DISEASES OF AQUATIC ORGANISMS Vol. 19: 193-202.1994 Published August 25 Dis. aquat. Org.

Microfluorometrical study of bioaccumulation of benzo(a)pyrene and marker xenobiotics in the bivalve Donaxtrunculus from clean and polluted sites along the Mediterranean shore of Israel

V. Bresler, L. Fishelson

Institute for Nature Conservation Research and Department of Zoology. Tel Aviv University, Ramat Aviv 69978, Israel

ABSTRACT: This study focused on bioaccumulahon of benzo(a)pyrene [B(a)P]from a polluted envi- ronment and 2 xenobiotic markers under experimental conditions in various organs of the marine bivalve Donax trunculus. The cationic dye acndine orange (AO) and anionic dye fluorescein (FLU) were used as markers. Test molluscs were collected from 3 sites along the Israeli Mediterranean shore: a relatively clean site, an oil-polluted site and a site polluted by chemical effluents from a polyvinyl- chloride (PVC) factory. B(a)P was determined in tissue samples using benzene extraction and solid- surface luminescent analysis. Bivalves from the oil-polluted site contained 55.6 ng B(a)P g-' wet wt. B(a)P concentration varied among body compartments: > total soft tissues > kidney > shell >mantle edge = gds. In experiments, living bivalves from these 3 sites were kept in seawater with 10 PM A0 or 100 pM FLU for 24 to 96 h. Accumulation and distribution of A0 and FLU in the molluscs was determined by vital contact microfluorometry and from ethanolic extraction of the markers. Following exposure to the markers, D. trunculus from the clean site showed A0 concentrations as follows: edge > kidney > hepatopancreas > > central part of the mantle. For FLU the order was: ludney > gills > mantle edge > hepatopancreas > central part of the mantle. D. trunculus from polluted sites demonstrated a decrease in A0 concentration and an increase in FLU concentration, especially in specimens from the site polluted by the PVC factory. Experimental thermal stress produced a drastic decrease in A0 bioaccumulation. The observed bioaccumulation of B(a)P, A0 and FLU reflects the environmental quality and can thus be used for monitoring.

KEY WORDS: Bivalve mollusc Donax trunculus - Bioaccumulation of xenobiotics . Defense mecha- nisms against xenobiotics

INTRODUCTION ations (Couch et al. 1979, Lowe 1988, Moore 1988, Triebskorn et al. 1991, Yawetz et al. 1992) and mass Molluscs are numerically and energetically important mortality (Okland 1992, Fishelson et al. unpubl.). in numerous benthic communities. Many are filter- Along the Mediterranean shore of Israel, the bivalve feeding and long-lived, and they are in constant con- Donax trunculus occurs in population densities of up to tact with various xenobiotics dissolved in water or as- 2 kg biomass m-2 (Fishelson et al. unpubl.). Following sociated with bottom sediments or suspended material. a local long-term oil spill along the Mediterranean Some molluscs accumulate considerable quantities of shore, mass mortality of the bivalve Mastra corallina potentially toxic anthropogenic or natural xenobiotics occurred, the shells of which covered the shore, along with no noticeable toxic effect (Dindal & Wurzinger with lower mortality of Venus spp., Angulus planatus 1971, Neff et al. 1976, Roberts 1976, Eriksson et al. and Ensis spp. The latter 3 species dwell in deep water 1986, 1989, Paasivara et al. 1992). Others, upon expo- and dig deeper into the sand than D. trunculus. How- sure to anthropogenic pollutants, exhibit various phys- ever the shallow-water D. trunculus population was iological, histopathological, bio- or histochemical alter- apparently unaffected, as only a few died. Atomic ab-

0 Inter-Research 1994 194 Dis. aquat. Org. 19: 193-202. 1994

sorption analysis of the soft tissues of D. trunculusfrom quickly transported to the laboratory in plastic contain- this oil-polluted site showed a marked increase in the ers with ambient seawater. The specimens were kept content of some trace metals, especially chrome, lead there for a short time (2 to 10 d) in aerated aquaria with and copper, that were probably derived from oil (Fish- seawater at 25 "C without substrate and without artifi- elson et al. unpubl.). cial feeding. For comparison, 23 live specimens of the The main biologically active, cytotoxic, mutagenic, bivalve Mactra corallina - a species which is very carcinogenic components of oil are polycyclic aromatic sensitive to given pollutions - were taken from the hydrocarbons (PAHs), which are often accumulated by same clean site where D. trunculus was sampled. molluscs (Neff et al. 1976, Couch et al. 1979, Kauss & Test chemicals. Xenobiotic markers in the tissues of Hamdy 1991). Accumulation and distribution of heavy molluscs were directly quantified with contact fluores- metals in various molluscs, including Donax trunculus, cent microscopy and microfluorometry. Two fluores- have been studied and are well established (Hornung cent xenobiotic markers were used: acridine orange & Oren 1981, George 1984, Romeo & Gnassia-Barelli (AO; N,N,N1,N'-tetramethyl-3,6-acridinediamine; pKb = 1988, Jenner et al. 1991); however, the accumulation 10.4) was selected as a cationic lysosomotropic com- and biological activity of PAHs derived from oil have not pound (Wittekind 1973, Swanson 1989, Murphy 1990) been sbdiec? ic exposed L). truncu!us, whose apparznt and %=rcscc:n (FLU; 3',6'-dihydrcxy sp:rc[ischenzo- resistance to oil spills has not been investigated. En- furan-l(3M)S1-xanthenl-3-one,disodium salt; pK, = 7.0) zymes for detoxification of xenobiotics have been stud- was selected as an anionic analogue of numerous ied, but in many hosts, especially invertebrates, other natural and anthropogenic weak organic acids which protective systems may be important in antichemical are constantly present in the environment (Zanker & defense (Harborne 1982, Bresler et al. 1985, 1990, Peter 1958, Perdue & Gjessing 1989) and as a marker Triebskorn et al. 1991). Therefore, the present study for determining the viability of plasma membranes attempted to explore the sensitivity of a new method- (DuBois et al. 1980, Bowen 1981, Pieters et al. 1989). ological approach for detection of physiological, bio- In preliminary tests, Lhe optimal concentrations of chemical or cytomorphological mechanisms of antixeno- A0 and FLU for microfluorometry and vital micro- biotic defense. For this purpose methods were adapted scopic investigations were found to be 10 and 100 pM and developed encompassing vital light microscopy, 1-' respectively. Subsequently, sufficient A0 or FLU luminescent microscopy, microfluorometry and special was added to seawater to maintain the predetermined fluorescent probes. exposure level of A0 at 10 PM and FLU at 100 PM.In The availability of modern fluorescent methods and additional experiments to study possible run-out of probes facilitates the detailed study of cellular organi- accumulated marker, the tested molluscs were trans- sation, function and metabolism (Goldberg 1989, Taylor ferred for 24 h into clean seawater for depuration after & Wang 1989, Bresler et al. 1990, Darzynkiewicz & 96 h incubation with A0 or FLU. Kapuscinski 1990). The present study investigated Thermal stress produces marked alterations in cell accumulation and distribution of benzo(a)pyrene [B(a)P] structures and metabolism in molluscs (Moore 1976). and xenobiotic markers in Donax trunculus from both Therefore, the effect of high temperature (24 h incuba- relatively clean and polluted sites along the Medi- tion at 35 "C) on A0accumulation by Donax trunculus terranean shoreline of Israel, and the reactions of these from the clean site was also studied. molluscs to oil and other pollutants. Analytical procedures. After incubation with A0or FLU, and after depuration, the valves of the molluscs were opened by cutting the adductor muscles. Inten- MATERIAL AND METHODS sity of fluorescence was determined by vital contact rnicrofluorometry on the natural surface of: mantle Test molluscs and experimental design. In this study edge along the ; central part of mantle; gills; approximately 450 specimens of adult Donax trunculus renocardial area (kidney); hepatopancreas (digestive with a mean length of 24.4 * 0.5 mm (* 95% confidence diverticula); ; and extrapallial surface of limit) were used, some of which (n = 165) were col- the nacreous shell layer. Autofluorescence intensity lected from a relatively clean site along the Israeli was also measured. For each specimen, 10 separate Mediterranean shore (Akko), and others from sites measurements were taken from both periostracum and polluted by local long-term oil spills (n = 165, Kiryat shell, and 20 measurements from each of the other Yam) or by chemical effluents from a PVC factory tissues. For each separate experiment (site of collec- (n = 135, Frutarom). These 3 stations have been used tion, type of marker, time of incubation) 15 specimens and characterized by Yawetz et al. (1992) and Fishel- of Donax trunculus were used. son et al. (unpubl.). All collections were performed The technique of vital microfluorometry was de- from July to October. Samples of live D, trunculus were scribed in detail by Bresler et al. (1975, 1979, 1985, Bresler & Fishelson: Bioaccurnulation of benzo(a)pyrene and xenobiotic markers 195

1990) and Bresler & Nikiforov (1977).A contact objec- RESULTS tive of 25 X 0.75 was used for microfluorometry of Donax trunculus with a rectangular photometric diaphragm During incubation from 24 to 96 h with A0or FLU at that had an of 75 X 75 pm in the plane of the ob- 25°C no mortality of Donax trunculus was observed ject. The electric current from the photomultiplier tube and no marked alteration occurred in their behavior measured the intensity of fluorescence at 530 nm in ar- (opening of valves, syphonal activity, movement). Dur- bitrary units (a.u.).Fluorescence of accumulated marker ing 24 h incubation at 35°C (experimental thermal was calculated as the difference between the experi- stress) 15 of the 25 incubated D. trunculus from the mentally measured fluorescence and autofluorescence. clean site died while others drew in their syphons and For cuvette fluorometry, after incubation with markers partially closed their valves. and rnicrofluorometry, whole soft bodies of specimens or The maximal amount of B(a)P equivalents was found samples of the mantle edge along the pallial line, central in soft body and shells of Donax trunculus from the oil- part of the mantle, renocardial area, and hepatopan- polluted site; bivalves from the PVC factory site con- creas were rapidly dissected, weighed and transferred tained approximately half as much B(a)P, and those into 30 % ethanol. Fluorochrome concentration in these from the relatively clean site had only traces in their extracts was determined by microcuvette fluorometry, soft bodies (Table 1). In specimens from polluted sites as described earlier (Sernetz & Thaer 1973, Bresler the B(a)P distribution in various tissues in decreasing & Nikiforov 1977, Bresler et al. 1975, 1990). In each order was: hepatopancreas >> total soft body > kidney determination 12 bivalves were used for whole soft body > shell > mantle edge = gills (Table 1). B(a)P concen- samples and 12 for tissue samples. tration was highest in sediments from the oil-polluted Tissue samples for determination of B(a)P equiva- site, and lowest in the sediment from the relatively lents were prepared from Donax trunculus from all clean site (Table 1). collection sites as described above and extracted with Microfluorometrical data demonstrated that the benzene. For each determination a total of 15 speci- distribution of A0 in the tissues of Donax trunculus mens were used for both whole soft body and shell from both the relatively clean and polluted sites was samples, and 12 to 15 were used for samples of various very irregular (Table 2): the maximal fluorescence tissues. Samples of the sediment present permanently intensity of A0 was observed in numerous large on the bottom of containers holding collected molluscs (50 + 9 pm) bodies along the pallial line of the were also investigated. Nine samples of such sedi- mantle (Fig. 1); the lowest activity was observed on ment from each collection site were weighed, dried the shell surface. In the central part of the mantle, and extracted with benzene. B(a)P in benzene extracts gills, hepatopancreas and kidney, A0 was detected was determined by room-temperature solid-surface as green diffuse fluorescence of the cytoplasm and fluorescence, at wavelength pair excitation/emission orange or red fluorescence of lysosomes and related 380/430 nm (Hurtubise 1981, Hurtubise et al. 1989). structures; in the kidney A0 was also present in the B(a)P equivalent includes B(a)P, pyrene and fluoran- lumen of tubules. thene (Krahn et al. 1987). A steady-state level of A0 accumulation in the tis- For rnicrofluorometry and fluorometry results, means sues of Donax trunculus from the clean site was ob- and 95% confidence limits were calculated. When served within 48 h of incubation (Fig. 2). necessary the correlation coefficient (r)and regression After depuration, a statistically significant decrease equation were calculated. of A0 content in the mantle edge and hepatopancreas

Table 1. Concentration of benzo(a)pyrene equivalents [B(a)Pj in the tissues of Donaxtrunculus (ng g-' wet weight) from a relatively clean site and polluted sites along the Mediterranean shore of Israel (mean * 95 % confidence lunit, n = number of individuals tested). All differences between samples from clean and polluted sites are significant at the 95 % level

Sample Relatively clean Oil polluted PVC-factory-polluted B(a)P n B(a)P n B(a)P n

Sedunent Whole mollusc Whole soft body Shell Hepatopancreas Renocardial area Mantle Gills Dis. aquat. Org. 19: 193-202, 1994

of Donax trunculus from the clean site was observed, whereas in D. trunculus from polluted sites the A0 content in these tissues was unchanged (Table 2). A0 accumulation in the tissues of D. trunculus decreased drastically after 24 h incubation at 35 "C (Tables 2 & 3, experimental thermal stress). E .??v! F-.? 0'4'4 The distribution of FLU in various tissues of Donax -cue -m- mcum 2 trunculus from both clean and polluted sites also 2 flflfl ++l+ fltlfl fl .d 'S-?? 9"? =?F9 4 showed a very irregular pattern (Tables 4 & 5): the g 3cuw NNP- 9-N 'C mmcu mmm mmm m- a, highest fluorescence intensity was detected in reno- a cardial tissues, and the lowest in the central part of the mantle. After depuration, a significant decrease in fluorescence intensity in all soft tissues, except the renocardial area, was obsenred (Table 4). Quantitative determination of A0 or FLU concen- tration after 96 h incubation in various tissues of bi- valves by ethanolic extraction showed that in Donax trunculus from the clean site, the mantle edge along the pallial line contained about 3000 pg A0 g-' wet tissue weight (ww), the renocardial area (kidney) about 1000 pg g-I ww, and total soft tissues about 400 1-19 g-' ww (Table 3). All tissues of D. trunculus from the clean site contained only small amounts of FLU after 96 h incubation: about 2.6 1-19 g-' ww in the renocardial area and about 0.364 pg g-' ww in total soft tissues (Table 5). After depuration total soft tissues contained only 0.18 pg g-' ww (data not shown in Table 5). There is a strong correlation (r = 0.9) between the data on A0 or FLU determined by ethanolic extraction and by microfluorometry. Comparison of data from Tables 2 to 5 shows sig- nificant differences between Donax trunculus sam- pled from relatively clean and polluted sites. First, A0 accumulation in the mantle edge, gills and hepatopancreas of molluscs from polluted, especially the PVC-factory-polluted, sites was less than in the same tissues of D. trunculusfrom the relatively clean site. Second, FLU accumulation in the mantle, gills and hepatopancreas of D. trunculus from polluted, especially the PVC-factory-polluted, sites was greater than in the same tissues of specimens from the rela- tively clean site. After 96 h ~ncubation, the reno- cardial area of D, trunculus from the oil-polluted site contained the highest concentration of FLU; a lower concentration was observed in molluscs from the clean site, and the lowest was found in specimens from the PVC-factory-polluted site (Tables 4 & 5). Third, during depuration, run-out of FLU from man- tle, gills and hepatopancreas of D. trunculus from polluted, especially the PVC-factory-polluted, sites was weaker than from the same tissues of molluscs from the clean site. However, after depuration, no run-out of FLU or A0 from the renocardial area of D. trunculus from both clean and polluted sites was observed. Bresler & Fishelson: Bioaccumulation of benzo(ajpyrene and xenobiotic markers 197

Fig. 1. Donax trunculusincubated with 10 IJM acridine orange for 24 h showing numerous intensely fluo- rescent large bodies (arrows) in the mantle edge along pallial line. Vital contact fluorescent microscopy; ob- jective 25 X 0.75, ocular x5, photo- magnification X 4

DISCUSSION (1979) demonstrated a similar uptake pattern in using I4C-labeled B(a)P. These results show that PAH Information on PAH accumulation from oil-polluted accumulation in the various molluscs studied is tissue- sites as well as on PAH distribution in these molluscs specific. In this study the soft body of D. trunculusfrom is very limited. Our data show that some typical the oil-polluted site had from 65 to 98 ng B(a)P g-' ww. PAHs [B(a)Pequivalents] were accumulated by Donax Kauss & Hamdy (1991) showed that in whole soft trunculusfrom the oil-polluted site. Their distribution bodies of from highly polluted sediments the in the tissues of D. trunculuswas very irregular, show- maximal concentration of B(a)P equivalent was up to ing concentrations as follows: hepatopancreas > total 1220 ng g-' dry weight. Such concentrations may be soft body > kidney > shell > mantle edge = gills. Neff cytotoxic and would be important markers to unmask & Anderson (1975) described a similar distribution of any signs of cytopathological and cytogenetic alter- B(a)P in the Rangia cuneata, and Couch et al. ations produced by accumulated PAHs.

Table 3. Acridine orange (AO) content (pg g-' wet wt) in various tissues of Donax trunculusfrom the clean site, oil-polluted site and site polluted by effluents of the polyvinylchloride (PVC) factory, and in the same tissues of Mactra corallinafrom the clean site of the Mediterranean coast of Israel. A0 content was deternlined by ethanolic extraction after 96 h incubation and micro- cuvette fluorometry (mean i 95 % confidence 11mit)

Species and Mantle Mantle Gills ffidney Hepato- Total soft sampling origin edge center pancreas tissues

D. trunculus 2987.0+150.0 147.5i37.0 450.1*57.7 985.0i80.8 590.0k53.4 385i36.1 from clean site D. trunculus 2487.5 + 300.3' 101.2 17.6 391.2 + 48.3 923.7 111.4 491.2 + 112.2 311.0 46.3 from oil-polluted site i i i D. truncdusfrom PVC- 2271.2 385.4' 78.7 21.9' 330.0 i 82.4 771.2 126.8' 445.0 87.5' 280.7 35.8' factory-polluted site * * i * i D. trunculusfrom clean site; 507.5 49.2' 22.2 3.5' 31.5 + 4.9' 41.0 6.5' 14.7 3.5' 46.5 2.0' acute thermal shock applied * i i * i M. coraLlina 383.7 t 48.3' 206.2 58.6' 612.5 + 87.2' 1420.0 78.6' 812.0 52.6' 491.7 25.1' from clean site i i * i 'Statistically significant (p < 0.05) from control (the same tissue of D. trunculusfrom the clean site) 198 Dis. aquat. Org. 19: 193-202, 1994

. . . . ?F? L?Ncq ??S -8- 000 0-0 0-0 ++I+ +I*# +I#+I $5 '?a!? =?O? =??W (n2 ocoo --?E1 -N.- ... . . E .v,? NE? ??? +m- crmm mwco .2 +I+++ ++I$( +I+# "!S* clcl? .--=? g mmo cow- 'C omcu v) p.2- aa +

.. a.. ??v, v,?? *-c 3: 000 000 m-m +I+I+ +++I ++I+I 9'09 =??P ?9cq Nt-o m ++a Time of incubation (hours) - N +m N .2 G z- Fig. 2. Donax trunculus. Accumulation of acridine orange in A 2 & .-d various tissues of molluscs from the clean site along the Israeli 2.g Mehterranean shore as a functlon of incubation time. Con- B y, d ?b ?cl."' centration of acndme orange in seawater is 10 PM. (1) Mantle .-G -2 m $22 2 m,, +#+I **+I +(+(+I edge, (2) central part of the mantle, (3) gills. (4) hepato- $2 $2?'?V F*? ?V9 pancreas, (5) renocardial area. Vertical bars indicate 95 % 5 E -WO v)mcr wdm N W-P) C-lP-W confidence limit a, E gm .+ 4 N Pi 5: 0 5 g G ...... m The relationship between the concentration cf con- 5 ?cl(- qcqq -m C 2 taminants in the organism or its tissues and that in the 32 v, 0-0 4.30 m-W v 3 2 +++I H++ ++I+I a surrounding water or sediment is known as the biocon- "2 3:: 22: GQ) centration factor (BCF). Recently, attention has been + v NW P)(DN .-V) 5 g focused on correlations of BCF with various physico- *P) 5 l1 chemical properties of xenobiotics (Kauss & Hamdy :0 C - 4; .. S.. 2 1991, Niimi & Lee 1992). However, the influence of 444 'D?=? cl"!'? : z :g QIL 000 000 w-m f .O various biological factors on BCF requires more study. " - -2 #+,+I ++I+ +I+I+ g 3 a, a, 5% 4.9 9%. "!qv BCF for B(a)P in various tissues of Donax trunculus 2 2 Or00 "Wd &Q)" .S a, 2%zp_ from the oil-polluted site was very low: for the whole '9 JL soft body it was 0.173 (wet weight basis). This is inter- .C:a g 2 esting, as PAHs are very Lipophilic. In short-term ex- .-c 8 2 E S ... S.. periments on oysters with I4C-labeled B(a)P, BCF was "v) 9kcu ??O .-V? r[l E m cNc OWN VP)W G$ +#+I +I++ +I## a. very high (Couch et al. 1979).However, many authors 5; 5 ?-V TO'? ??c! .X found very low (usually < 1) BCF for mussel/sediment Ta Za, coon woco P-"W ZJT; - P) 4mcc crco* " PAHs under field conditions (Roesijadi et al. 1978, Heit z; .S .S -m d .d4 c et al. 1980, Elder & Dressler 1988, Kauss & Hamdy C E.E! 5 2 1991). Due to the low water solubility of PAHs, during 2 long-term exposure they interact with dissolved F! 5 4 e 2-c 5 .-.- " d .- organic carbon and are absorbed by fine suspended -2 - 2%: g g~ & BEg ""r .- matter and bioaccumulated by plankton (Kauss - Q) m m C2 X 8 c Harndy 1991).Therefore, under field conditions, PAHs E0 and other lipophllic xenobiotics penetrate into mol- a, E 5 .-d luscs mainly in the particulate-associated form via the V) c E U digestive canal, especially the hepatopancreas, and -(U E! S -1 h2 3 only a small fraction extens in dissolved form via the a, -0 2 .M.- 5 gills (Brodtman 1970, Dindal & Wurzinger 1971, Kauss ? A 'G U P zc Q & Hamdy 1991). a, d 0 - .-4 3 U 8 2 B(a)P accumulation in the hepatopancreas of Donax 0 E v) 'm '- c - C .- .E 2 0 trunculus from polluted sites also indicates that the m= 5 Q U %g a rate of B(a)P detoxification in the hepatopancreas is E2 G 5 2 ?F slower than the rate of its uptake and bioaccumulation. Bresler & Fishelson: Bioaccumulation of benzo(a)pyreneand xenobiotic markers 199

Table 5. Fluorescein (FLU) content (1.19 g-' wet wt) in various tissues of Donax trunculus from the clean site, oil-polluted site and site polluted by effluents of the polyvinylchloride (PVC) factory, and in the same tissues of Mactra corallina from the clean site of the Mediterranean coast of Israel. FLU content was determined by ethanolic extraction after 96 h incubation and micro- cuvette fluorometry (mean + 95 % confidence limit)

Species and Mantle Mantle Gills Kidney Hepato- Total soft sampling origin edge center pancreas tissues

D trunculus 0.57 t 0 06 0 06 k 0 006 0.70 t 0.08 2.60 0.29 0.10 0.035 0.364 0 033 from clean site * * * D. trunculus from 1.16 t 0.05' 0.10 0.033' 0 94 0.16' 3.75 0.62' 0.32 0.132' 0.455 + 0.047' oil-polluted site * * * *

D' trunculus fromPVC- 1.62 0.37' 1.30 0.25' 1.39 0.48' 1.66 0.35' 0.46 0.15' 0.595 0.099' factory-polluted site * * * * * * M.corallina from clean site 'Statistically significant (p < 0.05) from control (the same tissue of D. trunculus from the clean site)

Yawetz et al. (1992) showed that the significant in- Therefore, the level of A0 accumulation in the cell is crease in cytochrome P450 content in D. trunculus determined by its specific structure and function, i.e. from the same oil-polluted site was accompanied by by production and massive intracellular accumulation a drastic decrease in 7-ethoxyresorufin 0-deethylase of the mucoproteins, or by the presence of numerous catalytic activity. Consequently, PAH distribution and lysosomes. Our data show that in Donax trunculus the BCF values in molluscs from polluted sites are tissue- main part of the accun~ulated A0 is bound by large specific and possibly dependent upon specific uptake bodies distributed along the pallial line. Thus, in this pathways. tissue, marker binds with some intracellular mucopro- Bioaccumulation and distribution of A0 and FLU teins, and such binding is inhibited significantly by were very different in the various tissues of molluscs. pollution and especially by pollution from the PVC For AO, BCF (wet weight basis) was high considering factory. Xenobiotic markers probably compete with its octanol/water partition coefficient: in Donax trun- environmental pollutants for binding sites within the culus from the relatively clean site BCF was 1118.7 in intracellular mucoproteins. Binding of xenobiotics with the mantle edge along the pallial line, 368.9 in the structures which contain mucoproteins is a known kidney, and 55.2 in the central part of the mantle. In defense mechanism against environmental pollutants contrast, for FLU, BCFs were very low, decreasing in flatworms and molluscs (Dindal & Wurzinger 197 1, from 0.08 in the hdney to 0.0018 in the central part of Triebskorn et al. 1991). This defense mechanism is well the mantle. Mactra corallinafrom the same clean site developed In D. trunculus, but it is undeveloped in had different BCF values; for AO, BCF was 531.8 in Mactra corallina. the kidney, 143.7 in the mantle edge, and 77.2 in the In other tissues of Donax trunculusA0 is accumu- central part of the mantle, and for FLU it was 0.14 in lated mostly by lysosomes and related structures, and the kidney and 0.03 in the central part of the mantle. such accumulation is also inhibited by environmental Consequently, the data for D. trunculus and M. coral- pollutants. Saito et al. (1991) and Dierickx & Van de lina suggest that BCF values for A0 and FLU are Vyver (1991) showed that accumulation of another species-specific. lysosomotropic marker, neutral red, was inhibited A non-ionized form of A0 penetrates through significantly by various anthropogenic xenobiotics in plasma membranes; in the cells A0 binds with some experiments in vitro. Lysosomal accumulation of xeno- proteins, especially mucoproteins, and nucleic acids, biotics is believed to defend the cells against some and is also accumulated by cell compartments with low pollutants (George 1984, Jenner et al. 1991). Using pH, such as lysosomes and related structures (Witte- A0 as a marker shows that this defense mechanism kind 1973, Swanson 1989, Darzynkiewicz & Kapus- is well developed in both D. trunculus and Mactra cinski 1990, Murphy 1990). As intralysosomal pH and, corallina. accordingly, lysosomal accumulation of cationic chem- Our data show that B(a)P and especially A0 and FLU icals depend on cellular energetic metabolism, each were concentrated by the molluscan kidney. Harrison cell injury will induce a proportional decrease in (1962) postulated the presence of active excretory the accumulation of lysosomotropic markers (Swanson processes for anionic xenobiotics in the kidney of the 1989, Dierickx & Van de Vyver 1991, Saito et al. 1991). rufiens.The kidney of other molluscs 200 Dis. aquat. Org.

can concentrate organic anions such as paraamino- This system of ecotoxicological analysis should be hippurate and phenol red (Harrison & Martin 1965, incorporated into studies of population stability exam- Martin et al. 1965, Martin & Harrison 1966). In verte- ining numerical changes in community structures and brates the renal proximal tubules, l~verand choroid determining intrinsic markers for organ~smalhealth. plexus, and Malpighian tubules, contain 2 transport systems: one for active elimination of organic acids and one for active elimination of organic bases (Weiner Acknowledgements. This work was supported by a grant from the Ministry of Sclence and Technology for early moni- 1973, Bresler et al. 1975, 1979, 1985, 1990, Maddrell toring of environmental pathology and genotoxicity In water 1980, Moller & Sheikh 1982). Similar defense systems ecosystems. The authors thank Dr E Sahar and M. Z. Zuk- may exist in the tubules of the molluscan kidney, and Rlmon for their contribution to this study and Mrs C. Shaplro in D. trunculus these systems were probably inhibited and MS N. Paz for typing and help in edlting the manuscript. by pollutants from the PVC factory. The contribution made by the anonymous referees is grate- fully acknowledged. Deposition of B(a)P and marker xenobiotics was also found in the periostracum and shell of Donax truncu- lus. Bias & Karbe (1985) found a loosely bound fraction LITERATURE CITED cf cadr?.i-m in the pericstracurr. cf the freshxilter =us- Blas, R., Karbe, L. (1985). Bioaccumulation and partitlonlng of sel Dreissena polymorpha. The mechanisms whereby cadmium within the freshwater , Dreissena poly- such xenobiotics are deposited into the shell have not rnorpha Pallas. Int. Rev. ges. Hydrobiol. 70. 113-115 been studied. Bowen, I. D. (1981).Techniques for demonstrating cell death. A sharp decrease in A0 accumulation in all soft In: Bowen, I. D., Lockshin, R. A. (eds.) Cell death in tissues of Donax trunculus after experimental thermal biology and pathology. Chapman and Hall, London, p. 379-444 stress demonstrated that all the above-mentioned mech- Bresler, S. E., Bresler, V. M,, Kazbekov, E. N., Niklforov, A. A., anisms of A0accumulation were dependent on cellular Vasilyeva, N. N. (1979).On the active transport of organlc metabolism and nativity of cell structures. acid (fluorescein) in the choroid plexus of the rabbit. BIO- It is axiomatic that the plasma membrane of intact chim. Biophys. Acta 550: 110-119 Bresler, V. M., Belyaeva, E. A., Mozhaeva, M. G. (1990). A living cells is inpermeable to water-soluble organic comparative study on the system of actlve transport of anions, including FLU. Therefore, determination of cell organic acids in Malplghian tubules of Insects. J. Insect permeability to anionic dyes is widely used to detect Physiol. 36: 259-270 cell injury or changes in cell viability (DuBois et al. Bresler, V. M,, Bresler, S. E., Nikiforov, A. A. (1975).Structure 1980, Bowen 1981, Pieters et al. 1989).Increase in FLU and active transport in the plasma membrane of the tubules of frog kldney. Biophys. Biochim. Acta, 406: accumulation in the mantle, gills and hepatopancreas 526-537 of Donax trunculus from all polluted sites reflects an Bresler, V. M,, Mozhaeva, M. G., Belyaeva, E. A. (1985). A increase in plasma membrane permeabihty, i.e. the comparative study of the system of active transport of presence of cell injury produced by environmental organlc acids in Malpighian tubules in the troplcal cock- roach, Blaberus giganteus. Compar. Biochem Physiol. pollutants. 80A: 393-397 We draw the following conclusions from our study: Bresler, V. M., Nikiforov, A. A. (1977). Double dependence of (1) Bioaccumulation and BCF values for B(a)P, A0 the active transport of organic acids into the proximal and FLU are compound-dependent, tissue-specific and tubules of surviving frog kidney on Na+. Biochlm. Bio- possibly species-specific. phys Acta 468. 81-89 Brodtman, N. V Jr (1970). Studies on the assinulation of (2) Under field conditions BCF values for B(a)P are l,l,l,l-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) by very low, and B(a)P in the tissues of Donax trunculus Crassostrea v~rginlca Gmelln. Bull. envlron. Contam. from polluted sites is accumulated mainly in the Toxicol. 5. 455-462 hepatopancreas, hdney and shell. Couch, J. A., Courtney, L. A., Wlnstead, J. T., Foss, S. S. (1979). The American (Crassostrea virginica) as an (3) Under experimental conditions the level of A0 indicator of carcinogens in the aquahc environments. In: and FLU accumulation in the tissues of Donax truncu- Animals as monitors of environmental pollutions Nahonal lus depends on the site of collection, i.e, environmental Academy of Sciences, Washington, DC, p. 65-84 conditions, and probably reflects the action of pollu- Darzynkiewicz, Z., Kapuscinski, J. (1990). Acridine orange: a tants on cell viability. versatile probe of nucleic aclds and other cell constituents. In: Melamed, M M,,Lindmo, T , Mendelson, M (eds ) Flow (4) The ludney of Donax trunculus and Mactra coral- cytometry and sorting. Wiley-Liss, New York, p 291-314 lina may contain at least 2 separate transport systems Dierickx, P. J., Van de Vyver, I. E. (1991). Correlation of the to eliminate anionic and cationic xenobiotics. neutral red uptake lnhibltlon assay of cultured fathead (5) Donax trunculus are able to depos~tmost of the minnow fish cells with fish lethality tests. Bull. envlron. Contam. Toxicol. 46. 649-653 accumulated A0 into large bodies in the mantle edge Dindal, D L, Wurzinger, K.-H. (1971). Accumulation and along the pahal line, as well as B(a)P, A0 and FLU into excretion of DDT by the terrestnal snad, Cepaea hor- their shells. tensis. Bull. envlron. Contam. Toxicol 6: 362-371 Bresler & Fishelson: Bioaccumulation c ~fbenzo(a)pyrene and xenobiotic markers 201

DuBols, M,, Bowman, P,, Meck, R.,Daniel, C. (1980).Stimula- Academic Press, London, p. 353-386 tion of protein synthesis in cytochalasin B-enucleated Martin. A. W., Steward. D. M,, Harrison, F. M. (1965). Urine human fibroblasts by dexamethasone and insulin. Exp. formation in a pulmonate land , Achatina fulica. Cell Res. 125: 363-367 J exp. Biol. 42: 99-123 Elder, J F., Dressler, R. V. (1988). Accumulation and bio- Moller. J. V., Sheikh, M. 1. (1982). Renal organic anion trans- concentration of polycyclic aromatic hydrocarbons in a port system: pharmacological, physiological and bio- nearshore estuarine environment near a Pensacola chemical aspects. Pharmacol. Rev. 34: 315-358 (Florida) creosote contamination site. Environ. Pollut. 49- Moore, M. N. (1976). Cytochemical demonstration of latency 117-132 of lysosomal hydrolases in digestive cells of the common Eriksson, J., Meriluoto, J., Lindholm, T (1986). Can cyano- mussel, A4ytilus edulis, and changes induced by thermal bacteria peptide toxins accumulate in aquatic ? stress. Cell Tissue Res. 175: 279-287 In. Megusar, F., Gautar. M. (eds.)Perspectives in microbial Moore, M. N. (1988). Cytochemical responses of the lyso- ecology. Proc. Fourth Int. Symp. Microbial Ecology. somal system and NADP H-fernhemoprotein reductase in Ljubljana, 24-29 August 1986. Slovene Soc. Microbiol., molluscan digestive cells to environmental and experi- Ljublyana, p 655-658 mental exposure to xenobiotlcs. Mar. Ecol. Prog. Ser. 46. Eriksson, J., Meriluoto, J., Lindholm, T (1989). Accumula- 81-89 tion of a peptide toxin from cyanobacterium Oscillatoria Murphy, R. (1990). Ligand binding. endocytosis, and process- agardhi in the freshwater mussel Anodonta cygnea. ing. In: Melamed, M., Lindmo, T., Mendelson, M. (eds.] Hydrobiologia 183: 211-216 Flow cytometry and sorting. Wiley-Liss, New York, George. S. G. (1984). Intracellular control of Cd concentra- p. 355-366 tions in marine mussels. Mar. environ. Res. 14: 465-468 Neff, J. M,, Anderson, J. (1975). Accumulation, release and Goldberg, M. C. (ed.) (1989). Luminescence applications in distribution of benzo(a)pyrene-C14 in the clam Rangja biological, chemical, environmental and hydrological cuneata. Proc. Conf, Prevention and Control of Oil sciences. ACS Symposium Series 383. American Chemical Pollution. Am. Petroleum Institute, U.S. EPA, U.S. Coast Society, Washington, DC Guard, Washington, DC, p. 469-471 Harborne, J. B. (1982). Introduction to ecological blochem- Neff, J. M,, Cox, B. A., Dixit, D., Anderson, J. (1976). Accu- istry. Academic Press, London mulation and release of petroleum-derived aromatlc Harrison, F. M. (1962). Some excretory processes in the hydrocarbons by four species of marine animals. Mar. Biol. abalone, Hallotjs rufescens. J. exp. Biol. 39: 179-192 38: 279-289 Harrison. F. M., Martin, A. W. (1965). Excretion in the Niimi, A. J., Lee, H. B. (1992). Free and conjugated concen- dofleini. J. exp. Biol. 42: 71-98 trations of nine resin acids in rainbow trout (Oncorhynchus Heit, M., Klusek, C. S., Miller, K. M. (1980) Trace elements, mykiss) following waterborne exposure. Environ. Toxicol. radionuclide, and polycyclic aromatic hydrocarbon con- Chem. 11. 1403-1407 centration in mussel from Northern Lake Oldand. J. (1992). Effects of acidic water on freshwater snail. George. Environ. Sci. Technol. 14: 465-468 results from a study of 1000 lakes throughout Norway. Hornung, H., Oren, 0. H. (1981). Heavy metal in Donax trun- Envlron. Pollut. 78: 127-130 culus L. () in Haifa Bay, Mediterranean (Israel). Paaslvara, J., Rantalainen, A.-L , Welling, L., Herve, S., Mar. environ. Res. 4: 195-201 Heinonen, P. (1992). Organochlorines as environmental Hurtubise, R. J. (1981). Solid surface luminescence analysis. tainting substances: taste panel study and chemical analy- Marsel Dekker, New York sis of incubated mussels. Water Sci. Technol. 25 105-113 Hurtubise, R. J., Ramasamy, S. M., Bello, J. M., Burrell, G. J., Perdue, E. M., Gjessing, E. T. (eds.) (1990) Organic acids in Citta, L. A. (1989). Applications and interactions in solid- aquatic ecosystems. Wiley & Sons. Chichester surface luminescence analysis. In: Goldberg, M. C. (ed.) Pieters, R., Huismans, D, Leyva, A., Veerman, A. (1989). Luminescence applications in biological, chemical, envi- Comparison of the rapid automated MTT-assay with a dye ronmental and hydrological sciences. American Chemical exclusion assay for chemosens~tivity testing in childhood Society, Washington, DC, p. 155-166 leukaemia. Br. J. Cancer 59: 217-220 Jenner, H. A., Hemelraad, J., Marquenie, I. M., Noppert, F. Roberts, D. (1976). Mussel and pollution. In: Bayne, B. L. (ed ) (1991). Cadmium kinetics in freshwater (Unionidae) Marine mussels: the11 ecology and physiology. Cambridge under field and laboratory conditions. Sci. total Environ. Univ. Press, Cambridge 108: 205-214 Roesijadi, G., Anderson, J. W., Blaylock, J. W. (1978). Uptake Kauss, P B., Hamdy, Y S (1991). Polycyclic aromatic hydro- of hydrocarbons from marine sediments contaminated carbons in superficial sediments and caged mussels of with Prudhoe Bay crude oil: influence of feeding type, of the St. Mary's River, 1985. Hydrobiologia 219: 37-62 test species and availability of polycyclic aromatic hydro- Krahn, M. M., Burrows, D. G., MacLeod, W. D., Malins, D. C. carbons. J. Fish Res. Bd Can. 35: 608-614 (1987). Determination of individual metabolites of aro- Romeo, M., Gnassia-Barelli, M. (1988). Donax trunculus and matic compounds in hydrolized bile of English sole Venus verrucosa as bioindicators of trace metal concentra- (Parophrys vetulus) from polluted sites in Puget Sound, tions in Mauritanian coastal water. Mar. Biol. 99: 223-227 Washington. Arch. environ. Contam. Toxicol. 16: 511-522 Saito, H., Iwam~,S., Shigeoka T. (1991). In vjtro cytotoxicity of Lowe, D. M. (1988). Alterations in cellular structure of Mytilus 45 pesticides to goldfish GF-scale (GFS) cells. Chemo- edulis resulting from exposure to environmental contami- sphere, 23: 525-537 nants under field and experimental conditions. Mar. Ecol. Sernetz, M,, Thaer, A. A. (1973). Microcapillary fluorometry Prog. Ser. 46: 91-100 and standardization for microscope fluorometry. In: Thaer, Maddrell. S. H. P. (1980). Characteristics of epithelia1 trans- A. A., Sernetz, M. (eds.) Fluorescence techniques in cell port in insect Malpighian tubules. Curr. Top. Membr. biology. Springer-Verlag, Berlin. p. 41-49 Transp. 14: 427-463 Swanson, J. (1989). Fluorescent labeling of endocytic com- Martin, A. W., Harrison, F. M. (1966). Excretion. In: Wilbur, partments. In: Taylor, D. L., Wang, Y.-L. (eds.) Fluorescent K. M-, Yonge. C. M. (eds.) Physiology of . Vol. 2: microscopy of living cells in culture. Methods in cell 202 Dis. aquat. Org. 19: 193-202, 1994

biology, Vol. 29. Academic Press, San Diego, p. 137-152 Wittekind, D. (1973). Fluorescence properties of the mono- Taylor, D. L., Wang, Y.-L. (eds.) (1989). Fluorescent micro- aminoacridines and some 2-aminoacridine-derivatives. In: scopy of living cells in culture. Methods in cell biology, Thaer, A. A., Sernetz, M. (eds.) Fluorescence techniques Vol. 29. Academic Press, San Diego in cell biology. Springer-Verlag, Berlin, p. 95-105 Triebskorn, R.. Kohler. H-R., Zahn, T., Vogt, G., Ludwig, M., Yawetz, A., Manelis, R., Fishelson, L. (1992). The effects of Rumpf, S., Kratzrnann, M,, Alberti, G., Storch, V. (1991). Aroclor 1254 and petrochemical pollutants on cytochrome Invertebrate cells as targets for hazardous substances. Z. P450 from digestive gland microsomes of four species of angew. Zool. 78: 277-287 Mediterranean molluscs. Comp. Biochem. Physiol. 103C: Weiner, I. M. (1973). Transport of weak aclds and bases. In: 607-614 Weiner, I. M. (ed).Handbook of physiology, Sect. 8, Renal Zanker, V., Peter, W. (1958). Die prototropen Formen des physiology. Plenum Press, Washington, p. 521-554 Fluoresceins. Chem. Ber. 91: 572-580

Responsible Subject Editor: A. K. Sparks, Seattle, Manuscript first received: July 13, 1993 Washington, USA Revised version accepted: May 5, 1994