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Responses of the green-lipped mussel Perna viridis (L.) to suspended solids
Marine Pollution Bulletin 45 (2002) 157–162 www.elsevier.com/locate/marpolbul
Responses of the green-lipped mussel Perna viridis (L.) to suspended solids P.K.S. Shin *, F.N. Yau, S.H. Chow, K.K. Tai, S.G. Cheung Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
Abstract Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes ðp > 0:05Þ in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant ðp < 0:001Þ with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly ðp < 0:001Þ related to exposure time. Gill damage, however, was significantly greater in treatments ðp < 0:001Þ. Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mussel; Suspended solids; Survival; Physiology; Behaviour; Morphological changes
1. Introduction Marine coastal developments often involve extensive reclamation from the sea. To provide fill material for land formation, dredging of marine sand is a common practice. Dredging is also required for maintenance of navigation channels due to siltation. One of the main impacts caused by dredging is the increase in suspended solids in the water body and high levels of suspended solids can also transport contaminants, such as heavy metals, nutrients and organic compounds, through the aquatic systems (USEPA, 1986). As a non-toxic stressor, suspended solids can reduce light penetration when in suspension, thus affecting primary production of algae, macrophytes and seagrasses (Lloyd, 1987). Adverse effects on organisms can also occur due to mechanical abrasion of gills, reduction in feeding rates, behaviour and increased susceptibility to diseases (Richardson, 1985; Doeg and Milledge, 1991; Newcombe and Mac-
*
Corresponding author.
Donald, 1991; Leverone, 1995). Upon deposition, suspended solids can cause adverse effects by smothering benthic organisms and their habitats, which, in turn, reduce the food supply and refuge for many bottomdwelling animals (Hogg and Norris, 1991; Rice and Hunter, 1992). Suspension-feeding bivalves are particularly vulnerable to the effects of elevated levels of suspended solids owing to their filtering mechanism in the water column. Studies on physiological responses of bivalves to increasing suspended sediment concentrations show a decrease in clearance rate (Bricelj and Malouf, 1984; Ward and MacDonald, 1996; Bacon et al., 1998), oxygen consumption (Grant and Thorpe, 1991; Alexander et al., 1994) and growth (Bricelj et al., 1984; MacDonald et al., 1998). In Hong Kong, the green-lipped mussel (Perna viridis) is widely distributed in coastal waters and has been applied as a bioindicator for trace metals and organochlorines (Phillips and Yim, 1981; Phillips, 1985; Phillips and Rainbow, 1988), PAHs (Xu et al., 1999) and hypoxia (Wu and Lam, 1997). Continuous coastal development in Hong Kong involves seabed dredging and
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extraction of marine sand for reclamation of land (Wu et al., 1998). The adverse effects of suspended solids on the marine biota are thus of concern. In this study, we explored lethality as well as physiological, behavioural and morphological responses of the green-lipped mussel to elevated levels of suspended solids. We investigated whether the green-lipped mussel can be used as a bioindicator of the effects of suspended solids in the marine environment.
2. Materials and methods 2.1. Experimental setup Suspended solids used in all laboratory experiments were collected from a clean site in Port Shelter, on the eastern side of Hong Kong. The sediment was wet sieved through a 63 lm mesh and particles of less than 63 lm were retained, oven-dried at 80 °C and ground before use in subsequent experiments. To maintain particles in a homogeneous suspension, a water current was generated by installing one submersible venturi pump (SICCE NOVAâ HI-TECH LINE) and three 9 cm long rectangular air stones connected to an air compressor (JUNâ , ACO-300A) in the test tank ð29:5 cm 19 cm 19:5 cmÞ with 8 l of filtered seawater. The actual concentration of suspended solids in the seawater was calibrated with measurement of turbidity using a HACH RA710XR Turbidmeter. The green-lipped mussels, P. viridis, with shell length between 65 and 73 mm, were collected from Tolo Harbour on northeastern waters of Hong Kong, and acclimated for 7 d prior to the experiments. During acclimation, the mussels were fed with the green microalga Dunaliella tertiolecta on the first and fourth day only. 2.2. Mortality A 96 h test was used to determine mortality of the green-lipped mussel by using suspended particles with nominal concentrations of 0, 1250, 1500, 1750 and 2000 mg l1 prepared from the oven-dried, < 63 lm sediment. Three replicates of each concentration were used, each of which contained 10 mussels. The test tanks were placed randomly and maintained at approximately 18 °C in the laboratory. During the test period, the mussels were not fed and water in the tank unchanged. Mortality of the mussels, and water temperature, salinity, pH, dissolved oxygen (DO) and turbidity in the test tanks were recorded daily. 2.3. Physiological responses A 28-d test was used to evaluate the physiological responses of the green-lipped mussel exposed to nominal
concentrations of 0, 250, 500, 750 and 1000 mg suspended solids l1 which were prepared from oven-dried, < 63 lm sediment. For each concentration, two replicates were used, each of which contained 15 mussels. The mussels were exposed to suspended solids in the first 14 d of the experiment and transferred to filtered seawater only in the following 14 d. During the test period, water in the tanks was changed twice in every 7 d, and the test mussels were fed with the green microalga D. tertiolecta at the same time. Water temperature, salinity, pH and DO in the test tanks were recorded every two days and in situ turbidity was measured with a HACH RA710XR Turbidmeter twice every 7 d. The following physiological responses were investigated during the experiment: (a) Oxygen consumption – For each suspended solids concentration, a total of seven mussels from the two replicates were randomly removed from the test tanks 24 h after feeding and the rate of oxygen consumption by each mussel was measured individually using a sealed 760 ml glass container. Filtered seawater was aerated for 1 h prior to the start of the measurement. The fully aerated seawater was then added to the container with the individual mussel. The set up was allowed to settle for 15 min. DO content in the container was measured with a DO meter (YSI model 58) at the start of the measurement and after 1.5 h. At the conclusion of the measurement, the volume of the seawater in the container was recorded using a measuring cylinder and that occupied by the mussel was measured by displacement. The rate of oxygen consumption per test mussel ðVO2 ; mg O2 h1 Þ was calculated (Eq. (1); Widdows and Johnson, 1988): VO 2 ¼
60½Cðt0 Þ Cðt1 ÞðVg Vm Þ ; ðt1 t0 Þ1000
ð1Þ
where CðtÞ is the concentration of oxygen in seawater (mg O2 l1 ) at time t; t0 ; t1 are the time (min) for start and completion of the measurement, Vg is the volume of the container (ml), Vm is the volume of the test mussel (ml). Upon completion of the measurement, the mussels were placed back into the respective test tanks. Measurement of oxygen consumption was undertaken on day 7, 14, 21 and 28 of the experiment. (b) Clearance rate – Clearance rate, defined as volume of seawater filtered by the mussels, was estimated by measuring the removal of the microalga D. tertiolecta from seawater in a glass chamber containing the test individuals. A fixed volume of 400 ml of the microalgae was placed into the chamber with five mussels collected randomly from the two replicates of each suspended solids concentration (treatment). The concentration of the microalgae was examined by counting the number of cells with a Sedgewick Rafter cell counter. This initial concentration was ensured to be about 2 104 cells ml1 ,
P.K.S. Shin et al. / Marine Pollution Bulletin 45 (2002) 157–162
so as to prevent production of pseudofaeces (Wang et al., 1993). At the conclusion of 45 min, the concentration of the microalgae in the chamber was measured again using the cell counter. A total of three replicates were performed. Clearance rate per test mussel (CR, l h1 ) was calculated (Eq. (2); Coughlan, 1969) 60V ½ðln C0 ln Ct Þ ; ð2Þ nt1000 where V is the volume of the water in the chamber (ml), C0 is the initial concentration of the microalgae in the chamber with mussels (cells ml1 ), Ct is the final concentration of the microalgae in the chamber with mussels (cells ml1 Þ, n is the number of test mussels used in measurement, and t is the duration of the measurement (min). Upon completion of the measurements, the mussels were placed back into the respective test tanks. Measurement of clearance and absorption rates was undertaken on day 7, 14, 21 and 28 of the experiment. (c) Dry gonosomatic index (DGSI) – For each suspended solids concentration, a total of four mussels from the two replicates were randomly removed from the test tanks on day 7, 14, 21 and 28 of the experiment, and sacrificed for determination of somatic and gonad tissue weight, after examination of gill damage (see Test of Morphological Responses). Individual mussel was dissected and the fresh tissues were removed from the shell. The soma and gonad were separated and dried to constant weight at 90 °C on pre-weighed aluminium foil. The DGSI was calculated (Eq. (3); Cheung, 1991)
CR ¼
DGSI ¼
dry wt: of gonad dry wt: of gonad þ dry wt: of soma 100%:
ð3Þ
2.4. Behavioural responses Byssus production – Mussels were wrapped individually in transparent plastic paper along the dorso-ventral axis of the shell. The plastic paper was fixed to the mussel using non-toxic Aron Alphaâ instant glue. In this orientation, the shell valves could open freely and byssal secretion was not affected (Seed and Richardson, 1999). At the start of the test, all byssus threads were removed from the mussels and six replicates were placed in each suspended solids treatment. The number of new byssus threads produced by individual mussels and attached onto the plastic paper was counted and subsequently removed on day 7, 14, 21 and 28 of the experiment. 2.5. Morphological responses Gill damage – The four mussels, prior to determination of somatic and gonad tissue weight, were carefully
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opened and the morphology of the gill filaments were examined using a light microscope at 100 magnification. The degree of gill damage was scored based on a scale of 1–6 according to the percentage of area damaged: scale 1– no damage, 2 – very slightly damaged ð> 0–20%Þ, 3 – slightly damaged ð> 20–40%Þ, 4 – damaged ð> 40–60%Þ, 5 – seriously damaged ð> 60–80%Þ, and 6 – very seriously damaged ð> 80–100%Þ. Gills were examined on day 7, 14, 21 and 28 of the experiment. 2.6. Data treatment Data obtained from the various tests were analysed using two-way ANOVA (Treatment Exposure time). Percent data of DGSI were arcsine transformed prior to the analysis.
3. Results During the experimental period for the various tests, water temperature was 16–18 °C, salinity 32–33%, pH 8.0–8.3 and DO 7:0–7:2 mg l1 . The actual mean concentration of suspended solids (SS) in the different treatments, as estimated from the measurements of in situ turbidity, was 59–73% of the nominal concentrations used in the test tanks. For the nominal SS concentrations of 0, 1250, 1500, 1750 and 2000 mg l1 used in the mortality test, the actual mean concentrations were found to be 0, 750, 900, 1050 and 1200 mg l1 . For the nominal concentrations of 0, 250, 500, 750 and 1000 mg l1 used in the sublethal tests, the actual mean concentrations were measured at 0, 180, 300, 440 and 600 mg l1 . All data reported herein are the measured SS concentrations. Results of the mortality test showed that all mussels (100%) survived in different SS treatments during the test period of 96 h. For the experiments on sublethal physiological responses, oxygen consumption rates of the mussels under the five SS treatments are presented (Table 1). The mean oxygen consumption rate ranged from 0.54 to 1.15 mg O2 h1 . However, no significant statistical changes ðp > 0:05Þ in oxygen consumption were found among treatments and exposure time. Similar results were obtained for DGSI in the test mussels. Changes in the DSGI were minimal, with a range of means from 19.0% to 27.6% (Table 2). In terms of measurements of clearance rate, an apparent decrease was noted on day 14 at the end of the exposure period and increase after the mussels were returned to clean, seawater for all suspended solids treatments (Fig. 1). The mean clearance rate ranged from 0.043 to 0:067 l h1 . Results of ANOVA indicated that only exposure time was significant ðp < 0:001Þ in affecting the clearance rate of the mussels.
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Table 1 Mean ð S:E:Þ oxygen consumption rate ðmg O2 h1 Þ of P. viridis exposed to SS treatments (n ¼ 7) Day
0 mg l1
180 mg l1
300 mg l1
440 mg l1
600 mg l1
7 14 21a 28a
0:64 0:07 0:76 0:04 0:67 0:09 0:96 0:14
0:70 0:02 1:15 0:17 0:86 0:10 0:64 0:06
0:65 0:05 0:79 0:20 0:56 0:11 0:68 0:13
0:61 0:10 0:72 0:03 0:65 0:13 0:55 0:08
0:89 0:15 0:68 0:06 0:54 0:20 0:65 0:09
a
Test mussels transferred to filtered seawater.
Table 2 Mean ( S:E:) DGSI (%) of P. viridis exposed to SS treatments (n ¼ 4) Day
0 mg l1
180 mg l1
300 mg l1
440 mg l1
600 mg l1
7 14 21a 28a
20:6 4:2 26:3 0:9 25:6 5:3 22:6 2:0
19:0 3:4 24:9 1:7 24:3 7:1 20:3 3:2
22:2 4:9 23:2 3:2 27:3 3:4 23:4 0:8
25:0 3:4 20:2 2:2 26:7 0:8 20:7 3:1
22:7 2:1 25:0 4:1 21:4 2:5 27:6 6:4
a
Test mussels transferred to filtered seawater.
Fig. 1. Changes in clearance rate (mean SE) of P. viridis over test period (n ¼ 3).
For the physiological response, the mean number of byssus production ranged from 7.0 to 33.0 per mussel. Significant changes ðp < 0:001Þ in byssus production were found with exposure time only (Fig. 2). A clear dose–response relationship was noted (Fig. 3) for gill damage scores and thus was significant difference ðp < 0:001Þ among treatments.
4. Discussion The results of the present study demonstrate that P. viridis can tolerate high concentrations ð1200 mg l1 Þ of suspended solids without mortality within a test period
Fig. 2. Changes in byssus production (mean SE) of P. viridis over test period (n ¼ 6).
of 96 h. Such an adaptation of survival in waters with high sediment loadings is attributed to the high efficiency of particle rejection by its labial palps in the mantle cavity (Seed and Richardson, 1999). In P. viridis, material in the inhalant water is thickly bound up with mucus and, in the anterior regions of the mantle cavity, virtually all surfaces are concerned with rejection of these muscu-bound strings of particulate material except for the finest particles (Morton, 1987). Preferential sizedependent rejection of larger particles could be of significant adaptive value in the natural environment to counteract high suspended material in the water (Defossez and Hawkins, 1997) and such effective particle
P.K.S. Shin et al. / Marine Pollution Bulletin 45 (2002) 157–162
Fig. 3. Changes in scale of gill damage (mean SE) of P. viridis over test period (n ¼ 4).
sorting mechanism thus allows the green-lipped mussel to widely distribute in turbid, tropical estuarine areas (Huang et al., 1985). In sublethal tests, there were no significant changes in oxygen consumption and somatic and gonad tissue weight (as expressed in DGSI) in the green-lipped mussel among treatments and as a function of exposure time. This further supports the adaptive survivorship of P. viridis that the animal maintains a relative stable physiological state under varying environmental conditions from heavy sediment loadings to filtered seawater. The mean DGSI in this study ranged from 19.0% to 27.6%. A DGSI of greater than 10% indicates potential spawning in the green-lipped mussel (Cheung, 1991), suggesting that reproduction of P. viridis is not affected by high suspended solids within the test period. The clearance rate and byssus production in the test mussels, however, varied significantly with exposure time. In our results, the clearance rate of P. viridis decreased when the mussels were exposed to suspended solids treatments, then increased and leveled when the test mussels were transferred to clean seawater (Fig. 2), suggesting a possible recovery from the exposure to sediment loadings. Byssus production is sensitive to mechanical agitation (Young, 1985) and water velocity (Dolmer and Svane, 1994). The number of byssus threads produced was initially higher when P. viridis were transferred from field to the perplex plates in the laboratory, and byssus production subsequently decreased (Seed and Richardson, 1999). Newly detached blue mussels Mytilus edulis were stimulated to produce more byssus during the first 24 h of attachment (Young, 1983). Similar observations were noted in this
161
study (Fig. 3), with higher byssus production when the test mussels were first exposed to the suspended solids treatments (day 7) and later transferred to seawater (day 21), suggesting that byssus production could be related to exposure to a new environment. Most literature has reported the instantaneous clearance rate of bivalves in a mixture of algae and suspended sediments and showed a clear dose–response relationship (Bricelj and Malouf, 1984; Ward and MacDonald, 1996; Bacon et al., 1998). In this study, measurements of clearance rate in P. viridis were undertaken in algal solutions with concentrations less than the threshold for pseudofaeces production (Wang et al., 1993), after the mussels were exposed to various suspended solids treatments. Our aim was to investigate whether or not prolonged exposure of the mussel to high suspended solids would result in morphological damages of the gill structure, which, in return, would affect clearance rate. Indeed, our results show a clear dose–response in gill damage as a function of increasing suspended solids concentrations. This suggests that the morphology of the ctenidia can be a good indicator for sublethal stress caused by exposure to high sediment loadings. The present results also show that even there are damages in the gill structure, there is no significant dose–response in clearance rate within the test period. This implies that the mussel may have compensatory mechanisms, such as a temporary increase in ciliary beating rates, in order to cope with impairment of gill structure. Gill area and pumping rate in mussels are closely related (Jones et al., 1992). Hence, seriously damaged gill filaments could reduce the effective gill surface area in P. viridis and may affect its pumping rate. Our results also show no observable repair of gill damage when the test mussels were transferred from suspended solids treatments to clean seawater even after 14 d. This indicates that whilst P. viridis can survive in a high suspended solids environment, morphological damages of the ctenidia may eventually exert sublethal effects resulting in reduced activities in feeding, respiration and even growth in the longer term.
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Bricelj, V.M., Malouf, R.E., de Quillfeldt, C., 1984. Growth of juvenile Mercenaria mercenaria and the effect of resuspended bottom sediments. Marine Biology 84, 167–173. Cheung, S.G., 1991. Energetics of transplanted populations of the green-lipped mussel Perna viridis (Linnaeus) (Bivalve: Mytilacea) in Hong Kong: I. Growth condition and reproduction. Asian Marine Biology 8, 117–131. Coughlan, J., 1969. The estimation of filtering rate from the clearance of suspensions. Marine Biology 2, 356–358. Defossez, J.M., Hawkins, A.J.S., 1997. Selective feeding in shellfish: size-dependent rejection of large particles within pseudofaeces from Mytilus edulis, Ruditapes philippinarum and Tapes decussates. Marine Biology 129, 139–147. Doeg, T.J., Milledge, G.A., 1991. Effects of experimentally increased concentrations of suspended sediment on macroinvertebrate drift. Australian Journal of Marine and Freshwater Research 42, 519– 526. Dolmer, P., Svane, I., 1994. Attachment and orientation of Mytilus edulis L. in flowing water. Ophelia 40, 63–74. Grant, J., Thorpe, B., 1991. Effects of suspended sediment on growth, respiration, and excretion of the soft-shell clam (Mya arenaria). Canadian Journal of Fisheries and Aquatic Science 48, 1285–1292. Hogg, I.D., Norris, R.H., 1991. Effects of runoff from land clearing and urban development on the distribution and abundance of macroinvertebrates in pool areas of a river. Australian Journal of Marine and Freshwater Research 42, 507–518. Huang, Z.G., Lee, S.Y., Mak, P.M.S., 1985. The distribution and population structure of Perna viridis (Bivalvia: Mytilacea) in Hong Kong waters. In: Morton, B. (Ed.), Proceedings of the Second International Workshop on the Malacofauna of Hong Kong and southern China, Hong Kong, 1983. Hong Kong University Press, Hong Kong, pp. 465–471. Jones, H.D., Richards, O.G., Southern, T.A., 1992. Gill dimensions, water pumping rate and body size in the mussel Mytilus edulis L. Journal of Experimental Marine Biology and Ecology 155, 213–237. Leverone, J.R., 1995. Growth and survival of caged adult bay scallops (Argopecten irradians concentricus) in Tampa Bay with respect to levels of turbidity, suspended solids and chlorophyll A. Florida Science 58, 216–227. Lloyd, D.S., 1987. Turbidity as a water quality standard for salmonid habitats in Alaska. North American Journal of Fisheries Management 7, 34–45. MacDonald, B.A., Bacon, G.S., Ward, J.E., 1998. Physiological response of infaunal (Mya arenaria) and epifaunal (Placopecten magellanicus) bivalves to variations in the concentration and quality of suspended particles II. Absorption efficiency and scope of growth. Journal of Experimental Marine Biology and Ecology 219, 127–141. Morton, B., 1987. The functional morphology of the organ of the mantle cavity of Perna virdis (Linnaeus, 1758) (Bivalvia: Mytilacea). American Malacological Bulltein 5, 159–164. Newcombe, C.P., MacDonald, D.D., 1991. Effects of suspended sediment on aquatic ecosystems. North American Journal of Fisheries Management 11, 72–82.
Phillips, D.J.H., 1985. Organochlorines and trace metals in green lipped mussels Perna viridis from Hong Kong waters: a test of indicator ability. Marine Ecology Progress Series 21, 251–258. Phillips, D.J.H., Rainbow, P.S., 1988. Barnacles and mussels as biomonitors of trace elements: A comparative study. Marine Ecology Progress Series 49, 83–93. Phillips, D.J.H., Yim, Y.S.S., 1981. A comparative evaluation of oysters, mussels and sediment as indicators of trace metals in Hong Kong. Marine Ecology Progress Series 6, 285–293. Rice, S.A., Hunter, C.L., 1992. Effects of suspended sediment and burial on scleractinian corals from west central Florida patch reefs. Bulletin of Marine Science 51, 429–442. Richardson, B.A., 1985. The impact of forest road construction on the benthic invertebrate fauna of a coastal stream in southern New South Wales. Bulletin of Australian Society of Limnology 10, 65–88. Seed, R., Richardson, C.A., 1999. Evolutionary traits in Perna viridis (Linnaeus) and Septifer virgatus (Weigmann) (Bivalvia: Mytilidae). Journal of Experimental Marine Biology and Ecology 239, 273–287. USEPA, 1986. Quality Criteria for Water. EPA-440/5-86-001. US Environmental Protection Agency, Office of Water, Washington, DC. Wang, C., Zhou, X., Cheng, R., 1993. The effects of pollutants on the filtration rate of Perna viridis (Bivalvia: Mytilidae). In: Proceedings of the First International Conference on the Marine Biology of the South China Sea, Hong Kong, 28 October–3 November, 1990. In: Morton, B. (Ed.),. The Marine Biology of the South China Sea, vol. 1. Hong Kong University Press, Hong Kong, pp. 253–260. Ward, J.E., MacDonald, B.A., 1996. Pre-ingestive feeding behaviours of two sub-tropical bivalves (Pinctada imbricata and Arca zebra): Responses to an acute increase in suspended sediment concentration. Bulletin of Marine Science 59, 417–432. Widdows, J., Johnson, D., 1988. Physiological energetics of Mytilus edulis: scope for growth. Marine Ecology Progress Series 46, 113–121. Wu, R.S.S., Cheung, R.Y.H., Shin, P.K.S., 1998. The ‘Beneficial Uses’ approach in coastal management in Hong Kong: a compromise between rapid urban development and sustainable development. Ocean & Coastal Management 41, 89–102. Wu, R.S.S., Lam, P.K.S., 1997. Glucose-6-phosphate dehydrogenase and lactate dehydrogenase in the green-lipped mussel (Perna virdis): possible biomarkers for hypoxia in the marine environment. Water Research 31, 2797–2801. Xu, L., Zhang, G.J., Lam, P.K.S., Richardson, B., 1999. Relationship between tissue concentrations of polycyclic aromatic hydrocarbons and DNA adducts in green-lipped mussels (Perna viridis). Ecotoxicology 8, 73–82. Young, G.A., 1983. Response to, and selection between, firm substrata by Mytilus edulis. Journal of Marine Biological Association, United Kingdorm 63, 653–659. Young, G.A., 1985. Byssus-thread formation by the mussel Mytilus edulis: effects of environmental factors. Marine Ecology Progress Series 24, 261–271.
Responses of the green-lipped mussel Perna viridis (L.) to suspended solids P.K.S. Shin *, F.N. Yau, S.H. Chow, K.K. Tai, S.G. Cheung Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong
Abstract Laboratory experiments were conducted to investigate the lethal and sublethal effects of suspended solids on the survival and physiological, behavourial and morphological changes of the green-lipped mussel Perna viridis collected from Tolo Harbour, Hong Kong. Results showed that P. viridis survived in all test conditions of suspended solids from 0 to 1200 mg/l over a period of 96 h. Physiological responses of the green-lipped mussel under 14-d exposure of suspended solids from 0 to 600 mg/l, followed by 14-d recovery in natural seawater, revealed no significant changes ðp > 0:05Þ in oxygen consumption and dry gonosomatic index for treatments in different concentrations of suspended solids and exposure time. Changes in clearance rate were only found to be significant ðp < 0:001Þ with exposure time. Responses in behavourial and morphological changes of the green-lipped mussel were also studied under similar experimental treatments and exposure time. Byssus production was significantly ðp < 0:001Þ related to exposure time. Gill damage, however, was significantly greater in treatments ðp < 0:001Þ. Present findings suggested that P. viridis could tolerate a high level of suspended solids in the laboratory. There were dose-dependent effects of suspended solids on morphology of gill filaments. Implications of survival and responses of the green-lipped mussel to suspended solids in the marine environment are discussed. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mussel; Suspended solids; Survival; Physiology; Behaviour; Morphological changes
1. Introduction Marine coastal developments often involve extensive reclamation from the sea. To provide fill material for land formation, dredging of marine sand is a common practice. Dredging is also required for maintenance of navigation channels due to siltation. One of the main impacts caused by dredging is the increase in suspended solids in the water body and high levels of suspended solids can also transport contaminants, such as heavy metals, nutrients and organic compounds, through the aquatic systems (USEPA, 1986). As a non-toxic stressor, suspended solids can reduce light penetration when in suspension, thus affecting primary production of algae, macrophytes and seagrasses (Lloyd, 1987). Adverse effects on organisms can also occur due to mechanical abrasion of gills, reduction in feeding rates, behaviour and increased susceptibility to diseases (Richardson, 1985; Doeg and Milledge, 1991; Newcombe and Mac-
*
Corresponding author.
Donald, 1991; Leverone, 1995). Upon deposition, suspended solids can cause adverse effects by smothering benthic organisms and their habitats, which, in turn, reduce the food supply and refuge for many bottomdwelling animals (Hogg and Norris, 1991; Rice and Hunter, 1992). Suspension-feeding bivalves are particularly vulnerable to the effects of elevated levels of suspended solids owing to their filtering mechanism in the water column. Studies on physiological responses of bivalves to increasing suspended sediment concentrations show a decrease in clearance rate (Bricelj and Malouf, 1984; Ward and MacDonald, 1996; Bacon et al., 1998), oxygen consumption (Grant and Thorpe, 1991; Alexander et al., 1994) and growth (Bricelj et al., 1984; MacDonald et al., 1998). In Hong Kong, the green-lipped mussel (Perna viridis) is widely distributed in coastal waters and has been applied as a bioindicator for trace metals and organochlorines (Phillips and Yim, 1981; Phillips, 1985; Phillips and Rainbow, 1988), PAHs (Xu et al., 1999) and hypoxia (Wu and Lam, 1997). Continuous coastal development in Hong Kong involves seabed dredging and
0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 1 ) 0 0 2 9 8 - 3
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extraction of marine sand for reclamation of land (Wu et al., 1998). The adverse effects of suspended solids on the marine biota are thus of concern. In this study, we explored lethality as well as physiological, behavioural and morphological responses of the green-lipped mussel to elevated levels of suspended solids. We investigated whether the green-lipped mussel can be used as a bioindicator of the effects of suspended solids in the marine environment.
2. Materials and methods 2.1. Experimental setup Suspended solids used in all laboratory experiments were collected from a clean site in Port Shelter, on the eastern side of Hong Kong. The sediment was wet sieved through a 63 lm mesh and particles of less than 63 lm were retained, oven-dried at 80 °C and ground before use in subsequent experiments. To maintain particles in a homogeneous suspension, a water current was generated by installing one submersible venturi pump (SICCE NOVAâ HI-TECH LINE) and three 9 cm long rectangular air stones connected to an air compressor (JUNâ , ACO-300A) in the test tank ð29:5 cm 19 cm 19:5 cmÞ with 8 l of filtered seawater. The actual concentration of suspended solids in the seawater was calibrated with measurement of turbidity using a HACH RA710XR Turbidmeter. The green-lipped mussels, P. viridis, with shell length between 65 and 73 mm, were collected from Tolo Harbour on northeastern waters of Hong Kong, and acclimated for 7 d prior to the experiments. During acclimation, the mussels were fed with the green microalga Dunaliella tertiolecta on the first and fourth day only. 2.2. Mortality A 96 h test was used to determine mortality of the green-lipped mussel by using suspended particles with nominal concentrations of 0, 1250, 1500, 1750 and 2000 mg l1 prepared from the oven-dried, < 63 lm sediment. Three replicates of each concentration were used, each of which contained 10 mussels. The test tanks were placed randomly and maintained at approximately 18 °C in the laboratory. During the test period, the mussels were not fed and water in the tank unchanged. Mortality of the mussels, and water temperature, salinity, pH, dissolved oxygen (DO) and turbidity in the test tanks were recorded daily. 2.3. Physiological responses A 28-d test was used to evaluate the physiological responses of the green-lipped mussel exposed to nominal
concentrations of 0, 250, 500, 750 and 1000 mg suspended solids l1 which were prepared from oven-dried, < 63 lm sediment. For each concentration, two replicates were used, each of which contained 15 mussels. The mussels were exposed to suspended solids in the first 14 d of the experiment and transferred to filtered seawater only in the following 14 d. During the test period, water in the tanks was changed twice in every 7 d, and the test mussels were fed with the green microalga D. tertiolecta at the same time. Water temperature, salinity, pH and DO in the test tanks were recorded every two days and in situ turbidity was measured with a HACH RA710XR Turbidmeter twice every 7 d. The following physiological responses were investigated during the experiment: (a) Oxygen consumption – For each suspended solids concentration, a total of seven mussels from the two replicates were randomly removed from the test tanks 24 h after feeding and the rate of oxygen consumption by each mussel was measured individually using a sealed 760 ml glass container. Filtered seawater was aerated for 1 h prior to the start of the measurement. The fully aerated seawater was then added to the container with the individual mussel. The set up was allowed to settle for 15 min. DO content in the container was measured with a DO meter (YSI model 58) at the start of the measurement and after 1.5 h. At the conclusion of the measurement, the volume of the seawater in the container was recorded using a measuring cylinder and that occupied by the mussel was measured by displacement. The rate of oxygen consumption per test mussel ðVO2 ; mg O2 h1 Þ was calculated (Eq. (1); Widdows and Johnson, 1988): VO 2 ¼
60½Cðt0 Þ Cðt1 ÞðVg Vm Þ ; ðt1 t0 Þ1000
ð1Þ
where CðtÞ is the concentration of oxygen in seawater (mg O2 l1 ) at time t; t0 ; t1 are the time (min) for start and completion of the measurement, Vg is the volume of the container (ml), Vm is the volume of the test mussel (ml). Upon completion of the measurement, the mussels were placed back into the respective test tanks. Measurement of oxygen consumption was undertaken on day 7, 14, 21 and 28 of the experiment. (b) Clearance rate – Clearance rate, defined as volume of seawater filtered by the mussels, was estimated by measuring the removal of the microalga D. tertiolecta from seawater in a glass chamber containing the test individuals. A fixed volume of 400 ml of the microalgae was placed into the chamber with five mussels collected randomly from the two replicates of each suspended solids concentration (treatment). The concentration of the microalgae was examined by counting the number of cells with a Sedgewick Rafter cell counter. This initial concentration was ensured to be about 2 104 cells ml1 ,
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so as to prevent production of pseudofaeces (Wang et al., 1993). At the conclusion of 45 min, the concentration of the microalgae in the chamber was measured again using the cell counter. A total of three replicates were performed. Clearance rate per test mussel (CR, l h1 ) was calculated (Eq. (2); Coughlan, 1969) 60V ½ðln C0 ln Ct Þ ; ð2Þ nt1000 where V is the volume of the water in the chamber (ml), C0 is the initial concentration of the microalgae in the chamber with mussels (cells ml1 ), Ct is the final concentration of the microalgae in the chamber with mussels (cells ml1 Þ, n is the number of test mussels used in measurement, and t is the duration of the measurement (min). Upon completion of the measurements, the mussels were placed back into the respective test tanks. Measurement of clearance and absorption rates was undertaken on day 7, 14, 21 and 28 of the experiment. (c) Dry gonosomatic index (DGSI) – For each suspended solids concentration, a total of four mussels from the two replicates were randomly removed from the test tanks on day 7, 14, 21 and 28 of the experiment, and sacrificed for determination of somatic and gonad tissue weight, after examination of gill damage (see Test of Morphological Responses). Individual mussel was dissected and the fresh tissues were removed from the shell. The soma and gonad were separated and dried to constant weight at 90 °C on pre-weighed aluminium foil. The DGSI was calculated (Eq. (3); Cheung, 1991)
CR ¼
DGSI ¼
dry wt: of gonad dry wt: of gonad þ dry wt: of soma 100%:
ð3Þ
2.4. Behavioural responses Byssus production – Mussels were wrapped individually in transparent plastic paper along the dorso-ventral axis of the shell. The plastic paper was fixed to the mussel using non-toxic Aron Alphaâ instant glue. In this orientation, the shell valves could open freely and byssal secretion was not affected (Seed and Richardson, 1999). At the start of the test, all byssus threads were removed from the mussels and six replicates were placed in each suspended solids treatment. The number of new byssus threads produced by individual mussels and attached onto the plastic paper was counted and subsequently removed on day 7, 14, 21 and 28 of the experiment. 2.5. Morphological responses Gill damage – The four mussels, prior to determination of somatic and gonad tissue weight, were carefully
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opened and the morphology of the gill filaments were examined using a light microscope at 100 magnification. The degree of gill damage was scored based on a scale of 1–6 according to the percentage of area damaged: scale 1– no damage, 2 – very slightly damaged ð> 0–20%Þ, 3 – slightly damaged ð> 20–40%Þ, 4 – damaged ð> 40–60%Þ, 5 – seriously damaged ð> 60–80%Þ, and 6 – very seriously damaged ð> 80–100%Þ. Gills were examined on day 7, 14, 21 and 28 of the experiment. 2.6. Data treatment Data obtained from the various tests were analysed using two-way ANOVA (Treatment Exposure time). Percent data of DGSI were arcsine transformed prior to the analysis.
3. Results During the experimental period for the various tests, water temperature was 16–18 °C, salinity 32–33%, pH 8.0–8.3 and DO 7:0–7:2 mg l1 . The actual mean concentration of suspended solids (SS) in the different treatments, as estimated from the measurements of in situ turbidity, was 59–73% of the nominal concentrations used in the test tanks. For the nominal SS concentrations of 0, 1250, 1500, 1750 and 2000 mg l1 used in the mortality test, the actual mean concentrations were found to be 0, 750, 900, 1050 and 1200 mg l1 . For the nominal concentrations of 0, 250, 500, 750 and 1000 mg l1 used in the sublethal tests, the actual mean concentrations were measured at 0, 180, 300, 440 and 600 mg l1 . All data reported herein are the measured SS concentrations. Results of the mortality test showed that all mussels (100%) survived in different SS treatments during the test period of 96 h. For the experiments on sublethal physiological responses, oxygen consumption rates of the mussels under the five SS treatments are presented (Table 1). The mean oxygen consumption rate ranged from 0.54 to 1.15 mg O2 h1 . However, no significant statistical changes ðp > 0:05Þ in oxygen consumption were found among treatments and exposure time. Similar results were obtained for DGSI in the test mussels. Changes in the DSGI were minimal, with a range of means from 19.0% to 27.6% (Table 2). In terms of measurements of clearance rate, an apparent decrease was noted on day 14 at the end of the exposure period and increase after the mussels were returned to clean, seawater for all suspended solids treatments (Fig. 1). The mean clearance rate ranged from 0.043 to 0:067 l h1 . Results of ANOVA indicated that only exposure time was significant ðp < 0:001Þ in affecting the clearance rate of the mussels.
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Table 1 Mean ð S:E:Þ oxygen consumption rate ðmg O2 h1 Þ of P. viridis exposed to SS treatments (n ¼ 7) Day
0 mg l1
180 mg l1
300 mg l1
440 mg l1
600 mg l1
7 14 21a 28a
0:64 0:07 0:76 0:04 0:67 0:09 0:96 0:14
0:70 0:02 1:15 0:17 0:86 0:10 0:64 0:06
0:65 0:05 0:79 0:20 0:56 0:11 0:68 0:13
0:61 0:10 0:72 0:03 0:65 0:13 0:55 0:08
0:89 0:15 0:68 0:06 0:54 0:20 0:65 0:09
a
Test mussels transferred to filtered seawater.
Table 2 Mean ( S:E:) DGSI (%) of P. viridis exposed to SS treatments (n ¼ 4) Day
0 mg l1
180 mg l1
300 mg l1
440 mg l1
600 mg l1
7 14 21a 28a
20:6 4:2 26:3 0:9 25:6 5:3 22:6 2:0
19:0 3:4 24:9 1:7 24:3 7:1 20:3 3:2
22:2 4:9 23:2 3:2 27:3 3:4 23:4 0:8
25:0 3:4 20:2 2:2 26:7 0:8 20:7 3:1
22:7 2:1 25:0 4:1 21:4 2:5 27:6 6:4
a
Test mussels transferred to filtered seawater.
Fig. 1. Changes in clearance rate (mean SE) of P. viridis over test period (n ¼ 3).
For the physiological response, the mean number of byssus production ranged from 7.0 to 33.0 per mussel. Significant changes ðp < 0:001Þ in byssus production were found with exposure time only (Fig. 2). A clear dose–response relationship was noted (Fig. 3) for gill damage scores and thus was significant difference ðp < 0:001Þ among treatments.
4. Discussion The results of the present study demonstrate that P. viridis can tolerate high concentrations ð1200 mg l1 Þ of suspended solids without mortality within a test period
Fig. 2. Changes in byssus production (mean SE) of P. viridis over test period (n ¼ 6).
of 96 h. Such an adaptation of survival in waters with high sediment loadings is attributed to the high efficiency of particle rejection by its labial palps in the mantle cavity (Seed and Richardson, 1999). In P. viridis, material in the inhalant water is thickly bound up with mucus and, in the anterior regions of the mantle cavity, virtually all surfaces are concerned with rejection of these muscu-bound strings of particulate material except for the finest particles (Morton, 1987). Preferential sizedependent rejection of larger particles could be of significant adaptive value in the natural environment to counteract high suspended material in the water (Defossez and Hawkins, 1997) and such effective particle
P.K.S. Shin et al. / Marine Pollution Bulletin 45 (2002) 157–162
Fig. 3. Changes in scale of gill damage (mean SE) of P. viridis over test period (n ¼ 4).
sorting mechanism thus allows the green-lipped mussel to widely distribute in turbid, tropical estuarine areas (Huang et al., 1985). In sublethal tests, there were no significant changes in oxygen consumption and somatic and gonad tissue weight (as expressed in DGSI) in the green-lipped mussel among treatments and as a function of exposure time. This further supports the adaptive survivorship of P. viridis that the animal maintains a relative stable physiological state under varying environmental conditions from heavy sediment loadings to filtered seawater. The mean DGSI in this study ranged from 19.0% to 27.6%. A DGSI of greater than 10% indicates potential spawning in the green-lipped mussel (Cheung, 1991), suggesting that reproduction of P. viridis is not affected by high suspended solids within the test period. The clearance rate and byssus production in the test mussels, however, varied significantly with exposure time. In our results, the clearance rate of P. viridis decreased when the mussels were exposed to suspended solids treatments, then increased and leveled when the test mussels were transferred to clean seawater (Fig. 2), suggesting a possible recovery from the exposure to sediment loadings. Byssus production is sensitive to mechanical agitation (Young, 1985) and water velocity (Dolmer and Svane, 1994). The number of byssus threads produced was initially higher when P. viridis were transferred from field to the perplex plates in the laboratory, and byssus production subsequently decreased (Seed and Richardson, 1999). Newly detached blue mussels Mytilus edulis were stimulated to produce more byssus during the first 24 h of attachment (Young, 1983). Similar observations were noted in this
161
study (Fig. 3), with higher byssus production when the test mussels were first exposed to the suspended solids treatments (day 7) and later transferred to seawater (day 21), suggesting that byssus production could be related to exposure to a new environment. Most literature has reported the instantaneous clearance rate of bivalves in a mixture of algae and suspended sediments and showed a clear dose–response relationship (Bricelj and Malouf, 1984; Ward and MacDonald, 1996; Bacon et al., 1998). In this study, measurements of clearance rate in P. viridis were undertaken in algal solutions with concentrations less than the threshold for pseudofaeces production (Wang et al., 1993), after the mussels were exposed to various suspended solids treatments. Our aim was to investigate whether or not prolonged exposure of the mussel to high suspended solids would result in morphological damages of the gill structure, which, in return, would affect clearance rate. Indeed, our results show a clear dose–response in gill damage as a function of increasing suspended solids concentrations. This suggests that the morphology of the ctenidia can be a good indicator for sublethal stress caused by exposure to high sediment loadings. The present results also show that even there are damages in the gill structure, there is no significant dose–response in clearance rate within the test period. This implies that the mussel may have compensatory mechanisms, such as a temporary increase in ciliary beating rates, in order to cope with impairment of gill structure. Gill area and pumping rate in mussels are closely related (Jones et al., 1992). Hence, seriously damaged gill filaments could reduce the effective gill surface area in P. viridis and may affect its pumping rate. Our results also show no observable repair of gill damage when the test mussels were transferred from suspended solids treatments to clean seawater even after 14 d. This indicates that whilst P. viridis can survive in a high suspended solids environment, morphological damages of the ctenidia may eventually exert sublethal effects resulting in reduced activities in feeding, respiration and even growth in the longer term.
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