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Appropriate experimental design for transplanting mussels (Mytilus sp.) in analyses of environmental stress: an example in Sydney Harbour (Australia)
Journal of Experimental Marine Biology and Ecology 297 (2003) 253 – 268 www.elsevier.com/locate/jembe
Appropriate experimental design for transplanting mussels (Mytilus sp.) in analyses of environmental stress: an example in Sydney Harbour (Australia) P.J.C. Honkoop *, B.L. Bayne, A.J. Underwood, S. Svensson Centre for Research on Ecological Impacts of Coastal Cities, Marine Ecology Laboratories A11, University of Sydney, Sydney, NSW 2006, Australia Received 11 December 2002; received in revised form 28 July 2003; accepted 3 August 2003 This paper is dedicated to Dr. Peter Donkin.
Abstract Some locations in Sydney Harbour (Sydney, Australia) contain large amounts of contaminants (heavy metals and hydrocarbons), sometimes in concentrations thought to affect biological systems. In order to estimate effects of sediment-bound contaminants on the physiology of organisms living above the sediment, the rates of clearance and respiration and the efficiency of absorption of mussels, Mytilus sp., living in a contaminated location were measured, the scope for growth (SfG) was calculated and compared to that of mussels living in uncontaminated locations. Two different models were proposed to explain expected differences. The first was that the contaminants at the impacted location reduced the SfG of local mussels; the second was that at the contaminated location only those mussels survived that had a small SfG (genetic differences between populations might be a reason for differential survival). To test which model applied, mussels were transplanted between the contaminated and uncontaminated locations. Moving and disturbing mussels (handling, tagging and caging) required the inclusion of two control treatments. These treatments were essential for a proper evaluation of the results but have generally not been included in similar studies. Effects of moving were estimated by translocating mussels from the uncontaminated and from the contaminated location to similar locations. To estimate effects of disturbance, mussels in the experimental locations were given the same treatment as the experimental mussels, but were returned to the place of origin. It was predicted that translocating and disturbing mussels would have no effect on the SfG, which would be similar to that of mussels at the place of origin. As expected, SfG was smaller (because rate
* Corresponding author. Present address: Department of Marine Ecology and Evolution, Royal Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands. Tel.: +31-222-369492; fax: +31-222-319674. E-mail address: [email protected] (P.J.C. Honkoop). 0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2003.08.001
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of clearance was reduced) in undisturbed mussels at the contaminated location compared with those of mussels at the uncontaminated location. Because there were significant effects of disturbance on the SfG of mussels at the contaminated location, it was concluded that this difference was not caused by differences between the amounts of contaminants in the two locations, but was caused by other confounding factors (physical disturbance by crabs and fouling organisms). That the interpretation of the results would have been different if proper controls were not included is discussed in this paper and the importance of proper experimental controls is stressed. D 2003 Elsevier B.V. All rights reserved. Keywords: Transplant experiment; Control treatments; Contaminants; Scope for growth; Environmental stress; Mussels
1. Introduction Mussels (Mytilus spp.) are commonly used as bio-indicators to monitor contamination by chemicals, sedimentation, etc., in marine areas (e.g. Martin, 1985; Widdows et al., 1990, 1995). For various reasons, mussels are particularly suited to use as test organisms in so-called Mussel Watch Programs (Widdows and Donkin, 1992; Sericano et al., 1995). Because contaminants affect physiological processes, physiological variables of mussels are measured to calculate their energy budget and to estimate the potential toxic effects of contaminants on any of the physiological traits. Measuring these variables and combining them into a single energy equation was first applied to bivalves by Widdows and Bayne (1971) and became rapidly very popular under the name scope for growth (SfG, Bayne and Widdows, 1978). If the gain of energy per unit time is negative or positive but small, it is thought that animals are stressed. If it is large, it is thought that the animals are in good condition and have energy available for growth and reproduction. Mussels rapidly accumulate metals. In contrast to most organic compounds, heavy metals were relatively easily depurated from tissues when animals were transplanted to clean locations or when the concentrations of contaminants declined (Okazaki and Panietz, 1981). Despite the ability to remove metals from tissues or to detoxify and isolate them from metabolic processes (see the review by Rainbow, 1995), heavy metals negatively affect growth of mussels and other bivalves (Manley et al., 1984; Din and Ahamad, 1995). Several metals have been associated with reduced growth as indicated by decreased rates of clearance (Widdows and Johnson, 1988) and/or increasing rates of respiration (Manley, 1983; Cheung and Cheung, 1995). Organic toxic compounds also accumulate rapidly in mussels. They negatively affect feeding (Bayne et al., 1982; Widdows et al., 1990) by reducing ciliary activity (Donkin et al., 1997). They also increase respiration (Bayne et al., 1982; Widdows et al., 1990), but generally have no effect on absorption efficiency (but see Bayne et al., 1982). For a review of the effects of organic contaminants on physiological energetics, see Widdows and Donkin (1991). Among other things, the ability of mussels to accumulate and concentrate heavy metals and hydrocarbons makes them useful as biomonitors of pollution (Phillips, 1977; Rainbow, 1995).
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Heavy metals are often accumulated in sediments and can be redissolved in the overlying water during periods of turbulence or as a result of biological activities. Generally, loads of heavy metal contaminants are largest in the small particles of the sediment for two main reasons: (1) their relatively large surface area and (2) the strong chemical binding properties of clay particles (Fo¨rstner et al., 1982). The sediments in bays in Sydney Harbour (Sydney, NSW, Australia) consist of 50 –90% of clay and mud ( < 63 Am), particularly in the upper ends of embayments (Irvine and Birch, 1998). In shallow parts of these embayments, resuspension of clay and mud is common under certain weather conditions (Taylor and Birch, 1999; Hellou et al., 2002) and ingestion of contaminants by filter-feeding bivalves is therefore likely. Polycyclic aromatic hydrocarbons (PAHs) are also common contaminants in Sydney Harbour. With few exceptions, levels of contamination are similar throughout Sydney Harbour. Although the concentration of these hydrocarbons is among the largest reported for estuaries around the world (Birch and Taylor, 2000), adverse biological effects are thought to occur only occasionally (McCready et al., 2000). One aim of this study was to compare the SfG of mussels (Mytilus sp.) living in areas contaminated with heavy metals and/or hydrocarbons with that of mussels living in nonor less contaminated, control sites. Blackwattle Bay and Darling Harbour (Cockle Bay) are among the embayments in Sydney Harbour containing the largest concentrations of heavy metals in the < 63 Am size-class of the surface sediment, especially in those parts of the embayments furthest away from the main river (Birch and Taylor, 1999). The metals thought to have a possible detrimental or toxic concentration in these embayments are Cu, Zn, Pb and Cd. In Blackwattle Bay, Cu, Zn and Cd are at 50 times the background concentration (Taylor and Birch, 1999). Concentrations of hydrocarbons are also large in Blackwattle Bay and Darling Harbour (Birch and Taylor, 2000). Different groups of contaminants can have antagonistic (Stro¨mgren, 1986) or synergistic or additive (Widdows and Johnson, 1988; Widdows and Donkin, 1992; Donkin et al., 1997) effects on the health of animals. This means that a single compound may not necessarily have a detectable effect or has only a small effect, but, in combination with one or more others, it can have a significant negative effect on physiological processes. The combination of a mixture of heavy metals and hydrocarbons in Blackwattle Bay and Cockle Bay and the fact that resuspension of sediments occurs regularly, potentially increasing the rate of uptake of contaminants by bivalves (Harrison, 1979), makes these bays suitable to study possible effects on biological processes. Concentrations of contaminants are significantly smaller towards the main channel of Port Jackson and (supposedly unimpacted) control locations could be found in this area. By comparing the SfG of mussels living in contaminated locations with that of mussels living in ‘clean’ control locations, the first hypothesis we tested was that the SfG of mussels living in impacted locations would be less than that of mussels living in ‘clean’ control sites. If the SfG of mussels in a contaminated location is smaller than that of mussels living in an uncontaminated location, two models can explain these observations. The first model is that the SfG of mussels in contaminated locations is simply reduced by the contaminants. The second model is that the mussels with reduced SfG are the only ones to survive in contaminated areas. From these models, a number of hypotheses can be formulated and
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they need to be tested in order to find out which model is most realistic. The first hypothesis, following from the first model, is that if mussels from a contaminated location were taken to an uncontaminated location, their SfG would increase and match that of mussels living in that uncontaminated location. Moreover, if mussels from an uncontaminated location were taken to a contaminated location, their SfG would reduce and match that of that of mussels living in that contaminated location. The second hypothesis, from the second model, is that if mussels from a contaminated location were moved to an uncontaminated location, their SfG would not change, but would remain similar to what it was at the place of origin. Also, if mussels were taken from an uncontaminated to a contaminated location, their SfG would not change, but would continue to match that of mussels living at the place of origin. To test the two hypotheses, the following groups of mussels need to be included in an experiment and their SfG determined: mussels at an uncontaminated location, mussels at a contaminated location, mussels taken from an uncontaminated to a contaminated location and mussels taken from a contaminated to an uncontaminated location. Transplanting mussels from one place to another necessitates that they are disturbed and moved. Moving animals from one place to another can change their physiology or behaviour, thereby confounding results (Chapman, 1986) and disturbing animals can reduce SfG considerably (unpublished data). Therefore, treatments to estimate effects of moving and disturbing animals need to be included. To control for any effect of moving and establishing mussels, mussels must be translocated from an uncontaminated to another uncontaminated location and from a contaminated location to another contaminated location. To control for any effect of disturbance itself, mussels from uncontaminated and from contaminated locations must be disturbed like the other experimental groups, but returned to their location of origin. The prediction concerning the control treatments was that they would not have any effect on SfG and that mussels from the translocation and disturbance treatment would have a similar SfG to those of the undisturbed mussels at the place of origin. Thus, if mussels are to be reciprocally transplanted between uncontaminated and contaminated locations, the following eight treatments must be examined: (1) undisturbed mussels at a contaminated location; (2) mussels transplanted from a contaminated to an uncontaminated location; (3) mussels translocated to another contaminated location; (4) disturbed mussels at the contaminated location; (5) undisturbed mussels at an uncontaminated location; (6) mussels transplanted from an uncontaminated to a contaminated location; (7) mussels translocated to another uncontaminated location; and (8) disturbed mussels at the uncontaminated location. The concept of a (reciprocal) transplant experiment is not new and is regularly used in the fields of ecology, toxicology and aquaculture (e.g. Theisen, 1982; Dickie et al., 1984; Widdows et al., 1984; Mallet et al., 1987, 1990; Mallet and Carver, 1989; Scanes, 1993; Myrand and Gaudreault, 1995; Iglesias et al., 1996). Except for the study of Scanes (1993), all those studies lack the proper controls as designated by Chapman (1986) and Underwood and Peterson (1988), i.e. the tests for evaluating the disturbance (handling) of experimental animals and for the evaluation of moving of animals out of their habitat to a new habitat. What is new and innovative in this study is the inclusion of proper control treatments. This paper describes an experimental transplantation and considers the problems for interpretation where some of the necessary controls are lacking.
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2. Materials and methods 2.1. Experimental sites and treatments Two locations in Sydney Harbour (33j52VS, 151j12VE), were chosen for this experiment, Blackwattle Bay (BWB) to represent a contaminated location and Mort Bay (MB) to represent a relatively uncontaminated location (Fig. 1). At each location, physiological components of SfG were measured for undisturbed mussels (undisturbed
Fig. 1. Map of Sydney Harbour (Sydney, NSW Australia) and the locations used in this experiment. BWB = Blackwattle Bay, supposedly polluted location; MB = Mort Bay, supposedly ‘clean’ control location; DH = Darling Harbour, translocation control for BWB; and LB = Lavender Bay, translocation control for MB.
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treatment, U) and for groups of mussels transplanted (transplant treatment, TP) from Blackwattle Bay to Mort Bay and vice versa. At each location, groups of mussels were disturbed (disturbance treatment, D) and returned to their place of origin. To control for moving the animals (translocation treatment, TL), two additional locations were selected. According to published data (Taylor and Birch, 1999; Birch and Taylor, 2000), Darling Harbour (DH) had similar levels of contaminants to those in BWB and could serve as a second contaminated location. Lavender Bay (LB) had levels of contamination comparable to those of MB and was, therefore, used as a second relatively uncontaminated bay (Fig. 1). In total, there were thus eight treatments: two undisturbed treatments (BWB; MB), two disturbed treatments (BWB to BWB; MB to MB) and two translocated treatments (BWB to DH; MB to LB) and two transplantations (BWB to MB; MB to BWB). To achieve replication, within each location, two sites 25 m apart were selected. 2.2. Collection and handling of mussels At each of the two sites at BWB and MB, clumps of mussels were collected from the sides of floating jetties. Clumps were carefully removed with a large paint scraper and transferred to the laboratory where mussels with a shell-length of 4– 5 cm were selected for the experiment and carefully removed from the clumps by cutting the byssal threads with a pair of scissors. The shells were carefully cleaned and fouling organisms removed. Shells were air-dried and tagged with shellfish tags using Superglue, then placed in an aquarium tank with 450 l seawater for 3 days to recover from handling. Ten tagged mussels from the same site of origin were placed into each of three cages. One cage was placed back at the site of origin (disturbed treatment), one cage was placed at one of the sites of the location serving as a control for moving mussels (translocation control) and one cage was placed at one of the sites at the location of transplantation. The cylindrical cages were made from polypropylene mesh (mesh size 5 5 mm) with a diameter of 10 cm and a height of 20 cm. The cylinders were closed with polypropylene lids. All cages were placed at the sites on 18 June 2001. Thus, at each of two sites in BWB, there was one cage with mussels transplanted from one of the sites from MB, one cage with mussels from the disturbed treatment and undisturbed mussels. At each of the two sites at MB, there was one cage containing mussels from one of the sites at BWB, one cage with mussels from the disturbed treatment and undisturbed mussels. At each of the two sites at DH, there was one cage translocated from one of the two sites of BWB, and at each of the two sites at LB, there was one cage translocated from one of the two sites of MB. For physiological measurements, animals were collected starting at 24 September 2001. Every second day, animals were randomly chosen from each of the eight groups. Because the mussels were always submerged, cages or clumps (undisturbed treatment) were removed from the jetty and transferred under water to buckets. The buckets were then transported to the laboratory. Cages were always kept under water. In the laboratory, cages were transferred to an aquarium containing 450 l of recirculating seawater. From each group, five tagged or untagged (undisturbed
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treatment) individuals were carefully removed from the cages or clumps keeping them submerged all the time. Fouling organisms (if any) were very carefully removed. During the next 2 days, five animals from each group were used for physiological measurement making sure that animals were only used in one measurement (rate of respiration or clearance) per day. The mussels were fed twice a day with a mixture of algae (see below) making sure that the concentration of algae never exceeded 35,000 cells ml 1. 2.3. Physiological measurements In order to calculate SfG, the following variables were measured for each individual: rates of respiration (RR or oxygen consumption) and clearance (CR or the rate by which particles were removed from the water) and efficiency of absorption (AE or the efficiency by which nutrients were absorbed). 2.3.1. Food Mussels were fed with a mixture of two species of algae, Nannochloropsis sp. and Tetraselmis sp. Frozen algal paste (Premium 3600 Instant Algae) was obtained from Reed Mariculture and did not contain any cryopreservatives. From the paste, a stock solution containing 1.75 108 cells ml 1 was prepared, with a volume ratio of 10 Nannochloropsis/1 Tetraselmis. This stock solution was made fresh every 2 days. To determine the amount of particulate organic material (POM) per 10,000 algae, 10 ml of algal stock was transferred to each of six centrifuge tubes, centrifuged for 10 min with a Sanyo Harrier 18/80 refrigerated centrifuge. The supernatant was decanted and the pellet redissolved in 5 ml 3.2% iso-osmotic ammonium-formate to remove salt. The samples were centrifuged at 6000 rpm for 10 min and the supernatant decanted. The pellet was then transferred to porcelain crucibles and dried for 48 h at 80 jC, a period and temperature sufficient to remove all ammonium-formate and to dry the samples to constant weight. They were transferred to a desiccator, cooled to room temperature and weighed. Then the samples were ashed overnight at 540 jC. The oven was switched off in the morning and the samples were transferred to a desiccator when they still had a temperature of about 200 jC. They were cooled to room temperature and weighed. The loss of weight during incineration was due to the loss of organic material (POM). All weighings were done to the nearest 0.01 mg with a Sartorius R200D precision balance. POM amounted to 1.7 10 4 mg per 10,000 algae. 2.3.2. Rate of respiration (RR) and respired energy (R) Consumption of oxygen (Amol h 1) of each individual was measured as described by Bayne (1999). Using six respirometers simultaneously (a run), RR was measured from five individual mussels and one blank (no animal). Animals that remained closed within the respirometer and from which the consumption of oxygen would be severely underestimated were excluded from further analysis. Each run contained a random mixture of mussels of different groups. After consumption of oxygen was measured, the animals were returned to the aquarium tank and placed into a small container in
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order to collect faeces for determination of the absorption efficiency. For each individual, the amount of respired energy (R) was calculated by multiplying RR by the energy content of 1 Amol O2, 0.450 J Amol 1 (Gnaiger, 1983). 2.3.3. Rate of clearance (CR) and consumed energy (C) For each mussel, rate of clearance was calculated according the method of Coughlan (1969) and as described by Bayne et al. (1999). The number of cells used in other studies is generally about 30,000 cells ml 1. We used the small Nannochloropsis (2 Am) rather than the generally used larger Isochrysis (5 Am) and, therefore, used a larger number of cells, about 55,000 cells ml 1 (POM = 0.925 mg l 1). Production of pseudofaeces at this concentration was observed only occasionally and, when it happened, the animals were excluded from further analyses. After the rate of clearance was measured, the animals were returned to the aquarium tank and placed into a small container in order to collect faeces for the determination of the absorption efficiency. The amount of ingested or consumed energy (C) was calculated for each individual by multiplying CR by the amount of organic material (POM) per litre in each bucket at the beginning of each run and assuming an energy content of algal material of 23 J mg 1 (Widdows and Johnson, 1988; Widdows et al., 1990). 2.3.4. Efficiency of absorption (AE) Faeces from individual mussels were collected, making sure that no pseudofaeces were produced and mixed with the faeces (Iglesias et al., 1998). At least two samples from each individual were collected whenever sufficient material was produced either in the aquarium, the oxygen-consumption chamber or the bucket used for the determination of the rate of clearance. Faecal pellets were transferred to centrifuge tubes and treated as described for the determination of POM. After centrifuging, however, pellets were transferred to pre-weighed (W1) platinum crucibles. Food was pipetted directly from the stock solution into the centrifuge tubes and treated as described for faeces. Each day, four replicate samples of food were taken. Crucibles were dried for 48 h at 80 jC, transferred to a desiccator, cooled to room temperature and weighed (W2). Then the samples were ashed overnight at 540 jC, cooled to room temperature in a desiccator and weighed (W3). The relative amount of organic material of each sample {(W2 W3)/(W2 W1)} was then calculated. Absorption efficiency was calculated according the method of Conover (1966). 2.3.5. Correction of rates to a standard body weight To correct for differences in physiological rates caused by differences in body weight among mussels, all physiological rates were converted to a standard rate according the allometric relationship described by Bayne and Newell (1983) and Bayne et al. (1999). For RR and CR, a value of b = 0.66 was used. After physiological variables were measured, the ash-free dry mass of each mussel was determined. Shells were opened and the tissues transferred to porcelain crucibles. They were dried for 48 h, cooled in a desiccator and weighed. Subsequently they were ashed, cooled in a desiccator and weighed again. The AFDM was the weight loss during incineration. The average AFDM per mussel (n = 131 mussels) was 0.298 F 0.069 g. The standardised weight (Wst) was set at 0.300 g.
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2.3.6. Calculation of SfG The scope for growth (SfG) was calculated as: SfG ¼ A R; where A is the absorbed energy and defined as C AE (consumed energy absorption efficiency), and R is respired energy.
3. Results In BWB and MB, significant effects of treatment on the ln-transformed rate of clearance (CR) were observed (Table 1). A-posteriori tests revealed that the CR of undisturbed mussels at BWB was significantly slower than that of the control and transplant treatments, which were similar (Fig. 2A). The undisturbed mussels at Mort Bay had a much faster CR than did the undisturbed mussels at BWB. Mussels transplanted from BWB to MB (TPBWB) had a similar CR to that of disturbed mussels at MB, but significantly slower than all other treatments, which were not statistically different (Fig. 2B).
Table 1 Analyses of differences among treatments (U, D, TL and TP) on rate of clearance (CR), efficiency of absorption (AE), rate of respiration (RR) and scope for growth (SfG) of mussels living at or originating from Blackwattle Bay (prediction 1) and Mort Bay (prediction 2) Source of variation
df
Prediction 1
Prediction 2
Mean square
F-ratio
P
Mean square
F-ratio
P
Ln-transformed CR Treatment Site within treatment Residual
4 5 40
3.37 0.16 0.13
21.70 1.17
< 0.01 >0.30
0.67 0.09 0.06
7.55 0.93
< 0.05 >0.45
AE Treatment Site within treatment Residual
4 5 40
0.02 0.01 0.01
1.38 1.39
>0.35 >0.20
0.02 0.02 0.01
0.72 1.93
>0.60 >0.10
Ln-transformed RR Treatment Site within treatment Residual
4 5 40
0.02 0.20 0.04
0.08 4.96
>0.95 < 0.01
0.11 0.12 0.08
0.95 1.54
>0.50 >0.15
SfG Treatment Site within treatment Residual
4 5 40
15.35 0.62
< 0.01 >0.65
1.98 1.56
>0.20 >0.15
2809 183 296
1332 673 431
Significant effects are in bold. In all cases, except for SfG (prediction 1, see text) where P < 0.01 was used to identify significant differences, Cochran’s test for homogeneity of variances was not significant ( P >0.05).
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Fig. 2. Mean ( F S.E.; n = 10) rates of clearance (CR, A and B), efficiency of absorption (AE, C and D), rate of respiration (RR, E and F) and scope for growth (SfG, G and H) of mussels of different treatments (Undisturbed (U), Disturbed (D), Translocation (TL) and Transplantation (TP)). Mussels were living at or originating from Blackwattle Bay (BWB, left-hand graphs and open bars) or Mort Bay (MB, right-hand graphs and dashed bars).
No significant effects of treatment on the absorption efficiency were observed (Table 1; Figs. 2 C and D). The average value of AE amounted to 0.78 F 0.01 (mean F S.E.). Mussels living in or originating from BWB all had similar rates of respiration (Fig. 2E). There were, however, differences between sites within location, but, because we had no hypothesis about site-differences and because site was a random factor in the experimental design, these differences will not be explored any further. Differences in rate of respiration between the groups of mussels living at or originating from MB (Fig. 2E) were larger, but not significantly so, than those for BWB (Table 1).
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Scope for growth showed a similar pattern to that observed for CR (compare Figs. 2A and B with Figs. 2G and H). The SfG for undisturbed mussels at BWB (UBWB) was significantly smaller than that for all other groups living at or originating from BWB (Fig. 2G). As for CR, the SfG of these latter groups were not statistically different (Table 1). The pattern in SfG observed for mussels living at or originating from MB (Fig. 2H) was similar to that of CR (Fig. 2B) although the difference among the translocation treatment and all others seems to be larger. Differences were, however, not statistically significant (Table 1).
4. Discussion 4.1. General comments on physiological variables Significant effects of treatment on rate of respiration (RR) and clearance (CR) were observed, but not on efficiency of absorption (AE). This is in accordance with the findings presented in most environmental studies (see references in Introduction for a selection of a small number of a vast body of papers). Generally, the most contaminant or stresssensitive variable is CR followed by RR. This pattern could also be observed in the results presented in this paper; the largest differences among groups were observed for CR and the resulting SfG showed a similar pattern to that for CR, suggesting that this is the main factor determining differences in SfG. The sensitivity of CR to environmental stresses is the reason that measuring CR alone has been proposed (Donkin et al., 1997). It is known that the main factor affecting AE of bivalves is the amount and quality of food; the more food and the poorer the quality the smaller is AE (e.g. Bayne et al., 1993; Cranford, 1995; Albentosa et al., 1996; Iglesias et al., 1996; Barille´ et al., 1997). In contrast, AE is generally insensitive to contaminants. The average value for AE was 0.78 F 0.01 (mean F S.E.), a relatively large value associated with excellent availability and good quality of food. In the field, these values are, generally, only observed during spring algal blooms (Bayne and Widdows, 1978; Loo, 1992). Because the gut-passage time of food in mussels is short, only a few hours, and the time to respond to a change in quality of food is also short (Cranford, 1995), in this study, the large AE value most likely reflects the quality of the food used in our measurements and can, therefore, not be considered to reflect field-values. Comparing the values for SfG found in this study with those published by others is difficult for a number of reasons, the main one being the difference in quality and quantity of food available or used in different studies. The largest value we found in literature was 87 J g 1 h 1 (Gardner, 2000); all other values were much smaller (e.g. Bayne and Widdows, 1978; Widdows et al., 1984, 2002). Considering that we calculated SfG for mussels with a standardised weight of 0.3 g, our values were large and the mussels (except UBWB) appeared only to have experienced mild environmental stress. 4.2. Effects of sediment-bound contaminants and evaluation of control treatments Comparing the scope for growth of mussels living at different locations and relating it to levels of contaminants is done in many studies. Here, SfG and its components in
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mussels at the contaminated location of Blackwattle Bay were compared to those of mussels living at Mort Bay, a relatively uncontaminated location. If only the SfG of mussels living at these locations had been compared, the results would have confirmed the hypothesis that the SfG from mussels at BWB would be smaller than that of mussels living at MB. The SfG for the undisturbed treatment at BWB was about five times smaller than that at MB (Figs. 2G and H). This difference was mainly due to a slower rate of clearance (Figs. 2A and B) and a faster rate of respiration (Figs. 2E and F). As expected, absorption efficiency was not affected. These differences, however, could also have been caused by innate (possibly genetic) differences between the two populations. To investigate this, a reciprocal transplant (including proper control treatments) is probably the best procedure. If, after a period of exposure of transplanted individuals to a different habitat, the variables of interest are not similar to those of local conspecifics, it can be concluded that either differences have a genetic origin or that physiological adaptation is constrained, for example, by unknown biochemical and/or behavioural traits. Mussels, however, have proved in other studies to be quite adaptable (Widdows and Donkin, 1992). An alternative explanation is that the period of exposure was not long enough (Widdows et al., 1984). The reciprocal transplant has been used in a number of studies. Some have identified genetic differences between populations (Dickie et al., 1984; Mallet et al., 1987; Johannesson et al., 1990; Kautsky et al., 1990; Myrand and Gaudreault, 1995), others have not (Theisen, 1982; Scanes, 1993; Iglesias et al., 1996) or have suggested alternative explanations for observed differences (Widdows et al., 1984). To control for (genetic) differences between the BWB and MB populations, a reciprocal transplant was done here. The hypothesis tested was that animals transplanted to another location would show a similar CR, AE, RR and SfG as the local mussels. This hypothesis was only partly supported. Mussels transplanted from BWB to MB had a larger CR and SfG and a similar AE and RR than the mussels at the place of origin. Although AE, RR and SfG were not significantly different, CR was smaller than that of mussels living at MB. The variables of the reciprocal transplant, from MB to BWB were, however, similar to those of mussels at MB (the location of origin) and thus significantly larger than those of the mussels living at the location to which they were transplanted (BWB). Mussels from BWB might be quicker to respond to a change in habitat and could adjust faster than the mussels from MB (Widdows et al., 1984), but these differences could also reflect differences between rates of uptake and of depuration of contaminants (Widdows and Donkin, 1992). The fact that mussels from BWB were able to adjust their physiology towards the values of the mussels at MB indicated that mussels at BWB were affected by environmental factors. Their SfG improved after transplantation to a supposedly cleaner location. The lack of a response of the MB mussels transplanted to BWB did not confirm nor reject this statement. To control for disturbance of mussels due to experimental procedures, a treatment generally neglected in most studies was added, viz. the disturbance treatment D. In BWB, CR and SfG increased compared to the undisturbed treatment (AE and RR were again not statistically different) and became similar to all other experimental treatments. CR and SfG from this disturbance-control treatment at MB became significantly smaller. The only explanation for this pattern is that the CR and SfG of undisturbed mussels at BWB were
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reduced by factors not considered in the experimental treatments. A possible source of disturbance is the presence of crabs and fish in the ‘undisturbed’ clumps of mussels causing the mussels to reduce feeding-rate by full or partial closure of their shells. Although not completely comparable, an effect like this has been reported. Growth of pea crab-infested mussels was limited mainly due to a reduced rate of feeding (Bierbaum and Shumway, 1988). A reason for the reduced CR and SfG at BWB and not at MB is that clumps of mussels at BWB were much denser and larger than at MB and the mussels were severely overgrown with barnacles, sponges, seaweed and encrusting algae. These factors can reduce growth considerably (Dittman and Robles, 1991; Svane and Ompi, 1993; Ricciardi et al., 1995). Because experimental mussels were thoroughly cleaned and fouling organisms removed before they were placed in cages, the possible disturbing effects on caged mussels would have been absent or, at least, less severe. The result was that the removal of fouling organisms from the caged mussels at BWB made them similar to the relatively unfouled mussels at MB. Comparing SfG of mussels from which fouling organisms have been removed could test this proposition. Treatments would include cleaned mussels, mussels overgrown by fouling organisms and controls for cleaning. The disturbance of mussels at MB resulted in a significantly reduced rate of clearance (Fig. 2B). Although this was not reflected in a significantly reduced SfG, it might be possible that handling and caging mussels indeed negatively affect physiological traits. If so, the effect of fouling organisms in BWB was even larger than described in the previous paragraph. The last added control treatment was to control for the effect of removing animals from their habitat to a similar habitat somewhere else. It might be that only this fact causes a physiological change, rather than factors associated with a different habitat (as proposed in the hypothesis; Chapman, 1986). Therefore, groups of mussels were translocated from the experimental locations to similar locations. Finding the right location for a translocation is, however, difficult because there is no way to test the assumption that the experimental locations are similar in all aspects. That may be the reason for the observed increased CR and SfG from the groups of mussels translocated from MB to Lavender Bay (Figs. 2B and H). Darling Harbour appeared to be similar to BWB as the translocation treatment was not different from other treatments (except the undisturbed treatment, but see previous paragraph). Therefore, the interpretation of the effects of translocation is that observed differences among treatments were not a consequence of moving animals from their original location to another. To summarise, had the proper controls not been included, our conclusion would have been that there were differences between the physiology of mussels at the presumably contaminated location of BWB and the presumably uncontaminated location of MB with, as predicted, a smaller SfG at BWB than at MB. The controls, particularly the control for disturbance, however, made clear that the differences were not caused by the differences among locations, such as presumed differences in the concentration of contaminants in the sediment. Differences must have been due to other factors, probably differences in the physical disturbance of mussels between the locations. Generally, but not always (Scanes, 1993), proper controls for moving and disturbing experimental animals have been lacking (Widdows et al., 1984; Iglesias et al., 1996; Nasci et al., 1999; Webb and Keough, 2002) or not replicated (Widdows et al., 1980– 1981,
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1990; Bayne et al., 1982; Din and Ahamad, 1995). This does not, however, necessarily imply that conclusions from such studies were invalid. To unconfound the effects of moving and disturbance, proper controls are essential in studies like the current one and the final conclusion is totally dependent on them (Chapman, 1986; Underwood and Peterson, 1988). Therefore, the statement that a transplantation procedure is a valuable monitoring tool for environments (Nasci et al., 1999) is only true if the right control treatments are included in the study.
Acknowledgements Thanks to Shannon Long, Michelle Button, Amy Palmer and Craig Myers for help during measurements and Bill Maher and Gavin Birch for their help and advice. We are much indebted to Bryan Skepper from Sydney Fish Markets, Sebastian Schmid from Sydney Aquarium, Pamela Wood from ‘‘The Anchorage’’ and David Ashton for their permission to connect cages to their pontoons. This study was supported by funds from the Australian Research Council through the Special Centres Program. [SS]
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Appropriate experimental design for transplanting mussels (Mytilus sp.) in analyses of environmental stress: an example in Sydney Harbour (Australia) P.J.C. Honkoop *, B.L. Bayne, A.J. Underwood, S. Svensson Centre for Research on Ecological Impacts of Coastal Cities, Marine Ecology Laboratories A11, University of Sydney, Sydney, NSW 2006, Australia Received 11 December 2002; received in revised form 28 July 2003; accepted 3 August 2003 This paper is dedicated to Dr. Peter Donkin.
Abstract Some locations in Sydney Harbour (Sydney, Australia) contain large amounts of contaminants (heavy metals and hydrocarbons), sometimes in concentrations thought to affect biological systems. In order to estimate effects of sediment-bound contaminants on the physiology of organisms living above the sediment, the rates of clearance and respiration and the efficiency of absorption of mussels, Mytilus sp., living in a contaminated location were measured, the scope for growth (SfG) was calculated and compared to that of mussels living in uncontaminated locations. Two different models were proposed to explain expected differences. The first was that the contaminants at the impacted location reduced the SfG of local mussels; the second was that at the contaminated location only those mussels survived that had a small SfG (genetic differences between populations might be a reason for differential survival). To test which model applied, mussels were transplanted between the contaminated and uncontaminated locations. Moving and disturbing mussels (handling, tagging and caging) required the inclusion of two control treatments. These treatments were essential for a proper evaluation of the results but have generally not been included in similar studies. Effects of moving were estimated by translocating mussels from the uncontaminated and from the contaminated location to similar locations. To estimate effects of disturbance, mussels in the experimental locations were given the same treatment as the experimental mussels, but were returned to the place of origin. It was predicted that translocating and disturbing mussels would have no effect on the SfG, which would be similar to that of mussels at the place of origin. As expected, SfG was smaller (because rate
* Corresponding author. Present address: Department of Marine Ecology and Evolution, Royal Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, Texel, The Netherlands. Tel.: +31-222-369492; fax: +31-222-319674. E-mail address: [email protected] (P.J.C. Honkoop). 0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2003.08.001
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of clearance was reduced) in undisturbed mussels at the contaminated location compared with those of mussels at the uncontaminated location. Because there were significant effects of disturbance on the SfG of mussels at the contaminated location, it was concluded that this difference was not caused by differences between the amounts of contaminants in the two locations, but was caused by other confounding factors (physical disturbance by crabs and fouling organisms). That the interpretation of the results would have been different if proper controls were not included is discussed in this paper and the importance of proper experimental controls is stressed. D 2003 Elsevier B.V. All rights reserved. Keywords: Transplant experiment; Control treatments; Contaminants; Scope for growth; Environmental stress; Mussels
1. Introduction Mussels (Mytilus spp.) are commonly used as bio-indicators to monitor contamination by chemicals, sedimentation, etc., in marine areas (e.g. Martin, 1985; Widdows et al., 1990, 1995). For various reasons, mussels are particularly suited to use as test organisms in so-called Mussel Watch Programs (Widdows and Donkin, 1992; Sericano et al., 1995). Because contaminants affect physiological processes, physiological variables of mussels are measured to calculate their energy budget and to estimate the potential toxic effects of contaminants on any of the physiological traits. Measuring these variables and combining them into a single energy equation was first applied to bivalves by Widdows and Bayne (1971) and became rapidly very popular under the name scope for growth (SfG, Bayne and Widdows, 1978). If the gain of energy per unit time is negative or positive but small, it is thought that animals are stressed. If it is large, it is thought that the animals are in good condition and have energy available for growth and reproduction. Mussels rapidly accumulate metals. In contrast to most organic compounds, heavy metals were relatively easily depurated from tissues when animals were transplanted to clean locations or when the concentrations of contaminants declined (Okazaki and Panietz, 1981). Despite the ability to remove metals from tissues or to detoxify and isolate them from metabolic processes (see the review by Rainbow, 1995), heavy metals negatively affect growth of mussels and other bivalves (Manley et al., 1984; Din and Ahamad, 1995). Several metals have been associated with reduced growth as indicated by decreased rates of clearance (Widdows and Johnson, 1988) and/or increasing rates of respiration (Manley, 1983; Cheung and Cheung, 1995). Organic toxic compounds also accumulate rapidly in mussels. They negatively affect feeding (Bayne et al., 1982; Widdows et al., 1990) by reducing ciliary activity (Donkin et al., 1997). They also increase respiration (Bayne et al., 1982; Widdows et al., 1990), but generally have no effect on absorption efficiency (but see Bayne et al., 1982). For a review of the effects of organic contaminants on physiological energetics, see Widdows and Donkin (1991). Among other things, the ability of mussels to accumulate and concentrate heavy metals and hydrocarbons makes them useful as biomonitors of pollution (Phillips, 1977; Rainbow, 1995).
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Heavy metals are often accumulated in sediments and can be redissolved in the overlying water during periods of turbulence or as a result of biological activities. Generally, loads of heavy metal contaminants are largest in the small particles of the sediment for two main reasons: (1) their relatively large surface area and (2) the strong chemical binding properties of clay particles (Fo¨rstner et al., 1982). The sediments in bays in Sydney Harbour (Sydney, NSW, Australia) consist of 50 –90% of clay and mud ( < 63 Am), particularly in the upper ends of embayments (Irvine and Birch, 1998). In shallow parts of these embayments, resuspension of clay and mud is common under certain weather conditions (Taylor and Birch, 1999; Hellou et al., 2002) and ingestion of contaminants by filter-feeding bivalves is therefore likely. Polycyclic aromatic hydrocarbons (PAHs) are also common contaminants in Sydney Harbour. With few exceptions, levels of contamination are similar throughout Sydney Harbour. Although the concentration of these hydrocarbons is among the largest reported for estuaries around the world (Birch and Taylor, 2000), adverse biological effects are thought to occur only occasionally (McCready et al., 2000). One aim of this study was to compare the SfG of mussels (Mytilus sp.) living in areas contaminated with heavy metals and/or hydrocarbons with that of mussels living in nonor less contaminated, control sites. Blackwattle Bay and Darling Harbour (Cockle Bay) are among the embayments in Sydney Harbour containing the largest concentrations of heavy metals in the < 63 Am size-class of the surface sediment, especially in those parts of the embayments furthest away from the main river (Birch and Taylor, 1999). The metals thought to have a possible detrimental or toxic concentration in these embayments are Cu, Zn, Pb and Cd. In Blackwattle Bay, Cu, Zn and Cd are at 50 times the background concentration (Taylor and Birch, 1999). Concentrations of hydrocarbons are also large in Blackwattle Bay and Darling Harbour (Birch and Taylor, 2000). Different groups of contaminants can have antagonistic (Stro¨mgren, 1986) or synergistic or additive (Widdows and Johnson, 1988; Widdows and Donkin, 1992; Donkin et al., 1997) effects on the health of animals. This means that a single compound may not necessarily have a detectable effect or has only a small effect, but, in combination with one or more others, it can have a significant negative effect on physiological processes. The combination of a mixture of heavy metals and hydrocarbons in Blackwattle Bay and Cockle Bay and the fact that resuspension of sediments occurs regularly, potentially increasing the rate of uptake of contaminants by bivalves (Harrison, 1979), makes these bays suitable to study possible effects on biological processes. Concentrations of contaminants are significantly smaller towards the main channel of Port Jackson and (supposedly unimpacted) control locations could be found in this area. By comparing the SfG of mussels living in contaminated locations with that of mussels living in ‘clean’ control locations, the first hypothesis we tested was that the SfG of mussels living in impacted locations would be less than that of mussels living in ‘clean’ control sites. If the SfG of mussels in a contaminated location is smaller than that of mussels living in an uncontaminated location, two models can explain these observations. The first model is that the SfG of mussels in contaminated locations is simply reduced by the contaminants. The second model is that the mussels with reduced SfG are the only ones to survive in contaminated areas. From these models, a number of hypotheses can be formulated and
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they need to be tested in order to find out which model is most realistic. The first hypothesis, following from the first model, is that if mussels from a contaminated location were taken to an uncontaminated location, their SfG would increase and match that of mussels living in that uncontaminated location. Moreover, if mussels from an uncontaminated location were taken to a contaminated location, their SfG would reduce and match that of that of mussels living in that contaminated location. The second hypothesis, from the second model, is that if mussels from a contaminated location were moved to an uncontaminated location, their SfG would not change, but would remain similar to what it was at the place of origin. Also, if mussels were taken from an uncontaminated to a contaminated location, their SfG would not change, but would continue to match that of mussels living at the place of origin. To test the two hypotheses, the following groups of mussels need to be included in an experiment and their SfG determined: mussels at an uncontaminated location, mussels at a contaminated location, mussels taken from an uncontaminated to a contaminated location and mussels taken from a contaminated to an uncontaminated location. Transplanting mussels from one place to another necessitates that they are disturbed and moved. Moving animals from one place to another can change their physiology or behaviour, thereby confounding results (Chapman, 1986) and disturbing animals can reduce SfG considerably (unpublished data). Therefore, treatments to estimate effects of moving and disturbing animals need to be included. To control for any effect of moving and establishing mussels, mussels must be translocated from an uncontaminated to another uncontaminated location and from a contaminated location to another contaminated location. To control for any effect of disturbance itself, mussels from uncontaminated and from contaminated locations must be disturbed like the other experimental groups, but returned to their location of origin. The prediction concerning the control treatments was that they would not have any effect on SfG and that mussels from the translocation and disturbance treatment would have a similar SfG to those of the undisturbed mussels at the place of origin. Thus, if mussels are to be reciprocally transplanted between uncontaminated and contaminated locations, the following eight treatments must be examined: (1) undisturbed mussels at a contaminated location; (2) mussels transplanted from a contaminated to an uncontaminated location; (3) mussels translocated to another contaminated location; (4) disturbed mussels at the contaminated location; (5) undisturbed mussels at an uncontaminated location; (6) mussels transplanted from an uncontaminated to a contaminated location; (7) mussels translocated to another uncontaminated location; and (8) disturbed mussels at the uncontaminated location. The concept of a (reciprocal) transplant experiment is not new and is regularly used in the fields of ecology, toxicology and aquaculture (e.g. Theisen, 1982; Dickie et al., 1984; Widdows et al., 1984; Mallet et al., 1987, 1990; Mallet and Carver, 1989; Scanes, 1993; Myrand and Gaudreault, 1995; Iglesias et al., 1996). Except for the study of Scanes (1993), all those studies lack the proper controls as designated by Chapman (1986) and Underwood and Peterson (1988), i.e. the tests for evaluating the disturbance (handling) of experimental animals and for the evaluation of moving of animals out of their habitat to a new habitat. What is new and innovative in this study is the inclusion of proper control treatments. This paper describes an experimental transplantation and considers the problems for interpretation where some of the necessary controls are lacking.
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2. Materials and methods 2.1. Experimental sites and treatments Two locations in Sydney Harbour (33j52VS, 151j12VE), were chosen for this experiment, Blackwattle Bay (BWB) to represent a contaminated location and Mort Bay (MB) to represent a relatively uncontaminated location (Fig. 1). At each location, physiological components of SfG were measured for undisturbed mussels (undisturbed
Fig. 1. Map of Sydney Harbour (Sydney, NSW Australia) and the locations used in this experiment. BWB = Blackwattle Bay, supposedly polluted location; MB = Mort Bay, supposedly ‘clean’ control location; DH = Darling Harbour, translocation control for BWB; and LB = Lavender Bay, translocation control for MB.
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treatment, U) and for groups of mussels transplanted (transplant treatment, TP) from Blackwattle Bay to Mort Bay and vice versa. At each location, groups of mussels were disturbed (disturbance treatment, D) and returned to their place of origin. To control for moving the animals (translocation treatment, TL), two additional locations were selected. According to published data (Taylor and Birch, 1999; Birch and Taylor, 2000), Darling Harbour (DH) had similar levels of contaminants to those in BWB and could serve as a second contaminated location. Lavender Bay (LB) had levels of contamination comparable to those of MB and was, therefore, used as a second relatively uncontaminated bay (Fig. 1). In total, there were thus eight treatments: two undisturbed treatments (BWB; MB), two disturbed treatments (BWB to BWB; MB to MB) and two translocated treatments (BWB to DH; MB to LB) and two transplantations (BWB to MB; MB to BWB). To achieve replication, within each location, two sites 25 m apart were selected. 2.2. Collection and handling of mussels At each of the two sites at BWB and MB, clumps of mussels were collected from the sides of floating jetties. Clumps were carefully removed with a large paint scraper and transferred to the laboratory where mussels with a shell-length of 4– 5 cm were selected for the experiment and carefully removed from the clumps by cutting the byssal threads with a pair of scissors. The shells were carefully cleaned and fouling organisms removed. Shells were air-dried and tagged with shellfish tags using Superglue, then placed in an aquarium tank with 450 l seawater for 3 days to recover from handling. Ten tagged mussels from the same site of origin were placed into each of three cages. One cage was placed back at the site of origin (disturbed treatment), one cage was placed at one of the sites of the location serving as a control for moving mussels (translocation control) and one cage was placed at one of the sites at the location of transplantation. The cylindrical cages were made from polypropylene mesh (mesh size 5 5 mm) with a diameter of 10 cm and a height of 20 cm. The cylinders were closed with polypropylene lids. All cages were placed at the sites on 18 June 2001. Thus, at each of two sites in BWB, there was one cage with mussels transplanted from one of the sites from MB, one cage with mussels from the disturbed treatment and undisturbed mussels. At each of the two sites at MB, there was one cage containing mussels from one of the sites at BWB, one cage with mussels from the disturbed treatment and undisturbed mussels. At each of the two sites at DH, there was one cage translocated from one of the two sites of BWB, and at each of the two sites at LB, there was one cage translocated from one of the two sites of MB. For physiological measurements, animals were collected starting at 24 September 2001. Every second day, animals were randomly chosen from each of the eight groups. Because the mussels were always submerged, cages or clumps (undisturbed treatment) were removed from the jetty and transferred under water to buckets. The buckets were then transported to the laboratory. Cages were always kept under water. In the laboratory, cages were transferred to an aquarium containing 450 l of recirculating seawater. From each group, five tagged or untagged (undisturbed
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treatment) individuals were carefully removed from the cages or clumps keeping them submerged all the time. Fouling organisms (if any) were very carefully removed. During the next 2 days, five animals from each group were used for physiological measurement making sure that animals were only used in one measurement (rate of respiration or clearance) per day. The mussels were fed twice a day with a mixture of algae (see below) making sure that the concentration of algae never exceeded 35,000 cells ml 1. 2.3. Physiological measurements In order to calculate SfG, the following variables were measured for each individual: rates of respiration (RR or oxygen consumption) and clearance (CR or the rate by which particles were removed from the water) and efficiency of absorption (AE or the efficiency by which nutrients were absorbed). 2.3.1. Food Mussels were fed with a mixture of two species of algae, Nannochloropsis sp. and Tetraselmis sp. Frozen algal paste (Premium 3600 Instant Algae) was obtained from Reed Mariculture and did not contain any cryopreservatives. From the paste, a stock solution containing 1.75 108 cells ml 1 was prepared, with a volume ratio of 10 Nannochloropsis/1 Tetraselmis. This stock solution was made fresh every 2 days. To determine the amount of particulate organic material (POM) per 10,000 algae, 10 ml of algal stock was transferred to each of six centrifuge tubes, centrifuged for 10 min with a Sanyo Harrier 18/80 refrigerated centrifuge. The supernatant was decanted and the pellet redissolved in 5 ml 3.2% iso-osmotic ammonium-formate to remove salt. The samples were centrifuged at 6000 rpm for 10 min and the supernatant decanted. The pellet was then transferred to porcelain crucibles and dried for 48 h at 80 jC, a period and temperature sufficient to remove all ammonium-formate and to dry the samples to constant weight. They were transferred to a desiccator, cooled to room temperature and weighed. Then the samples were ashed overnight at 540 jC. The oven was switched off in the morning and the samples were transferred to a desiccator when they still had a temperature of about 200 jC. They were cooled to room temperature and weighed. The loss of weight during incineration was due to the loss of organic material (POM). All weighings were done to the nearest 0.01 mg with a Sartorius R200D precision balance. POM amounted to 1.7 10 4 mg per 10,000 algae. 2.3.2. Rate of respiration (RR) and respired energy (R) Consumption of oxygen (Amol h 1) of each individual was measured as described by Bayne (1999). Using six respirometers simultaneously (a run), RR was measured from five individual mussels and one blank (no animal). Animals that remained closed within the respirometer and from which the consumption of oxygen would be severely underestimated were excluded from further analysis. Each run contained a random mixture of mussels of different groups. After consumption of oxygen was measured, the animals were returned to the aquarium tank and placed into a small container in
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order to collect faeces for determination of the absorption efficiency. For each individual, the amount of respired energy (R) was calculated by multiplying RR by the energy content of 1 Amol O2, 0.450 J Amol 1 (Gnaiger, 1983). 2.3.3. Rate of clearance (CR) and consumed energy (C) For each mussel, rate of clearance was calculated according the method of Coughlan (1969) and as described by Bayne et al. (1999). The number of cells used in other studies is generally about 30,000 cells ml 1. We used the small Nannochloropsis (2 Am) rather than the generally used larger Isochrysis (5 Am) and, therefore, used a larger number of cells, about 55,000 cells ml 1 (POM = 0.925 mg l 1). Production of pseudofaeces at this concentration was observed only occasionally and, when it happened, the animals were excluded from further analyses. After the rate of clearance was measured, the animals were returned to the aquarium tank and placed into a small container in order to collect faeces for the determination of the absorption efficiency. The amount of ingested or consumed energy (C) was calculated for each individual by multiplying CR by the amount of organic material (POM) per litre in each bucket at the beginning of each run and assuming an energy content of algal material of 23 J mg 1 (Widdows and Johnson, 1988; Widdows et al., 1990). 2.3.4. Efficiency of absorption (AE) Faeces from individual mussels were collected, making sure that no pseudofaeces were produced and mixed with the faeces (Iglesias et al., 1998). At least two samples from each individual were collected whenever sufficient material was produced either in the aquarium, the oxygen-consumption chamber or the bucket used for the determination of the rate of clearance. Faecal pellets were transferred to centrifuge tubes and treated as described for the determination of POM. After centrifuging, however, pellets were transferred to pre-weighed (W1) platinum crucibles. Food was pipetted directly from the stock solution into the centrifuge tubes and treated as described for faeces. Each day, four replicate samples of food were taken. Crucibles were dried for 48 h at 80 jC, transferred to a desiccator, cooled to room temperature and weighed (W2). Then the samples were ashed overnight at 540 jC, cooled to room temperature in a desiccator and weighed (W3). The relative amount of organic material of each sample {(W2 W3)/(W2 W1)} was then calculated. Absorption efficiency was calculated according the method of Conover (1966). 2.3.5. Correction of rates to a standard body weight To correct for differences in physiological rates caused by differences in body weight among mussels, all physiological rates were converted to a standard rate according the allometric relationship described by Bayne and Newell (1983) and Bayne et al. (1999). For RR and CR, a value of b = 0.66 was used. After physiological variables were measured, the ash-free dry mass of each mussel was determined. Shells were opened and the tissues transferred to porcelain crucibles. They were dried for 48 h, cooled in a desiccator and weighed. Subsequently they were ashed, cooled in a desiccator and weighed again. The AFDM was the weight loss during incineration. The average AFDM per mussel (n = 131 mussels) was 0.298 F 0.069 g. The standardised weight (Wst) was set at 0.300 g.
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2.3.6. Calculation of SfG The scope for growth (SfG) was calculated as: SfG ¼ A R; where A is the absorbed energy and defined as C AE (consumed energy absorption efficiency), and R is respired energy.
3. Results In BWB and MB, significant effects of treatment on the ln-transformed rate of clearance (CR) were observed (Table 1). A-posteriori tests revealed that the CR of undisturbed mussels at BWB was significantly slower than that of the control and transplant treatments, which were similar (Fig. 2A). The undisturbed mussels at Mort Bay had a much faster CR than did the undisturbed mussels at BWB. Mussels transplanted from BWB to MB (TPBWB) had a similar CR to that of disturbed mussels at MB, but significantly slower than all other treatments, which were not statistically different (Fig. 2B).
Table 1 Analyses of differences among treatments (U, D, TL and TP) on rate of clearance (CR), efficiency of absorption (AE), rate of respiration (RR) and scope for growth (SfG) of mussels living at or originating from Blackwattle Bay (prediction 1) and Mort Bay (prediction 2) Source of variation
df
Prediction 1
Prediction 2
Mean square
F-ratio
P
Mean square
F-ratio
P
Ln-transformed CR Treatment Site within treatment Residual
4 5 40
3.37 0.16 0.13
21.70 1.17
< 0.01 >0.30
0.67 0.09 0.06
7.55 0.93
< 0.05 >0.45
AE Treatment Site within treatment Residual
4 5 40
0.02 0.01 0.01
1.38 1.39
>0.35 >0.20
0.02 0.02 0.01
0.72 1.93
>0.60 >0.10
Ln-transformed RR Treatment Site within treatment Residual
4 5 40
0.02 0.20 0.04
0.08 4.96
>0.95 < 0.01
0.11 0.12 0.08
0.95 1.54
>0.50 >0.15
SfG Treatment Site within treatment Residual
4 5 40
15.35 0.62
< 0.01 >0.65
1.98 1.56
>0.20 >0.15
2809 183 296
1332 673 431
Significant effects are in bold. In all cases, except for SfG (prediction 1, see text) where P < 0.01 was used to identify significant differences, Cochran’s test for homogeneity of variances was not significant ( P >0.05).
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Fig. 2. Mean ( F S.E.; n = 10) rates of clearance (CR, A and B), efficiency of absorption (AE, C and D), rate of respiration (RR, E and F) and scope for growth (SfG, G and H) of mussels of different treatments (Undisturbed (U), Disturbed (D), Translocation (TL) and Transplantation (TP)). Mussels were living at or originating from Blackwattle Bay (BWB, left-hand graphs and open bars) or Mort Bay (MB, right-hand graphs and dashed bars).
No significant effects of treatment on the absorption efficiency were observed (Table 1; Figs. 2 C and D). The average value of AE amounted to 0.78 F 0.01 (mean F S.E.). Mussels living in or originating from BWB all had similar rates of respiration (Fig. 2E). There were, however, differences between sites within location, but, because we had no hypothesis about site-differences and because site was a random factor in the experimental design, these differences will not be explored any further. Differences in rate of respiration between the groups of mussels living at or originating from MB (Fig. 2E) were larger, but not significantly so, than those for BWB (Table 1).
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Scope for growth showed a similar pattern to that observed for CR (compare Figs. 2A and B with Figs. 2G and H). The SfG for undisturbed mussels at BWB (UBWB) was significantly smaller than that for all other groups living at or originating from BWB (Fig. 2G). As for CR, the SfG of these latter groups were not statistically different (Table 1). The pattern in SfG observed for mussels living at or originating from MB (Fig. 2H) was similar to that of CR (Fig. 2B) although the difference among the translocation treatment and all others seems to be larger. Differences were, however, not statistically significant (Table 1).
4. Discussion 4.1. General comments on physiological variables Significant effects of treatment on rate of respiration (RR) and clearance (CR) were observed, but not on efficiency of absorption (AE). This is in accordance with the findings presented in most environmental studies (see references in Introduction for a selection of a small number of a vast body of papers). Generally, the most contaminant or stresssensitive variable is CR followed by RR. This pattern could also be observed in the results presented in this paper; the largest differences among groups were observed for CR and the resulting SfG showed a similar pattern to that for CR, suggesting that this is the main factor determining differences in SfG. The sensitivity of CR to environmental stresses is the reason that measuring CR alone has been proposed (Donkin et al., 1997). It is known that the main factor affecting AE of bivalves is the amount and quality of food; the more food and the poorer the quality the smaller is AE (e.g. Bayne et al., 1993; Cranford, 1995; Albentosa et al., 1996; Iglesias et al., 1996; Barille´ et al., 1997). In contrast, AE is generally insensitive to contaminants. The average value for AE was 0.78 F 0.01 (mean F S.E.), a relatively large value associated with excellent availability and good quality of food. In the field, these values are, generally, only observed during spring algal blooms (Bayne and Widdows, 1978; Loo, 1992). Because the gut-passage time of food in mussels is short, only a few hours, and the time to respond to a change in quality of food is also short (Cranford, 1995), in this study, the large AE value most likely reflects the quality of the food used in our measurements and can, therefore, not be considered to reflect field-values. Comparing the values for SfG found in this study with those published by others is difficult for a number of reasons, the main one being the difference in quality and quantity of food available or used in different studies. The largest value we found in literature was 87 J g 1 h 1 (Gardner, 2000); all other values were much smaller (e.g. Bayne and Widdows, 1978; Widdows et al., 1984, 2002). Considering that we calculated SfG for mussels with a standardised weight of 0.3 g, our values were large and the mussels (except UBWB) appeared only to have experienced mild environmental stress. 4.2. Effects of sediment-bound contaminants and evaluation of control treatments Comparing the scope for growth of mussels living at different locations and relating it to levels of contaminants is done in many studies. Here, SfG and its components in
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mussels at the contaminated location of Blackwattle Bay were compared to those of mussels living at Mort Bay, a relatively uncontaminated location. If only the SfG of mussels living at these locations had been compared, the results would have confirmed the hypothesis that the SfG from mussels at BWB would be smaller than that of mussels living at MB. The SfG for the undisturbed treatment at BWB was about five times smaller than that at MB (Figs. 2G and H). This difference was mainly due to a slower rate of clearance (Figs. 2A and B) and a faster rate of respiration (Figs. 2E and F). As expected, absorption efficiency was not affected. These differences, however, could also have been caused by innate (possibly genetic) differences between the two populations. To investigate this, a reciprocal transplant (including proper control treatments) is probably the best procedure. If, after a period of exposure of transplanted individuals to a different habitat, the variables of interest are not similar to those of local conspecifics, it can be concluded that either differences have a genetic origin or that physiological adaptation is constrained, for example, by unknown biochemical and/or behavioural traits. Mussels, however, have proved in other studies to be quite adaptable (Widdows and Donkin, 1992). An alternative explanation is that the period of exposure was not long enough (Widdows et al., 1984). The reciprocal transplant has been used in a number of studies. Some have identified genetic differences between populations (Dickie et al., 1984; Mallet et al., 1987; Johannesson et al., 1990; Kautsky et al., 1990; Myrand and Gaudreault, 1995), others have not (Theisen, 1982; Scanes, 1993; Iglesias et al., 1996) or have suggested alternative explanations for observed differences (Widdows et al., 1984). To control for (genetic) differences between the BWB and MB populations, a reciprocal transplant was done here. The hypothesis tested was that animals transplanted to another location would show a similar CR, AE, RR and SfG as the local mussels. This hypothesis was only partly supported. Mussels transplanted from BWB to MB had a larger CR and SfG and a similar AE and RR than the mussels at the place of origin. Although AE, RR and SfG were not significantly different, CR was smaller than that of mussels living at MB. The variables of the reciprocal transplant, from MB to BWB were, however, similar to those of mussels at MB (the location of origin) and thus significantly larger than those of the mussels living at the location to which they were transplanted (BWB). Mussels from BWB might be quicker to respond to a change in habitat and could adjust faster than the mussels from MB (Widdows et al., 1984), but these differences could also reflect differences between rates of uptake and of depuration of contaminants (Widdows and Donkin, 1992). The fact that mussels from BWB were able to adjust their physiology towards the values of the mussels at MB indicated that mussels at BWB were affected by environmental factors. Their SfG improved after transplantation to a supposedly cleaner location. The lack of a response of the MB mussels transplanted to BWB did not confirm nor reject this statement. To control for disturbance of mussels due to experimental procedures, a treatment generally neglected in most studies was added, viz. the disturbance treatment D. In BWB, CR and SfG increased compared to the undisturbed treatment (AE and RR were again not statistically different) and became similar to all other experimental treatments. CR and SfG from this disturbance-control treatment at MB became significantly smaller. The only explanation for this pattern is that the CR and SfG of undisturbed mussels at BWB were
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reduced by factors not considered in the experimental treatments. A possible source of disturbance is the presence of crabs and fish in the ‘undisturbed’ clumps of mussels causing the mussels to reduce feeding-rate by full or partial closure of their shells. Although not completely comparable, an effect like this has been reported. Growth of pea crab-infested mussels was limited mainly due to a reduced rate of feeding (Bierbaum and Shumway, 1988). A reason for the reduced CR and SfG at BWB and not at MB is that clumps of mussels at BWB were much denser and larger than at MB and the mussels were severely overgrown with barnacles, sponges, seaweed and encrusting algae. These factors can reduce growth considerably (Dittman and Robles, 1991; Svane and Ompi, 1993; Ricciardi et al., 1995). Because experimental mussels were thoroughly cleaned and fouling organisms removed before they were placed in cages, the possible disturbing effects on caged mussels would have been absent or, at least, less severe. The result was that the removal of fouling organisms from the caged mussels at BWB made them similar to the relatively unfouled mussels at MB. Comparing SfG of mussels from which fouling organisms have been removed could test this proposition. Treatments would include cleaned mussels, mussels overgrown by fouling organisms and controls for cleaning. The disturbance of mussels at MB resulted in a significantly reduced rate of clearance (Fig. 2B). Although this was not reflected in a significantly reduced SfG, it might be possible that handling and caging mussels indeed negatively affect physiological traits. If so, the effect of fouling organisms in BWB was even larger than described in the previous paragraph. The last added control treatment was to control for the effect of removing animals from their habitat to a similar habitat somewhere else. It might be that only this fact causes a physiological change, rather than factors associated with a different habitat (as proposed in the hypothesis; Chapman, 1986). Therefore, groups of mussels were translocated from the experimental locations to similar locations. Finding the right location for a translocation is, however, difficult because there is no way to test the assumption that the experimental locations are similar in all aspects. That may be the reason for the observed increased CR and SfG from the groups of mussels translocated from MB to Lavender Bay (Figs. 2B and H). Darling Harbour appeared to be similar to BWB as the translocation treatment was not different from other treatments (except the undisturbed treatment, but see previous paragraph). Therefore, the interpretation of the effects of translocation is that observed differences among treatments were not a consequence of moving animals from their original location to another. To summarise, had the proper controls not been included, our conclusion would have been that there were differences between the physiology of mussels at the presumably contaminated location of BWB and the presumably uncontaminated location of MB with, as predicted, a smaller SfG at BWB than at MB. The controls, particularly the control for disturbance, however, made clear that the differences were not caused by the differences among locations, such as presumed differences in the concentration of contaminants in the sediment. Differences must have been due to other factors, probably differences in the physical disturbance of mussels between the locations. Generally, but not always (Scanes, 1993), proper controls for moving and disturbing experimental animals have been lacking (Widdows et al., 1984; Iglesias et al., 1996; Nasci et al., 1999; Webb and Keough, 2002) or not replicated (Widdows et al., 1980– 1981,
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1990; Bayne et al., 1982; Din and Ahamad, 1995). This does not, however, necessarily imply that conclusions from such studies were invalid. To unconfound the effects of moving and disturbance, proper controls are essential in studies like the current one and the final conclusion is totally dependent on them (Chapman, 1986; Underwood and Peterson, 1988). Therefore, the statement that a transplantation procedure is a valuable monitoring tool for environments (Nasci et al., 1999) is only true if the right control treatments are included in the study.
Acknowledgements Thanks to Shannon Long, Michelle Button, Amy Palmer and Craig Myers for help during measurements and Bill Maher and Gavin Birch for their help and advice. We are much indebted to Bryan Skepper from Sydney Fish Markets, Sebastian Schmid from Sydney Aquarium, Pamela Wood from ‘‘The Anchorage’’ and David Ashton for their permission to connect cages to their pontoons. This study was supported by funds from the Australian Research Council through the Special Centres Program. [SS]
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