Bioconcentration of bromo- and chlorobenzenes by fish (Gambusia affinis)

War. Res. Vol. 31, No. 1. pp. 61-68, 1997

Pergamon PII: S0043-1354(96)00229-1

Copyright © 1996 ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/97 $17.00 + 0.00

B I O C O N C E N T R A T I O N OF BROMO- A N D C H L O R O B E N Z E N E S BY FISH (GAMBUSIA AFFINIS) YUPADEE CHAISUKSANT*, Q I M I N G YUO and DES W. CONNELL Faculty of Environmental Sciences, Griffith University, Nathan, QLD 4111, Australia (First received December 1995)

Abstract--l~;ioconcentration factors, uptake rate constants (kl), and clearance rate constants (k2) were determined for bromo- and chlorobenzenes with the mosquito fish (Gambusia aff~nis). The values of k~ and k2 on a lipid weight basis ranged from 3500 to 47,000 d -~ and 1.50 to 0.27 d -~, respectively. The rate constants e~:hibit good nonlinear correlations with the n-octanol/water partition coefficient (Kow).The bioconcentration factors reach a maximum at pentachlorobenzene (log Kow= 5.2) and then decline. The bioconcentration factors on a lipid weight basis (KBL)correlated well with Kow.The bioconcentration kinetics can be described by equivalent mass transfer models and experimental data indicate that the mass transfer process in the lipid phase is the rate-limiting step in the bioconcentration process. Copyright © 1996 Elsevier Science Ltd Key words---bioconcentration, bromobenzenes, chlorobenzenes, mosquito fish, n-octanol/water partition coefficient, mass transfer process

human adipose tissue (Howard, 1989; Davies and Mes, 1987). The objectives of this study were to investigate the kinetics and bioconcentration factors of bromo- and chlorobenzenes with fish, and to compare the bioconcentration data of bromobenzenes with those of chlorobenzenes. This information would be used to assess the importance of passive diffusion and other processes in the bioconcentration process.

INTRODUCTION Bioconcentration of organic chemicals by aquatic biota is an important factor in the assessment of the potential hazard of chemicals to the environment (Hawker and Connell, 1986). The parameter used to quantify bioconce~tration in aquatic biota is the bioconcentration factor (KB) which is defined as the ratio of the concentration of the chemical in the biota (CB) to that in water (Cw) at equilibrium. The bioconcentration factor is usually measured in the laboratory using fish but alternatively it can be evaluated using quantitative structure activity relationships (QSARs) (e.g. Mackay, 1982; Veith et al., 1983). Most bioconcentration measurements have been carried out with chlorohydrocarbon pesticides, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs), but few on halobenzenes (Veith et al., 1979; Konemann and van Leeuwen, 1980; Gobas et al., 1986, 1989; Shaw and Connell, 1987). Konemann (1981) have derived a relationship between log Kow and log KB for chlorobenzenes in :fish. The halobenzenes are widely utilised in household, agriculture, and industry (Sittig, 1985; Richardson and Gangoli, 1994). These substances are lipophilic and degrade slowly, and so remain for relatively long periods in the environment after their use. Residues have been found in natural water (Rogers et al., 1989), soil and sediments (Kaiser et al., 1990), fish (Ofstad et al., 1978), dairy products, and animal and

THEORETICALBACKGROUND Kinetics o f bioconcentration

A first-order kinetic model which uses a single compartment to represent an organism is the most common model for bioconcentration kinetics with nonbiodegradable lipophilic compounds (Moriarty, 1975; Connell, 1988). The biotic concentration, CB (CBL for lipid-based), after an exposure time period of t, can be expressed as CB = Cw(k,/k2)(1 - e -k2')

(1)

where CB is the biotic concentration and Cw the aqueous concentration of the chemical, and k~ (kLI for lipid-based) and k2 the rate constants of the uptake and clearance processes, respectively. These kinetic rate constants and the bioconcentration factor have been found to correlate well with the n-octanol/water partition coefficient, Kow in the range log Kow value of 2.5~.0 (Connell and Hawker, 1988; Banerjee and Baughman, 1991).

*Author to whom all correspondence should be addressed [Fax: +61 7 3875 7459]. 61

Y. Chaisuksant et al.

62

Mass transfer process It is generally regarded t h a t the m e c h a n i s m o f b i o c o n c e n t r a t i o n o f lipophilic chemicals is a passive diffusion of the organic chemicals between water a n d the organism. The b i o c o n c e n t r a t i o n kinetics can therefore be related to relevant mass transfer models t h a t are equivalent to the first-order kinetics (Barber et al., 1988; G o b a s et al., 1986; M a c k a y a n d Hughes, 1984). T h e kinetic constants, kl a n d k2, are expressed in terms o f either mass transfer rate or mass transfer resistance. In the f o r m u l a t i o n of Barber et al. (1988), it is assumed t h a t the mass transfer resistance in the internal phase o f the fish is negligible a n d the relevant c o n c e n t r a t i o n gradient is between the interlamellar water a n d the aqueous p o r t i o n of the capillary b l o o d of the gills. F o r h y d r o p h o b i c chemicals, it is also assumed that the tissue-phase mass transfer resistance is negligible a n d the water-phase mass transfer is the rate limiting step. Similar conclusions were reported by G o b a s et al. (1986). The b i o c o n c e n t r a t i o n kinetic constant, k~, is linearly p r o p o r t i o n a l to the waterphase diffusivity o f a chemical ( G o b a s et al., 1986) or the external water-phase mass transfer coefficient which is linearly p r o p o r t i o n a l to the water-phase diffusivity (Barber et al., 1988). Mass transfer correlations generally show t h a t the mass transfer coefficient is p r o p o r t i o n a l to D n, where D is the diffusivity, a n d n = 0.5-1. MATERIALS AND EXPERIMENTAL METHODS

Materials Chlorobenzenes: 1,4-dichlorobenzene (1,4-diCB), 1,2,3trichlorobenzene (1,2,3-triCB), 1,3,5-trichlorobenzene (l,3,5-triCB), 1,2,3,4-tetrachlorobenzene (1,2,3,4-teCB), 1,2,3,5-tetrachlorobenzene (1,2,3,5-teCB), pentachlorobenzene (pentaCB), hexachlorobenzene (hexaCB); and bromobenzenes: 1,4-dibromobenzene (1,4-diBB), 1,2,4tribromobenzene (1,2,4-triBB), 1,2,4,5-tetrabromobenzene (1,2,4,5-teBB), were purchased from Aldrich Chemical Company and were all stated to be a minimum of 97% pure. Acetone and diethyl ether (analytical grade) and hexane (commercial grade) were obtained from Ajax Chemical, and analysis indicated these solvents did not contain impurities which would cause interference with sample analysis. Nitrogen gas (ultra high purity grade) used for evaporation was from Commonwealth Industrial Gases (CIG). Glass fibre filters (GF/C) from Whatman were heated overnight at 450°C before use. Anhydrous sodium sulfate (BDH) and florisil (Mallinckrodt Chemicals) were activated by heating overnight at 450°C and 600°C, respectively, and stored in

airtight glass bottles. The latter was deactivated prior to use by adding 5% (w/w) of distilled water.

Fish The mosquito fish (Gambusia a~nis) captured from local streams were acclimated in glass aquaria containing dechlorinated tap water for at least 2 weeks before use in experiments. During the acclimation period they were fed daily with commercial fish food at a level of approximately 1% of body weight per day (OECD, 1992). The fish were not fed for 24 h prior to or during the bioconcentration experiments. Adult fish, both male and female, with an average length of 2.75 cm (n = 300, SD = 0.32) and average weight of 1.90 mg (n = 300, SD = 0.06) were used in the experiments. Bioconcentration experiments Bioconcentration experiments were carried out using the semistatic system at 23.1°C (n = 8, SD = 1.1) (OECD, 1981). The pH of the exposure water was 7.6 (n = 64, SD = 0.2). Three experiments were carried out with fish (10/jar) exposed in five tightly closed glass jars (5L) to one of three concentrations of a mixture of eight chemicals giving a total of 150 fish. This was then repeated at a different time to give a duplicate set of results. The exposure water contained one of three concentrations of eight chemicals prepared from acetone stock solutions as shown in Table 1. The highest concentration used consisted of a mixture with each chemical present in a concentration equal to 1/20 of the LCs0. The amount of acetone used in the final exposure was 0.1 mL per jar giving a concentration of 20 ppm (v./v.). This was not found to cause any mortalities in the controls. The water containing the test chemicals was renewed at each exposure concentration at 12-h intervals. The dissolved oxygen (DO) in the exposure water was found to be sufficient for test organisms during 12-h intervals. The average-observed DO values in the exposure water at the beginning and end of the renewal were 9.4ppm (n = 64, SD = 0.2) and 8.5 ppm (n = 64, SD = 0.3), respectively. The used water (500 mL sample) was extracted with hexane at each renewal period to determine the level of chemical remaining in the water after the exposure period. A total of five organisms, i.e. one from each jar, were randomly selected for removal from each exposure concentration at 12, 24, 36, 48 and 96 h from the start oftbe experiment. The control was treated in the same manner to that described above except acetone (0.1 mL) only was added. The experiments were conducted twice for each set of test chemicals concentrations. The exposed organisms were wrapped in aluminium foil and stored at - 2 0 ° C in sealed glass containers for later analysis. Clearance The organisms were exposed to the chemicals as outlined above except no sampling was undertaken. At 96 h-time periods the fish were transferred from each exposure concentration into clean water which was renewed every 24 h. During the experiment the fish were fed once daily. Five randomly selected individuals were removed from the

Table 1. Nominal and actual concentrations of chloro- and bromobenzenes in exposure water Experiment 1 Experiment 2 Experiment 3 Nominal Actual' Nominal Actual' Nominal Actual, Chemical Concentration (/~g1- t) 1,4-diCB 200 233.0 + 23.9 100 101.0 + 9.1 50 57.0 + 4.1 1,4-diBB 40 45.3 + 6.2 20 30.8 5:6.5 10 22.0 + 1.4 1,2,3-triCB 140 132.2 + 18.9 70 80.8 + 4.8 35 38.5 + 2.4 1,2,3,5-teCB 30 20.1 + 4.0 15 12.0 + 1.6 7.5 8.4 -I- 1.1 1,2,4-triBB 20 17.7 -I- 2.3 10 10.8 :t: 2.4 5 7.3 + 1.0 pentaCB 12 15.7 + 2.7 6 6.2 + 1.1 3 3.3 + 1.0 hexaCB 5 3.7 + 0.8 2.5 2.1 + 0.6 1.25 1.2 + 0.2 1,2,4,5-teBB 8 7.8 + 1.2 4 3.8 + 1.8 2 2.9 + 0.5 • Mean + standard deviation (SD); n = 16.

Bromo- and chiorobenzenes in mosquito fish clean water for each exposure treatment at 6, 12, 24, 48 and 96 h from the transfer time and were treated in the same manner to those in 1he uptake experiment.

Isolation of bromo- gInd chlorobenzenes The stored organisms (n = 5) were first thawed and dried on paper, then were weighed (approximately 2 g) and ground in 10 mL of laexane and 15 g of anhydrous sodium sulfate. Appropriate internal standards were added before the grinding process. The homogenate was then filtered with GF/C glass fibre and washed with 50 mL of hexane before making up the volume of the hexane extract to 60 mL with hexane. The extract (40 mL sample) was concentrated to 3 mL by evaporation under a gentle stream of ultrapure nitrogen in a water bath set at 40°C. The chloro- and bromobenzene fractions were isolated by loading the concentrate into a glass column (450 × 1.5 mm) containing deactivated florisil (10 g) topped with 2 g of anhydrous sodium sulfate which was pre-wetted with hexane, and eluted with 65 mL of 5% diethyl ether in hexane. The eluate was concentrated and stored at - 2 0 ° C in glass vials for later analysis by gas chromatography.

Analytical methods Analysis of chloro- and bromobenzenes in the final concentrate was conducted using a Tracor Gas Chromatograph (model 560) fitl:ed with a linearizer, Hewlett Packard 3390A integrator and 63Ni electron capture detector. The fused silica capillary column (DB5, 0.25 #m thickness, J&W Scientific) was 30 m by 0.25 mm ID. The carrier gas was ultra high purity helium (CIG) at a flow rate of 0.5 mL min -~ with 10% of methane in argon (CIG) as the make-up gas. The oven temperature was 50-250°C, with 10°C rain -~ program rate while those of the injector and detector were 220 and 300°C, respectively. A standard simple injection volume (1 #L) was used. Identification of chlLoro- and bromobenzenes was carried out by comparison of the retention times of the peaks in the samples relative to the internal standards (1,3,5-triCB and 1,2,3,4-teCB) with the relative retention times of standard reference chemicals. A calibration curve was prepared using 1,3,5-triCB and 1,2,3,4-teCB as the internal standards. All plots were linear with a correlation coefficient of 99% or better with least squares method. Recoveries of each chloro- and bromobenzene from fish tissue were determined by spiking five tissue samples (3 g) with hexane solution containing a known amount of each chemical. The samples were treated by the procedure as described above and the estimated percent recoveries of each compound were: 72 (l,4-diCB), 83 (1,4-diBB), 73 (I,2,3triCB), 70 (l,2,3,5-teCB), 68 (l,2,4-triBB), 74 (QCB), 77 (HCB), and 78 (1,2,4,5-teBB). For tissue blanks, l:hree separate analyses of fish tissue that had not been exposed to chloro- and bromobenzenes

63

were conducted using the procedure outlined above. No chloro- and bromobenzenes were found in these blanks. Lipid content was determined using an aliquot (20 mL) of the hexane extract from the isolation step. By evaporating this solution to dryness under a gentle stream of nitrogen gas at room temperature in a pre-weigbed beaker, the fish lipid content was estimated after weighing to a steady weight. The fish tissue concentrations (CBL) were all expressed on a lipid weight basis. The mean lipid content was 3.1% of fish weight (n = 300, SD = 0.9). RESULTS AND DISCUSSION

Observed bioconcentration factors and kinetic rate constants Plots o f bioconcentration and clearance o f chloroand b r o m o b e n z e n e s are shown in Fig. 1. All c o m p o u n d s were found to fit first-order kinetics with high correlation coefficients (r 2 > 0.90). The clearance rate constant values (k2) obtained from the slopes o f the linear regression lines for plots o f In Ca against time were averaged from three sets o f experiments and shown in Table 2. These values were then used with equation (1) to determine the uptake rate constants in terms o f wet weight, k~, and lipid weight, kL~. The r 2 values in all cases ranged from 0.89 to 0.99 while the intercepts were at acceptable values. The mean values o f k], kL~, Ks and Kat are also summarized in Table 2. The bioconcentration factors for the chlorobenzenes are comparable to those reported by K o n e m a n n and van Leeuwen (1980) with guppy, and Oliver and Niimi (1983) with rainbow trout as shown in Table 3. F o r 1,4-diBB, the kinetic values are also in the same order o f magnitude o f those with guppy reported by G o b a s et al. (1989) (wet weight-based kt and k2 o f 130 and 1.41 d ~while in this study 272 and 1.26 d-~, respectively were found). The bioconcentration factor for 1,2,4,5-tetrabromobenzene (Ka 2800) is consistent with that found by Oliver and Niimi (1985) (KB 3700--8200).

Relationship between bioconcentration factor, uptake and clearance rate constants and the n-octanol/water partitional coefficient Typical plots o f uptake rate constants (as logs) against log Kow are shown in Fig. 2. It was found that

Table 2. Observed bioconcentrationfactors and kinetic rate constants for the test chemicals

Chemical 1,4-diCB 1,4-diBB 1,2,3-triCB 1,2,3,5-teCB 1,2,4-triBB pentaCB hexaCB 1,2,4,5-teBB

log Kow 3.44 3.89 4.07 4.67 4.98 5.20 5.77 6.04

Uptake rate constant (_+SD)a d -, Wet wt Lipid wt k~ ku( x 104) 112 _ 20 272 + 67 470 + 10 631 _ 96 1040 + 320 1520 + 600 1850 5- 700 900 5- 270

+ Standard deviation; n = 6. bWet weight-based bioeoneentration factor. ~Lipid-based bioconcentration factor.

0.35 5- 0.04 1.04 + 0.29 1.07 __.0.21 2.00 + 0.70 2.62 5- 0.82 3.40 5- 1.20 4.70 + 1.80 2.37 5- 0.38

Clearance rate constant (5-SD)a d -, Wet wt Lipid wt k2 k2 1.44 5- 0.55 1.26 + 0.25 1.10 + 0.09 0.48 5- 0.05 0.58 + 0.07 0.38 + 0.07 0.49 5- 0.03 0.32 + 0,09

1.50 _ 0.67 1.32 + 0.32 0.90 _+0.18 0.45 + 0.07 0.40 + 0.07 0.27 + 0.05 0.46 5- 0.13 0.30 + 0.05

Bioconcentration factor (Ke)b (__SD)~

Bioconcentration factor (KaL)c (_+SD)~(× 104)

78 __. 12 220 __.54 430 _+70 1320 + 300 1800 + 220 4000 + 980 3730 5- 1,100 2800 + 40

0.25 _ 0.68 0.78 + 0.41 1.20 + 0.24 4.30 5- 0.92 6.64 + 2.00 12.40 + 2.30 10.00 + 1.20 8.24 + 2.30

64

Y. Chaisuksant et al.

~

Bloconcentratlo n - ) ~ - ~ - C t a a r a nee

-J

, Bloconc entr|tlon.)/t(_~Clearanc ¢

•

4 A

o

E 3

,0/ / / "

"I

. ,,0,BB

o 0.5

E 3

3-

0

2-

~

0 50

100

Time

2.S

E

o

150

0

200

100

/" 1,2,3-MCB

150

200

(hr)

Time

1.0' ( - B l o c o n c e n t r a t l o n ~ [ < - ~ Clearance

Clearance

/

0.8

-I

2.0

0'0

(hr)

/ ~C-Bloconcentratlon ~

¢

l~

1-

0.0 0

Tf

'

h ~ a •C B o 1,2,4,5-teB8

U 1,2,4-triBB

10/ 1

1'1

,an.C.

E

o.6

¢j

0.4

0.2

0.5

O0

~h 0

50

"100 Time

150

0.0

200

5'0

(hr)

100 Time

lS0

200

(hr)

Fig. 1. Plots of lipid-based concentrations (CBL) of 1,4-diCB, 1,4-diBB, 1,2,3-triCB, 1,2,3,5-teCB, 1,2,4-triBB, pentaCB, hexaCB, and 1,2,4,5-teBB in fish versus time period for bioconcentration and clearance (C, zero).

there is an acceptable linear relationship over the range from 1,4-diCB to hexaCB, excluding 1,2,4,5teBB, as expressed by the following regression equation:

alternatively by the following quadratic expression that fits all data including 1,2,4,5-teBB: log kLI = -- 2.23 + 2.44 log Ko, -- 0.22(Iog/(Low) 2 (n = 8, r 2 = 0.95)

log kLI = 2.11 + 0.46 log Kow (n = 7, r 2 = 0.95) (2) The log kL~-log Kow correlation can be described

(3)

The uptake rate constant of 1,2,4,5-teBB (log Ko, = 6.04) is less than that expected by linear

Table 3. Physico-chemical characteristics of bromo- and chlorobenzenes

Molecular Chemical log/(Low' log KaLb weight 1,4-diCB 3.44 3.40 147.01 1,4-diBB 3.89 3.90 235.83 1,2,3-triCB 4.07 4.10 181.44 1,2,3,5-teCB 4.67 4.63 215.88 1,2,4-triBB 4.98 4.82 314.73 pentaCB 5.2 5.10 250.32 hexaCB 5.77 5.00 284.76 1,2,4,5-teBB 6.04 4.91 393.64 'From Chessells et al. (1992). Wrhis study based on lipid weight. 'Calculated by the Le Bas method (Miller et al., 1985). ~From Gavezzotti (1985).

No. of halogen atoms

Molar volume,c Vm (era3 mol -I )

Molecular free surface area,~ Sm (A2)

Aqueous solubility,' Sw (in log) (/zg L-')

Lipid solubility,' SL (in log) (/zg L-')

2 2 3 4 3 5 6 4

137.8 142.6 158.7 179.6 165.9 200.5 221.4 189.2

141.0 157.2 158.1 175.2 182.4 192.3 209,4 207,6

4.77 4.92 4.36 3.72 3.86 3.51 2.69 1,60

8.54 9.22 8.62 8.57 9.07 8.65 8.69 7.90

Bromo- and chlorobenzenes in mosquito fish

O~)

--

authors found that the log k2 was best fitted to the following polynomial in log Ko.:

hex,CeJ

s'° 1

484

pentaCBJ

]

m

log k2 = - 5.27 + 5.34 log Ko, - 1.65(Iog Ko,)2 + 1.85 x 10-1(log Kow)3

1,2,4,5-teBB

/~"

4.2"1

//1,2,3,5-,.CB

-

I 1,4"dlOE; _~j~"

3.8]

y

65

1,2,3-trlCB

6.9 x 1 0 - 3 ( l o g K o . ) 4

(n = 45, r 2 = 0.79)

The log Ks and log KBL values were plotted against the log Ko, values and the linear regression equations obtained by excluding hexaCB and 1,2,4,5-teBB were

(ll)

3,4 ~ . 1'4;'dl,CB . . . . .

(6)

0.4"

log KB = 0.92 log Ko, -- 1.21

_o*°'°° -0.8

log KBL = 0.93 log Ko, + 0.25

,., .

.

4

.

.

.

S

;

(n = 6, r 2 = 0.99)

(7)

7

log Kow Fig. 2. Plots of the lipid-based uptake rate constant (kL]) (a) and clearance rate constant (k2) (b) versus Ko, for bromoand chlorobenzenes with fish.

extrapolation from the rest of the chemicals. This is in accordance with the decline in log kL~ when the log Ko, value exceeds 5.5-6.0 as observed by Connell and Hawker (1988) with fish and Mortimer and Connell (1993) with crabs. This can probably be attributed to decreasing solubility in lipid and/or other factors such as aqueous solubility and membrane permeability (Miller, 1985; Gobas et al., 1986, 1988; Mortiraer and Connell, 1993). The plot of the clearance rate constants, as log k2, against log Ko, exhibited a good linear relationship with compounds having log Ko, values from 3.4 to 5.2, excluding hexaCB and 1,2,4,5-teBB (Fig. 2). The linear regression equation is as follows:

According to Connell (1990), plots of 1ogKBL versus log Ko, should have a slope of unity and an intercept of zero if octanol perfectly represents biota lipid. In this equation the intercept of 0.25 is close to zero on the scale that the log KBt values are expressed, and the slope of 0.93 is close to unity. In addition, this equation is consistent with, for example, those obtained by Konemann and van Leeuwen (1980) for chlorobenzenes with guppy. The values log KBt for hexaCB and 1,2,4,5-teBB derived by linear extrapolation from the linear relationship previously reported, equation (7), deviate from the actual experimental values. When a polynomial expression is fitted a more satisfactory equation describing the full data set is obtained (r 2 0.99). The KBL values reported by Konemann and van Leeuwen (1980) for fish reach a maximum at hexachlorobenzene (2.9 x 105) with a KBLof pentaCB

6.5

log k2 = 1.75 - 0.44 log Kow (n = 6, r 2 = 0.97)

s.0. (4)

:~

This relationship is similar to those reported by Konemann and van Leeuwen (1980) for chlorobenzenes with fish and ldortimer and Connell (1994) with the crab. Alternatively, the log kr-log Ko, relationship can be described by a quadratic equation that fits all data including hexaCB and 1,2,4,5-teBB (Fig. 2). Thus

~ -

log k2 = 4.27 - 167 log Ko. + 0.15(log Ko.)2

(n = 8, r 2 = 0.90) (5) This relationship is comparable to that established by Connell and Hawker (1988) with fish for chlorinated hydroc~Lrbons having log Ko, 2-9. Those

a.s.

a.0.

0.5

log Kow Fig. 3. Plots of bioconcentration factor against Ko, of bromo- and chlorobenzenes based on wet weight (a) and lipid weight (b) with linear regression and quadratic expressions.

66

Y. Chaisaksant et al.

Table 4. Linear correlationships between lipid-based bioconcentration factor (KBt) and physico-chemical properties (P); log KaL = a P + b P V,~ Sm log Sw log SL/Sw

a

b

r2

n

Equation no.

0.025 0.033 - 1.02 1.10

0.27 - 1.27 8.95 --0.6

0.86 0.99 0.86 0.95

7 6 6 6

(8) (9) (10) (11)

a = Regressioncoefficient,b = intercept,r2= correlationcoefficient, n = number of the test compounds. of 2.6 x 105. In this study the maximum was at pentaCB (/(BE 1.26 X 105). Generally, a maximum log KB value has been found at log Kowof about 6 and then a gradual decrease of log KB value with the log Kow values greater than 6 (Connell and Hawker, 1988; Bruggeman et al., 1984; Gobas et al., 1989). It is noteworthy that this decline results in a loss of biological activity of hexaCB and 1,2,4,5-teBB, particularly evident with toxicity. HexaCB has been found to lack toxic effect on fish, crustaceans, and annelid worms since its aqueous solubility is less than a probable toxic concentration (Laska et al., 1978; Nebeker et al., 1989). Relationships o f bioconcentration parameters with other physiological parameters

Apart from hydrophobicity, expressed as log Kow, other physicochemical parameters as summarized in Table 3 were also investigated for their relationship to bioconcentration characteristics in fish. Good linear relationships were found between log/(BE and molar volume calculated by the Le Bas method (Miller et al., 1985); IogKBL and molecular free surface area as determined by the Gavezzotti (1985); log KBL and aqueous solubility. These relationships can be described by the regression equations shown in Table 4. In a similar way to the relationships between the bioconcentration factor and log Kow, a more satisfactory relationship is observed with the full data set using quadratic expressions giving r 2 values ranging from 0.86 to 0.97.

log KBL(Br) = -- 0.43 + 2 . 9 9 B r - 0 . 4 1 ( B r ) 2 ( n = 3 , r 2=1.00)

(15)

The correlation with three points with the bromocompounds requires further verification with more compounds; however, the line produced is in accordance with that of chlorocompounds. The overall correlations of log KB with molecular volume (Vm) and free surface area (S~) suggest that the increased molecular size of the bromocompounds has resulted in an increased hydrophobicity of these compounds since the molar volume exhibits a wide divergence between the two groups for the same number of halogen substituents. These results suggest that the increased size of the bromine atom, reflected in increased molecular surface area and volume, results in increased lipophilicity (reflected by the log Kow values) giving a trend of higher uptake rate constants and lower clearance rate constants. Mass transfer process

The basic model for bioconcentration is based on passive diffusion with the assumption that mass transfer resistance in the fish is assumed to be negligible. The assumption that the lipid-phase mass transfer resistance is much less than that in the external water phase was tested with the experimental data by plotting log k~ against log Dw, the waterphase diffusivity, using data from this study and Konemann and van Leeuwen (1980). The diffusivities of the chemicals were estimated from the Wilke and Chang (1955) correlation (Brodkey and Hershy, 1988). The relationships between lipid-based uptake rate constant and the water-phase diffusivity are as follows: log kLl = -- 68.17 -- 7.90 log Dw (this study; n = 8, r 2 = 0.84)

(16)

log kr~ = - 56.75 - 6.62 log Dw (Konemann and van Leeuwen (1980);

Influence o f halogen type on bioconcentration

n = 6, r 2 = 0.62)

The 1ogKBL of chloro- and bromobenzenes separately correlate well with molecular weight and number of halogen atoms. The following equations were found: log gilL(El) =

-

2.61 + 5.54 x 10 -2 M W

- - 1 . 0 0 × 10 - 4 M W 2 ( n = 5 , r 2=0.99)

(12)

log KBL(Br) -~ -- 3.80 q- 4.84 x 10 -2 MW -6.67x

l0 -SMW:

( n = 3 , r 2=1.00)

(13)

log KBt(CI)= 1.11 + 1.37 C1 - 0.12(C1) 2 (n = 5, r 2 = 0.99)

(17)

A negative slope was obtained instead of a positive slope. This indicates that the lipid-phase mass transfer resistance may be important. Mackay and Hughes (1984) pointed out that the overall mass transfer resistance consists of those in both the water and the lipid phases. The relative importance of the two mass transfer steps can be analyzed by the following two-phase mass transfer model. The bioconcentration kinetics is then described by the two-film mass transfer model. aCa/c3t = k~(C~ -- C~i)

= kL(CBi - C~) (14)

= K(C.

-

G/K~)

(18)

Bromo- and chlorobenzenes in mosquito fish

where Cwi and CBi are water-phase and biota-phase concentrations at the interface, respectively; kw and ke are the mass transfer coefficients (per unit volume) in the water and the lipid phases, respectively; and K is the overall mass transfer coefficient which can be expressed as 1/K := 1/kw + 1/(kL'KB)

(19)

where 1/kw and 1/(kL'KB) represent the mass transfer resistance in the water and the lipid phases, respectively; and 1/K is the overall mass transfer resistance. The above model is equivalent to the first-order bioconcentration kinetics model, with kt = K and k2 = K/KB and, to t]ae models of Gobas et aL (1986), Mackay and Hughes (1984) and Barber et al. (1988). Therefore, they all should correlate the bioconcentration data equally well. If we make the assumption that the lipid-phase mass transfer resistance is negligible, 1/kw >> 1/(kL"KB)

(20)

then k~ = K = k w . It was found that this is inconsistent with experimental data. If we make the assumption that the. opposite case is true, 1/kw<< l/(kL.KB)

(21)

then kt = K = kL'KB

and k:~ = K/K8 = kL

(22)

This suggests that the plots of log k~ against Iog(DL'KB) and log k2 against IOgDL should show positive slopes, where DL is the lipid-phase diffusivity. The following linear relationships show that this is the case. log kLl = 7.49 + 061 1og(DL'KBL) (this study; n = 8, r 2 = 0.84)

(23)

log kLi = 6.03 + 0.40 Iog(DL'KBL) (Konemann and van Leeuwen (1980); n = 6 , r 2=0.76)

(24)

log k2 = 53.28 + 5 44 log DL (this study; n = 8, r 2 = 0.66)

(25)

log k2 = 95.15 + 9 73 log DL (Konemann and van Leeuwen (1980); n = 6 , r 2=0.98)

(26)

This indicates that the mass transfer resistance in the lipid phase is impc,rtant in the bioconcentration process. WR3UI--C

67 CONCLUSIONS

The uptake and clearance rate constants and the bioconcentration factors of bromo- and chlorobenzenes with fish are related to the log Kow values over the range of about 3 to about 6. For more hydrophobic compounds a decrease in bioconcentration occurred. The uptake and clearance rate constants (as logs) correlate well with log Kow using linear and quadratic expressions. These relationships are similar to those reported previously in fish and other aquatic organisms. The bioconcentration factor also showed a good relationship with other physico-chemical properties, i.e. molar volume, molecular free surface area, aqueous solubility, and lipid solubility. Because of the inconsistency of the experimental data with the assumption that the lipid-phase mass transfer resistance is negligible compared to that in the external water phase, the mass transfer resistance in the lipid phase is most likely rate limiting in the bioconcentration process. REFERENCES

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