Induction of micronuclei in gill tissue of Mytilus galloprovincialis exposed to polluted marine waters

Marine Pollution Bulletin Marine Pollution Bulletin, Volume 2 I, No. 2, pp. 74-80. 1990. Printed in Great Britain.

I)O25-326X/90 $3.00+0.00 © 1990 Pergamon Press plc

Induction of Micronuclei in Gill Tissue of Mytilus galloprovincialis Exposed to Polluted Marine Waters R. SCARPATO*, L. MIGLIORE*, G. ALFINITO-COGNEq"-FI t and R. BARALE* *Dipartimento di Scienze dell'Ambiente e del Territorio, Universitd di Pisa, Via S. Giuseppe 22, Pisa; t Dipartimento di Biornedicina Sperimentale Infettiva e Pubblica, Universitd di Pisa, Via Volta 4, Pisa; *Istituto di Zoologia, Universit~ di Ferrara, Via Borsari 46, Ferrara, Italy

The micronucleus assay in gill tissue of the mussel Mytilus galloprovincialis has been developed in our Laboratory to assess the mutagenic activity of compounds present in marine environments. The sensitivity of the test was assessed by performing mutagenic treatment for 48 h with the two standard compounds vincristine (VCR) (0.005, 0.01, 0.02 mg I-l), and benzo(a)pyrene (BaP) (0.025, 0.075, 0.225, 0.675 mg l-I). Since both tested chemicals produced significant increases in the number of micronucleated cells, animals were directly exposed to marine waters: mussels grown in clean water (control sample) were transferred to polluted areas and then collected weekly. Micronuclei frequencies of sampled mussels were significantly higher than the value of the control group.

Aquatic pollution results from different sources of contamination, principally due to human technological activities. Large quantities of chemicals are discharged daily into marine environments by industrial and domestic effluents. Although pollutants are usually present in water at relatively low concentrations, high levels of toxicity may nonetheless be reached in the aquatic ecosystem (Bascomb, 1982). Therefore, ecotoxicological studies have been developed to better evaluate the effects of chemical contamination on aquatic life and, consequently, their implications for human health. In addition, since many of these xenobiotics are known or suspected mutagens/carcinogens, mutagenicity tests have also been applied to aquatic toxicology (de Raat et al., 1985). In this respect, the possibility of using aquatic organisms as indicators of water pollution has also been considered (Phillips, 1978). At present, some of the most frequently studied animals are the mussels Mytilus edulis and Mytilus galloprovincialis. These organisms are easily able to accumulate and concentrate chemical pollutants, in particular heavy metals, pesticides and 74

polycyclic aromatic hydrocarbons (PAHs) in their tissues. High levels of mercury, copper, and zinc were detected in mussels from the Adriatic Sea (Mikac & Picer, 1985; Martincic et al., 1987a,b). Comparative determination of cadmium concentration was performed in M. edulis and M. galloprovincialis, respectively collected from Irish and Ligurian coasts (Nolan, 1985). Bioaccumulation of organochlorine compounds and PAHs by mussels was also observed in both laboratory experiments (exposure to diesel oil hydrocarbons, organochlorine insecticides, sediments, polychlorinated biphenyls) (Livingstone et al., 1985; Herranz-Santos & Ruiz-Amil, 1985; Pruell et al., 1986; Suteau et al., 1987), and in investigations of natural environments (Deival et al., 1986; Rainio et al., 1986; Shaw et al., 1986). In recent years, some authors have successfully employed aquatic organisms in cytogenetic studies, for the in vivo detection of chromosome aberrations (CAs), sister-chromatid exchanges (SCEs) or micronuclei (MNs) (Alink et aL, 1980; Pesch et al., 1981 ; Hooftman & de Raat, 1982; Siboulet et al., 1984; A1-Sabti, 1985; Das & Nanda, 1986; Gola et al., 1986). However, the micronucleus (MN) assay seems to be a fast and sensitive test since it is able to detect genomic damage due to both clastogenic effects and alterations of the mitotic spindle (Migliore et al., 1987). MNs have the appearance of intracytoplasmic chromatine masses resembling the main nucleus, and are easily observed in interphasic cells (Fig. 1). Therefore, to detect the presence of mutagenic activity in marine environments, we have developed the MN assay in gill tissue of M. galloprovincialis. In the present work, in order to assess the suitability and the sensitivity of the methodology, MNs induction in gill cells of mussels after treatment with the two chemical compounds vincristine (VCR) (Lilly) and benzo(a)pyrene (BaP) (Sigma) is reported. Moreover, the results of an in situ assay performed by transferring mussels from a different site to polluted areas are also reported.

Volume 21/Number 2/February 1990

Fig. 1 Micronucleated cells in gill tissue of Mytilus galloprovincialis.

Materials and methods Animals Individuals of Mytilus galloprovincialis (shell length about 4 cm) were collected from two different mussel farms in the La Spezia Gulf (Ligurian Sea), one at Palmaria Isle and the other near the inner side of the La Spezia port breakwater. Chemical treatment During the experiments, both control and treated samples were maintained at 18°C in aquaria containing 20 1. of synthetic sea water and fed with Liquifry N.1. Treatment of mussels was performed for a total of 48 h (two inputs of mutagens with water replacement every 24 h). VCR (0.005, 0.01, 0.02 mg 1-~) was directly added to the water, while BaP (0.025, 0.075, 0.225,

0.675 mg 1-~) was first dissolved in an appropriate amount of dimethylsulphoxide (DMSO). The DMSO maximum concentration in water was 0.075%0, well below the 0.25%o level which was found to produce no cytogenetic or toxic effects (Jaylet et aL, 1986a). In addition, the BaP control group was exposed to the same DMSO amount of treated samples. Five and six mussels were used respectively for each VCR and BaP concentration. A whole cell cycle is required for MNs formation and, since 48 h is a sufficient time for a significant proportion of gill cells to have completed one cell replication (Dixon et al., 1982), gill harvesting was 48 h after the end of treatment. In situ assay Because of their lower spontaneous MNs frequency, mussels from Palmaria Isle were then chosen as control 75

Marine Pollution Bulletin ~8

sample to perform an in situ assay. Several animals from this station were placed in two polluted areas of the Tuscan coast (the wet-dock of Livorno port and the Fiume Morto estuary), and collected weekly to compare their MNs frequencies to the basal level of the control sample assessed at the start of the experiment (time of collection from Palmaria Isle).

15--

1

12--

9

Slides preparation Gills were removed, enzymatically digested by a solution of dispase (Boehringer Mannheim) in modified (20%o) Hank's Balanced Salt Solution (1.5 mg ml -~) for 20 min. at 37°C and filtered to obtain a cellular suspension. After centrifugation at 1000 r.p.m, for 10 min., the pellet was fixed in Carnoy's solution for at least 20 min., dropped onto clean glass slides, and then stained in 3% Giemsa/distilled water. The majority of the cell population recovered is composed by the one cell type (large, roundish with a large nucleus), accompanied by a minor second cell type easily distinguishable for its smaller size and the darker nucleus. A third cell type, the fusiform obtainable with other tissue disaggregation methods previously used by us, is not recovered. We performed cytogenetic analysis only on the first homogeneous cell population. 1000 cells per slide (2000 cells per animal) with preserved cytoplasm were scored for MNs frequency under an oil-immersion objective (× 1000 final magnification); MNs identification was based on the criteria proposed by Countryman & Heddle (1976).

6--

3 -!I'

o o

I 0.005

i 0.015

001

0.02

Mg I-~

5°I / :~

4o-

\ 38

I

i i

20

I0

0

t 02

I 0.4

I 0.6

J 0.8

Mg I - '

Statistical analysis The data were statistically processed by regression analysis and one-way analysis of variance (ANOVA). In the latter case, in order to stabilize the variance of MNs frequencies, the angular transformation arcsin~/p was used. The statistical values reported in Tables 1 and 2 were obtained by comparing each experimental point to the respective control, and by comparing between them the two controls in Table 1 and the five last samplings at Fiume Morto estuary in Table 2. In addition, on the basis of regression analysis, a coefficient of mutagenic activity was calculated in the following way: Mutagenic activity (MA)--

Increment per Xm (Ym--Yo) Intercept -(Yo)

where x m is the arithmetic mean of independent variable, Ym is the corresponding value and Yo is the value for x = 0. Besides, for a better assessment of MA appropriate data transformations were required.

Fig. 2 Induction of MNs in gills of Mytilus galloprovincialis treated with (a) VCR (0.005, 0.01, 0.02 mg 1-~), (b) BaP (0.025, 0.075, 0.225, 0.675 mg 1-'). The points are the mean of 5 and 6 mussels in (a) and (b) respectively. 2000 cells per animal were scored. TABLE 1 Frequencies of MNs in the gills of Mytilusgalloprovincialistreated with two chemical mutagens. MNs values are the mean of 5 mussels for VCR and 6 mussels for BaP. Dose (mg 1-1)

No. of MNs (mean ± SD)

F value*

Significance (probability)

VCR

0.005 0.01 0.02

5.70:1:2.39 11.00±2.24 13.75±3.09

5.45 55,80 67,98

<0.05 <0.001 <0.001

BaP

0.025 0.075 0.225 0.675

24.75 ± 9.35 37.17±6.97 30.83 ± 9.67 25.17±6.88

34,64 156,50 45,44 58,47

<0.001 <0.001 <0.001 <0.001

16.25

<0.005

Mutagen

VCR control

2.90± 1.24 1

BaP control

6.75 ± 1.97

J

Results

*Data were statistically processed by A N O V A using the arcsin~/p transformation.

The results of mutagenic treatments are given in Fig. 2 and Table 1. Data show that both VCR and BaP produce an increase in the frequency of micronucleated cells in treated animals: the average values of all tested groups are significantly higher than the mean of the control sample (2.90+1.24%o for VCR and 6.75 + 1.97%o for BaP). VCR and BaP maximum MNs frequencies were respectively obtained with a concentration of 0.02 mg 1-I (13.75 + 3.09%0) and 0.075 mg 1-1

(37.17 + 6.97%0), while at the two highest BaP doses a reduction in the mean level of micronuclei was detected (30.83 + 9.67%0 at 0.225 mg 1-' and 25.17 + 6.88%0 at 0.675 mg l-J). This decrease may be due to the strong clastogenic effect of the mutagen which would delay or stop the division of most damaged cells. Moreover, MNs frequencies have also shown a dependence on VCR and BaP concentrations. A better linear relationship was calculated, for VCR, by using the dose square

76

Volume 21/Number 2/February 1990

root and the MNs natural logarithm (t=7.056, p=0.01950), and, for BaP, by using the natural logarithm of both variables (t = 6.765, p = 0.00660) (Fig. 3). Table 3 reports the regression equations (expressed by untransformed values) and the relative MA coefficients (BaP MA---- 3.315, VCR MA = 1.954). These data seem to suggest a more marked efficiency of BaP in inducing micronucleated cells. Figures 4, 6 and Table 2 show the results of the weekly samplings performed at Livorno port and the Fiume Morto estuary. At Livorno port it was not possible to continue the experiment after the third week. At the Fiume Morto estuary, mussels were sampled up to the fourth week, then at 12th, and following this, from the 15th to the 18th week of exposure. Except in the case of the first collection, the MNs mean values were always higher than the basal level of the control sample (i:2.2+0.91%o); already from the second week, MNs frequencies of mussels from both areas, as a function of time, began to rise significantly. At the Flume Morto estuary these increases were

TABLE 2 Frequencies of MNs in the gills of M. galloprovincialis exposed to polluted areas. MNs values are the mean of 3 mussels, except for the control (5 mussels).

Weeks of No. of MNs exposure (mean 5: SD)

Site Fiume Motto estuary

3.00 5:1.00 6.175:1.61 9.505:2.00 9.005:3.46 5.505:1.00 4.335:1.04 6.83 :t: 1.89 5.665:1.44 7.66 5:1.52

control

2.205:0.91

1 2 3

Livorno port

1.34 t9.32 48.03 21.46 17.99 8.28 22.72 15.65 34.33

2.505:1.00 7.175:3.88 9.50+2.00

control

0.20 10.74 48.03

N.S.

2.55

<0.005 <0.001 <0.005 <0.01 <0.05 <0.005 N.S. <0.01 <0.005

N.S. <0.05 <0.001

2.20:t:0.91

*Data were statistically processed by ANOVA using the arcsin~/p transformation.

3.7

2.8

(Q)

b

1 2 3 4 12 ] 15 ] 16 17 18

Significance (probability)

F value*

(b)

25

34

2.2

3II

1.9

218

L6

2.5 /

1.3

y= 1.05_+ I 1.58X

/ 0/

-I 0

2.2

t = 7.056 p = 0.01950

', 0.05

I 006

J 009

E 0.12

I 015

119

-230

I -190

I -150

Mg 1-=

I -I10

i -70

; -50

I io

Mg I - I

Fig. 3 Linear regression ( y = a + b x ) for the (a) VCR and (b) BaP transformed data (as described in the text). The t value gives the significance of the angular coefficient.

12

15 (a)

(b) I

12

i

I

8

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6

!/

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9

6

I0

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4

o

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2

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0 Weeks

2 of exposure

:5

0 Weeks

2 of exposure

Fig. 4 Induction of MNs in the gills of Mytilus galloprovincialis after (a) 3 weeks of exposure to the water in Livorno port, (b) 4 weeks of exposure to water in the Fiume Morro estuary. The points are the mean of 3 mussels, except for the week 0 (5 mussels). 2000 cells per animal were scored.

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Marine Pollution Bulletin

detected up to the 18th week. The top mean value in the two groups was observed at the third week (9.50+2.00%o for both), but the following sampling already showed a decline in MNs frequency, and from the 12th to 18th week the mean values ranged between 4.33+ 1.04%o in the 15th and 7.66+ 1.52%o in the 18th week. In Table 3 the values of the regression analysis relative to the first month of exposure are reported for both sites. The equations indicate, as shown in Fig. 5, that the best significant linear relationships were obtained between the square of both variables, and between the MNs square root and time, for Livorno port (t= 8.773, p=0.01274) and Fiume Morto estuary (t=5.539, p = 0.01159) respectively. Furthermore, the comparison of MA values indicates a weak prevalence of Livorno port (Livorno port MA = 1.825, Fiume Motto estuary M A = 1.543), but it is still more interesting to observe that these values represent the 55.1 and 46.5% of BaP MA, and the 93.4 and 79.0% ofVCR MA.

Discussion VCR is leading to cytes MN mutagens

a typical non-disjunctional direct mutagen positive results in in vitro human lymphoassay (Migliore et aL, 1987). Other direct were tested on mussels by cytogenetic

32

-

methods and the compounds used were generally found to give positive results. In fact, significant increases of micronuclei were detected in gill cells of M. galloprovincialis exposed for 48 h both to different concentrations of heavy metals previously solubilized in nitrilotriacetic acid trisodium salt (Gola et al., 1986), and to mitomycin C (MMC) (0.5× 10 7, 10-7 M ) (Majone et aL, 1987). Also the frequency of SCEs in gills of M. edulis (Dixon et al., 1982) and in developing eggs of M. galloprovincialis (Brunetti et al., 1986) was increased by treatment with MMC doses ranging from 6 × 10 - 9 tO 6 x 10-6 M and mercury chloride (0.03 mg 1-~) respectively. BaP is an important component in the class of PAHs, and its environmental impact on the aquatic ecosystem has been discussed above. This compound is converted to several mutagenic and carcinogenic metabolites by cytochrome P-450 enzymes and the epoxide hydrase function (Jones et aL, 1979). The clastogenic effects of BaP detected by cytogenetic methods have been widely shown in aquatic organisms, but only for vertebrate systems. Induction of CAs after treatment for 4 and 6 days with BaP 10 mg 1-1 was observed in gill tissue of the fish Notobranchius rachowi (Hooftman, 1981). In particular, high levels of MNs were detected in circulating erythrocytes of two amphibians, the newt Pleurodeles

IOO

(b)

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80

2.6

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7: 0

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x

t =8.775 p = 0 01274

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Weeks of exposure

Fig. 5 Linear regression ( y = a + b x ) for the (a) Fiume Morro estuary and (b) Livorno port transformed data (as described in the text). The t value gives the significance of the angular coefficient.

TABLE 3 Regression analysis for the data reported in Figs 2, 4. Regression

Equation

Intercept

Slope

Vincristine (MN vs dose)

y = e (~+b,'~)

1.05

11.58

Benzo(a)pyrene (MN vs dose)

y = e a xb

3.38

Livorno port (MN vs time)

y - . ] a + bx'-

Fiume Morto (MN vs time)

y = (a+bx) 2

e~#- - 1

M A = 1.954

0.0065

(~-xb/~ ~,

M A = 3.315

3.22

9.99

(.~a+ bx~)/,/a)- 1

MA ~ 1.825

1.48

0.44

[bx.,)bxm+ 2a)]/a"

MA = 1.543

*The first column gives the expression to calculate the MA.

78

Mutagenic activity*

Volume 21/Number 2/February 1990

tC

T

8

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2, flii!!!!iliiiii!!iiii:! :i~!:i:!:i:ii:!!!i:i}!!!::! 12

15

16

17

18

Weeks of exposure

Fig. 6 Variation of MNs frequency during the in situ assay at the Fiume Morto estuary. Bars represent the values of Table 2 relative to each of the last five sampling performed weekly.

walt and the axolotl Ambystoma mexicanum, both exposed for various times to different concentrations of BaP (Grinfeld et al., i986; Jaylet et al., 1986). Concerning the ability of mussels to produce mutagenic metabolites of BaR our data are confirmed by the evidence that in many bivalves the microsomal mixedfunction oxygenase (MFO) and other related enzymes are present. BaP hydroxylase functions and the MFO were detected in Mytilus edulis, and the maximum of activity was especially found in the digestive gland (Livingstone & Farrar, 1984b). Metabolites of BaP produced by the cytochrome P-450 monooxygenase and the epoxide hydrase enzyme were also identified in Mytilus edulis (Stegeman, 1985). In addition, high levels of cytochrome P-450 were detected in mussels exposed for different times to diesel oil (Livingstone et al., 1985; Livingstone, 1987). Therefore, the induction of the MFO in marine mussels seems to occur particularly in response to the presence of BaP or other PAHs in water. In this respect, extracts of mussels exposed in natural environment to oil pollution have given positive results by the Salmonella typhimurium fluctuation test (TA 98 and TA 100 strains) without S9-Mix. This suggests that the conversion of pre-mutagens generally present in crude oil to direct agent is a consequence of animal metabolism (Khadim & Parry, 1984). On the other hand, the inability of the postmitochondrial fraction of the Mytilus galloprovincialis digestive gland treated with BaP to produce mutagenic metabolites in the Ames test (TA 98 strain) (Britvic & Kurelec, 1986) might be explained as a lack of in vivo induction of the BaP metabolism, rather than an effective absence of cytochrome P-450 enzymes in the marine mussel. Extracts of mussels from a mutagenically clean site placed in a polluted area, and then collected after 1 month of exposure, were found to give positive results in different bacterial and yeast mutagenicity assays (Parry etal., 1980). The delayed appearance of a significant increase in the number of micronucleated cells osberved here for both sites at the first weekly sampling, may be explained in terms of necessary time for mussels to bioaccumulate chemical pollutants until the mutagenically active threshold-concentration and statistically evident genetic

damage have been reached. The decrease of MNs frequencies detected after the 4th week is probably due both to system saturation, which may have attained the maximum level of sensitivity to that water pollution degree after 1 month of exposure, and to the dilution consequent on the replication of gill cells. Nevertheless, the presence of organic compounds particularly toxic towards tissue proliferation may be excluded since the continuous formation of MNs is an index of cellular division. On the other hand, the differences detected between the MNs mean values of the five samples collected from the 12th to 18th week were not found to be statistically significant by ANOVA test (F value: 2.55). For this reason, it is unlikely that these fluctuations reflect qualitative/quantitative temporary variations in water pollutants, or a dependence on exposure time; they are more probably the consequence of individual variability. Therefore, our data seem to indicate that mussels placed in polluted marine areas, and then collected after relatively short times (3-4 weeks), increase their MNs basal level as a function of time. Furthermore, it is also possible that during long-term exposures to polluted environments, mussels may reduce their MNs frequencies to a lower level than the maximum expected value, but to a level, however, generally higher than the original spontaneous frequency. In this respect, it was found that the MNs levels in mussels treated for 48 h with mitomycin C (MMC) did not return to the control value, at least up to the 51st day after the treatment (Majone etal., 1987). Nevertheless, according to these results, the significant difference (P < 0.005) found between the control groups of chemically treated mussels (see Table 1) may be explained as a consequence of the environmental characteristics of the two collection areas: the first station, located in open-sea, is probably less polluted than the second which is enclosed between the dockyards and the military port, and thus may be more directly subjected to the effects of both harbour activities and industrial effluents.

Conclusions This paper confirms the previous evidence of positivity of the methodology towards direct mutagens and also shows the ability of mussels to detect genetic damage produced by indirect agents such as BaP; therefore the MN test in gill tissue of Mytilus galloprovincialis appears to be a suitable system for the assessment of mutagenic activity in marine environments. In addition, an in situ assay was proposed by using, in relatively short-term exposures, mussels from a different location rather than by sampling natural populations from the selected areas. In this respect, the major advantages which derive from this procedure are: L monitoring of marine sites in which mussels might not spontaneously grow, as occurs, for example, in the wetdock of Livorno port; 2. avoiding the possibility that animals, subjected to long-term exposure, may reduce MNs frequencies in spite of the continuous presence of genotoxic compounds in their environment; and 3. 79

Marine Pollution Bulletin

using only one group of mussels for the monitoring of several areas; in particular, it allows a better comparison of mutagenic activity in many different environments since it is detected by employing animals from the same reference site. Alink, G. M., Frederix-Wolters, E. M. H., Van der Gaag, M. A., Van de Kerkhoff, J. F. J. & Poels, C. L. M. (1980). Induction of sister-chromatid exchanges in fish exposed to Rhine water. Mut. Res. 78, 369374. AI-Sabti, K. (1985). Carcinogenic-mutagenic chemicals induced chromosomal aberrations in the kidney cells of three cyprinidis. Comp. Biochem. Physiol. C. 82,489-493. Bascomb, W. (1982). The effects of waste disposal on the coastal waters of Southern California. Environ. Sci. Technol. 16,226-235. Britvic, S. & Kurelec, B. (1986). Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine mussel Mytilus galloprovincialis. Comp. Biochem. Physiol. C. 85, 111-114. Brunetti, R., Gola, I. & Majone, E (1986). Sister-chromatid exchange in developing eggs of Mytilus galloprovincialis Lmk. (Bivalvia). Mur Res. 174,207-211. Countryman, P, I. & Heddle, J. A. (1976). The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mur Res. 41,321-332. Das, R. K. & Nanda, N. K. (1986). Induction of micronuclei in peripheral erythrocytes of fish Heteropneustes fossilis by mitomycin C and paper mill effluent. Mur Res. 175, 67-71. Deival, C., Fournier, S. & Vigneault, Y. (1986). Polychlorinated biphenyl residues in some marine organisms from the Bale des Anglais (Bale Comeau, Quebec, Saint-Lawrence Estuary). Bull. Environ. Contain. Toxicol. 37,823-829. De Raat, W. K., Haustveit, A. O. & de Krenk, J. E (1985). The role of mutagenicity testing in the ecotoxicological evaluation of industrial discharges into the aquatic environment. Fd. Chem. Toxic. 23, 33-41. Dixon, D., Kadim, M. & Parry, J. M. (1982). The detection of mutagens in the marine environment using the mussel Mytilus edulis. In Mutagens in Our Environment (Vainio & Sorsa, eds), pp. 297-312. Alan R. Liss, New York. Gola, I., Brunetti, R., Maione, F. & Levis, A. G. (1986). Applications of the micronucleus test to a marine organism treated with NTA and insoluble heavy metals. AttiAG132, 95-96. Grinfeld, S., Jaylet, A., Siboulet, R., Deparis, P, & Chouroulinkov, I. (1986). Micronuclei in red blood cells of the newt Pleurodeles waltl after treatment with benzo(a)pyrene: dependence on dose, length of exposure, post-treatment time, and uptake of the drug. Environ. Mutagenesis 8, 41-51. Herranz-Santos, M. J. & Ruiz-Amil, M. (1985). Pyruvate kinase of mussel (Mytih~s edulis L.) foot. Influence of organochlorine insecticides. Cornp. Biochem. PhysioL C. 81,375-377. Hooftman, R. N. (1981). The induction of chromosome aberrations in Notobranchius rachowi (Pisces: cyprinodontidae) after treatment with ethyl methanesulphonate or benzo(a)pyrene. Mut. Res. 91,347-352. Hooftman, R. N. & de Raat, W. K. (1982). Induction of nuclear anomalies (micronuclei) in the peripheral blood erythrocytes of the eastern mudminnow Umbra pygmae by ethyl methanesulphonate. Mut. Res. 10,1, 147-152. Jaylet, A., Deparis, P., Ferrier, V., Grinfeld, S. & Siboulet, R. (1986a). A new micronucleus test using peripheral blood erythrocytes of the newt Pleurodeles waltl to detect mutagens in fresh-water pollution. Mut. Res. 164, 245-257. Jaylet, A., Deparis, P. & Gaschignard, D. (1986b). Induction of micronuclei in peripheral erythrocytes of axolotl larvae following in vivo exposure to mutagenic agents. Mutagenesis 1, 211-215. Jones, C. A., Moore, B. P., Cohen, G. M. & Bridges, J. W. (1979). Metabolism of benzo(a)pyrene to oxidative and conjugative metabolites

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