Toxicity of bile from fish exposed to PCP-spiked sediment

talc ELSEVIER

Aquatic Toxicology 30 (1994) 171-182

TgxICOLgGY

Toxicity of bile from fish exposed to PCP-spiked sediment Maria Andr6asson*, G6ran Dave Department of Zoophysiology, Universityof G6teborg, Medieinaregatan 18, 413 90 G6teborg, Sweden (Received 30 August 1993; revision received 25 January 1994; accepted 16 April 1994)

Abstract

In order to evaluate if toxicity of the bile may be used to establish lethal and sublethal exposure of fish to pollutants, rainbow trout (Oncorhynchus rnykiss) were exposed to PCPspiked sediment (4, 8, 16, 32 and 64 mg PCP/kg w.w.) for 1 or 2 days, sacrificed and their bile tested for toxicity to Daphnia magna. Exposure was monitored daily by Daphnia toxicity tests of sediment and water. The 24-h EC50s, expressed as mg PCP/kg sediment w.w., were 20 + 6.4 for sediment and 9.6 + 5.1 for water phase tests, respectively. The corresponding 24-h LC50 value for sediment to rainbow trout was ca 16 mg/kg w.w. Bile toxicity was tested directly and after hydrolysis by acid and by fl-glucuronidase. Bile and bile extract of surviving trout exposed to the median lethal concentration of sediment (16 mg PCP/kg w.w.) were 10 times more toxic than control fish. Hydrolyzed bile extract of fish exposed to 4 mg PCP/kg w.w. was 10-50 times more toxic than that of control fish. Thus, toxicity tests of extracts of hydrolyzed bile were more sensitive than toxicity tests based on water and sediment. Therefore, at least for chemicals which accumulate in the bile, toxicity tests of the bile may be used to establish sublethal exposure in fish.

Key words: Bile; Daphnia; Fish; PCP; Rainbow trout; Sediment; Toxicity

1. Introduction

N u m e r o u s xenobiotics concentrate in aquatic organisms and in sediment (Lee, 1992). In fish, m a n y xenobiotics accumulated in the bile as part o f the depuration process, and analysis o f fish bile has been r e c o m m e n d e d as a tool in water quality m o n i t o r i n g (Lech et al., 1973; S t a t h a m et al., 1976; Oikari and K u n n a m o - O j a l a , 1987, F6flin and Wachtmeister, 1989; Wachtmeister et al., 1991). The general objective o f the present study was to investigate if the toxicity o f fish bile m a y be used as a sensitive environmental indicator o f water pollution. M o r e

* Corresponding author. 0166-445X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0166-445X(94)00033-M

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precisely the toxicity of bile of rainbow trout exposed to PCP-spiked sediment was investigated, and quantified in tests with Daphnia magna. Furthermore, Daphnia tests were used to investigate if hydrolysis (enzymatic and acidic) affected the toxicity of the bile. The gall bladder was chosen as the tissue of concern because of its capacity to concentrate xenobiotics (opus cited). Rainbow trout was used because this species is commercially available and extensively used in physiological research (Love, 1980; Gall, 1992). The selection of pentachlorophenol (PCP) as a model xenobiotic was based on the extensive knowledge on its biliary excretion in fish (Kobayashi and Akitake, 1975a,b; Glickman et al., 1977; Kobayashi, 1979; Niimi and McFadden, 1982; McKim et al., 1986) and its acute toxicity to fish and invertebrates (Davis and Hoos, 1975; Anonymous, 1987; Mayer and Ellersieck, 1986).

2. Materials and methods

2.1. Fish exposure Rainbow trout (Oncorhynchus mykiss), weighing 80-140 g, and acclimated to laboratory conditions for 4 days without feeding, were exposed for up to 48 h to sediment (19% d.w.) spiked with PCP to nominal concentrations of 0, 4, 8, 16, 32 and 64 mg PCP/kg of sediment (w.w.). Each duplicated treatment comprised five fish in 50 1 of water and sediment (10% w.w.). Toxicant exposure was monitored by daily measurements of sediment and water toxicity to Daphnia magna, as described below. Temperature, pH and dissolved oxygen was recorded daily in all treatments. All fish exposed to 32 and 64 mg PCP/kg of sediment died within 6 h, and only 4 of 10 fish exposed to 16 mg/kg were alive after 24 h. Those exposed to 0, 4 and 8 mg/kg survived, were removed by a dip net, and the bile was separated from the gall bladder by a syringe. Bile from all fish (N = 5) in each duplicated treatment was pooled. Toxicity tests with D.magna were used to quantify bile toxicity and fish exposure through tests with sediment and water. All D. magna exposures were made at 20 + I°C with recordings of immobility in the different concentrations (dilution factor 0.5) after 24 and 48 h according to ISO (1989). The 24- and 48-h EC50s and their 95% confidence limits were determined by probit analysis or moving average according to Peltier and Weber (1985). Sediment toxicity was tested by taking 5 g of the sediment from each duplicated treatment (concentration) and dilute it with standardized, reconstituted water (ISO, 1989) to 50 ml. The toxicity of the overlying water was tested in 50 ml samples of the water phase. After equilibration for 1 h, twenty 4-6 d old daphnids were added. Sediment and water toxicities were both expressed as nominal 24-h EC50-values in mg PCP/kg sediment (w.w.) in the sediment.

2.2. Chemicals Technical grade sodium pentachlorophenate (MW288) manufactured by Fluka AG,

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Switzerland, was used to prepare toxicant solutions and was adjusted to the appropriate pH by addition of 0.1 M HCI. Adjustment of bile pH was made with 0.5 M HC1. The acetate buffer (0.4 M; pH 3.5) used to control pH during extraction of the bile was made from analytical grade sodium acetate and acetic acid, and the organic solvent used for bile extraction, a 1 : 3 mixture of acetone and hexane, was prepared from analytical grade (>99.5%) solvents. The fl-glucuronidase, type H-1 from Helix pomatia with 416,800 units/g solid, was obtained from Sigma Chemical Company, St. Louis, MO 63178, USA. The reference toxicant used in the Daphnia tests, potassium dichromate, (K2Cr2OT), was analytical grade (ISO, 1989). 2.3. Sediment characteristics

Surface sediment was collected with an Ekman dredge from an unpolluted site at 12 m depth in lake Stora Delsj6n in G6teborg on November 19, 1991. An aliquot was sent to the ICM-laboratory in Uppsala for chemical analysis. The sediment was stored at 4°C for 4 weeks before it was used for fish exposure. A test with D. magna was made to verify that the sediment was non-toxic. Furthermore, an experiment with PCP-spiking of the sediment was made with D. magna to determine suitable concentrations for fish exposure, and to study the effect of ageing (for 1, 2, 3 and 5 d) at different sediment-water ratios (0, 5, 10 and 20% sediment) on the toxicity of PCPspiked sediment. The PCP concentrations in the spiked sediment ranged from 8000 to 16 mg/kg w.w. (dilution factor 0.5). 2.4. Bile toxicity

The bile samples were extracted according to the method described by Oikari (1986). To a 250/11 sample of bile in a glass centrifuge tube, adjusted to pH 3.5 with HC1, 1.25 ml deionized water and 0.5 ml acetate buffer were added. For acidic hydrolysis aliquots of bile (250 ltl) were adjusted to pH 3.5 with 0.5 M HC1 (20 ill), diluted with deionized water to 1.5 ml, heated to 70°C for 3 h and cooled to room temperature, whereafter 0.5 ml acetate buffer was added. The enzymatic hydrolysis was carried out at pH 5.0 in 0.5 M HC1 (6.5 ill) and 1.25 ml water with 2000 units of fl-glucuronidase (which included 25 units of sulfatase). After incubation for 3 h at 40°C on a shaking water bath, pH was lowered to pH 3.5 (with 13.5/A 0.5 M HC1) and 0.5 ml acetate buffer was added. After addition of acetone : hexane (500/11), the sample was shaken on a cyclomixer (Heidolph) for 1 min. The two phases were allowed to separate over night at 4°C, centrifuged for 20 min at 1000 g, and the organic phase was removed and tested after serial dilution. Newborn daphnids (6-24 h old) were taken from laboratory cultures. Exposures were made with 2 x 2.5 ml test volumes and 2 x 10 daphnids per concentration. Test concentrations of bile ranged from 2.5 ml/1 to 80 ml/1 with a dilution factor of 0.5. Bile extracts were tested after evaporation of the organic solvent at 40-60°C for 30 min followed by addition of standardized, reconstituted water (ISO, 1989). Test concentrations of extracts corresponded to 0.04-30 ml bile/1 with a dilution factor of 0.5. A solvent control corresponding to 200/ll evaporated extracting solvent, a dilution

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water control and a test series with the reference toxicant, K2Cr207, were tested simultaneously. To determine the influence of hydrolysis and extraction on the toxicity of PCP in bile, an experiment with spiked bile was performed. A series of concentrations of PCP in bile (25, 50, 100 and 200 mg/1) were prepared and the samples were then extracted directly, after acid hydrolysis and after enzymatic hydrolysis, respectively, as described above. The concentration series (dilution factor 0.5) of extracted bile was subjected to serial dilution and tested for toxicity. Bile and bile extract toxicity was expressed as EC50 related to the volume of bile, i.e. as ml bile per liter of water required for immobilization of 50% of the test organisms.

3. Results and discussion 3.1. Sediment characteristics and sediment P C P toxicity to D. magna

Chemical analysis of the control sediment, showed that it was not contaminated with metals or nutrients. Furthermore, tests with D. magna showed that the sediment was not toxic (Table 1). Thus, the only toxicant to consider in this study is PCP, which was spiked to the sediment. The analyzed sediment sample had a dry weight of 8.6%, while the sediment sample used for testing had a dry weight of 19%. During the exposures of fish to sediment the reference toxicant had a 24-h EC50 for D. magna corresponding to 1.0 mg/1 K2Cr207 (95% CL of 0.8-1.2), and when the bile was tested it was 0.9 (0.(~1.2). These values (1.0 and 0.9) are both within the limits of 0.9-2.0 accepted by ISO (1989) for this test. The effect of sediment-water ratios and of ageing on the toxicity of the spiked sediment to D. magna is shown in Table 2. These results showed that the sedimentTable 1 Chemical and toxicologicalcharacteristics of the sediment matrix from Lake Delsj6n Parameter

Measured value

Dry weight (d.w.) Loss on ignition Total-Nitrogen Total-Phosphorous Mercury (Hg) Cadmium (Cd) Lead (Pb) Chromium (Cr) Nickel (Ni) Copper (Cu) Zinc (Zn) 24-h EC50 for D. magna 48-h EC50 for D. magna

8.6% w.w. 34% d.w. 0.65% d.w. 0.10% d.w. 0.33 mg/kg d.w. 0.9 mg/kgd.w. 69 mg/kg d.w. 34 mg/kg d.w. 25 mg/kg d.w. 38 mg/kg d.w. 186 mg/kg d.w. > 32% of w.w. >32% of w.w.

a

Guideline

0.15-0.3 mg/kg d.w.a 0.7-2.0 mg/kg d.w.a 30-100 mg/kg d.w.a 25-75 mg/kg d.w.a 30-75 mg/kg d.w.a 25-50 mg/kg d.w.a 175-300 mg/kg d.w)

Defined as 'Moderatelyhigh concentrations'accordingto the Swedish EPA, Public Advice 1990:13.

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water ratio was important for the effect of PCP. There was a dose-response relationship between the proportion of sediment and 24-h EC50, i.e. a doubling in the proportion of sediment doubled the toxicity. This was expected because more PCP was added with increasing proportions of sediment. The 24-h EC50 values for D. magna without sediment of 0.7-1.6 mg/1, during day 1 to 5 were slightly higher than that found previously (0.5 mg/1) by Berglind and Dave (1984). At least part of this discrepancy m a y be due to the difference in age (size) of D. magna and biomass-water ratio used in these two studies. The effect of time (ageing) on the toxicity of PCP in the sediment was less clear.

3.2. Toxicant exposure to fish During exposure of the fish the daily measurements of dissolved oxygen, p H and temperature ranged from 94 to 100% of air saturation, 6.4 to 7.1 and from 10.4 to 13.5°C, respectively. The toxicity of sediment and water was monitored by Daphnia tests and the results are presented in Table 3. The sediment was less toxic than the water. The higher toxicity of water m a y be due to a higher proportion of fine particles in the Daphnia tests with water. Furthermore, the filtering of particles by D. magna could be important in this respect because lipophilic chemicals in water are preferentially bound to smaller particles. Sediment toxicity values (24-h EC50s) found in the Daphnia test (13 + 10, mean + SD in Table 2) were slightly lower than in the fish exposure experiment (20 + 6.4, sediment exposure grand mean + SD in Table 3), both with 10% sediment. This m a y be due to the equilibration of the spiked sediment and water prior to sampling of the sediments. In spite of this difference, and considering the precision of the Daphnia toxicity test (ISO, 1989), we conclude that the sediment test with Daphnia (Table 2) provided relevant guidance for the exposure of fish to sediment (Table 3). Results from the water tests with Daphnia in the fish exposure experiment were more variable than those from the sediment tests (Table 3). A possible reason for the

Table 2 Toxicity of PCP-spiked sediment to D. magna at different concentrations of sediment in water, and the effect of storage Days after spiking 24-hEC50, expressedas mg PCP/kg sediment, w.w.

1 2 3 5 mean _+SD

0% sedimenta

5% sediment

10% sediment

20% sediment

1.2 (0.9-1.5) 1.6 (1.2-2.1) 0.7b 1.1b 1.2 +_0.4

34b

44 (33-58) 22 (18 27) 47 (36452) 37 + 11

12b 27b 5b 9b 13 _+10

9.2b 6.1b 6.1b 4.6b 6.5 + 1.9

aToxicity of PCP in water without sediment expressed as mg/l. bAn approximate EC50 was estimated.

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Table 3 Toxicity of PCP-spiked sediment and PCP-equilibrated water to Daphnia magna during fish exposure D a y of experiment a

-1

0

1

2

3

4

24-h EC50 for D. magna, expressed as m g PCP/kg sediment, w.w.

Replicate

l 2 mean 1 2 mean 1

2 mean 1 2 mean 1 2 mean 1 2 mean

G r a n d m e a n + SD

Sediment exposure

Water exposure

15 (12 18) b 15 (13-18) 15 (12-18) 13(11 16) 15 (13-18) 14 (12-17) 22 (18 27) 21 (18-25) 22 (18 26) 21 (17-24) 24 (20 29) 22 (18 27) 35 (26-50) 26 (22 31) 31 (24-40) 16c 16c 16 20 + 6.4

4.8 (3.5 5.7) b 7.2 (6.1 8.4) 6.0 (4.8-7.0) 3.0c 3.4 c 3.2 12 (10 14) 12 (10-14) 12 ( 1 ~ 1 4 ) 12 (10 14) 9.4 (7.2 12) 11 (8.4-13) 16c 16 > 64 d --<64 d ca 64 9.6 + 5.1 d

"Fish were added on day 0 (series 1) and day 3 (series 2). b 95% confidence limits are shown in parentheses. cA n approximate EC50 is given. d 3/20 immobilized in highest concentration (64 m g PCP/kg sediment, w.w.). e 13/20 immobilized in highest concentration (64 m g PCP/kg sediment, w.w.). fResults from day 4 (ca. 64) excluded (N = 5).

Table 4 Mortality of rainbow trout exposed to various concentrations of PCP in sediment (mg PCP/kg w.w.) D a y of experiment (hours of exposure)

0 1 (24 h)

(48 h) 3" (0 h) 4 a (24 h) 2

Sediment concentration (mg PCP/kg, w.w.) 0

4

8

16

0/10 0/10 0/10 0/10 0/10

0/10 0/10 0/10

0/10 0/10 0/10

-

"Exposure started on day 3. b6/10 dead within 24 h in concentrations 16 m g PCP/kg, w.w. 10/10 dead within 6 h in concentrations 32 and 64 m g PCP/kg, w.w.

32

64 -

-

10/10 0/10 6/10 b

10/10 0/10 10/10 ~

10/10 0/10 10/10 c

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decreasing water toxicity may be uptake of PCP from water by the fish followed by conjugation, and/or fish mucus-mediated precipitation of dissolved PCP. 3.3. Toxicity offish bile after P C P exposure

The mortality of fish during exposure to PCP-spiked sediment is shown in Table 4. The rainbow trout 24-h LC50 for PCP in sediment was approximately 16 mg PCP/kg sediment w.w., which is close to the 24-h EC50s for Daphniaof 13 and 20 mentioned above. The toxicity of fish bile (24- and 48-h EC50) is shown in Table 5 and 6. The bile toxicity increased in proportion to the PCP concentration in the sediment. Furthermore, the toxicity of the extract increased after hydrolysis of the PCP-conjugates to free-PCP. Toxicity values after acid and enzymatic hydrolysis were similar. Exposure of rainbow trout to half the 24-h LC50 concentration (8 mg PCP/kg sediment, w.w.) for 48 h resulted in a bile that was more toxic to Daphniathan bile from control fish. Hydrolyzed bile extract was much more toxic than original bile and bile extract in PCP-exposed fish, and in the 4 mg PCP/kg w.w. sediment exposure (25% of fish LC50) hydrolyzed bile extract was 10-50 times or more toxic than that from control fish (2.4-0.6 versus >25, Tables 5 and 6). The reason for the higher toxicity of

Table 5 Toxicity of bile and bile extracts (24-h EC50 for D. magna) from rainbow trout exposed to PCP-spiked sediment Exposure of fish (mg PCP/kg sediment, w.w.)

Replicate

O"

1 2 mean 1 2 mean 1 2 mean 1 and 2

4a

8"

16b

Toxicity of bile (ml bile/l)c

Toxicity of bile extract (ml bile/l)c Direct extraction

Acid hydrolysis prior to extraction

Enzymatic hydrolysis prior to extraction

34 (24-50) d 32 (23~,6) 33 (24~-8) 28 (21-37) 28 (21-37) 28 (21-37) 28 (20-41) 15(10 21) 22 (15 31) 0.74 (0.544).96)

> 20 e > 30 >25 >20 > 30 > 25 > 20 >30 > 25 3.0 (2.3~,.0)

> 20 > 30 >25 2.3 (1.7-3.1) 2.6 (1.8-3.6) 2.4 (1.8-3.4) 0.9 (0.6-1.4) 1.5(1.0-2.2) 1.2 (0.8-1.8) 1.4 (1.0-1.9)

> 20 > 30 >25 1.0 (0.8-1.4) 2.2 (1,6-3.0) 1.6 (1,2-2.2) 0.3 (02-0.4) 1.4(1.0 2.0) 0.8 (0.6-1.2) 0.2 (0.2 0.4)

a Five fish were exposed for 48 h in each of two replicated aquaria with PCP-spiked sediment concentrations as indicated. Biles from all fish in the same replicate were pooled. bBiles from 5 surviving fish (out of 10) exposed for 24 h were pooled. cToxicity of bile and bile extract is expressed as the EC50 for D. magna in ml bile/1. dNinety five percent confidence limits given in parentheses. e Highest concentration of bile or bile extract immobilized less than 50% meaning that EC50 is higher than this concentration.

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the hydrolyzed bile extract must be the higher toxicity of free-PCP compared to PCP-conjugates. Toxicity of control bile spiked with PCP was also tested (Table 7), and did not show such profound differences. The equal or slightly higher toxicity of the fl-glucuronidase hydrolyzed extract compared to the acid + heat hydrolyzed extract (Table 5 and 6) is consistent with previous findings showing that PCP is mainly excreted as a glucuronide in fish bile (Glickman et al., 1977; Kobayashi, 1979; Melancon and Lech, 1984; Oikari et al., 1984). One exception to this was found in fish exposed to the highest concentration (16 mg PCP/kg sediment, w.w.) in which both the bile and the bile extract were much more toxic compared to that from fish exposed to lower concentrations, in spite of the shorter exposure period (24 versus 48 h). The bile from these fish was also more toxic than control bile when tested directly on D. magna. Our interpretation of this discrepancy is, that during short-term, high concentration exposure (16 mg/kg for 24 h), the capacity of the fish to conjugate PCP had been exceeded resulting in the uptake of free PCP in the bile. Therefore, the difference between bile extract and hydrolyzed bile extract was less in these fish than in fish exposed to lower concentrations. Thus, we conclude that toxicity tests with bile and bile extract may be used to detect toxicity at near lethal short-term exposure to PCP, but that tests with hydrolyzed bile extract should be used to detect exposure at lower concentrations for longer periods. 3.4. Concentrations o f P C P in sediment, water and fish bile

The toxicity tests with PCP-spiked bile did not indicate any substantial impact of the hydrolytic procedures on the toxicity of the extract (Table 7). Calculated 24-h Table 6 Toxicity of bile and bile extracts (48-h EC50 for D. magna) in rainbow trout exposed to PCP-spiked sediment Exposure o f fish (mg PCP/kg sediment, w.w.)

Replicate

0a

8a

1 2 mean 1 2 mean 1

2

8 (6-1o)

16b

mean 1 and 2

7 (5-10) 0.4 (0.3~.6)

4a

"~ See Table 5.

Toxicity of bile (ml bile/l) c

17 (12-26) d 16(12-24) 16 (12-25) 12 (9-18) 16 (12-22) 14 (10-20) 6 (4-10)

Toxicity of bile extract (ml bile/l) c Direct extraction

Acid hydrolysis prior to extraction

Enzymatic hydrolysis prior to extraction

> 20 e > 30 >25 > 20 >30 >25 > 20 > 30 >25 1.6 (1.2 2.2)

> 20 > 30 >25 0.8 (0.4-2.0) 1.6 (1.0-2.5) 1.2 (0.7-2.2) 0.7 (0.4-1.2) 1.2 (0.9-1.8) 1.0 (0.7-1.5) 0.7 (0.4-1.0)

> 20 > 30 >25 0.5 (0.4-0.8) 0.8 (0.5 1.4) 0.6 (0.4-1.1) 0.2 (0.0-0.2) 0.6 (0.4-1.0) 0.4 (0.2-0.6) 0.2 (0.1-0.2)

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Table 7 Toxicity of PCP-spiked fish bile to D. magna Spiked PCP in bile (rag/l)

0 25 50 100 200

Exposure

24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

Toxicity of bile extract to D. magna, EC50 (ml bile/l) Direct extraction

Acid hydrolysis prior to extraction

Enzymatic hydrolysis prior to extraction

> 40 > 40 > 40 28 (16-84) 40 13 (248) 18 (14-24) 9.0 (4.0-16) 7.6 (5.1-10) 3.9 (2.4-5.4)

> 40 23 32 (25-55) 19 (1245) 23 > 40 17 (13-23) 5.5 (2.6-8.8) 7.9 (6.0-11) 2.8 (1.34.6)

> 40 20 40 18 (12-25) 28 (2242) 15 (11-22) 6.9 (5.0-10) 1.2 5.4 (3.8-7.0) 2.6 (1.6-4.2)

EC50s for PCP after these treatments ranged from 0.5 to 2 mg/1 (spiked concentrat i o n x EC50 expressed in ml bile/l: 1000), which is consistent with the 24-h EC50 determined for PCP in water of 1.2 + 0.4 mg/1 (mean + SD in Table 2). Therefore, we assume that the extraction of PCP from bile was efficient and that the effect of the hydrolytic procedures used was negligible. This is important for the interpretation of results from bile toxicity tests of exposed fish. If major difference in toxicity due to hydrolytic treatment (acid + heat or enzyme) is found in exposed fish, then this must be due to conjugation in vivo during exposure. Toxicity of PCP-spiked bile (Table 7) revealed that the relationship between the PCP-concentration and toxicity was adequately described by the equation: In (PCP in bile, mg/1) = a + b x In (24-h EC50, ml bile/l) Relationships derived from the different treatments of bile prior to toxicity testing were similar. The best relationship was found for the combined data (direct extraction, extraction after acid + heat hydrolysis and extraction after enzymatic hydrolysis) from the 24-h EC50 tests. Consequently, this equation, with a = 7.0 and b = -0.93 (r = -0.90, N = 10, P < 0.0005), was used to determine PCP concentrations in bile of fish after exposure to PCP. The concentrations of free PCP, free PCP+total conjugates of PCP and free PCP+glucuronide PCP in exposed fish estimated from 24-h EC50s for D. magna are shown in Table 8. At a sediment concentration of PCP of 16 mg/kg w.w., the bile concentration of free + glucuronide PCP in rainbow trout estimated by this method was 4690 mg PCP/l. This exposure concentration was close to the 24-h EC50 for D. magna of 1.2 mg/l (Table 2). Therefore, the bile-water ratio at this concentration was estimated to be 3900 (4690:1.2). This ratio is somewhat lower than that deter-

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Table 8 Concentrations of free PCP and conjugated PCP (as m g PCP/I) in bile from rainbow trout exposed to PCP-spiked sediment estimated from 24-h bile fraction toxicity to Daphnia" PCP fraction of bile

Sediment exposure concentration, mg PCP/kg sediment (w.w.) 0

4

8

16

Free PCP

< 53

< 53

< 53

380

Free + conjugated PCP Free + glucuronide PCP

< 53 < 53

467 506

889 1295

771 4,690

"Equations for determinations of PCP concentrations in fish bile from bile toxicity to D. magna were derived from tests with PCP-spiked bile shown in Table 7. The equation for the combined data from 24-EC50 tests with directly extracted bile, extracts of acid + heat hydrolyzed bile and extracts of enzymatically hydrolyzed bile were as follows: In (PCP in bile, rag/l) = 7.0-0.93 In (24-h EC50, ml bile/l), r = -0.90, N = 10, P<0.0005. Bile toxicity in

PCP-exposed fish (24-h EC50s, ml bile/l) inserted into this equation are given in Table 5.

mined previously for rainbow trout exposed for 24 h to 0.1 mg PCP/1 by Statham et al. (1976) of 5360 and less than half of the ratio of 9030 determined by M c K i m et al. (1986) after exposure to 0.001 mg PCP/1. Thus, the test concentration and duration of exposure are important in determining the bile-water ratio for PCP as well as for other toxicants. Nevertheless, this comparison between concentrations of PCP and bile-water ratios determined by toxicity tests and chemical analysis is remarkably consistent, considering that they were determined independently by different researchers at different times using different methods for exposure and analysis.

3.5. Bile toxicity of unknown pollutants In investigations with wastewaters or sediments with unknown toxicants sediment, water and bile toxicity tests could be used to establish transfer of toxicants from sediment to water and into the fish. I m p o r t a n t factors to consider in such investigations are the bile-water ratio and the toxicity of the chemical, because these factors will influence the detection limit for specific chemicals. Our comparison of the toxicity of bile, water and sediment showed that bile which was hydrolyzed prior to extraction was more toxic than water and sediment samples. Therefore, toxicity of hydrolyzed bile extracts m a y be a more sensitive measurement of environmental pollutants than toxicity of water and sediment. The major limitation is that all chemicals are not stored in the bile to the same extent. However, a large number of chemicals of environmental concern are concentrated in the bile of fish, and chemical analysis of fish bile has been recommended as an indicator of water quality (Lech et al., 1973; Statham et al., 1976; F6rlin and Wachtmeister, 1989; Wachtmeister et al., 1991). The potential sensitivity of the bile toxicity approach can be estimated f r o m the present results as follows. The 24-h LC50 of rainbow trout for PCP in sediment was 16 mg/kg sediment w.w., and fish exposed to 1/4 of this concentration (4 mg/kg) had

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bile toxicities after hydrolysis which were at least 10 to 50 times higher than that in unexposed fish (24- and 48-h EC50s o f hydrolyzed bile extracts shown in Table 5 and 6). Thus, if bile and sediment concentrations are correlated, then the bile toxicity test could detect sediment concentrations which are 40 to 200 times lower than the 24-h LC50 concentration, i.e. 0.4-0.08 m g P C P / k g sediment w.w., which is equivalent to 2-0.4 m g P C P / k g sediment d.w. P C P sediment quality and cleanup standards for the State o f Washington reported by G i n n and P a s t o r o k (1992), were 0.36 and 0.69 mg/kg d.w., respectively. Thus, the bile toxicity test would be sensitive e n o u g h to detect P C P - c o n t a m i n a t i o n equivalent to these quality and cleanup standards. However, it is i m p o r t a n t to consider that our estimated sensitivity o f the bile toxicity a p p r o a c h is based on results with only one chemical (PCP) and under short-term l a b o r a t o r y conditions. With other chemicals, mixtures and m o r e realistic exposure conditions, the use o f the bile toxicity m e t h o d m a y be m o r e or less sensitive. The m a j o r advantage o f this bile toxicity test m e t h o d is that all c o m p o u n d s that accumulate in the bile are expressed in p r o p o r t i o n to their toxicity.

Acknowledgements We t h a n k Eva Nilsson and Birgitta Vallander for skilled technical assistance and the Swedish Environmental Protection A g e n c y for financial support.

References Anonymous (1987) Canadian Water Quality Guidelines. Canadian Council of Resource and Environment Ministers. Water Quality Objectives Division, Water Quality Branch, Environment Canada, Ottawa, Ont. Berglind, R. and G. Dave (1984) Acute toxicity of chromate, DDT, PCP, TPBS, and zinc to Daphnia magna cultured in hard and soft water. Bull. Environ. Contain. Toxicol. 33, 63-68. Davis, J.C. and R.A.W. Hoos (1975) Use of sodium pentachlorophenate and dehydroabietic acid as reference toxicants for salmonid bioassays. J. Fish. Res. Board Can. 32, 411~16. F6rlin, L. and C.A. Wachtmeister (1989) Fish bile analysis for monitoring of low concentrations of polar xenobiotics in water. In: Chemicals in the Aquatic Environment, edited by L. Landner. Springer, Berlin, pp. 150-164. Gall, G.A.E., guest editor (1992) The Rainbow Trout. Proc. Syrup. 4~7 September 1990, Univ. Sterling, Scotland. Aquaculture 100(1-3), 332 pp. Elsevier, Amsterdam. Ginn, T.C. and R.A. Pastorok (1992) Assessment and management of contaminated sediments in Puget Sound. In: Sediment Toxicity Assessment, edited by G.A. Burton, Jr. Lewis, Chelsea, MI, pp. 371~01. Glickman, A.H., C.N. Statham, A. Wu and J.J. Lech (1977) Studies on the uptake, metabolism and disposition of pentachlorophenol and pentachloroanisole in rainbow trout. Toxicol. Appl. Pharmacol. 41:649 658. ISO (1989) Water quality - determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea). ISO 6341(E), International Organization for Standardization. Kobayashi, K. (1979) Metabolism of pentachlorophenol in fish. In: Pesticide and Xenobiotic Metabolism in Aquatic Organisms, edited by M.A.Q. Khan. ACS Symp. Ser. 99, 131-143. Kobayashi, K. and H. Akitake (1975a) Studies on the metabolism of chlorophenols in fish. I. Absorption and excretion of pentachlorophenol by goldfish. Bull, Jpn. Soc. Sci. Fish. 41, 87-92.

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M. Andrkasson, G. Dave~Aquatic Toxieology 30 (1994) 171 182

Kobayashi, K. and H. Akitake (1975b) Studies on the metabolism of chlorophenols in fish. II. Turnover of absorbed PCP in goldfish. Bull. Jpn. Soc. Sci. Fish. 41, 93-99. Kobayashi, K., H. Akitake and T. Tomiyama (1977) Studies on the metabolism of pentachlorophenate, a herbicide, in aquatic organisms. III. Isolation and identification of a conjugated PCP yielded by a shellfish, Tapesphilippinarum. Bull. Jpn. Soc. Sci. Fish. 36, 103 108. Kobayashi, K., H. Akitake and K. Manabe (1979) Relation between toxicity and accumulation of various chlorophenols in goldfish. Bull. Jpn. Soc. Sci. Fish. 45, 173 175. Lech, J.J., S.K. Pepple and C.N. Statham (1973) Fish bile analysis: a possible aid in monitoring water quality. Toxicol. Appl. Pharmacol. 25, 430-434. Lee, H. (1992) Models, muddles, and mud: predicting bioaccumulation of sediment-associated pollutants. In: Sediment Toxicity Assessment, edited by G.A. Burton, Jr. Lewis, pp. 267-293. Love, R.M. (1980) The Chemical Biology of Fishes, Vol. 2: Advances, 1968 1977. Academic Press, New York. Mayer, F.L. and M.R. Ellersieck (1986) Manual of acute toxicity: interpretation and data base for 410 chemicals and 66 species of freshwater animals. U.S. Fish Wildl. Serv. Resour. Publ. 160, 579 pp. McKim, J.M., P.K. Schmieder and R.J. Erickson (1986) Toxicokinetic modeling of [~4C]pentachlorophenol in the rainbow trout (Salmo gairdneri). Aquat. Toxicol. 9, 59-80. Melancon, M.J. and J.J. Lech (1984) Metabolism of [14C]-2-methylnaphthalene by rainbow trout (Salmo gairdneri) in vivo. Comp. Biochem. Physiol. 79C, 331-336. Niimi, A.J. and C.A. McFadden (1982) Uptake of sodium pentachlorophenate (NaPCP) from water by rainbow trout (Salmo gairdneri) exposed to concentrations in the ng/l range. Bull. Environ. Contam. Toxicol. 28, 11-19. Oikari, A.O.J. (1986) Metabolites of xenobiotics in the bile of fish in waterways polluted by pulpmill effluents. Bull. Environ. Contam. Toxicol. 36, 429-436. Oikari, A. and T. Kunnamo-Ojala (1987) Tracing of xenobiotic contamination in water with the aid of fish bile metabolites: a field study with caged rainbow trout (Salmo gairdneri). Aquat. Toxicol. 9, 327-341. Oikari, A., E. An/is, G. Kurzynski and B. Holbom (1984) Free and conjugated resin acids in the bile of rainbow trout, Salmo gairdneri. Bull. Environ. Contam. Toxicol. 33, 233-240. Peltier, H.W. and C.I. Weber (1985) Methods for measuring the acute toxicity of effluents to freshwater and marine organisms, 3rd edition. US EPA/600/4-85/013, Appendix E. U.S. Environmental Protection Agency, Cincinnati, OH, pp. 170-216. Statham, C.N., M.J. Melancon and J.J. Lech (1976) Bioconcentration of xenobiotics in trout bile: a proposed monitoring aid for some waterborne chemicals. Science 193, 680-681. Wachtmeister, C.A., L. F6rlin, K.C. Arnoldsson and J. Larsson (1991) Fish bile as a tool for monitoring aquatic pollutants: studies with radioactively labelled 4,5,6-trichloroguaiacol. Chemosphere 22, 39-46.