The freshwater mussel (Anodonta anatina) in monitoring of 2,4,6-trichlorophenol: Behaviour and environmental variation considered

Chemosphere, Vol. 32, No. 2, pp. 391--403, 1996

Pergamon

0045-6535(95)00333-9

Copyright © 1996 Elsevier Science Ltd Printed in Great Britain, All rights reserved 0045-6535/96 $15.00+0.00

THE FRESHWATER MUSSEL (Anodonta anatina) 1N MONITORING OF 2,4,6-TRICHLOROPHENOL: BEHAVIOUR AND ENVIRONMENTAL VARIATION CONSIDERED

Viii P.M. Englund ~* and Mikko P. Heino 2

~Department of Biosciences, Division of Animal Physiology, P.O.Box 17, FIN-O0014, University of Helsinki 2Department of Ecology and Systematics, Division of Population Biology, P.O.Box 17, FIN-O0014, Universio, of Helsinki (Received in Germany 7 March 1995; accepted 27 August 1995)

ABSTRACT

The effects of 2,4,6-trichlorophenol, hurnic acid and food particles (Selenastrum capricornotum) on the valve movements of Anodonta anatina were studied under laboratory conditions using a factorial experimental design. None of the treatments affected the average valve openness or the number of valve movements. 2,4.6TCP affected the short-term variability of the valve openness, but in a way depending on the presence of humic acid and food particles. Humic acid increased the accumulation of 2,4,6-TCP, while the presence of food particles and the valve openness had no effect.

INTRODUCTION

Freshwater mussels are well suited for biomonitoring because they are reasonably sized, largely sessile, long-lived, widely distributed, available at large quantities and relative tolerant to xenobiotics. They are widely used in the monitoring of water quality (Green et al., 1989; Storey and Edward, 1989; Mtikelti and Oikari, 1990; Muncaster et al., 1990; Mtikelti et al., 1991). Different biomonitoring strategies include analyses of tissue levels of contaminants of native (Kinney et al., 1994) or transplanted mussels (Herve, 1991), or the use of valve movement responses as a biological early warning system (Kramer et al., 1989; Borcherding and Volpers, 1994). However, the use of mussels for biomonitoring presumes an understanding of the underlying biological phenomena, which, if not taken into account, may produce artefactual results. For example, the rate of accumulation and the amount of different xenobiotics vary between different mussel species (Hemelraad, 1988; Becker et al., 1992; Metcalfe-Smith, 1994; Roseman et al., 1994) or even between the sexes (MetcalfeSmith, 1994). Hinch and Green (1989) have shown that the source of the mussels can affect the uptake of

391

392 metals. Under natural conditions, Englund and Heino (1994a) have shown differences in valve openness between two unionid species. It is possible that differences in filtration rates between mussel species or mussels originating from different bodies of water may affect the accumulation of xenobiotics. Since we do not have enough detailed information about the behaviour of mussels and the accumulation of xenobiotics under various conditions, we need further studies to be able to evaluate the value of mussels as indicators of environmental perturbations.

Chlorophenols are found in sulphite pulp and paper industry effluents. Because they are lipophilic, they can readily accumulate in freshwater animals (Manahan, 1992). The freshwater mussel, Anodonta anatina (Linn6), is routinely used in monitoring chlorophenols in Finnish freshwaters, and 2,4,6-trichlorophenol is the main chlorophenolic compound found in mussels incubated near sulphite pulp mills (Herve, 1991). However, Finnish freshwaters display a wide variation in trophic state and in humic acid concentration. These factors may effect the accumulation rate and biodegradation of xenobiotics (e.g. Robinson and Novak, 1994). The purpose of this study was twofold: firstly, to discover whether valve movements, and the presence of humic acids or food particles affect the bioaccumulation of 2,4,6-trichloropbenol and secondly, to investigate whether 2,4,6trichlorophenol, humic acid, and food particles affect the valve movements of A. anatina.

MATERIAL AND METHODS

Animals

Freshwater mussels, A. anatina, were collected from Lake Kirmustenj/irvi in the winter of 1994 from a depth of two meters. Lake Kirmustenjiirvi is a mesohumic, eutrophic and unpolluted lake situated in southern Finland. The shell length of the mussels was, on average, 89 mm. The animals were stored in an aquarium at +4"C with constantly flowing active carbon filtered tap water (chemical composition is given in Table 1). The mussels were not fed during storage.

Table 1. Chemical composition of the experimental water. colour, mg Pt/l conductivity, mS/m pH, range alkalinity, retool/1 bicarbonate, mg/1 (mmol/l) hardness, °dH nitrate, NO;, mg/l (mol/l)

0 13.9 8.0-8.2 0.64 38.8 (0.63) 3.0 0.28 (0.0004)

sulphate, SO42-, mg/l (mmol/1) chloride, mg/l (retool/l) calcium, mg/l (mmol/l) magnesium, mg/l (retool/l) potassium, mg/l (mmol/1) sodium, mg/1 (mmol/l) aluminium, mg/l (retool/l)

7.5 (0.07) 6.4 (0.18) 17.1 (0.42) 1.6 (0.07) 1.4 (0.03) 5.2 (0.22) 0.06 (0.003)

393

Experimental set-up and statistical analyses

A 23 factorial experimental design with two replicates was used. The treatments were 2,4,6-TCP, humic acid and food particles (described in detail below). At the end of the experiment, the animals in each treatment with 2,4,6-TCP in water were divided into three groups according to their weighted valve openness. Thus, this grouping represents a nested effect.

Analysis of variance (ANOVA) was applied to evaluate the effects of 2,4,6-TCP, humic acid, and food particles on the valve movements, i.e. the average valve openness, the activity score, and the autocorrelation coefficients (lag 1) of the time series, consisting of 30 min averages of the aforementioned variables. Autocorrelation coefficients describe" the interdependency of successive observations in the time series. A nested ANOVA was used to analyse the effects of humic acid and food particles (main effects) and the weighted valve openness (nested effect) on the 2,4,6-TCP accumulation. Logarithmic transformations of 2,4,6-TCP concentrations and the activity scores were needed to ensure homoscedasticity and normality of residuals. The proportion of dead mussels in treatments was analysed using a generalised linear model with logit link function and binomial error structure.

Exposures

The treatments were: 2,4,6- trichlorophenol (TCP) (Fluka 91340), humic acid (Aldrich-Chemie H1,675-2), and unicellular green algae as food particles (Selenastrura capricornutum). The TCP concentration in the water was 20.0 ~g/1 ( + 3 #g/l). The TCP was dissolved in a small amount of I N NaOH before being added to the water of the aquaria. The humic acid concentration was 100 mg Pt/l as colour (TOC about 12 mg/l), a median humic acid concentration of Finnish lakes (Kortelainen, 1993). At the beginning of the experimem, algal suspension was added to aquaria to a concentration of 109 cells/l; an amount which, according to Sprung and Rose (1988) and Mersch et al. (1993) should cause a response of altering valve movements. An amount equalling l0 s cells/l of suspension was added daily to the aquaria.

Mussels were acclimated to the experimental temperature ( + 18 ° C) gradually over a period of 5-7 days. Twelve randomly selected mussels were placed into each experimental group. Four experimental groups were exposed simultaneously in steel aquaria (47 x 47 x 75 cm) containing 100-L aerated active carbon filtered tap water. The exposures, lasting 10 days, were run in four batches. The treatments were randomly allocated to test aquaria in both space and time. Constant photoperiod (L12:D12) was used with dim light. In all exposures water pH ranged between 8.0 and 8.2.

394 Each mussel was put into a small glass vial ( 0 100 mm) containing washed sand (grain size 0.8-1.2 mm), they it could maintain its natural upright position. Valve movements were recorded using the digital monitoring system described by Englund et al. (1994), which measures three relative states of the valves: closed (siphons not visible), open (siphons fully extended), and the half-open position. The weighted valve openness describes the relative openness of the valves over one hour, with time in the half-open position weighted by one half relative to the time when the valves were fully open. The weighted valve openness varied between 0 and 100%. The activity score equals the number of valve movements per hour.

At the end of the exposures, the soft tissues were dissected, wrapped in aluminium foil, and frozen in liquid nitrogen. The frozen tissues were stored at -20 ° C for later analysis. The soft parts of each mussel were weighed and homogenised using an Ultra Turrax homogenizer. The animals in each aquarium were ranked according to their weighted valve openness. The homogenates from the three animals with the highest valve openness were pooled into one sample, and the homogenates from the three animals with the lowest valve openness were pooled into another. Remaining animals ("moderately open") were pooled into the third homogenate. Due to mortality, this pooled homogenate resulted from three to six animals. The homogenates were stored at -20 ° C.

Chemical analysis

The homogenised tissues were freeze-dried, and measured amounts of 2,3,6-trichlorophenol were added as internal standard. The sample was extracted in a Soxhlet apparatus with petroleum-ether:acetone: hexane:ether for six hours. The solvent was evaporated and the residue weighed to give the fat content. The residue was dissolved in 0.1 M KCO3 and washed with hexane. Acetic anhydride was added to residue and the acetylated chlorophenols were extracted into the hexane phase. The f'mal determinations of 2,4,6-TCP were done using twin-column quartz capillary gas chromatography with EC detectors (stationary phases SE-54 and OV- 1701) at The Institute for Environment Research, University of Jyv~iskyl~i.

The 2,4,6-trichlorophenol (2,4,6-TCP) concentration in the water of the test aquaria was determined by gas chromatography (Hewlett-Packard 5890A). A 10 mL sample of aquarium water was acidified with H~.SO4. The 2,4,6-TCP was extracted into 50 mL of hexane (p.a. quality, Merck 4367) by shaking in a separatory funnel (five minutes). The hexane layer was removed, a further aliquot of 50 mL of hexane was added and the procedure repeated. The combined hexane phases were evaporated under an air stream to a volume of 2.0 mL. One ;zL of this hexane solution was injected into a gas chromatograph fitted with an HP 5 column (cross-linked 5 % phenyl silicone gum phase capillary column) and EC detector. The temperature program was 100°C initially, increasing at 10 ° C/min to 180 ° C. Helium was used as carrier gas. The injector temperature was 190 ° C.

395 RESULTS AND DISCUSSION

2,4,6-TCP accumulation

The average lipid content of the experimental mussels was 4.9% (SD 0.8), and the dry matter content was 8.9% (SD 1.2). A total of 15 mussels (7.8%) died during the experiment. However, there were no significant (P < 5 %) differences in mortality between the treatments. The 2,4,6-TCP concentration used (20 ~g.'l~ was obviously much iower than the LCs~ value of 2,4,6-TCP. Kaila and Saarikoski (1977) raported that the eight-day LC,,, value of 2.4.6-TCP tbr crayfish (Astacusfluviatilis) was 38-39 mg/l.

Toxicity of chlorophenols depends on pH due to the effects of pH on the degree of their ionisation (Kaila and Saarikoski, 1977). It is generally known that an ionizable compound is in non-ionized form, when the pH is below the pK,, -value. Hence, in our experiment 2,4,6-TCP was in ionized form, because the pH (8.08.2) was much higher than the pK~ -value. Non-ionized forms penetrate biological membranes more easily than the ionized tbrms (Kaila and Saarikoski, 1977),

Lipophilicity of xenobiotics and the lipid content of the organ or the organisms affect the bioaccumulation of xenobiotics (M/ikel~i and Oikari, 1990; Manahan, 1992; Pellinen et al., 1994). This was also the case in our experiment: the higher the lipid content of the mussels, the higher the absolute 2,4,6-TCP burden in the animal (Fig. 1). However, the effect of lipids can be excluded if the TCP -content is calculated relative to the lipid content or dry weight. The latter is true only when the lipid content and the dry weight are positively correlated, as was the case in this study.

Water pH and the amount of dissolved organic matter may strongly affect the bioavailability of hydrophobic compounds (Manahan, 1992). McCarthy (1983), Kukkonen and Oikari (1989, 1991) and Robinson and Novak (1994) pointed out that chemicals sorbed to suspended organic matter (humic acids) have a greatly reduced bioavailability. However, this was in contrast to our results: when dissolved humic acid was added, the 2,4,6-TCP concentration of the mussels was significantly higher than in the treatments with no humic acid addition (Tables 2 and 3). Since the 2,4,6-TCP was under ionised form, due to the high pH (8.0-8.2), it may have been absorbed more effectively to the humic acid, forming particles which mussels can filter. This could be the reason humic acid increased the accumulation of 2,4,6-TCP. Roberts (1972) suggested that organochlorine accumulated mainly via the ingestion of contaminated food. However, in our study, the food particles did not affect the accumulation of 2,4,6-TCP. Because the food particles (S. capricornutum) did not affect the weighted valve openness or the number of valve movements, it is possible that mussels did not ingest these food particles. Unfortunately, we did not check the stomach content of the mussels. Recently, Hart and

396 Santer (1994) found that S. capricornutum is an inferior or inadequate diet for two copepods. They postulated that the dietary inadequancy of this alga is caused by low digestibility or some biochemical deficiency.

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Lipid content (g) Figure 1. The effect of lipid content on 2,4,6-TCP accumulation. The absolute TCP-burden is correlated with the lipid content of the animal, which is not seen in the relative TCP-content.

397 Table 2. F-statistics of ANOVA for the 2,4,6-TCP concentration (ng/g dw) in A. anatina. The valve openness is a three level nested factor. Effect

df

HA food food*HA valve openness

1, 1, 1, 8,

8 8 8 12

F

P

21.5 0.43 0.77 1.02

0.002 0.532 0.406 0.470

Table 3. Average 2.4,6-TCP concentrations and bioconcentration factors in tissues of A. anatina. The presence of food particles had no effect on the 2,4,6-TCP concentrations, hence the data is pooled. The standard error of the mean is in parentheses.

Humic acid 0 mg Pt/1 100 mg Pt/l Controls

2,46-TCP concentration ng/g dw ug/g lipid

BCF

1046 (34) 1404 (67) < 20

1130

22.6 (1.0) 28.2 (1.4) < 0.4

1140

At the end of the experiment, :he average 2,4,6-TCP concentration of the mussels was 1225 ng/g DW (SD 258), or 25.4 ~g/g lipid (SD 5.0). The weighted valve openness did not affect the accumulation of 2,4,6TCP (Table 2). M/ikelii and Oikari (1990) observed that pentachlorophenol and 3,4,5-trichloroquaiacol accumulated rapidly into these mussels (A. anatina), reaching a steady state by 24 hr. In addition, Miikelii et al. (1991) found that the uptake rate of chlorophenols increased when the water temperature or concentration of chlorophenols increased. However, they stated that these factors did not change the bioconcentration factor. Thus, even though the weighted valve openness differed between the individual mussels, it is probable that the concentration of 2,4,6-TCP reached the steady state level in our long-term 10-day experiment.

Food particles, 2,4,6-TCP, and the pattern of valve movements

The average weighted valve openness was 46.9% (SE 1.8) and the average activity score was 5.7 valve movements per hour (SE 0.4). In studies under field conditions, the weighted valve openness varied between 56.7% (SE 3.5) and 89.3% (SE 1.3), and the activity score varied between 12.5/h (SE 1.2) and 28.5/h (SE 1.6) (Englund and Heino, 1994a,b, 1996). Hence under field conditions, both the weighted valve openness and the

398 activity score were higher than in the aquaria. It is probable that artificial conditions alter the natural pattern of valve movements.

The mussels in all test aquaria exhibited a rhythm of valve movements which was correlated with the dark-light regime (12D: 12L) of the aquaria (Fig. 2). Mussels responded to changes in light intensity by increasing the rate of valve movements. Darkness increased the weighted valve openness and the activity score. The same kind of photoperiodic rhythm in valve movement of the unionid mussel Ligumia subrostrata was noticed by McCorkle et al. (1979) in the laboratory. We have demonstrated diurnal rhythms of valve openness in A. anatina and Unio tumidus under field conditions in previous studies (Englund and Heino, 1994a,b, 1996) Moreover, McCorkle-Shirley (1982) revealed that the sodium flux in Corbiculafluminea was connected with the photoperiod. However, Morton (1970) stated that A. cygnea possessed a rhythm of adductor activity that was apparently unrelated to any environmental stimulus.

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Figure 2. The daily pattern of valve movement. The data is pooled from all the treatments. The period of darkness is indicated by horizontal bars. The standard error of valve openness (o) lies between 1.7 and 3.3 %, and of activity score (13) between 0.12 and 0.50.

399 Many authors have demonstrated that bivalves change their pattern of valve movement in response to the presence of xenobiotics, changes in temperature or light intemity, or altered particle concentration in the water (e.g. Sal~inki et al., 1974; Salfinki and V.-Balogh, 1989; Jgrgensen, 1990; Bayne et al., 1993; Ham and Peterson, 1994). This valve movement response has been used as a biological-early warning system (Slooff et al., 1983; Kramer et al., 1989; Borcherding, 1992a, b; Borcherding and Volpers, 1994; Matthias and R6mpp, 1994). This method is based on the ability to distinguish the altered behaviour caused by a stressor from the normal behaviour fluctuations.

Slooff et al. (1983) noticed that the detection limits in valve movement patterns in Dreissena polymorpha varied significantly between the different xenobiotics. For example, the detection limit for pentachlorophenol (PCP) was 140-520 gg/l. On the other hand, Borcherding (1992a,b) demonstrated that the threshold for PCP-induced changes in valve movement pattern in D. polymorpha was only 20-50 ~zg/l. Ham and Peterson (1984) found that the tolal residue chlorine fluctuations ranging between 20 and 70/~g/1 changed the valve movement pattern in Corbiculafluminea in field conditions. However, we did not notice any changes in the pattern of valve movements in our experiment caused by 2,4,6-TCP at 20/xg/1. In fact, none of the treatments (2,4,6-TCP, humic acid, or food particles) had statistically significant effects on the weighted valve openness or the activity score (Table 4). Only the autocorrelation coefficients of the weighted valve openness time series were affected by interactions involving 2,4,6-TCP (TCP*Humic acid and TCP*Food particle) (Table 4). On the other hand, the time series of activity scores were not affected. The autocorrelation coefficients of the w, :ighted valve openness were higher and less variable (0.939, ,';,D 0.018) than those of the activity score (0.553, SD 0.118).

Table 4. F-statistics of ANOVA for the valve movement ofAnodonta anatina, rl denotes the autocorrelation coefficient with lag one. df = 1, 8. Effect

Weighted valve openness F P

Activity score F P

rl(weighted valve openness) F P

rt(activity score) F P

HA food TCP food*HA TCP*HA TCP*food TCP*food*HA

1.01 0.13 0.00 2.32 1.11 0.92 0.01

1.07 0.75 0.05 0.62 2.76 0.01 0.00

0.24 0.72 0.45 0.50 6.34 7.68 0.92

0.65 0.04 0.19 2.74 0.03 0.19 0.19

0.345 0.733 0.955 0.166 0.323 0.365 0.924

0.331 0.412 0.830 0.453 0.135 0.931 0.984

0.638 0.419 0.522 0.500 0.036 0.024 0.365

0.442 0.847 0.674 0.137 0.873 0.674 0.677

400 The detection limits are extremely important when evaluating early warning systems based on valve movement patterns. Oikari et al. (1985) stated that the environmental concentrations of total chlorophenols varied between 0.58-2.58 ,g/1 in a "clean site", and near a pulp or paper mill, the concentration was 3-28 ,g/l. If the detection limits of the mussels for xenobiotics are much higher than those concentrations found in the environment, or if the detection limits vary over a wide range (Sloof et al., 1983), it will decrease their value for biological monitoring. On the other hand, mussels could be suitable for monitoring some specific substances, if we can distinguish the "normal behaviour" from the stress-induced behaviour. In this respect, it would be very important to take into account species-specific differences in the valve movement pattern.

Despite the fact that freshwater mussels fullfill many criteria of ideal biomonitoring organism, their thorough complexity as living organisms interacting with their surrounding environment should not be forgotten. For instance, many abiotic and biotic phenomena affect the valve movement pattern of mussels (Slooff et al., 1983; Kramer et al., 1989; Borcherding, 1992a, b; Borcherding and Volpers, 1994; Matthias and R6mpp, 1994; Englund & Heino, 1994a, b). Accordingly, the bioaccumulation may also change because of altered filtration efficiency. Moreover, chemical interactions between substances may affect their bioaccumulation or toxicity (Manahan, 1992). The accumulation kinetics of various xenobiotics in freshwater mussels is still poorly understood and much more attention needs to be paid to interactions between behaviour, physiology and bioaccumulation.

ACKNOWLEDGEMENTS

We would like to thank Dr. K. Pynn6nen for critical comments on the manuscript and Dr. R. Kristoffersson and Dr. J. Saarikoski for their helpful discussions. Dr. K. Lahti kindly provided the algal cultures. This study was financed by the Maj and Tor Nessling Foundation.

REFERENCES

Bayne, B.L., Iglesias, LI.P., Hawkins, A.J.S., Navarro, E., Heral, M., & Deslons-Paoli, J.M. 1993: Feeding behaviour of the mussel, Mytilgs edulis: Responses to variations in quantity and organic content of the seston. J. Mar. Biol. Ass. U.K. 73:813-829. Becker, K., Merlini, L., de Bertrand, N., de Alencastro, L.F. & Tarradellas, J. 1992: Elevated levels of organotins in Lake Geneva: bivalves as sentinel organism. Bull. Envir. Contain. Toxicol. 48:37-44.

401 Borcherding, J. 1992a: Another early warning system for the detection of toxic discharges in the aquatic encironment based on valve movements of the freshwater mussel Dreissena polymorpha. In: Neumann. D. & Jenner, H.A. (eds.), Limnoiogie aktuell - Vol. 4 - "The zebra mussel Dreissena polymorpha"., pp. 127146. Borcherding, J. 1992b: Die Schalenbewegung der Muschel Dreissena polymorpha als Monitorsystem zur Gew~isseriJberwachung. In: Steinh~iuser, K. G. and Hansen P.-D. (eds.), Schriftenreihe des Vereins fOr Wasser-. Boden- und Lufthygiene - Vol. 89 - "Biologische Testverfahren"., pp. 361-373. Borcherding, J. & Volpers, M. 1994: The "Dreissena-monitor"

- First results on the application of this

biological early warning system in the continuous monitoring of water quality. Wat. Sci. Tecb. 2 9 199-201. Englund, V.P.M. & Heino, M.P. 1994a: Valve movement ofAnodonta anatina and Unio tumidus (Bivalvia, Unionidae) in a eutrophic lake. Ann. Zool. Fennici 31:257-262. Englund, V.P.M. & Heino, M.P. 1994b: Valve movement of freshwater mussel Anodonta anatina: a reciprocal transplant experiment between two lakes. Ann. Zool. Fennici 31:417-423. Englund, V.P.M., Heino, M.P., & Melas, G. 1994: Field method for monitoring valve movements of bivalved molluscs. Wat. Res. 28:2219-2221. Englund, V.P.M. & Heino, M.P. 1996: Valve movement of freshwater mussel Anodonta anatina: a reciprocal transplant experiment between lake and river. Hydrobiologia, accepted. Green, R.H., Bailey, R.C., Hinch, S.G., Metcalfe, J.L. & Young, V.H. 1989: Use of freshwater mussels (Bivalvia:Unionidae) to monitor the nearshore environment of lakes. J. Great Lakes Res. 15:635-644. Ham, K.D. & Peterson, M.J. 1994: Effect of fluctuating low-level chlorine concentrations on valve-movement behavior of the asiatic clam (Corbiculafluminea). Environ. Toxicoi. Chem. 13:493-498. Hart, R.C. & Santer, B. 1994: Nutritional suitability of some uni-algal diets for freshwater calanoids: unexpected inadequances of commonly used edible greens and others. Freshwat. Biol. 31:109-116. Hemelraad, J. 1988: Accumulation and effects of cadmium in freshwater clams (Unionidae). Ph.D. dissert., University of Utrecht, Utrecht. Herve, S. 1991: Mussel incubation method for monitoring organochlorine compounds in freshwater recipients of pulp and paper industry. PhD dissert., Faculty of Science, University of Jyv~iskylL Research report No. 36.95 pp. + 11 App. Hinch, S.G. & Green, R.H. 1989: The effects of source and destination on growth and metal uptake in freshwater clams reciprocally transplanted among southcentral Ontario lakes. Can. J. Zool. 67:855-863. Jergensen, C.B. 1990: Bivalve filter feeding: hydrodynamics, bioenergetics, physiology and ecology. Olsen & Olsen, Fredensborg. 140 pp. Kaila, K. & Saarikoski J. 1977: Toxicity of pentachlorophenol and 2,3,6-trichlorophenol to the crayfish (Astacusfluviatilis L.). Environ. Pollut. 12:119-123. Kinney, R.M., Manos, C.G.Jr., Mills, E.L., MeUina, E., & Lisk, D.J. 1994: Zebra mussels (Dreissena polymorpha) as a biomonitoring tool for Sr 9° contamination. Chemosphere 28:729-735.

402 Kortelainen, P. 1993: Content of total organic carbon in Finnish lakes and its relationship to catchment characteristics. Can. J. Fish. Aquat. Sci. 50:1477-1483. Kramer, K.J.M., Jenner, H.A. & de Zwart, D. 1989: The valve movement response of mussels: a tool in biological monitoring. Hydrobiologia 188/189:433-443. Kukkonen, J. & Oikari, A. 1989: Effects of humus concentrations on benzo[a]pyrene accumulation from water to Daphnia magna: Comparison of natural waters and standard preparations. Sci. Tot. Environ. 79:197-207. Kukkonen, J. & Oikari, A. 1991: Bioavailability of organic pollutants in boreal waters with varying levels of dissolved organic material. Wat. Res. 25:455-463. Mtikelti, P. & Oikari, A.O.J. 1990: Uptake and body distribution of chlorinated phenolicsin the freshwater mussel, Anodonta anatina L. Ecotoxicol. Environ. Safety 20:354-362. Mtikela, P., Pettinen, T., Kukkonen, J. & Oikari, A.O.J. 1991: Accumulation and depuration of chlorinated phenolics in the freshwater mussel (Anodonta anatina L.). Ecotoxicol. Environ. Safety 22:153-163. Manahan S.E. 1992: Toxicological chemistry. 2nd ed. Lewis Publishers, Boca Raton, Ann Harbour, London, Tokyo. 449 pp. Matthias, U. & R6mpp, S. 1994: Erprobung des Dreissena-Monitors, eines neuen Biotestsystems mit der Zebramuschel (Dreissenapolymorpha), in der Rhein-GiitemeBstationKarlsruhe. Acta Hydrochim. Hydrobiol. 22:161-165. McCarthy, J.F. 1983: Role of particulate organic matter in decreasing accumulation of polynuclear aromatic hydrocarbons by Daphnia magna. Arch. Environ. Contain. Toxicol. 12:559-568. McCorkle, S., Shirley, T.C. & Diem, T.H. 1979: Rhythms of activity and oxycegen consumption in the common pond clam, Ligumia subrostrata (Say). Can. J. Zool. 57:1960-1964. McCorkle-Shirley, S. 1982: Effects of photoperiod on sodium flux in C0rbicula fluminea (Mollusca:Bivalvia). Comp. Biochem. Physiol. 71A:325-327. Mersch, J., Morhain, E. & Mouvet, C. 1993: Laboratory accumulation and depuration of copper and cadmium in the freshwater mussel Dreissenapolymorpha and the aquatic moss Rhynchostegium ripariodes. Chemosphere 27:1475-1485. Metcalfe-Smith, J.L. 1994: Ingluence of species and sex on metal residues in freshwater mussels (Family Unionidae) from the St. Lawrence river, with implications for biomonitoring programs. Environ. Toxicol. Chem. 13:1433-1443. Morton, B. 1970: The rhythmical behaviour ofAnodonta cygnea L. and Uniopictorum L. and its biological significance. Forma et Functio. 2:110-120. Muncaster, B.W., Hebert, P.D.N. & Lazar, R. 1990: Biological and physical factors affecting the body burden of organic contaminants in freshwater mussels. Arch. Environ. Contain. Toxicol. 19:25-34.

403 Oikari, A.O.J., Holmbom, B., An/is, E., Miilunpalo, M., Kruzynski, G. & Castr6n, M. 1985: Ecotoxicological aspects of pulp mill and paper mill effluents discharged to an inland water system: distribution in water, and toxicant residues and physiological effects in caged fish (Salmo gairdneri).

Aquat,

Toxicol. 6:219-239. Pellinen, J., Ruokolainen, M., M~ikel/i, P. & Taskinen, J. 1994: Organic halogen compounds, EOX, in mussels from a clean lake and a pulp mill recipient. Chemosphere 29:1515-1526. Roberts. D. 1972: The assimilation and chronic effects of sublethal concentrations of endosulphan on condition and spawning in the common mussel, Mytilus edulis. Mar. Biol. 16:119-125. Robinson, K.G. & Novak, J.T. 1994: Fate of 2,4,6,-trichloro-(~4C)-phenol bound to dissolved humic acid. Wat, Res. 28:445-452. Roseman, E.F,, Mills, E.L., Rutzke, M., Gutenmann, W.H. & Lisk, D.J. 1994. Absorption of cadmium from water by North American zebra and quagga mussels (Bivalvia:Dreissenidae). Chemosphere 28: 737-743. Sal~nki, J., Lukacsovics, F. & Hiripi, L. 1974: The effect of temperature variations on the rhythmic and periodic activity of the freshwater mussel (Anodonta cygnea L.). Ann. Biol, Tihany 41:69-79. Sal~inki. J. & V.-Balogh, K. 1989: Physiological background for using freshwater mussels in monitoring copper and lead pollution. Hydrobiologia 188/189:445-454. Slooff. W., de Zwart, D. & Marquenie, J.M. 1983: Detection limits of a biological monitoring system for chemical water pollution based on mussel activity. Bull. Environ. Contam. Toxicol. 30:400-405. Sprung, M. & Rose, U. 1988: Influence of food size and food quality on the feeding of the mussel Dreissena

polymorpha. Oecologia 77:526-532. Storey, A.W. & Edward, D.H.D. 1989: The freshwater mussel, Westralunio carteri Iredale, as a biological monitor of organochlorine pesticides. Aust. J. Mar. Freshwat. Res. 40:587-593.