Effects of heavy metals on oxygen consumption and ammonia excretion in green-lipped mussels (Perna viridis)

Pergamon

0025-326X(95)00137-9

Marine Pollution Bulletin, Vol. 31, Nos 4-12, pp. 381-386, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All fights reserved 0025-326X/95 $9.50 + 0.00

Effects of Heavy Metals on Oxygen Consumption and Ammonia Excretion in Green-Lipped Mussels (Perna viridis) S. G. C H E U N G and RICHARD Y. H. CHEUNG Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

Physiological responses (oxygen consumption and ammonia excretion) of the green-Hpped mussel (Perna viridis) exposed to four concentrations of Cd (0.15, 0.32, 0.70 and 1.50 ppm) and four concentrations of Zn (0.60, 1.29, 2.79 and 6.00 ppm) were monitored for 21 days. Changes occurred in the ammonia excretion rates and O:N ratios with individual mnsseis experiencing different metal concentrations over time. Oxygen consumption rate decreased significantly with time for Cd-exposed individuals and also decreased significantly with the interaction between the concentration of Zn and time for Zn-exposed individuals. Although low O:N ratios ( < 3 0 ) were obtained in most of the treatments, no predictable correlation was found between concentrations of metals and values of O:N obtained. The value of using O:N ratio as a stress index is questioned.

In recent years, the emphasis on toxicity testing in marine organisms has been moving towards sublethal tests, as they can provide much more relevant information in assessing the long-term effects of pollutants imposed on the ecosystem. Various sublethal tests and indices from cellular to physiological levels within an organism have been developed (Bayne et al., 1985). Two methods that have received much attention are the scope for growth and the O:N ratio. Scope for growth represents the amount of energy available for growth and reproduction in an organism, while the O:N ratio purports to indicate the relative utilization of protein in energy metabolism (Bayne et al., 1985). A high value of O:N is taken to represent a predominance of lipid and/or carbohydrate catabolism over protein degradation. In Mytilus edulis, O:N ratio values of 50 indicate a healthy condition, values less than 30 indicate a heavy reliance on protein as an energy source and a value of 7 represents a total reliance on protein (Widdows, 1985a). Correlations between the O:N ratio and pollution gradients have been reported (Widdows et aL, 1981, 1990), although other factors such as temperature (Widdows, 1978), ration (Bayne, 1973) and reproductive condition (Bayne et aL, 1976) may also influence the value of O:N. The application of physiological responses and the O:N ratio in pollution monitoring have been examined most extensively on the

temperate mussel, M. edulis. As a counterpart in subtropical and tropical regions, physiological studies on the green-lipped mussel (Perna viridis) in response to pollutants are scarce (Kxishnakumar et aL, 1987, 1990), although accumulated concentrations of metals and PCBs in body tissues have been extensively studied (Phillips, 1985; Chart et aL, 1990; Cheung & Wong, 1992) and the potential of this animal as a pollution biomonitor has been assessed (Rainbow, 1993). Since P. viridis occurs at very high density in polluted harbours in Hong Kong (Huang et al., 1985), the study of its physiological responses to sublethal availabilities of pollutants is helpful in predicting the ecological consequences of pollution in these harbours. This study attempts to examine the effects of Cd and Zn, which are major pollutants in the harbours of Hong Kong (EPD, 1994), on oxygen consumption and ammonia excretion (two components of the scope for growth equation) in P. viridis and to evaluate the usefulness of the O:N ratio in pollution biomonitoring.

Materials and Methods Individuals of P. viridis, with shell lengths between 60.00 and 65.85 mm, were collected at Ma Liu Shui in Inner Tolo Harbour and were allowed to acclimatize in the laboratory for 3 days. Nine experimental treatments included four concentrations of Cd (0.15, 0.32, 0.70 and 1.50 ppm), four concentrations of Zn (0.60, 1.29, 2.79 and 6.00 ppm) and one control. Metal solutions were prepared using either ZnC12 or CdC12 and artificial seawater. Inherent concentrations of Cd and Zn in artificial seawater were 11.0 ppb and 0.70 ppb, respectively (Chan, 1988). The highest concentrations of metal solutions prepared were the LCs0 values at 96 h obtained for this animal (Chan, 1988), and the lowest concentrations were one-tenth that of the LCs0 values. Nineteen individuals were maintained in each treatment. Among them, 10 were individually marked and had their physiological responses (oxygen consumption and ammonia excretion) measured. Others were used as substitutes for marked individuals dying during the experiment. Salinity, temperature and dissolved oxygen levels in each treatment were monitored daily, and observed ranges were 27-309/00, 22.7-24.5°C and 6.007.55 ppm, respectively. 381

Marine Pogution Bulletin 1 O0

Oxygen consumption

The oxygen consumption rates of the 10 marked individuals from each treatment were determined on days 1, 6, 13 and 20. If an individual died, it was replaced by another individual maintained under the same conditions. Each individual was placed for 1 h inside a sealed container (0.80-1.37 1) with artificial seawater. The oxygen level in each container was measured at the start and after 1 h using a YSI polarographic oxygen meter. The time was chosen such that the oxygen level did not fall below 4.00 mg 1-1, the critical concentration above which the oxygen consumption rate of each mussel remains constant (pers. obs.). Two containers without mussels were used as controls. Oxygen consumption rate was expressed as milligrams of oxygen per hour, assuming that all the mussels were of the same size.

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From each treatment, the ammonia production of the 10 marked individuals was measured on days 2, 7, 14 and 21. Each individual was placed in a beaker containing 200 ml GF/C faltered seawater, and the amount of ammonia produced in 1.5 h was determined using the phenol-hypochlorite method (Widdows, 1985b).

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Significant mortality (90% in 21 days) was observed at 1.50 ppm Cd (Fig. 1). No individual survived 21 days at 2.79 ppm and 6.00 ppm Zn, and 63% mortality was also observed at 1.29 ppm Zn at the end of the experiment (Fig. 2).

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Fig. 1 Cumulative mortality of P. viridis exposed to various concentrations of Cd for 21 days at ca 24°C.

Ammonia excretion

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As the physiological responses of the individuals were repeatedly measured on four occasions, the responses determined at each time were not independent. Therefore, a MANOVA was conducted (yon Ende, 1993). The dependent variable were the physiological responses on each of the four dates, and the treatment was the metal concentration. Profile analysis was conducted to separately examine the effects of time, metal concentration and the interaction between time and concentration of metal. Individual ANOVAs were used to identify the particular time intervals in which the treatment effects were different (yon Ende, 1993). Whenever 100% mortality was encountered in the treatments, the data set was removed from the analysis.

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Oxygen consumption rates of mussels at all concentrations of Cd decreased from day 1 to day 6 (Fig. 3). Time was significant (MANOVA, p<0.005) in affecting oxygen consumption rate between day 1 and day 6 only (Table 1). Oxygen consumption rate was depressed by Zn (Fig. 4) and was the result of the interaction between time and concentration of Zn (MANOVA, p < 0.005). As in the case of Cd exposure, the changes in oxygen consumption rate were

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Volume 31/Numbers 4-12/April-December 1995 significantly apparent (Table 2).

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A m m o n i a excretion rate

A m m o n i a excretion rates o f mussels at all concentrations o f Cd (including the control) changed in a similar manner. Except at Cd concentrations o f 1.50 ppm, where a m m o n i a excretion rates decreased after I week, a m m o n i a excretion rates increased f r o m day 2 to day 14 and then decreased (Fig. 5). Interaction between time and concentration o f Cd was responsible for the changes in the rate o f a m m o n i a excretion ( M A N O V A , p < 0.005), which occurred most clearly in the first week (Table 3). At low concentrations o f Zn (control, 0.60 and 1.29 ppm), the a m m o n i a excretion rate increased until day 14 and then decreased (Fig. 6), while at higher Zn concentrations (2.79 and 6.00 ppm) the a m m o n i a

excretion rate was m u c h depressed. The interaction between time and concentration o f Zn was significant in affecting the a m m o n i a excretion rate ( M A N O V A , p < 0 . 0 0 5 ) , most apparently between day 2 and day 7 (Table 4). O : N ratios

O:N ratios were high in mussels exposed to elevated CA concentrations on day 2 (Fig. 7) and can be attributed to elevated oxygen consumption rates (Fig. 3). O:N values then decreased to a b o u t 10 at day 7 and remained low throughout the experiment (Fig. 7). The O : N ratio changed significantly with time and the relationship of time and the concentration of Cd ( M A N O V A , p < 0 . 0 0 5 ) , and the changes were most obvious in the first week (Table 5). The m e a n O : N ratio o f mussels exposed to Zn gradually decreased with time,

TABLE 1 ANOVA for the effects of time and Cd concentration on the oxygen consumption rate in P. viridis.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

0.59 4.93

4.66 4.66

0.20 4.93

0.13 0.13

1.53 38.13

0.223 0.000

0.16 0.07

5.57 5.57

0.05 0.07

0.15 0.15

0.34 0.46

0.794 0.500

0.18 0.01

0.92 0.92

0.06 0.01

0.03 0.03

2.40 0.32

0.084 0.578

Day I-Day 6

Time x concentration Time Day 6-Day 13

Time x concentration Time Day I3-Day 20

Time x concentration Time

*Bonferroni adjustment of = = 0.05/3 : 0.0167. TABLE 2

ANOVA for the effects of time and Zn concentration on t h e oxygen consumption ram in P. viridb.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

0.95 0.64

3.02 3.02

0.32 0.64

0.12 0.12

2.73 5.49

0.064 0.027

0.31 0.04

2.27 2.27

0.10 0.04

0.09 0.09

1.18 0.49

0.338 0.489

Day I-Day 6

Time x concentration Time Day 6-Day 13

Time x concentration Time

*Bonferroni adjustment of at= 0.05/2 = 0.025.

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Marine Pollution Bulletin TABLE 3 ANOVA for the effects of time and Cd concentration on the ammonia excretion rate in P. viridis.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

5994.11 39 409.81

1981.26 1981.26

1998.04 39 409.81

56.61 56.61

35.30 696.20

0.000 0.000

371.74 187.81

2615.40 2615.40

123.91 187.81

74.73 74.73

1.66 2.51

0.194 0.122

381.03 4713.54

3014.43 3014.43

127.01 4713.54

86.13 86.13

1.47 54.73

0.238 0.000

Day 1-Day 6 Time x concentration Time

Day 6--Day 13 Time x concentration Time

Day 13-Day 20 Time x concentration Time

*Bonferroni adjustment of ct= 0.05/3 = 0.0167.

TABLE 4 ANOVA for the effects of time and Zn concentration on the ammonia excretion rate in P. viridis.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

4840.58 5649.53

3126.37 3126.37

1613.53 5649.53

107.81 107.81

14.97 52.40

0.000 0.000

3711.74 336.96

4819.94 4819.94

1237.25 336.96

166.20 166.20

7.44 2.03

0.001 0.165

Day I-Day 6 Time x concentration Time

Day 6-Day 13 Time x concentration Time

*Bonferroni adjustment of ct= 0.05/2 = 0,025.

TABLE 5 ANOVA for the effects of time and Cd concentration on the O:N ratio in P. viridis.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

43 007.49 78 827.62

42 983.81 42 983.81

14 335.83 78 82Z62

1343.24 1343.24

10.67 58.68

0.000 0.000

71.94 18.28

832.62 832.62

23,98 18,28

26.02 26.02

0.92 0.70

0.442 0.408

84.12 143.87

256,55 256,55

28.04 143.87

8.02 8.02

3.50 17.95

0.027 0.000

Day I-Day 6 Time x concentration Time

Day 6-Day 13 Time x concentration Time

Day 13-Day 20 Time x concentration Time

*Bonferroni adjustment of ct= 0.05/3 = 0.0167.

TABLE 6 ANOVA for the effects of time and Zn concentration on the O:N ratio in P. viridis.* Source

Hypoth. SS

Error SS

Hypoth. MS

Error MS

F

Sig. of F

226,65 785.92

544.30 544.30

113.33 785.92

25.92 25.92

4.37 30.32

0.026 0.000

133.17 0.14

539.06 539.06

66.59 0.14

25.67 25.67

2.59 0.01

0.098 0.942

45.46 93.93

62.64 62.64

22.73 93.93

2.98 2.98

7.62 31.49

0.003 0.000

Day I-Day 6 Time × concentration Time

Day 6-Day 13 Time x concentration Time

Day 13-Day 20 Time x concentration Time

*Bonferroni adjustment of ct = 0.05/3 = 0.0167. 384

Volume 31/'Numbers4-12/April-December 1995 140

DF=3,73; p>0.05) and Zn (F=0.923; p > 0.05) as tested by ANOVA.

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Fig. 8 Mean O:N ratios (_+1 SE) of P. viridis exposed to various concentrations of Zn at ca 24°C. TABLE 7 Comparisons of tissue dry weights (mean-+ 1 SD) of P. viridis before and after exposure to various concentrations of Cd and Zn for 3 weeks. Dry weight (g) Before the experiment

0.6721 (-t-0.1645)

After the experiment

Cd 0.15 ppm 0.32 pprn 0.70 ppm Zn 0.60 ppm 1.29 ppm Control

0.4927 (+0.1344) 0.5107 (-+0.1621) 0.5035 (_4-0.1792) 0.4667 (+0.1613) 0.3853 (+0.1234) 0.4425 (_+0.1279)

except at 6.00 ppm Zn (Fig. 8), and remained low (about 10) throughout the experiment. Again, significant interaction was found between the effects of time and metal concentration on the O:N ratio, and the changes in O:N ratio were most apparent in the first and third week (Table 6).

A confounding factor affecting physiological responses in this experiment was starvation. This resulted in a gradual decrease in oxygen consumption rate in the control group. At the same time, the ammonia excretion rate increased in control mussels, indicating the heavy reliance on protein to provide energy, and thus low values of the O:N ratio were obtained. Nevertheless, the effect of heavy metals on these physiological variables can be discerned, especially for Zn. Perna viridis is able to maintain constant body concentrations of 100 I~g Zn g-1 dry weight when the external dissolved Zn levels are below the threshold concentration of 362 lxg Zn 1-~ (Chan, 1988). In this study, however, once the threshold concentration is exceeded, Zn had a marked inhibition effect on respiration and ammonia excretion. This is probably due to the interference with gaseous exchange and the inhibition of mitochondrial respiration (Krishnakumar et al., 1987). Inhibition of respiration by Cd has been reported in mud crabs (Collier et al., 1973), mudsnails (Forbes & Depledge, 1992) and mussels (Naimo et al., 1992) and has been attributed to mucus production because it reduces the efficiency of gaseous exchange (Naimo et al., 1992). A similar response was not observed in this study. This was possibly as a result of the large variations in the data. Oxygen consumption even seemed elevated after exposure to raised Cd for 1 week. The reason is unknown, but increased glycolysis in lobsters exposed to Cd has been reported by Gould (1980), interpreted as 'an augmented expenditure of energy reserves characteristic of a stress compensation process'. Low O:N ratios were obtained in all treatments, indicating that all groups of individuals were stressed. However, in comparison with the controls, high values of O:N were obtained in individuals exposed to Cd for 1 week. This was attributed to the high oxygen consumption rates. Moreover, there was no predictable correlation between O:N ratios and exposure to metal concentrations, although the ratio changed with metal concentration and time. The O:N ratio has also been reported to be a poor indicator of stress in freshwater molluscs (Russell-Hunter et al., 1983; Aldridge et al., 1987). As differences in the physiological responses and the O:N ratio among the groups were most apparent in the first week, and since any starvation problem will become more serious as the time of exposure increases, sublethal tests should be limited, therefore, to the first week of exposure to pollutants. The authors thank S. Y. Chan, H. Li and P. K. Yim for conducting the experiment and Prof. Philip Rainbow for his constructive comments on this manuscript. The project is supported by a strategic grant of the City University of Hong Kong.

Tissue dry weight

Although all treatments showed significant decreases in tissue dry weight with time (Table 7), there was no significant difference in tissue weight among treatments at the end of the experiment for both Cd (F=0.773;

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