. For chemicals with a high KO,,both kl and k2 increase, and the maxima of the uptake and elimination rate constant shift toward higher & , with increasingdiffusion coefficient in water. For chemicals with low KO,, neither kl nor k2 is affected by 0,. Both kl and k2 increase with increasing area (A),without affecting the maximum uptake and elimination rate constants, which is at a log KO, of ca. 3 and 2, respectively, independent of area. Figure 2A,B shows the influence 6, has on uptake and elimination rate constant. Both kl and k2 decrease, and the maxima of the uptake and elimination rate constant shift toward higher KO,, with increasing lipid path length for chemicals with a high KO,. Chemicals with a high KO, are not affected by 6,. The diffusion coefficient (D,)has the opposite effect as the lipid path length. For chemicals with a low KO,, both kl and k2 increase, and the maxima of the uptake and elimination rate constant shift toward lower KO,, with increasing diffusion coefficient in lipid. For chemicals with high KO,, neither kl nor k2 is affected by D,. 2772 a ENVIRONMENTAL SCIENCE &TECHNOLOGY / VOL. 29, NO. 11, 1995

5

A

4

2

3

5

2

;1 M

2

1

O l 0

2

4 6 log Kow

10

8

-1

B

J

-6 0

2

4 6 log Kow

8

10

FIGURE 1. Influence of the aqueous diffusion path length on (A) the uptake rata constant (kl and (B) the elimination rate constant (&z) as a function of the octanollwater partition coefficient (KJ. Rate constants are calculated according to the presented model, using a standard fish of 0.1 g and 5% lipid (0).A 10-fold higher aqueous diffusion path length is represented by (A),and a 10-fold lower one is represented by (A).

While kl is unaffected by a change in lipid content (a), k2 is affected in two ways. The elimination rate constant increases, and the maximum eliminationrate constant shifts toward higher &, with decreasing lipid content.

Results Uptake Rate Constants. Within each size class of the fathead minnow, uptake rate constants increased slightly with increasing hydrophobicity of the chemicals, the increasebeing more pronounced for the smallestfish (Table 3). Whereas differencesbetween uptake rate constants of TrCBz are not more than 2-fold between the different size classes, those for TCB and HCB are up to 10-fold. Elimination Rate Constants. Within each size class, elimination rate constants decreased with increasing hydrophobicity (Table 3). For each individual chemical, elimination rate constants decreased with increasing fish sue. Bioconcentration Factors. Bioconcentration factors between size classes differed no more than 5-fold for all chemicals, except for TCB and HCB, where differences of almost up to 2 orders of magnitude are observed (Table 3). Lipid-normalized bioconcentration factors, however, dif-

0

2

4 6 log Kow

8

10

4

B

0

2

4

6

8

10

log Kow

FIGURE 2. Influence of the lipid path length on (A) the uptake rate constant (k,) and (B) the elimination rate constant (6) as a function Rate constants are of the octanoliwater partition coefficient (L). calculated according to the presented model, using a standard fish of 0.1 g and 5% lipid (0).A 10-fold higher lipid path length is represented by (A),and a 10-fold lower one is represented by (A).

fered much more for all chemicals between the different size classes (Table 3). Galassi and Calamari (5) also measured large differences between lipid-normalizedbioconcentrationfactors of 1,2,3and 1,2,4-trichlorobenzenein different life stages of rainbow trout: eyed-egg, hatching, and alevin.

Discussion Model Validation. Uptake Rate Constant. The model of diffusivetransfer assumes that the uptake of hydrophobic chemicals is controlled by their resistance through the aqueous diffusion layer and thus can be described by eq 2. This assumptionwas examined by comparing calculated uptake rate constants and the present and earlier reported uptake rate constants. For this examination,onlyliterature studies were included that studied the elimination of hydrophobicchemicals (log&, > 3) by fish. For calculated uptake rate constants, eq 2 is used, which requires information on fish size, fish gill surface area, the diffusion coefficient of the chemical in the aqueous diffusion layer, and the thickness of the layer, which are given by reported values or determined using eqs 7, 8, and 11. Since uptake rate constants do not differ much for chemicals with a log KO, > 3, an imaginary compound having a molecular weight of 250 glmol was chosen for calculation of the aqueous diffusion coefficient. In general,

good agreement is observed between calculated and experimentaluptake rate constants (Table 4). In general, experimentaluptake rate constants are some higher than calculated ones (ratios 7 1,Table 4). The largest differences are found for uptake rate constants obtained in the present study. The reason for the largest differences being found for the smallest fathead minnow in the present study may be due to the fact that these fish are juvenile fish and may not have fully developed gtlls yet, whereas all other reported studies have used adult or larger fish. However, one would expect to find lower than predicted uptake rate constants in juvenile fish, which is not the case. A better explanation may be that uptake is not solely occurring by gills but also by skin. Lien and McKim (32)proposed that uptake through the skin may significantly contribute to the total uptake rate constant of oxygen and hydrophobic chemicals, in particular for small fish. If uptake of the hydrophobic chemicals by skin would contribute to the totaluptake, the relationship that is found between the uptake rate constants and KO, in the juvenile fathead minnow (Figure 3A) is comparable to either a decreased aqueous diffusion layer (Figure lA), an increased relative area, an increased diffusion coefficient in water, a decreased diffusion coefficient in lipid, or an increased lipid path length with decreasing weight. Since neither diffusion coefficients are likely to depend on the size of fish, an increased relative area, a decreased aqueous diffusion layer, or an increased lipid path length (of skin) with decreasing weight is likely. Since not only the uptake rate constants increase with decreasing weight but also a shift toward higher log KO, for the maximum uptake rate constant is observed, an increased relative area cannot explain the experimentalresults. A decreased aqueous diffusion layer or an increased lipid path length (of skin) with decreasing weight is thus more likely. Elimination Rate Constant. When it is assumed that elimination of hydrophobicchemicals is also controlled by diffusion through the aqueous layer, elimination rate constants will depend on D,, ,a, KO,,A, F, and a (eq 3). This assumption was examined by comparing calculated elimination rate constants and the present and earlier reported elimination rate constants. For this examination, only literature studies were included that studied the elimination of polychlorinated benzenes and biphenyls in fish and reported lipid content and weight of the fish (Table 5). The average ratio of the calculated and experimental elimination rate constants were determined for each study (Table5). For most studies, the model predicted elimination rate constants within a factor 4; for the smallest fathead minnow in the present study as well as for the trout studies, the model underestimated elimination rate constantswithin a factor 14 (Table 5). The elimination rate constants of the chemicals by the smallest fathead minnow in the present study showed one of the largest differences from the calculated elimination rate constants. Experimental elimination rate constants were in general higher than those calculated. Again, the reason for the large differences between calculated and experimentalelimination rate constants for the fathead minnow in the present study may be due to the fact that elimination is not solely occurring by gills but also by skin. When skin contributes in the exchange of chemicals,the total surface area for exchangewill increase, which will increase elimination rate constants. VOL. 29, NO. 11, 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY 12773

TABLE 3

Experimentally Debtmined Uptake Rate Constants &I), Elimination Rate Constants &z), Bioconcentration Factors (Kc),and hid-lllormalized Bioconcentration Factors (KL)for a Series of Polychlorinated Benzenes and Biphenyls in Different Size Classes of Fathead Minnow

a

TrCBz TeCBz pCBz TCB HCB

700 1400 2200 5900

TrCBz TeCBz pCBz TCB HCB

0.33 0.48 0.62 0.20 0.01

TrCBz TeCBz pCBz TCB HCB

3.32 3.48 3.56 4.46

TrCBz TeCBz pCBz TCB HCB

3.94 4.10 4.18 5.08

N Da

ND

ND

0.41 0.068 0.22 0.0996 0.0916 0.0741 Uptake Rate Constant ( 4 in Ukgdl 1000 1300 800 2000 1800 1200 3000 1800 1300 18 000 4700 3800 1 1 000 5900 6300 Elimination Rate Constant ( k in ~ l/d) 1 .o 0.50 0.50 0.40 0.50 0.14 0.24 0.10 0.07 0.25 0.01 0.01 ND 0.01 0.01 Bioconcentration Factor (log rC, in Ukg) 3.00 3.43 3.18 3.70 3.70 3.92 4.08 4.40 4.26 4.86 5.59 5.53 ND 6.18 6.11 Lipid-Normalized Bioconcenhation Factor (log KL in Ukgf,t) 4.00 4.47 4.31 4.70 4.74 5.05 5.08 5.44 5.39 5.86 6.63 6.66 ND 7.22 7.24

1.17 0.0147

0.67 0.0358

800 1200 1500 1500 1100

1300 2900 3500 1800 3400

0.22 0.19 0.15 0.03 0.05

0.18 0.10 0.03 0.03 0.01

3.56 3.80 4.00 4.71 4.34

3.75 4.26 4.40 4.81 5.68

5.39 5.63 5.83 6.54 6.17

5.20 5.71 5.85 6.26 7.13

ND, not determined.

TABLE 4

Experimental and Calculated (Eq 2)8 Uptake Rate Constants of Hydrophobic Chemicals (log KO, =- 3) in Different Fish Species species

F(10-3 kg)

log k1 ( M g d ) calc

log kr(4kg.d) exp

ratiob

fathead minnow fathead minnow fathead minnow fathead minnow fathead minnow fathead minnow guppy rainbow trout American flagfish fathead minnow topmouth gudgeon goldfish goldfish rainbow trout rainbow trout rainbow trout

0.044 0.068 0.22 0.41 1.12 0.67 0.1 0.75 2.5 2.5 4.5 4.5 4.5 9.0 200 750

3.17 3.10 2.93 2.84 2.68 2.76 3.05 2.75 2.57 2.57 2.48 2.48 2.48 2.37 1.90 1.71

3.34 3.48 3.26 3.11 3.18 3.54 3.00 2.77 2.46 2.59 2.51 2.89 2.78 2.46 2.40 2.11

1.48 2.40 2.14 1.86 3.16 6.03 0.89 1.05 0.78 1.05 1.07 2.57 2.00 1.20 3.16 2.51

a For the calculated uptake rate constants, 0 , = 2.1 x Ratio = kl(exp) i kl(calc).

lo-”

1

this this this this this this

study study study study study study

35 36 37 36 38 39 40 27 47 77

m2/s,for which a hydrophobic chemical with a molecular mass of 250 g/mol is used.

If elimination of the hydrophobic chemicals by skin would contribute to the total elimination rate, the relationship that is found between the elimination rate constants and KO, in the juvenile fathead minnow (Figure 3B) is comparable to either a decreased aqueous diffusion layer (Figure 1B), an increasedrelative area,an increaseddiffusion coefficient in water, a decreased diffusion coefficient in lipid, an increased lipid path length, or a decreased lipid percentage with decreasing weight. Since lipid percentage was found to increase with decreasing weight (Table I), an increase of relative area should not have affected the maximum in elimination rate constant, and neither dif-

2774

ref

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29. NO. 11, 1995

fusion coefficients are likely to depend on the size of fish either an increased lipid path length (of skin)or a decreased aqueous diffusion layer in water with decreasing weight is more likely to explain size differences. Other factors which may cause the generally higher experimental elimination rate constants than those calculated following the present model (Figure41, e.g., in trout, are growth dilution, transfer by reproduction, faecal elimination, or biotransformation (6, 46,47). Application of the Model. Some models make use of simple relationships between K,,, and Kc to describe bioconcentration factors. Other models, which in general

/I

/

loo00

I

loo 4

lo

5

7

6

log Kow

j

B

j

4L -5 - 5

6

7

FIGURE 3. Experimentally determined (A) uptake rate constants (k,) and (B) eliminationrate constants (kz)in six size classes of juvenile fathead minnow of a series of polychlorineted benzenes and biphenyls as a function of their octanol/water partition coefficient Fish sizes: 0.045 (B),0.068 (0),0.22 (0),0.41 (0),1.17 (A), and 0.67 g (A). TABLE 5

Average DifferencesB between Calculated and Experimental Elimination Rate Constants of Polychlorinated Benzenes and Biphenyls in Different Fish Species F

a kg) (kgdkg)

fathead minnow 0.045 fathead minnow 0.068 fathead minnow 0.22 fathead minnow 0.41 fathead minnow 1.17 fathead minnow 0.69 0.1 guppy American flagfish 2.5 goldfish 4.5 eel 40 rainbow trout 200 rainbow trout 900

0.238 0.0996 0.0916 0.0741 0.0147 0.0358 0.05 0.10 0.063 0.276 0.08 0.10

no. of chemicals av (n) ratiob 5

4 5

5 5 5

11 3 4 7 7 13

14 7.7 2.2 2.1 0.2 3.4 1.7 2.2 3.0 3.9 3.0 13

-2

0

-1

1

2

FIGURE 4. Experimental (0, kz (exp)) and calculated (&z(calc)) eliminationrate constants of polychlorinated benzenesand biphenyls. The solid line represents a line with slope unity and abscissa through the origin.

log Kow

species

-3

log k2 (calc.)

I

5

4

-4

ref this study this study this study this study this study this study 72, 43, 44 37 39 45 47 42

Individual calculated and experimental elimination rate constants Average ratio between experimental and calculated elimination rate constants: av ratio = l/n{Z[Mexp) t k2(calc)l}. a

of all studies are plotted in Figure 4.

have been developed for small fish, are used to describe bioconcentration kinetics in other, bigger fish. Allometric models are being used for size-related bioconcentration

-1

0

2

4

6

8

1

0

log Kow

FIGURE 5. Calculated uptake rate constants for guppy (top line, F = 0.1 g, a = 0.05 kgtdkg), eel (middle line, F = 40 g, a = 0.276 kgdkg), and rainbow trout (bottom line, F = 900g, a = 0.10 kgtdg), according to the present mass diffusive transfer model, following eq 2.

and are based on empirical relationships on, for example, frequency distributions of sizes and concentrations of contaminants (48, 49). For three fish species, calculated uptake and elimination rate constants are illustrated as a function of %, (Figures 5 and 6) and are compared to literature data. Guppy studies were performed in the laboratory at 20-25 "C, exposure times were approximately2-3 weeks, and elimination times were approximatelyseveralmonths(35,371. The trout study was also performed in the laboratory at 11 "C, exposure was by oral dosing, and elimination time was approximately 3 months (42). The eel study was performed in the field at ambient temperature, exposure time was approximately 1-2 years, and elimination times were approximately 8 years (45). It is clearly seen that large differences exist between the species, both for the uptake and for the elimination rate constants. Only small differences, however, exist between eel (40 g, 27.6% lipid) and rainbowtrout (900 g, 10%lipid) for the elimination rate constant (Figure 6B,C), despite the fact that the trout is much larger than the eel. The high lipid content in combination with the eel VOL. 29, NO. 11, 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY 12775

-5 -

-4

-6-

@

'

1

1

.

1

C

0-

N

24

-

-3-

M

0

-4

-

-5

: . , . , \

-6

lives of the hydrophobic PCBs are very long. During an 8-year depuration study of PCBs in eel, no indication of elimination apart from growth dilution was found, which confirms the present findings. The model predictions for the elimination rate constants in eel underestimate the observed elimination rate constants with an average 4-fold. The unusual shape of eel, having a very large skin to gill area, may explain these differences. It must be noted that the model predictions of the elimination rate constants did not include growth dilution, which may highly affect the concentrations of hydrophobic organic chemicals in fish (6). However, the presented elimination rate constants in eel and trout were corrected for growth. The model thus shows that although elimination of extremely hydrophobic chemicals is observed in small fish, no measurable elimination of these chemicals is to be expected in larger animals. Although earlier bioaccumulation models recognize and include the influence of fish size and anatomy (2,9,12-16, 501,the present model explicitly uses allometric relationships, diffusion coefficients, and diffusion path lengths, which may identify the different rate-determining parameters in uptake and elimination and relate them to weight, lipid content, and physical-chemical parameters. The model shows that exchange surface and lipid content are the main fish properties that determine bioconcentration kinetics. Molecular weight and KO, are the main physicalchemical properties that determine bioconcentration kinetics. The more general allometricrelationships may even get more precise when exact relationships are used, e.g., between weight and gill surface area (13). However, in particular for very small fish, the observed relationships may show large deviations, because of either the relative large contribution of skin to uptake and elimination (32) or to other physiological processes that affect bioconcentration kinetics. In conclusion, a simple bioaccumulation model for hydrophobic chemicals in fish is presented that is based on diffusive mass transfer (2). The model contains parameters that are either related to the fish weight (allometric functions) and fish lipid content or related to physicalchemical parameters (Kaw and M) of the hydrophobic organic chemicals. Skin uptake and elimination as well as physiological factors other than size-related parameters in the model, however, may also be important for bioconcentration kinetics.

Acknowledgments The authors gratefully acknowledge Johannes Tolls for his useful comments. weight thus results in equal elimination rate constants for hydrophobic chemicals as in larger but leaner rainbow trout. The three examples show that large differences are expected in elimination rate constants, which indeed have been found in some studies. A 10-fold increase in size will result in a 3-folddecrease in elimination rate constant. The model shows that large andlor fatty fish, such as eel and rainbow trout, have an elimination rate constant for TCB as low as 0.34 x d-l, while guppy has an elimination rate constant of 16 x low3d-l. This means that the biological half-life of TCB in the large andlor fatty fish is 2000 d (5.6 year), while that in guppy is 43 d. The recent study of de Boer et al. (45) indicates that biological half2776

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TECHNOLOGY i VOL. 29, NO. i i . i s s 5

Glossary Kaw ki

k2 KC KL Kil Cf C W

t

octanol/water partition coefficient (-1 uptake rate constant (L/kgd) elimination rate constant (d-l) bioconcentration factor (L/kg) lipid-normalized bioconcentration factor (L/ kgfat) lipidlwater partition coefficient (-1 concentration in fish (uglkg) concentration in water (UglL) time (d)

a 6, 6, Dm

Dw

F

d A M R S

T N rl V

lipid content (kgfat/kg) diffusion length of the lipid layer (m) diffusion length of the aqueous diffusion layer (m) diffusion coefficient in lipid (m2/s) diffusion coefficient in the aqueous diffusion layer or in water (m2/s) fish weight (kg) distance between two gill lamellae (m) gill surface area (m2) molecular weight (glmol) gas constant (=8.314 J/mol*K) aqueous solubility temperature (KJ Avogrado’s constant (6.0220 x lV3 mol-’) viscosity (PPs) molecular specific volume (m3/moi)

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Received for review February 8, 1995. Revised manuscript received July 3, 1995. Accepted July 10, 1995.@ ES950079L @Abstractpublished inAdvanceACSAbstracts, September 1,1995.

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