Relation between toxicity and accumulation of chlorophenols at various pH, and their absorption mechanism in fish

~

Pergamon

0043-13,~1(94)00189-8

Wat. Res. Vol.29, No. 2, pp. 431-442, 1995 Copyright © 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/95$7.00+ 0.00

RELATION BETWEEN TOXICITY A N D A C C U M U L A T I O N OF CHLOROPHENOLS AT VARIOUS pH, A N D THEIR ABSORPTION MECHANISM IN FISH TAKUOKISHINOl*~ and KUNIOKOBAYASHI2~ ~Environmental Science Laboratory, Ube College, 5-40 Bunkyochyo, Ube 755 and 2Laboratory of Fisheries Environmental Science, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi, Fukuoka 812, Japan (First received November 1993; accepted in revised form July 1994) Abstract--The relation between the acute toxicity and accumulation of 12 chlorophenols in goldfish exposed to media containing the chemicals at various pH, and also the transport mechanism of chlorophenols from the surrounding water in the fish were investigated. Both the acute toxicity and accumulation of chlorophenols increased with increasing number of substituted chlorine atoms, although the magnitude of their increase decreased with increasing pH. For each chloropbenol, both the toxicity and the amount accumulated in the surviving fish was almost unchanged in the range of pH < pKa, but abruptly decreased with increasing media pH in the range of pH > pKg. On the other hand, the amount found in the dead fish was little dependent on the pH of media. The distribution ratio (K) between the medium (compartment I) and fish body (compartment II) was calculated on the basis of the assumption that only the undissociated form migrates between the two compartments by diffusion and that the ratio of the concentration of the undissociated form in compartment I to that in compartment II is proportional to the partition coefficients in 1-octanol-water and n-heptane-water systems. A good correlation was observed between log bioconcentration ratio (BCR) and log K value calculated by using the partition coefficient in the 1-octanol-water system. From these results, it is concluded that the transfer of chlorophenols from media to fish body, in which the formation of intermolecular hydrogen bond between the hydroxyl group in the chemicals and the components in gill membranes plays an important role, is mainly caused by the passive diffusion of the undissociated form through the membranes, and the concentration of the chemicals in the fish body does not readily reach a lethal level with increasing pH of the medium owing to the increased conversion of the undissociated form to the dissociated form, resulting in the reduction of the toxicity ofchlorophenols in the fish. However, the BCR was a little higher than that estimated from the concentration of the undissociated form in the media. The reason was presumed that the pH near the outer surface of the gill membranes is lower than that of the media. Key words--chlorophenols, toxicity, bioconcentration, pH, absorption mechanism, compartment model, fish

INTRODUCTION It is well known that the toxicity of ionizable compounds in aquatic organisms depends significantly on their degree of ionization caused by the pH of surrounding water (CrandaU and Goodnight, 1959; Sills and Allen, 1971; Marking, 1975; Dalela et al., 1980; Holcombe et al., 1980; Saarikoski and Viluksela, 1981). Holcombe et al. (1980) reported that a decrease of the toxicity of 2,4-dichlorophenol to fathead minnow with an increase of pH is mainly attributed to the decrease of the concentration of the undissociated form. On the other hand, Saarikoski and Viluksela (1981) reported that not only the undissociated form but also the dissociated form contributes to the toxicity, because the decrease in the toxicity of bromo-, chloro- and nitro-substituted phenols to guppy with an increase of pH is not as *Author to whom all correspondence should be addressed. 431

great as estimated based on the decrease of the concentration of the undissociated form in the surrounding water. In our previous study on the absorption and excretion of pentachlorophenol (PCP) by goldfish (Kobayashi and Akitake, 1975a), the mortality in various concentrations of PCP occurred when PCP concentration in the fish body reached a level of about 100#g g-1 body weight. It was presumed, therefore, that the change in the toxicity of PCP to fish by varying the pH of media must be based upon the change in the accumulation of PCP by fish. It has been reported that the pH-dependent absorption curve of ionizable compounds does not correlate with their ionization curve (Kakemi et al., 1969; Jollow and Brodie, 1972; Rubery and Sheldrake, 1973; Marking, 1975; Gutknecht and Walter, 1980; Saarikoski et al., 1986). The bioaccumulation of organic compounds in aquatic organisms is predicted by using the partition coefficient in

432

TAKUOKlSHINO and KUNIOKOBAYASHI 1989). After purifying 3-CP by distillation (bp 214-217°C), 2,6-DCP, 3,5-DCP, 2,4,5-TCP, 2,4,6-TCP and 2,3,4,6-TCP by recrystallization from ligroin, and PCP from benzene, their purities were also confirmed to be about 100% by the same gas chromatography procedure described above.

solvent-water systems such as 1-octanol-water (Neely et al., 19742 Veith et al., 1979; Gossett et al., 1983; Davies and Dobbs, 1984). In our previous paper (Kishino and Kobayashi, 1980), a pH-dependent linear relationship was observed between the bioconcentration ratio (BCR) of PCP in fish and its distribution ratio in the 1-octanol-water system in the p H range from 5.5 to 10. The present study was undertaken to ascertain the relationship between the acute toxicity of chlorophenols and their accumulation in goldfish exposed to media containing the chemicals at various pH. The absorption mechanism o f chlorophenols from media in fish was also investigated using the partition coefficients in l-octanol-water and n-heptane-water systems.

Effect of p H on the acute toxicity and accumulation of chlorophenols Firstly, PCP which has been often used in acute toxicity tests for fish as a standard toxicant, was subjected to an experiment as follows. Tap water was passed through an activated charcoal filter, aerated for 30 min and adjusted to pH 5.5, 6, 7, 8, 9 and 10 with l M NaOH or l M HC1 solution, respectively. The proper amounts of PCP were added to each pH-water at several concentrations as shown in Fig. 1. Twenty goldfish, Carassius auratus, having a body weight of 1.3 _ 0.1 g were placed in each 501 of the media at 20-21°C. As shown in our previous papers (Kobayashi and Akitake, 1975b; Kobayashi et al., 1977), it takes about 5 h for PCP to be excreted into the gall bladder of goldfish as its non-toxic glucuronide after the fish are exposed to PCP-media. Therefore, this experiment was carried out within a 5 h period. The suitable numbers of surviving fish in each group were taken out from the media at l, 2.5 and 5 h exposure, killed by electrocution (100V, AC), weighed, after removing water with filter paper, and then pooled in whole bodies. The pooled specimens were subjected to the determination of PCP by 4-aminoantipyrine method or gas chromatography (ECD) after monochloroacetylation (Kishino, 1989). The dead fish found in the test media were also assayed for PCP. The variation of pH in the media was within +_0.2 during 5 h exposure. For the other chlorophenols, similar experiments were also carried out at pH 6, 8 and l0 of their media in several concentrations as shown in Fig. 3, using 30 goldfish (2.2 +_0.2 g).

MATERIALS AND METHODS Chlorophenols Phenol and 12 chlorophenols were used in this study as follows: 2-chlorophenol (2-CP); 3-chlorophenol (3-CP); 4chlorophenol (4-CP); 2,3-dichlorophenol (2,3-DCP); 2,4dichlorophenol (2,4-DCP); 2,5-dichlorophenol (2,5-DCP); 2,6-dichlorophenol (2,6-DCP); 3,5-dichlorophenol (3,5DCP); 2,4,5-trichlorophenol (2,4,5-TCP); 2,4,6-trichlorophenol (2,4,6-TCP); 2,3,4,6-tetrachlorophenol (2,3,4,6-TCP) and PCP. Phenol, 2-CP and 4-CP were obtained from Wako Pure Chemical Industries Ltd, Osaka, Japan and the other chlorophenols from Tokyo Chemical Industry Co. Ltd, Tokyo, Japan. The purities of phenol, 2-CP, 4-CP, 2,3-DCP, 2,4-DCP and 2,5-DCP were confirmed to be about 100% by gas chromatography after monochloroacetylation (Kishino, pHS.8

pH6

pH 7 •I - 3

100

>.

,2

50

0.3

== r~

pH8

pH9

pH

10

i

0.5 i

I

~03/~0"2/ .~

60

~'

40 I t / / 0 " 1

~"

20 l f / /

03

~ 0.3 0.2

I

0.2

0.5

03 0.5

3

0.2

2 ! 0.3 O~

3

o

0 1

2.5

§ 0 1

2.8

6 0 1

2.6

6 0 1

2.8

6 0 1

2.5

5 0 1 2.5

Exposure time (h) Fig. 1. Effect of pH on the acute toxicity and accumulation of PCP in goldfish. The values in the figures express the PCP concentrations of the media in mg l- 1. The survival rates were cumulatively calculated at the respective observation times (l, 2.5 and 5 h).

6

433

Toxicity, absorption of chlorophenols in fish pH

9

8

7

6 5.5

I

I

I

!

!

/

10

5 0

2 0

tration of the media (Table 2), i.e. the average values at pH 5.5, 6, 7, 8 and 9 were 90, 91, 95, 100 and 86/~g g-~ body weight, respectively. The results suggest that the fish begins to die when the PCP concentration in the fish body reaches a level over about 80 #g g - ~ body weight. The acute toxicity and accumulation of the tested chlorophenols (except PCP) in goldfish at pH 6, 8 and 10 are shown in Fig. 3. In most of the chlorophenols, the mortality of the fish was enhanced with increasing their concentrations in media, except the mortality in 3-CP and 4-CP media, which decreased with increasing the concentration in the ranges of 10--30 and 20-30 mg l - ~ at pH 6, 10-50 and 10-20 mg I at pH 8, and 50-100 and 30-50 mg l - t at pH 10, respectively, although the fish were almost dying. In all chlorophenols tested, however, the amount accumulated in surviving fish increased with both the exposure time and the concentration in the media at each pH. The 5-h LCs0 values and the BCRs of the respective chlorophenols were calculated from the data shown in Figs 1 and 3 and summarized in Table 3, including their pKa values reported in our previous paper (Kishino and Kobayashi, 1994). As shown in the table, both the toxicity and accumulation of the chlorophenols were only slightly affected by the pH of media in the range of pH < pKa, but abruptly decreased with increasing the pH of media in the range of pH > pkg. For example, those of 2,3,4,6TCP having the smallest pK a = 5.2 among the tested chlorophenols (except PCP) abruptly decreased with increasing the pH of the media from 6 to 10, i.e. the 5-h LCs0 values were 0.2-0.3 and 1-1.5 mg l-~ at pH 6 and 8, respectively, while no test fish died even in l0 mg l-J at pH 10, and the BCRs were 250, 59 and 2.0 at pH 6, 8 and 10, respectively. However, phenol having the largest p K a =9.9 showed only small changes in toxicity and BCR in the pH range from 6 to 10, i.e. the 5-h LCs0 values were 130, 125 and 300mg l -~ and the BCRs were 2.1, 1.9 and 1.4 at pH 6, 8 and 10, respectively. On the other hand, both the toxicity and accumulation of chlorophenols increased with increasing the number of the substituted chlorine atoms, although the magnitude of the increase decreased with increasing the pH of the media. The average concentration of each chlorophenol (except PCP) found in the dead fish during exposure to the test media in various concentrations, which were pooled in whole bodies at p H 6 , 8 and 10, respectively is shown in Table 4. As with PCP, the concentration of each chlorophenol found in the dead fish was almost independent of the pH of the media. The result suggests that the toxicity of ionizable compounds such as chlorophenols to aquatic organisms should be evaluated as their accumulated amounts in viva instead of the concentration in the media, because the LCs0 values depend largely on the pH of media (Table 3). -

0.5

0-2 . ,

i

t

I

I

50

I00

5O0

I000

Bioconcentration ratio Fig. 2. Relation between the 5-h LCs0 values and the bioconcentration ratios at 5 h exposure to 0.1 mg I - ~ PCP media at pH from 5.5 to 10. Absorption test for chlorophenols at various pH The test media containing the respective chlorophenols in the concentrations shown in Table 1 were prepared by the same procedure as described above. Ten goldfish having a body weight of 1.4 _+0.1 g were exposed to each 501 of the media for I h at 27-28°C. The amounts of chlorophenols accumulated in the fish were determined by the same procedure as described above. The variation of pH in the media was within +0.1 during 1 h exposure.

RESULTS

AND

DISCUSSION

Effect o f p H on the acute toxicity and accumulation o f chlorophenols The 5-h LCs0 values of PCP were roughly 0.15, 0.2, 0.4, 0.85 and 2.5mg l-J at pH 5.5, 6, 7, 8 and 9, respectively, but no test fish died even in 3 mg l PCP-medium at pH 10 (Fig. 1), i.e. the acute toxicity of PCP to fish abruptly decreased with increasing the pH of PCP-media. The amount of PCP accumulated in surviving fish increased almost linearly with both the exposure time and PCP concentration in the media at each pH, but decreased so abruptly with increasing pH of media that the amounts at 5 h exposure to 0.1 mg 1-~ PCP-media at pH 5.5, 6, 7, 8, 9 and 10 were 66.4, 58.4, 30.6, 11.8, 4.21 and 0.89 #g g-~ body weight, respectively. A good linear relationship was observed between the 5-h LCs0 values and the BCRs at 5 h exposure to 0.1 m g I - ~ PCP-media in the range of the tested pH (Fig. 2). The concentration of PCP found in the dead fish was almost independent of the pH and PCP concen-

1

~°

0 m

Amount in fish (~tg/g)

Survival (%) Amount in fish (~tg]g)

Survival (%) Amount in fish (~tg/g)

Survival (%)

.~

o~

Z

e~

7 0

0

Toxicity. absorption of chlorophenols in fish pH6

pH8

,oo pH6

pHlO

12,5-DcPI tO0

.g -~

._o,,_,.ol

o.5-1

-

435

O.- I0~ o

5o

pH8

pill0

12'I6-DCP i ..o.,._,clO-iO0~

1-30

5O 1

2O

i

3O

i

i

i

t

J

i

i

i

2O 1D

40O

20 5

lOO

._=

o 200

i

5o

0

0

l~",5-Dcg

.~

2,4,5-TCP

50

5O

4o

0

~-o

,

,

"~

G

,

,

80

o.z 2

2o

e,,,

'"

,

,

5

t

40

,

,

3 •

50

0.5 20 Io

<

5

[2 3

I2,4,6-TCPI 100

"~

5o

0

200

i

3

100

40

< 0

2.5

5

I 2.5

5 0 1 2.5

5

Exposure

0 1 2.5

5 0 1 2.5

5

I 2.5

5

time (h)

(b) Fig. 3. Effect of pH on the acute toxicity and accumulation of chlorophenols in goldfish. The values in the figure express the concentrations of chlorophenols in the media in mg l - i WR 29,2--D

436

TAKUOKIsmr~o and KUNIOKOBAYASHI

Table I. The concentration of phenol and chlorophenols in the absorption test media at various pH

at pH 5.5, respectively. The relative BCRs of 2,6DCP and 2,4,5-TCP at various pH as a function of (pH-pKa) are shown in Fig. 5. The fraction present in the undissociated form of weak acids such as chlorophenols at various pH can be calculated from equation (1). The dashed line in the figure expresses the fraction present in the undissociated form of weak acids. Fraction present in undissociated form

pH Chemical Phenol 2-CP 3-CP 4-CP 2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,5-DCP 2,4,5-TCP 2,4,6-TCP 2,3,4,6-TCP PCP

5.5

6

7

8

9

I0

l0 3 2 2 0.5 0.5 0.5 0.5 0.5 0.3 0.2 0.2 0.1

10 3 2 2 0.5 0.5 0.5 0.5 0.5 0.3 0.2 0.2 0.1

10 3 2 2 0.5 0.5 0.7 1 0.5 0.5 0.4 0.3 0.2

10 3 2 2 I I 1 2 I 0.7 0.7 0.7 0.4

l0 7 4 4 3 3 3 10 2 3 5 2 2

10 20 7 7 10 10 10 50 5 l0 20 10 l0

[H ÷] - K~ + [H +]

If the absorption of chlorophenols by fish is caused only by the passive diffusion of the undissociated form as described above, the pH profiles of their relative BCRs must be coincident in position with that of the undissociated form. However, the pH profiles were found to be shifted to the alkaline side of the undissociated form. The relationships between the BCRs of the chlorophenols and their distribution ratios in the n-heptane-water and 1-octanol-water systems at the respective pHs of 5.5, 6, 7, 8, 9 and 10 are shown in Fig. 6. A poor correlation was observed at pH 8, 9 and 10 in n-heptane-water, and also at pH 8 and 9 in 1-octanol-water. The above poor correlation between the BCRs of chiorophenols and their distribution ratios in solvent-water systems may be caused by some different pattern in the dissociation equilibrium of chlorophenols between fish-water and solvent-water systems, i.e. in the fish-water system two dissociation equilibriums are present in each of the outside (surrounding water) and inside (blood) of gill membranes, while in the solvent-water system a single dissociation

The values in this table are expressed in mg I ~ i

Absorption mechanism of chlorophenolsfrom media in

fish The BCRs of the tested chlorophenols in goldfish at 1 h exposure to their media shown in Table 1 as a function of the difference between pH and pKa (pH - pKa) are shown in Fig. 4. As described above, the BCR was little affected by the pH in the range of pH < pKa, but abruptly decreased with an increase of the pH in the range of pH > pKa. This result suggests that the transfer of chlorophenols from the media to the fish body is mostly performed in their undissociated forms. Both 2,6-DCP and 2,4,5-TCP are mainly present in the undissociated form at pH 5.5 because their pK~ values are 6.6 and 6.9, respectively and, as shown in Fig. 4, their BCRs sharply reduced in the pH range from the respective pK a to 10. Their relative BCRs were calculated dividing the BCR at each pH by that

Table 2. The amounts of PCP found in the dead fish during 5 h exposure to PCP-media at various pH Medium

(1)

Number of dead fish

Amount in dead tish (/~g g ~)

pH

(mg I ~)

Exposure time (h)

Found

Average*

Range

5.5

0.2 0.3 0.5 0.7 I 1

5 2.5 2.5 2.5 1 2.5

10 I 10 10 6 8

82 97 89 88 96 98

90

82-98

6

0.2 0.3 0.3 0.5 0.7 1 l

5 2.5 5 2.5 2.5 1 2.5

4 2 l0 9 10 3 10

99 87 88 86 82 115 98

91

82 115

7

0.5 0.7 I

5 2.5 2.5

l0 14 20

92 89 100

95

89-100

8

0.7 1 1

5 2.5 5

I 1 9

82 96 102

100

82-102

9

3

5

I

86

--

--

Twenty goldfish having a body weight of 1.3 + 0.1 g were placed in each 501 of the media at 20-21 C. A suitable number of surviving fish and the dead fish found in the media were taken out from the media at 1, 2.5 and 5 h exposure. *The value was calculated as total accumulated amount/total body weight in each pH group.

437

T o x i c i t y , a b s o r p t i o n o f c h l o r o p h e n o l s in fish Table 3. The 5-h LCso values and bioconeentration ratios (BCR) of phenol and chlorophenols at various pH in goldfish 5-h LCs0 (rag I i)

BCR

Chemical

pK,

pH 6

pH 8

pH 10

pH 6

pH 8

pH 10

Phenol 2-CP 3-CP 4-CP

9.9 8.3 8.9 9.2

130 70-100 50 50-70

125 100-150 50-70 50-70

2. I 3.7 I0 7.6

1.9 3.8 9.7 6.9

1.4 0.60 2.0 3.1

2,3-DCP 2,4-DCP 2,5-DCP 2,6-DCP 3,5-DCP 2,4,5-TCP 2,4,6-TCP 2,3,4,6-TCP PCP

7.6 7.8 7.3 6.6 8.1 6.9 6.0 5.2 4.7

10-20 5-7 5 15-30 2-3 0.7-1 1.5 0.2-0.3 0.2-0.3

10-20 7-10 7 >80 3-5 1.5-3 7 I-I.5 0.85

300 >500 > 100 10-30, 50, 100-200 > 100 > 100 > 100 > 100 20-40 >50 > 70 > 10 >3

44 40 57 52 53 61 160 250 584

31 33 29 18 41 22 40 59 l 18

2.4 2.4 1.4 0.84 4.4 1.8 1.8 2.0 8.9

The LCs0 values in this table are expressed as the approximate values (rag I i) calculated from the data in Figs 1 and 3. The 1]CRs in this table were obtained in goldfish at 5 h exposure to the respective media as follows: phenol (10mg I -~); 2-CP (5rag I-I); 3-CP (5rag 1-~); 4-CP (3mg I ~ ) ; 2,3-DCP (1 mg 1-~); 2,4-DCP (0.Smg I-r); 2,5-DCP (0.5mg 1 ~); 2,6-DCP ( I m g 1-t); 3,5-DCP (0.5mg 1~); 2,4,5-TCP (0.3 mg I -i); 2,4,6-TCP (0.3 mg I ~); 2,3,4,6-TCP (0.1 mg I ~); and PCP (0.1 mg I- ~). The PKd values in this table were quoted from our previous paper (Kishino and Kobayashi, 1994).

Table 4. The amounts of chlorophenols found in the dead fish during 5 h exposure to test media at p H 6 , 8 and 10 Amount in dead fish (/zg g t)

Medium Chemical Phenol

2-CP

3-CP

pH

(rag I i)

Exposure time (h)

Number of dead fish

Found

Average*

Range

6

50 I00 100 150 150 200

5 2.5 5 2.5 5 2.5

2 1 2 4 6 8

79 149 164 235 244 313

240

79-313

8

50 I00 100 150 150 200 200

2.5 2.5 5 2.5 5 2.5 5

1 1 3 7 7 5 6

72 150 173 226 236 313 199

223

72-313

l0

100 300 300

5 2.5 5

2 6 3

149 342 349

309

149-349

6

100 150 200

2.5 I I

10 16 20

187 264 259

245

187-264

8

150 200

2.5 1

13 14

320 268

294

268-320

10

500

5

1

192

--

--

6

10 10 30 30 50 50 70 I00

2.5 5 2.5 5 2.5 5 2.5 1

4 I I I 4 3 20 20

63 93 174 287 254 375 423 362

348

63-423

8

10 30 50 70 100

2.5 5 2.5 2.5 I

4 I I 13 20

73 220 270 378 355

329

73-378

conlmued

TAKUO KISHINO a n d KUNIO KOBAYASHI

438

Table 4--continued Medium Chemical

pH

(mg I i)

Found

Average*

Range

10

50 50 I00

2.5 5 5

62 67 117

80

62-117

10 20 20 50 70

5 2.5 5 2.5 2.5

58 89 149 242 393

327

58-393

10 20 30 30 50 70

5 2.5 2.5 5 5 2.5

322

67-443

2 12

67 69 121 173 366 443

10

30 30 50 50 200 200

2.5 5 2.5 5 2.5 5

5 5 I 5 8 10

104 120 106 165 442 451

299

104-451

6

7 10 20 30

5 5 2.5 1

2 4 10 18

213 293 338 330

321

213-338

10 20 30

5 2.5 I

2 10 16

211 329 332

321

211-332

6

5 7 10 10

5 5 I 2.5

1 10 1 10

119 241 213 266

247

119-266

8

7 10

5 2.5

195 240

235

195-240

6

3 5 7 10 10

5 5 2.5 1 2.5

1 5 8 4 10

81 122 135 114 166

138

81-166

5 7 7 10 20

5 2.5 5 2.5 I

1 2 4 10 12

73 90 145 133 138

131

73-145

6

15 20 30 30

5 2.5 I 2.5

350 351 329 431

373

329-431

8

50 80

321 367

353

321-367

6

3 4 5 7 7 10

5 2.5 2.5 1 2.5 1

8 13 17 3 10 19

149 142 140 115 149 126

138

115-149

3 4 5 7 7 10

5 2.5 2.5 I 2.5 I

3 7 17 5 10 18

147 135 152 119 140 118

134

118-152

4-CP

2,3-DCP

2,4-DCP

2,5-DCP

2,6-DCP

3,5-DCP

Amount in dead fish (/ag g ~) Number of dead fish

Exposure time (h)

3 I

2 I

continued

439

Toxicity, absorption of ¢hlorophenols in fish Table 4--continued Amount in dead fish (/~g g ')

Medium Chemical

2,4,5-TCP

2,4,6-TCP

2,3,4,6-TCP

pH

(mg I - ')

Exposure time (h)

Number of dead fish

Found

Average*

Range

10

40 100

2.5 I

12 20

171 176

174

171-176

6

0.7 1 2

5 2.5 1

4 5 3

61 62 51

59

51~2

8

2 3 5

2.5 2.5 1

5 8 17

81 75 70

73

70-81

6

0.7 2 3

5 5 2.5

2 6 l0

67 167 210

181

67-210

8

5 7 7 10

2.5 2.5 5 2.5

I 3 4 l0

79 154 148 173

157

79-173

6

0.3 0.3 0.5 0.7

2.5 5 2.5 2.5

2 8 4 10

37 68 65 80

69

37-80

8

1.5 1.5 2

2.5 5 2.5

4 9 13

52 62 58

58

52~2

Thirty goldfish having a body weight of 2.2 + 0.2 g was used in this experiment, *The value was calculated as total accumulated amount/total body weight in each pH group.

equilibrium is present in the water phase, because chlorophenols are present only in their undissociated forms in organic solvents. Assuming the model consisted of two compartments as shown in Fig. 7, in which the ionizable chemical agent (HA) partly ionizes as H A ~ A - + H + in both compartments, the distribution ratio (K) of the agent between the compartments I (surrounding water) and II (fish body) is expressed by equation (2): [HA]n + [A-]tl K = [HA], + [A-]I

(2)

where [HA]I + [A ]t and [HA],I + [A-]It are the total concentration of the agent in the compartments I and II, respectively. Since the dissociation constant (Kd) is expressed as [HA] K. (3)

[A-] [H+]

the substitution of equation (3) into (2) gives the following: [HA],, ([H+]n + K~)/[H+],, K = [HA]---~" ([H+I, + K.)/[H+]I

(4)

Assuming that only the undissociated form can migrate between both compartments (I and II) by diffusion and that the distribution ratio of undissociated form in compartment I to that in compartment II is proportional to the partition coefficient (P) in solvent-water systems such as n-heptane--water and l-octanol-water, the distribution ratio is expressed as

[HA]n

- -

[HAl,

= k P

(5)

where k is a proportionality constant. Substituting equation (5) as k = 1 into (4), equation (2) is finally transformed to K = P ([H+]I' + Ka)/[H+]n ([H+]l + Ka)/[H +]l

(6)

The K values were calculated from equation (6) for compartment I pH 5.5, 6, 7, 8, 9 and 10 and compartment II pH 7.4, referring to the literature (Endo and Onozawa, 1987) as the pH of blood in goldfish. The partition coefficients and dissociation constants reported in our previous paper (Kishino and Kobayashi, 1994) were used for the calculation of the K values. The results of the linear regression analysis for the K values and the BCRs presented in Fig. 4 are shown in Table 5. The Kh~pand Ko,~ in the table are the K values calculated using the partition coefficients in the n-heptane-water and 1-octanol-water systems, respectively. A good correlation was observed between log BCR and log/(oct, except it was a little low at pH 10 (r =0.719). On the other hand, a much poorer correlation of' r =0.454 and -0.210 was observed between log BCR and log Khep at pH 9 and 10, respectively. Phospholipids such as lecithin and cephalin, which are major components in cell membranes, play an important role in the membrane permeability of

440

TAKUO KISHINOa n d KUNIO KOBAYASHI

PeP

.O

o

2,4,5-TCP

~ ~ ~

10

2,6-DCP

t~-~

10-I . ! 'O

.o

1

~i0-2 0

-IJ al tw

.o 0.1

,I

I

I

I

I

I

I

I

I

I

@

I

~j10-3

4-;

=o

0C 000 r~

lOO

10-2 -,~ 4J

Undlssoclated form kok

QI ,,,e

2,4,S-TCp

10- 4

i

-

3-CP ~

t0

;0-3 ~

~

,

,i

i

0 1 2 pH - PKa

-I

~

3

Fig. 5. The relative bioconcentration ratios of 2,6-DCP and 2,4,5-TCP in goldfish at 1 h exposure to their media at various pH as a function of the difference between pH and pK~ (pH - pKa). The dashed line in the figure expresses the fraction present in the undissociated form at various pH. 0.1

|

i

i

-4

i

i

-2

i

i

0

i

,i

2

pH - -

i

i

containing lecithin-water systems, and also that the formation of the intermolecular hydrogen bond in 1-octanol and lecithin with the hydroxyl group of chlorophenols plays an important role in their transfer from water to solvents (Kishino and Kobayashi, 1994). These results demonstrate the rationality of equation (6), suggesting that the absorption of chlorophenols by fish is mainly caused by the passive diffusion of the undissociated form through gill membranes, and that the formation of intermolecular hydrogen bond between the hydroxyl group of

6

4

PKa

Fig. 4. The bioconcentration ratios of chlorophenols in goldfish at 1 h exposure to their media as a function of the difference between pH and pKa (pH -pK,).

chemicals. We reported that a good correlation is observed between both partition coefficients of chlorophenols in l-octanol-water and n-heptane

5

pH 5.5

4

1-Octmml

•

4 S

3

(

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~

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2

r=O~20~ j~"

pH7 4

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°

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pH 6

3

5 10

w

50 100 200

-2

~-~

~

-1

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1

r = 0.~0 -1

i

i

i

,

i

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3 Ir-Oetanol = 0.383 t 2

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i

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0

°°° o

o

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:

I

I

50 100

-2

o

0

Q

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o

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•

-2

o

oo

oo

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o o

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r = 0 ]07 i

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r=0.495 -2

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r=0.528

I 0%

°

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p H 10

2

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pH 9

2

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pH 8

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-3

I

i

L

i

I

2

5

10

0-2

0.5

Bioeoncentration

r = 0.310 i

I

i

2

ratio

Fig. 6. Relation between the distribution ratios (D) of chlorophenols in l-octanol-water and n-heptane-water systems and their bioeoncentration ratios in goldfish at 1 h exposure to the test media at the pH 5.5, 6, 7, 8, 9 and 10.

441

Toxicity, absorption of chlorophenols in fish

iiiiill

Compartment I

i!i!ii~i!;

(Surroundingwater)[~.~i!..:.":: [~] H+ + A- ~ H A

~

Compartment I1 (Fish body) HA ~

A" + H+

Biological membrane (Gill) Fig. 7. Distribution of ionizable chemicals between both compartments I and II (two compartments model). chlorophenols and the components of biological membranes, presumably gill membranes, plays an important role in the transfer of the chemicals from the surrounding water into fish body. As described above, however, the p H profiles of the relative BCRs were not coincident in position with that of the undissociated form, i.e. the pH profiles of the relative BCRs shifted to the alkaline side of the undissociated form in the media (Fig. 5). It is well known that the pH-dependent absorption curve of ionizable compounds does not correlate with their ionization curve (Kakemi et al., 1969; Jollow and Brodie, 1972; Gutknecht and Walter, 1980; Saarikoski et al., 1986). Saarikoski et al. (1986) have reported that the rate of absorption may not be directly determined by the pH in bulk water phase but by the p H in some compartment between the bulk phase and the interior of gill membranes, and that the mucous layer on the surface of gills possibly serves as the barrier of the compartment. The pH near the surface of cell membranes is estimated to be 0.3-0.4 units lower than that of the surrounding water (Finean et al., 1978). In addition, the concentration of CO2 near the outer surface of gill membranes is thought to be considerably higher because of respiration. Consequently, it is presumed that the pH near the outer surface of gill membranes is probably lower Table 5. Relation between the bioconcentration ratios (BCR) of chlorophenols by goldfish at 1 h exposure to the test media at various pH and the distribution ratios (K) of the chemicals between both compartments I and II Equation obtained f r o m Correlation pH linear regression analysis coefficient 5.5 log BCR =0.318 log Kh~p+0.834 0.914 6 log BCR =0.318 log Khcp+ 0.831 0.904 7 log BCR = 0.346 log Khcp + 0.809 0.891 8 log BCR = 0.326 log Khcp + 0.807 0.806 9 log BCR =0.188 log Khcp+ 0.612 0.454 10 log BCR = -0.106 log Khep --0.251 --0.210 5.5 log BCR =0.407 log K~t-0.183 0.953 6 log BCR = 0.420 log K~t- 0.229 0.954 7 log BCR = 0.495 log K ~ - 0.443 0.968 8 log BCR =0.515 log K~t- 0.447 0.935 9 log BCR = 0.457 log K~t - 0.387 0.882 10 log BCR =0.384 log K~t-0.676 0.719 The Khepand Ko~tin this table are the K values calculated by using the partition coefficientsin n-heptane--water and I-octanol-water, respectively,when the pH of compartment I is 5.5, 6, 7, 8, 9 and 10, and the pH of compartment I1 is 7.4.

than that of media, and then the concentration of the undissociated form of chlorophenols near the outer surface o f gill membranes becomes higher than that in the media, resulting in a higher BCR than expected on the basis o f the concentration of the undissociated form in the media. F r o m the above discussion, it is concluded that the transfer of chlorophenols from media to fish body is mainly caused by the passive diffusion of the undissociated form through the gill membranes, being affected by the formation of an intermolecular hydrogen bond between the hydroxyl group of the chemicals and the components of the gill membranes, and that the concentration of the chemicals in the fish body does not readily reach a lethal level with increasing pH of media owing to the increased conversion of the undissociated form to the dissociated form, resulting in the reduction of the toxicity of chlorophenols in the fish. If the absorption of chlorophenols by fish is caused by the passive diffusion of the undissociated form as described above, the p H profile of the BCR must be coincident in position with that of the undissociated form. However, the pH profile of the BCR shifted to the alkaline side of the undissociated form. The reason was presumed to be that the pH near the outer surface of the gill membranes is lower than that of the media.

REFERENCES Crandall C. A. and Goodnight C. J. (1959) The effect of various factors on the toxicity of sodium penta chlorophenate to fish. Limnol. Oceanogr. 4, 53-56. Dalela R. C., Saroj Rani, Sarita Rani and Verma S. R. (1980) Influence of pH on the toxicity of phenol and its two derivatives pentachlorophenol and dinitrophenol to some fresh water teleosts. Acta Hydrochim. Hydrobiol. 8, 623-629. Davies R. P. and Dobbs A. J. (1984) The prediction of bioconcentration in fish. Wat. Res. 18, 1253-1262. Endo T. and Onozawa M. (1987) Effects of pH and temperature of the uptake of oxolinic acid by goldfish. Nippon Suisan Gakkaishi 53, 551-555. Finean J. B., Coleman R. and Michell R. H. (1978) Membranes and Their Cellular Functions, 2nd Edn, pp. 22-41. Blackwell Scientific, Oxford. Gossett R. W., Brown D. A. and Young D. R. (1983) Predicting the bioaccumulation of organic compounds in marine organisms using octanol-water partition coefficients. Mar. Pollut. Bull. 14, 387-392. Gutknecht J. and Walter A. (1980) Transport of auxin (indoleacetic acid) through lipid bilayer membranes. J. Membr. Biol. 56, 65-72. Holcombe G. W., Fiandt J. T. and Phipps G. L. (1980) Effects of pH increases and sodium chloride additions on the acute toxicity of 2,4~tichlorophenol to the fathead minnow. Wat. Res. 14, 1073-1077. Jollow D. J. and Brodie B. B. (1972) Mechanisms of drug absorption and of drug solution. Pharmacology 8, 21-32. Kakemi K,, Arita T., Hori R., Konishi R., Nishimura K., Matsui H. and Nishimura T. (1969) Absorption and excretion of drugs--XXXIV. An aspect of the mechanism of drug absorption from the intestinal tract in rats. Chem. Pharm. Bull. 17, 255-261.

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TAKUO KISHINO and KUNIO KOBAYASHI

Kishino T. (1989) Studies on the action mode of chlorophenols in fish. Doctoral thesis, Kyusyu University, Fukuoka, Japan. Kishino T. and Kobayashi K. (1980) Studies on the metabolism of chlorophenols in fish--XIV. A study on the absorption mechanism of pentachlorophenol in goldfish relating to its distribution between solvents and water. Nippon Suisan Gakkaishi 46, 1165-1168. Kishino T. and Kobayashi K. (1994) Relation between the chemical structures of chlorophenols and their dissociation constants and partition coefficients in several solvent-water systems. War. Res. 28, 1547-1552. Kobayashi K. and Akitake H. (1975a) Studies on the metabolism of chlorophenols in fish--I. Absorption and excretion of PCP by goldfish. Nippon Suisan Gakkaishi 41, 87-92. Kobayashi K. and Akitake H. (1975b) Studies on the metabolism of chlorophenols in fish--II. Turnover of absorbed PCP in goldfish. Nippon Suisan Gakkaishi 41, 93-99. Kobayashi K., Kimura S. and Shimizu E. 0977) Studies on the metabolism of chlorophenols in fish--IX. Isolation and identification of pentachlorophenyl-fl-glucuronide

accumulated in bile of goldfish. Nippon Suisan Gakkaishi 43, 601~07. Marking L. L. (1975) Effects of pH on toxicity of antimycin to fish. J. Fish. Res. Bd Can. 32, 769-773. Neely W. B, Branson D. R. and Blau G. E. (1974) Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. Technol. 8, Ill3-1115. Rubery P. H. and Sheldrake A. R. (1973) Effect of pH and surface charge on cell uptake of auxin. Nat. New Biol. 244, 285-288. Saarikoski J. and Viluksela M. (1981) Influence of pH on the toxicity of substituted phenols to fish. Arch. environ. contain. Toxicol. 10, 747-753. Saarikoski J., Lindstrom R., Tyynela M. and Viluksela M. (1986) Factors affecting the absorption of phenolics and carboxylic acids in the guppy (Poecilia reticulata). Ecotox. Envir. Safety 11, 158-173. Sills J. B. and Allen J. L. (1971) The influence ofpH on the efficacy and residues of quinaldine. Trans. Am. fish. Soc. 100, 544-545. Veith G. D., DeFoe D. L. and Bergstedt B. V. (1979) Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Bd Can. 36, 1040-1048.