Effects of Cd and Zn on oxygen consumption and ammonia excretion in sipuncula (Phascolosoma esculenta)

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Ecotoxicology and Environmental Safety 72 (2009) 507–515 www.elsevier.com/locate/ecoenv

Effects of Cd and Zn on oxygen consumption and ammonia excretion in sipuncula (Phascolosoma esculenta)$ XiXiang Chena,b, ChangYi Lua,, Yong Yea a

State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China b School of Chemistry & Life Science, Quanzhou Normal University, Quanzhou 362000, China Received 1 July 2007; received in revised form 16 November 2007; accepted 29 November 2007 Available online 12 February 2008

Abstract Physiological responses (oxygen consumption and ammonia excretion) of the sipuncula (Phascolosoma esculenta) exposed to four concentrations of Cd (0.45, 0.96, 2.04, and 4.46 mg L1) and four concentrations of Zn (1.09, 2.34, 4.96, and 10.91 mg L1) were monitored for 21 days, respectively. Oxygen consumption rates of sipuncula at all concentrations of Cd decreased from day 1 to day 6. At low concentrations of Cd (0.45 and 0.96 mg L1), the oxygen consumption rate was promoted. Time and concentration were significant in affecting oxygen consumption rate, respectively. Oxygen consumption rate decreased significantly with time for Zn-exposed individuals and also decreased significantly with the interaction between the concentration of Cd and time for Cd-exposed individuals. Changes occurred in the ammonia excretion rates and O:N ratios with individual sipuncula experiencing different metal concentrations over time. Although low O:N ratios (o30) 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. r 2007 Elsevier Inc. All rights reserved. Keywords: Sipuncula (Phascolosoma esculenta); Oxygen consumption; Ammonia excretion; O:N ratio

1. Introduction Heavy metals are the most common pollutants appearing in many coastal areas worldwide, leading to losses in oceanic yield and hazardous effects on health when contaminants enter the food chain. During the last years, studies have been increasing to assess final fate of heavy metals in coastal environments, particularly in order to find methods to evaluate the environmental damage and to create models to predict the deleterious effects before it is already irreversible. In recent decades, due to industrialization and urbanization in coastal areas, many estuarine waters in the world have increasing contents of heavy metal (Flower, 1990; Li et al., 2007). $ The experiments described in this article were conducted in accordance with national and institutional guidelines for the protection of animal welfare. Corresponding author. Fax: +86 592 2185622. E-mail address: [email protected] (C.Y. Lu).

0147-6513/$ - see front matter r 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2007.11.019

Jiulong River Estuary mangrove wetland of Fujian province is an important natural reserve of mangrove in China. In recent years, rapid economic growth and development in the region has led to excessive release of heavy-metal pollutants into the wetland. According to the standard quality of marine sediment in China, Zn and Cd polluted the mangrove wetland of Jiulong River Estuary (Liu et al., 2006). The chemical concentration in the environment may not be enough to kill the organism. However, sub-lethal concentrations often affect the biochemistry of an organism. It is well known that, trace contaminants in aquatic ecosystem pose environmental hazard because of their great toxicity or persistence (Meador et al., 1995). In recent years, the emphasis on toxicity testing in marine organisms has been moving towards sub-lethal tests, as they can provide much more relevant information in assessing the long-term effects of pollutants imposed on the ecosystem. Various sub-lethal tests and indices from cellular to physiological levels within an organism have been developed

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X. Chen et al. / Ecotoxicology and Environmental Safety 72 (2009) 507–515

(Bayne et al., 1985). Physiological parameters are considered to identify integrated responses to diverse stressors imposed by trace metals, other pollutants, and natural environmental factors such as temperature, salinity, and pH (Depledge et al., 1995). According to Hebel et al. (1997), many physiological variables must be measured to obtain the impact of toxicants in the whole organism which determine the survival potential of individuals. 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). The O:N values are discussed in relation to the various species’ feeding habits. 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, 1985). Correlations between the O:N ratio and pollution gradients have been reported (Widdows et al., 1990; Chinni et al., 2002), although other factors such as temperature (Saucedo et al., 2004; Wu and Sun, 2006), salinity (Wu and Sun, 2006), and starvation (Comoglio et al., 2005) may also influence the value of O:N. Sipuncula Phascolosoma esculenta, a marine depositfeeding benthonic invertebrate, is a special species of China. P. esculenta has a wide geographical distribution in the mangroves region in south China (Li, 1989). It is a very abundant intertidal macro-invertebrate along the rocky shore and it is likely to be among the first animals to be affected by anthropogenic sources of pollutants. It is an edible marine species and has long been used as a special dish. Although there was much research about P. esculenta on its distribution, classification (Li, 1989), nutritive composition (Zhou et al., 2006), embryo, and larval development (Wu et al., 2006), there has not been any published research regarding the effects of heavy metals on physiological responses of P. esculenta. Therefore, it is interesting to investigate the effects on oxygen consumption, ammonia excretion, and O:N ratio of P. esculenta exposure to cadmium and zinc. The objective of the present study was to evaluate sublethal responses such as oxygen consumption, ammonia excretion, and O:N ratio of P. esculenta due to waterborne exposure to cadmium and zinc, which are major pollutants in the marine ecosystem (Chen, 1997), in short-term laboratory exposures. This information will provide valuable new data by which any increase in heavy-metal concentration in the study area can be properly evaluated and should form an integral component of any ecotoxicological risk assessment. It is helpful in predicting the ecological consequences of pollution in the mangroves region.

2. Materials and methods 2.1. Collection and maintenance Specimens of P. esculenta (2–4 g body weight) were collected from the mangrove area of Jiulong River Estuary, Fujian. After transport to the Environmental Science Research Center of Xiamen University, animals were kept in tanks with a 15–20 cm layer of sediment from the original habitat. The experimental conditions are similar to environmental conditions. Salinity, water temperature, and dissolved oxygen levels observed were in ranges of 10–20%, 20–30 1C, and 5.4–7.6 mg L1 in the environment, respectively. So, salinity, temperature, and dissolved oxygen levels were observed in the ranges of 13–17%, 23–25 1C, and 6.1–7.6 mg L1, respectively. The animals were allowed to acclimatize in the laboratory for 3 days.

2.2. Toxicant preparation The stock solution of heavy metals were prepared by dissolving in distilled water, respectively. Nine experimental treatments included four concentrations of Cd (0.45, 0.96, 2.04, and 4.46 mg L1), four concentrations of Zn (1.09, 2.34, 4.96, and 10.91 mg L1), and one control. Metal solutions were prepared using either cadmium chloride (CdCl2  2.5H2O) (analytical grade) or zinc sulfate (ZnSO4  7H2O) (analytical grade) and artificial seawater. An atomic absorption spectrometer, Vario 6 (Analytik Jena AG) with a deuterium background correction, equipped with a transversely heated graphite furnace atomizer was used for this work. Zn was analyzed by a flame AAS, while Cd was analyzed using graphite furnace AAS. The monitored Zn and Cd wavelengths were 213.9 and 228.8 nm, respectively, while the slit bandwidth was set at 0.5 nm for Zn and 0.8 nm for Cd. The highest concentrations of metal solutions prepared were the LC50 values at 96 h obtained for this animal (Chen et al., 2007), and the lowest concentrations were one-tenth that of the LC50 values. Sixty individuals were maintained in each treatment and control, respectively. Each treatment and control had three replicates.

2.3. Oxygen consumption The oxygen consumption rates (OCR) of the 60 individuals from each treatment and control were determined on days 1, 6, 13, and 20 by using bottle-water method. Two individuals were placed for 1 h inside a sealed container (500 mL) with the same solution of heavy metals. The dissolved oxygen (DO) in each container was measured at the start and after 1 h by the Winkler method. One container without sipuncula was used as control. Each treatment and control had three replicates. The oxygen consumption rate was calculated by the following formulation: OCR ¼

½ðDO0  DOt Þ  V mg g1 h1 , W t

where DO0 is the DO of the water at the start of the experiment (mg L1), DOt the DO of the water at the end of the experiment (mg L1), V the volume of the container (L), W the live wet weight of sipuncula (g), and t is the experimental time (h).

2.4. Ammonia excretion From each treatment, the ammonia production of the 20 individuals was measured on days 1, 6, 13, and 20. Two individuals were placed inside a sealed container (500 mL) with the same solution of heavy metals, and the amount of ammonia produced in 1 h was determined measured using the phenol–hypochlorite method of Solorzano (1969). One container without sipuncula was used as control. Each treatment and control had three replicates. Ammonia excretion rate (AER) was calculated by the following formulation: AER ¼

½ðN t  N 0 Þ  V mg g1 h1 , W t

ARTICLE IN PRESS X. Chen et al. / Ecotoxicology and Environmental Safety 72 (2009) 507–515 where N0 is the NH4-N concentration of the water at the start of the experiment (mg L1), Nt the NH4-N concentration of the water at the end of the experiment (mg L1), V the volume of the container (L), W the live wet weight of sipuncula (g), and t is the experimental time (h).

Table 2 Cumulative mortality of P. esculenta exposed to various concentrations of Zn for 21 days Days

2.5. Statistical analysis All results are expressed as mean7S.D. Statistic analysis was performed using SPSS software (SPSS13.0). All data were statistically analyzed by two-way analysis of variance (ANOVA) to determine the effects of Cd or Zn on the physiological responses (OCR and AER). Duncan’s multiple-range test was used to evaluate significant differences among treatments at a 0.05 significance level. And the figures were drawn with OriginPro 7.5.

3. Results 3.1. Mortality The mortality of P. esculenta, when exposed to different concentrations of Cd and Zn was studied, respectively. Significant mortality (88.33% in 21 days) was observed at 4.46 mg L1 Cd (Table 1). Significant mortality (81.67% in 21 days) was observed at 10.91 mg L1 Zn (Table 2). And 53.33% mortality was also observed at 1.29 mg L1 Cd at the end of the experiment (Table 1).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

3.2. Oxygen consumption rate

Concentrations of Zn (mg L1) 0.00

1.09

2.34

4.96

10.91

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.6770.03

0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.6770.03 3.3370.03 3.3370.03 3.3370.03 5.0070.00 5.0070.00 6.6770.03

0 0 0 0 0 0 0 0 0 0 0 0 0 3.3370.03 3.3370.03 6.6770.03 6.6770.03 8.3370.06 8.3370.06 10.0070.05 15.0070.05

0 0 0 0 0 0 0 0 0 0 0 1.6770.03 3.3370.03 6.6770.03 10.0070.05 15.0070.05 16.6770.03 16.6770.03 18.3370.03 20.0070.05 25.0070.05

0 0 0 0 0 0 3.3370.03 6.6770.03 6.6770.03 10.0070.05 16.6770.03 20.0070.05 33.3370.06 38.3370.06 38.3370.06 61.6770.03 68.3370.03 73.3370.03 78.3370.03 81.6770.03 81.6770.03

3.0

Days Concentrations of Cd (mg L1)

0.45 mg L-1 0.96 mg L-1 2.04 mg L-1 4.46 mg L-1

2.5 OCR (mg g-1h-1)

Table 1 Cumulative mortality of P. esculenta exposed to various concentrations of Cd for 21 days

control

Cd

Oxygen consumption rates of sipuncula at all concentrations of Cd decreased from day 1 to day 6 (Fig. 1). At lower concentrations of Cd (0.45 and 0.96 mg L1), the oxygen

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

509

2.0

1.5

0.00

0.45

0.96

2.04

4.46

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1.6770.03

0 0 0 0 0 0 0 0 0 0 0 0 0 0 5.0070.00 6.6770.03 8.3370.03 10.0070.00 11.6770.03 15.0070.00 18.3370.03

0 0 0 0 0 0 0 0 0 0 1.6770.03 3.3370.03 3.3370.03 6.6770.03 6.6770.03 11.6770.03 18.3370.03 20.0070.05 33.3370.14 38.3370.10 40.0070.09

0 0 0 0 0 0 0 0 0 0 3.3370.03 6.6770.03 6.6770.03 10.0070.00 13.3370.03 13.3370.03 25.0070.05 33.3370.06 36.6770.06 45.0070.09 53.3370.13

0 0 0 0 1.6770.03 11.6770.03 16.6770.03 31.6770.03 35.0070.00 35.0070.00 46.6770.03 46.6770.03 63.3370.03 63.3370.03 65.0070.05 80.0070.05 81.6770.06 81.6770.06 83.3370.03 88.3370.03 88.3370.03

1.0 0

2

4

6

8

10

12

14

16

18

20

Days Fig. 1. Mean OCR (7S.D.) of P. esculenta exposed to various concentrations of Cd.

consumption rate was promoted, which were 142.9% and 130.7% higher than the control, respectively, at 1 day of exposition. However, there were inhibition in oxygen consumption of 15.9% and 16.4% at higher concentrations of Cd (2.04 and 4.46 mg L1), respectively, relative to the control at 1 day of exposition. And then for each concentration, no differences have been observed showing lower values of control group with time. Time and concentration were significant (ANOVA, po0.05) in affecting oxygen consumption rate, respectively

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(Tables 3 and 4), and was the result of the interaction between time and concentration of Cd (ANOVA, po0.05). Oxygen consumption rate was depressed by Zn (Fig. 2). As in the case of Zn exposure, the changes in oxygen consumption rate were significantly apparent (Tables 5 and 6). 3.3. Ammonia excretion rate Ammonia excretion rates of sipuncula at all concentrations of Cd (including the control) changed in a similar manner. Except at Cd concentrations of 4.46 mg L1, where ammonia excretion rates decreased sharply after 6 days, ammonia excretion rates increased from day 1 to day

3.0 control 1.09 mg L-1 2.34 mg L-1 4.96 mg L-1 10.91 mg L-1

Zn 2.5 OCR (mg g-1h-1)

510

2.0

1.5

1.0

0.5 0

2

4

6

8

10

12

14

16

18

20

Days Table 3 ANOVA for the effects of time and Cd concentration on the OCR in P. esculenta Source

Type III sum of squares

d.f. Mean square

Corrected model 10.809a Intercept 156.377 Concentration 3.359 Time 4.952 Concentration  time 2.497 Error 0.893 Total 168.079 Corrected total 11.702 a

19 1 4 3 12 40 60 59

F

Fig. 2. Mean OCR (7S.D.) of P. esculenta exposed to various concentrations of Zn.

Sig.

0.569 25.476 0.00 156.377 7002.854 0.00 0.840 37.606 0.00 1.651 73.926 0.00 0.208 9.320 0.00 2.233E-02

R-squared ¼ 0.924 (adjusted R-squared ¼ 0.887).

Table 4 Multiple comparisons for the effects of time and Cd concentration on the OCR in P. esculenta

Table 5 ANOVA for the effects of time and Zn concentration on the OCR in P. esculenta Source

Type III sum of squares

Corrected model 9.504a Intercept 113.462 Time 0.725 Concentration 8.033 Time  concentration 0.746 Error 1.364 Total 124.330 Corrected total 10.868 a

d.f. Mean square 19 1 3 4 12 40 60 59

F

Sig.

0.501 14.673 0.00 113.462 3328.188 0.00 0.242 7.086 0.00 2.008 58.907 0.00 6.220E-02 1.825 0.08 3.409E-02

R-squared ¼ 0.875 (adjusted R-squared ¼ 0.815).

A Time (days)

N

Subset 1

Duncana

20 13 6 1 Sig.

15 15 15 15

2

1.384 1.435

3

1.435 1.536

0.35

2.103 1.00

0.07

B Concentration (mg L1)

N

Subset 1

a

Duncan

4.46 2.04 0.96 0.45 0.00 Sig.

12 12 12 12 12

2

3

3.4. O:N ratios

1.296 1.375 1.703 1.812 0.20

13 and then decreased (Fig. 3). Interaction between time and concentration of Cd was responsible for the changes in the rate of ammonia excretion (ANOVA, po0.05) (Tables 7 and 8). At low concentrations of Zn (control, 1.09, and 2.34 mg L1), the ammonia excretion rate increased until day 13 and then decreased (Fig. 4), while at higher Zn concentrations (4.96 and 10.91 mg L1) the ammonia excretion rate was much depressed. The interaction between time and concentration of Zn was significant in affecting the ammonia excretion rate (ANOVA, po0.05) (Tables 9 and 10).

0.08

1.812 1.886 0.24

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 2.233E-02. a Alpha ¼ 0.05.

O:N ratios were high in sipuncula exposed to elevated Cd concentrations on day 1 (Fig. 5) and can be attributed to elevated oxygen consumption rates (Fig. 1). O:N values then decreased to about 10 at day 6 and remained low (almost below 15) throughout the experiment (Fig. 5). The O:N ratio changed significantly with time and the relationship of time and the concentration of Cd (ANOVA,

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511

Table 6 Multiple comparisons for the effects of time and Zn concentration on the OCR in P. esculenta

Table 7 ANOVA for the effects of time and Cd concentration on the AER in P. esculenta

A

Source N

Time (days)

20 13 6 1 Sig.

15 15 15 15

2

1.265 1.314 1.366 1.555 1.00

0.17

B

Corrected model 80800.177a Intercept 1049089.019 Concentration 14185.311 Time 22018.460 Concentration  time 44596.405 Error 9876.278 Total 1139765.474 Corrected total 90676.455 a

Concentration (mg L1) N Subset 1 Duncana 10.91 4.96 2.34 1.09 0.00 Sig.

2

3

4

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 3.409E-02. a Alpha ¼ 0.05.

control 0.45 mg L-1 0.96 mg L-1 2.04 mg L-1 4.46 mg L-1

AER (µg g-1h-1)

19 1 4 3 12 40 60 59

4252.641 17.224 0.00 1049089.019 4228.924 0.00 3456.328 14.363 0.00 7339.487 29.726 0.00 3716.367 15.052 0.00 246.907

R-squared ¼ 0.891 (adjusted R-squared ¼ 0.839).

Table 8 Multiple comparisons for the effects of time and Cd concentration on the AER in P. esculenta A Time (days)

N

Subset 1

a

20 1 13 6 Sig.

Duncan

300

200

Sig.

5

12 0.915 12 1.100 12 1.268 12 1.706 12 1.886 1.00 1.00 1.00 1.00 1.00

250

F

d.f. Mean square

Subset 1

Duncana

Type III sum of squares

15 15 15 15

2

109.320 118.185 145.103 156.314 0.06

0.13

B

Cd

Concentration (mg L1)

N

Subset 1

Duncana

150

100

0.45 2.04 4.46 0.96 0.00 Sig.

12 12 12 12 12

2

3

118.870 120.627 123.585 138.323 0.49

1.00

159.746 1.00

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 246.907. a Alpha ¼ 0.05.

50 0

2

4

6

8

10

12

14

16

18

20

Days Fig. 3. Mean AER (7S.D.) of P. esculenta exposed to various concentrations of Cd.

po0.05), and the changes were most obvious in the first week (Tables 11 and 12). At low concentrations of Zn (control, 1.09, and 2.34 mg L1), the mean O:N ratio decreased until day 13 and then increased (Fig. 6), while at higher Zn concentration (10.91 mg L1), the O:N ratio decreased with time. Moreover, few changes occurred at 4.96 mg L1 Zn (Fig. 6). 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 (Tables 13 and 14).

4. Discussion As sub-lethal parameter for toxicity studies, it is generally accepted that oxygen consumption gives a good indication of the overall metabolic state of an animal under heavy metal poisoning (Vosloo et al., 2002). Changes in the respiratory rates resulting from exposure to harmful chemicals indicate some abnormality or adaptive response in at least one of the biochemical pathways or physiological processes governing the metabolic rate in whole organisms (Watenpaugh and Beitinger, 1985). However, this response is not completely homogeneous, since in some cases the metabolic rate of an organism under toxic stress decreased (Spicer and Weber, 1991; Varghese et al., 1992; Bambang

ARTICLE IN PRESS X. Chen et al. / Ecotoxicology and Environmental Safety 72 (2009) 507–515

512 270

control 1.09 mg L-1 2.34 mg L-1 4.96 mg L-1 10.91 mg L-1

240

AER (µg g-1h-1)

210

Table 10 Multiple comparisons for the effects of time and Zn concentration on the AER in P. esculenta

Zn

A N

Time (days)

180

1

150

Duncana

120 90 60

20 1 6 13 Sig.

15 15 15 15

0

124.720 132.539

3

132.539 138.847

0.08

2

4

6

8

10

12

14

16

18

153.509 1.00

0.15

Concentration (mg L1) N Subset

20

Days

1

Fig. 4. Mean AER (7S.D.) of P. esculenta exposed to various concentrations of Zn.

Table 9 ANOVA for the effects of time and Zn concentration on the AER in P. esculenta d.f. Mean square 19 1 3 4 12 40 60 59

F

Sig.

Duncana 10.91 4.96 0.00 2.34 1.09 Sig.

5154.248 36.997 0.00 1132782.759 8131.050 0.00 2230.047 16.007 0.00 18428.862 132.281 0.00 1460.427 10.483 0.00 139.316

R-squared ¼ 0.946 (adjusted R-squared ¼ 0.921).

2

3

4

12 82.335 12 110.460 12 159.746 12 159.990 12 174.486 1.00 1.00 0.96 1.00

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 139.316. a Alpha ¼ 0.05.

40 control 0.45 mg L-1 0.96 mg L-1 2.04 mg L-1 4.46 mg L-1

Cd 35 30

O:N

Type III sum of squares

Corrected model 97930.709a Intercept 1132782.759 Time 6690.141 Concentration 73715.448 Time  concentration 17525.120 Error 5572.627 Total 1236286.096 Corrected total 103503.337 a

2

B

30

Source

Subset

25 20

et al., 1995; Barbieri, 2007), increased (Calow, 1989), or showed changes along experimental time of exposure (Vosloo et al., 2002). In addition, oxygen consumption may fluctuate with changes in the rate of intake, internal transport, and tissue utilization of oxygen (Grobler et al., 1989). It is finally accepted that any change in metabolic rates depends on the nature, magnitude, and persistence of toxic effects. Therefore, studies on the effects of metabolic rates provides a clue to the chemicals mode of toxicity in addition to revealing the importance of sub-lethal effect (Watenpaugh and Beitinger, 1985). Even though, it is considered as an indirect measure of the physiological condition of the organisms and it is frequently used as a deleterious effect to metal exposure. The measurement of oxygen consumption not only indicates the metabolic rate but also provides an index for sub-lethal stress condition and for biomonitoring the potentially toxic chemicals that elicit this response (Palanivelu et al., 2005). With a view to understand the extent of such differences in oxygen consumption of animals, the

15 10 5 0

2

4

6

8

10

12

14

16

18

20

Days

Fig. 5. Mean O:N ratios (7S.D.) of P. esculenta exposed to various concentrations of Cd.

present investigation was undertaken to study the effect of heavy metals in the estuarine edible sipuncula P. esculenta. Starvation could affect the results too. 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 sipuncula, 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

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20

Table 11 ANOVA for the effects of time and Cd concentration on the O:N ratio in P. esculenta Type III sum of squares a

Corrected model 2553.563 Intercept 11085.717 Concentration 258.219 Time 989.184 Concentration  time 1306.160 Error 206.299 Total 13845.579 Corrected total 2759.863 a

F

d.f. Mean square 19 1 4 3 12 40 60 59

control 1.09 mg L-1 2.34 mg L-1 4.96 mg L-1 10.91 mg L-1

Zn

Sig.

15

134.398 26.059 0.00 11085.717 2149.442 0.00 64.555 12.517 0.00 329.728 63.932 0.00 108.847 21.105 0.00 5.157

O:N

Source

513

10

R-squared ¼ 0.925 (adjusted R-squared ¼ 0.890).

5 0

2

4

6

8

10

12

14

16

18

20

Days Table 12 Multiple comparisons for the effects of time and Cd concentration on the O:N ratio in P. esculenta

Fig. 6. Mean O:N ratios (7S.D.) of P. esculenta exposed to various concentrations of Zn.

A Time (days)

N

Subset 1

Duncana

6 13 20 1 Sig.

15 15 15 15

2

3

Source

9.985 10.571 13.603 0.48

1.00

20.211 1.00

B Concentration (mg L1)

N

Subset 1

a

Duncan

2.04 0.00 4.46 0.96 0.45 Sig.

12 12 12 12 12

11.684 11.899 12.682

0.32

Table 13 ANOVA for the effects of time and Zn concentration on the O:N ratio in P. esculenta

2

3

Corrected model 315.433a Intercept 6349.136 Time 95.580 Concentration 96.451 Time  concentration 123.402 Error 127.794 Total 6792.363 Corrected total 443.226 a

12.682 14.435 0.07

17.263 1.00

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 5.157. a Alpha ¼ 0.05.

discerned. In this study, Zn is a ubiquitous and nutritionally essential metal that is required for normal growth, development, and functioning of animal species. On the contrary, Cd is a non-essential trace metal that is toxic to animals. 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. Inhibition of respiration by Cd has been reported in white shrimp (Wu and Chen, 2004), and penaeid shrimps (Barbieri, 2007) and has been attributed to mucus production because it reduces the efficiency of gaseous exchange (Naimo et al., 1992). A similar response was

Type III sum of squares

d.f. Mean square

F

Sig.

19 1 3 4 12 40 60 59

5.196 1987.305 9.972 7.547 3.219

0.00 0.00 0.00 0.00 0.00

16.602 6349.136 31.860 24.113 10.283 3.195

R-squared ¼ 0.712 (adjusted R-squared ¼ 0.575).

observed in this study. Oxygen consumption even seemed elevated after exposure to low concentration of cadmium for 6 days. 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’. Concerning the direct effect of heavy metal on ammonia excretion, Wu and Chen (2004) have found an increase in the levels of ammonia excreted by Litopenaeus vannamei after 24 h of exposure to cadmium and zinc which is in coincidence with our results at low concentrations of Zn (control, 1.09, and 2.34 mg L1) until day 13. Barbieri (2007) has also found that after separate exposures to cadmium and zinc, elevations in ammonium excretion were obtained, which were 174.28% and 162.5% higher than the control, respectively. However, Vanegas et al. (1997) have neither found effects of cadmium nor of zinc in the ammonia excretion in juvenile Penaeus setiferus after 21 days of exposure on its iso-osmotic point. Several authors have proposed theoretical minimum values of the O:N ratio for strictly protein catabolism

ARTICLE IN PRESS X. Chen et al. / Ecotoxicology and Environmental Safety 72 (2009) 507–515

514

Table 14 Multiple comparisons for the effects of time and Zn concentration on the O:N ratio in P. esculenta A Time (days)

N

Subset 1

Duncana

13 6 20 1 Sig.

15 15 15 15

2

8.874 9.934 10.009 12.330 1.00

0.11

B Concentration (mg L1)

N

Subset 1

Duncana

2.34 1.09 4.96 10.91 0.00 Sig.

12 12 12 12 12

2

3

8.276 9.933 9.962 11.364 1.00

0.07

2001), but exposure to cadmium in Mysidopsis bahia produced an increase in the O:N ratio after 4 days of exposure (Carr et al., 1985). In the same sense, Vanegas et al. (1997) reported an increase in O:N ratio in P. setiferus exposed to sub-lethal concentrations of zinc and cadmium. 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 6 days. 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 (Russcll-Hnnter et al., 1983; Aldridge et al., 1987). In this sense, the value of using O:N ratio as a stress index is questioned. 5. Conclusions

11.364 11.899 0.47

Means for groups in homogeneous subsets are displayed. Based on Type III sum of squares. The error term is mean square (error) ¼ 3.195. a Alpha ¼ 0.05.

(Conover and Corner, 1968; Snow et al., 1971). Mayzaud and Conover (1988) concluded that low O:N ratios correspond to a period when protein is heavily used, while higher values are closely related to depletion of the lipid reserves. All these values are based on the same assumptions—1 g of protein requires 0.94 L of oxygen and 1 g of lipid requires 2.04 L of oxygen, both in oxidative metabolism, and for this reason the lipid content may be the most influential fraction affecting O:N ratio. All the theoretical computations of O:N ratio mentioned above are directly related to the nature of the amino acids entering the Krebs cycle, the type of fatty acid involved, and by the fate of each biochemical fraction being catabolized. In this context, the O:N ratio is considered as a direct measure that reflects changes on the energy requirements due to internal and external variables. Clearly, any variable that produces a modification in the combined variation of the metabolic processes of oxygen consumption and ammonia excretion will cause a change in the O:N ratio (Mayzaud and Conover, 1988). However, it is important to take into account that if both parameters vary in the same proportion, the O:N ratio would not change. According to our results, low O:N ratios in all treatments and control groups could be indicating a protein catabolism. However, high O:N values were obtained in individuals exposed to higher cadmium concentration which could indicate a lipid catabolism. Heterogeneous data of O:N ratio were reported by different authors. A reduction in the O:N ratio was registered for the mysid Praunus flexuosus exposed to copper (Garnacho et al.,

In the present study, we determined the effects of both heavy metals on oxygen consumption, ammonium excretion; O:N ratio of P. esculenta was also demonstrated. Results show that P. esculenta is a good test organism for studying heavy-metal pollution. Although low O:N ratios (o30) 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. Our future work will focus on both the acute effects of these heavy metals on P. esculenta at other biological levels such as histological and biochemical levels, and chronic effects on metabolism, and growth rates which are also very important for the sipuncula culture industry. Consequently, from an ecotoxicological point of view, this study represents relevant information for conservation of the natural resources in this vulnerable mangroves region since the studied species has a widespread local distribution along the mangroves region. Acknowledgments We thank all who assisted in this study, in particular G.C. Chen, for laboratory assistance. Thanks also to the anonymous reviewers for their valuable considerations. This work was supported by grants from National Natural Science Foundation of China (Project no. 40476040) and Provincial Natural Science Foundation of Fujian (Project no. D0410006) awarded to Y. Ye. References Aldridge, D.W., Payne, B.S., Miller, A.C., 1987. The effects of intermittent exposure to suspended solids and turbulence on three species of freshwater sipuncula. Environ. Pollut. 45, 17–28. Bambang, Y., Thuet, P., Charmantier-Daures, M., Trilles, J.P., Charmantier, G., 1995. Effect of copper on survival and osmoregulation of

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