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Metal concentrations in sediments and tilapia collected from inland waters of Hong Kong
PII: S0043-1354(98)00115-8
Wat. Res. Vol. 32, No. 11, pp. 3331±3340, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
METAL CONCENTRATIONS IN SEDIMENTS AND TILAPIA COLLECTED FROM INLAND WATERS OF HONG KONG M H. Y. ZHOU1, R. Y. H. CHEUNG2, K. M. CHAN3 and M. H. WONG1**
Institute for Natural Resources and Waste Management, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China; 2Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, PR China and 3Department of Biochemistry, Chinese University of Hong Kong, Shatin, Hong Kong, PR China 1
(First received November 1997; accepted in revised form February 1998) AbstractÐThis paper presents data on the concentrations of six heavy metals, copper (Cu), zinc (Zn), lead (Pb), nickel (Ni), cadmium (Cd) and chromium (Cr), in sediments and ®sh sampled from inland waters of Hong Kong. Sediments and tilapia (Tilapia mossambica) were collected from Shing Mun River (at Fo Tan, Tai Wai and Siu Lek Yuen), Lam Tsuen River (at Tai Wo) and Tai Po River (at Tai Po), as well as from a commercial ®sh pond (at Mai Po) for comparison. The total concentrations of the six metals in river sediments were signi®cantly (p < 0.05) higher than those collected from the ®sh pond. The highest metal concentrations of Cu, Zn, Ni, Cd and Cr were recorded in the sediment of Fo Tan at Shing Mun River, with the values of 449, 1220, 67.0, 4.03 and 85.4 mg kgÿ1 (dry weight), respectively. On average, the total metal contamination in sediments followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po>Mai Po. Dierent metals seemed to accumulate in dierent organs of ®sh (muscle, gill, skin and viscera). A substantial amount of heavy metals was observed in tilapia which lived in contaminated environment (Fo Tan and Tai Wai), when compared to those collected from the ®sh pond. Tilapia collected from Fo Tan contained the highest concentrations of Cu (168 mg gÿ1) and Zn (266 mg gÿ1). Signi®cant relationships (p < 0.05) between metals in dierent chemical fractions (exchangeable, carbonate, Fe±Mn oxide, organic and residual) of sediments and metals in ®sh organs (Cu in viscera, Zn in skin, Pb in all organs and Cr in muscle) were found. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐcopper, zinc, nickel, lead, cadmium, chromium, river sediment, tilapia
INTRODUCTION
Hong Kong is one of the most densely populated cities in the world. It supports over 6 million people with a total area of only 1068 km2. More than half of the population (3.7 million) resided on the shores of Victoria Harbor and the Kowloon Peninsula (north of Hong Kong Island) and the rest is scattered around the New Territories in several satellite cities such as Sha Tin and Tai Po (Williams, 1994). The main source of water pollution comes from the domestic sewage of the inhabitants and euents discharged by various industries. The untreated inorganic and organic waste running into the aquatic systems contributed to the degradation of the inland (EPD, 1995a,b) and coastal water systems (Morton, 1989; Phillips, 1989). The coastal water and sediments are signi®cantly contaminated by trace metals (Morton, 1989; Phillips, 1989; Wong et al., 1995; Wong, 1996), while inland water systems are facing the same problem (EPD, 1995a). More than two-third of the rivers are grossly polluted (EPD, 1995b). Monitoring of the water qual*Author to whom all correspondence should be addressed.
ity has been undertaken regularly by the Environmental Protection Department (EPD) of the Hong Kong Government. Since the enactment of the waste disposal regulations in the late 1980s, improvement in water quality in some rivers is evident. At present, a lot of eort has been put into tackling pollution at the source and improving the water quality of the 12 priority rivers including Shing Mun River, Lam Tsuen River and Tai Po River. However, there is a lack of data on the heavy metals in river sediment and in living organisms concerning the inland water systems of Hong Kong. In this project, sediments and ®sh (Tilapia mossambica) were collected from three major waterways namely Shing Mun River, Lam Tsuen River and Tai Po River of the New Territories, Hong Kong. The heavy metal contents in dierent geochemical phases of the sediments and in dierent parts of the ®sh were investigated. It was hoped that the obtained data could serve as a baseline to evaluate the metal levels in the river sediments and their eects on associated biological organisms. The analysis of the total metal concentrations only provides information of metal enrichment of
3331
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H. Y. Zhou et al.
the sediment, but not direct information on the biological eects of the metals. This is due to the fact that dierent chemical forms of a metal in the sediment will determine its behavior in the environment. On the contrary, sequential extraction which provides information on the speciation of metals can give detailed information concerning the mode of occurrence, metal mobility and bioavailability (Tessier et al., 1979). More than ten dierent sequential extraction procedures have been developed to study the geochemical phase of metals in aquatic sediments, soils, road dust and sludge (e.g. Tessier et al., 1979; Stover et al., 1976; Miller and McFee, 1983). Among these methods, the one proposed by Tessier et al. (1979) becomes a widely used method. The problems such as limited selectivity of extractants and overlap between the fractions can be avoided by careful control of the parameter in each step (Rauret et al., 1989). Tilapia are omnivores which have a diversi®ed food spectrum (Huet, 1994). Various kinds of food including crustaceans, debris, vascular plants, microalgae and mud could be found in the gut of tilapia (Wong et al., 1996). They can survive at adverse environmental conditions because their resistance to disease is strong, their respiratory demands are slight so that they can tolerate low oxygen and high ammonia levels. Even some freshwater tilapia are able to survive and grow over a wide range of salinities (Watanabe et al., 1987). It has been noted that heavy metals were accumulated in tilapia after they were fed with metal contaminated sludge (Wong and Chiu, 1993). They are commonly found in ®sh ponds and streams of Hong Kong and, therefore, they were chosen to study the eect of environmental impact on this widely distributed organism. The objectives of the present study were: (1) to identify and compare dierent forms of heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the sediments of Shing Mun River, Lam Tsuen River and Tai Po River, using sequential extraction to fractionate the metals contained in sediments into six groups: water-soluble, exchangeable, carbonate, Fe± Mn oxide, organic and residual fractions, (2) to determine whether the ®sh which live in heavily contaminated Shing Mun River would contain a substantial amount of these metals. Sediments and ®sh were also collected from a commercial ®sh pond at Mai Po for comparison. MATERIALS AND METHODS
Sediment and ®sh sampling A total of six sediments (each composed of three replicates) were collected using a box sediment grab. Five samples were collected from Shing Mun River (at Fo Tan, Tai Wai and Siu Lek Yuen), Lam Tsuen River (at Tai Wo) and Tai Po River (at Tai Po) (Fig. 1). Another sediment was collected from a commercial ®sh pond at Mai Po. All the river sediments except that from the ®sh pond
Fig. 1. Map of sampling locations. Sampling sites: (1) Tai Wai, (2) Fo Tan, (3) Siu Lek Yuen, (4) Tai Po, (5) Tai Wo and (6) Mai Po. Other places: (A) Fo Tan industrial estate, (B) Sha Tin sewage treatment plant and (C) Tai Po industrial estate. were black in color and with strong disgusting odour. They were sealed in polyethylene bags embedded in ice during transportation to the laboratory. They were then freeze-dried and passed through a 1 mm size sieve to separate the stones, leaves and dead invertebrates. The sediments were then ground into powder using a mortar and a pestle to ensure the particle size was less than 100 mesh. Tilapia were also collected from Shing Mun River (at Fo Tan and Tai Wai) and the ®sh pond (at Mai Po). Three tilapia were collected from each site and transported to the laboratory in an ice compartment. They were then divided into gill, skin, muscle and viscera and the freezedried samples were stored at 48C. The moisture contents of the ®sh tissue were determined according to weight loss before and after freeze-drying. Analysis of total metal contents 0.1 g sediment was digested using 5 ml conc. nitric acid at 1358C for 4 h. 2 ml hydrogenperoxide (30%) and conc. perchloric acid were then added and the temperature was maintained at 1508C until the liquor was clear and the particles turned white or gray. This digestion method is precisely an extraction rather than a total digestion method. However, it gave good recoveries (90210%) for all the six heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the standard reference material (NBS 2704 river sediment, National Bureau of Standards (now National Institute of Standards and Technology, NIST), U.S. Department of Commerce, Gaithersberg, MD: less than 100 mesh in size). 2 g ®sh samples were predigested with 5 ml conc. nitric acid at room temperature overnight, and then at 1358C until the liquor was clear (AOAC, 1990). A standard reference material (NBS 1566a oyster tissue, U.S. Department of Commerce National Bureau of Standards, Gaithersberg, MD 20899) was also used to verify the accuracy of metal determination. The recovery rates for all
Metal concentrations in sediments and tilapia heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the standard were within 90210%. The analyses were triplicated for each sediment and ®sh sample. All the digested liquors were ®ltered through an Advantec 5C ®lter paper and diluted to 25 ml with distilled water. They were stored in acid-rinsed polyethylene bottles at 48C prior to analysis. The heavy metal concentrations (Cu, Zn, Pb, Ni, Cd and Cr) were determined using an atomic absorption spectrophotometer (Allen et al., 1989). Chemical fractionation of metals in sediments The extraction scheme adopted (Tessier et al., 1979) in the present project separated the metals into water-soluble, exchangeable, carbonate, Fe±Mn oxide, organic and residual fractions, respectively. 1 g of the freeze-dried sediment was sequentially extracted with 15 ml deionized water, 8 ml 1 M magnesium chloride, 8 ml 1 M sodium acetate (adjusted to pH 5 with acetic acid), 5 ml 0.04 M hydroxylammonium chloride in 25% acetic acid, 5 ml 30% hydrogenperoxide (adjusted to pH 2 with nitric acid) and 5 ml mixture of conc. nitric acid and perchloric acid (2:1, v/v). After each successive extraction, the solution was centrifuged at 14 000 rpm for 20 min and then ®ltered through an Advantec 5C ®lter paper. The remaining residue was washed with deionized water before further extraction. The heavy metals were analyzed using the method described above. The metal forms that are extracted by deionized water, magnesium chloride, sodium acetate, hydroxylammonium chloride, hydrogenperoxide and mixture of acids are, respectively, water-soluble, exchangeable, carbonate, Fe±Mn oxide, organic and residual bound phases. Statistical analysis An ANOVA test followed by Duncan's multiple range test were used to determine any signi®cant dierences (at p < 0.05) in terms of metal concentrations of ®sh and sediment between dierent sampling sites
RESULTS
Among all the metals monitored, Cd was the lowest (1.67±4.03 mg kgÿ1), followed by Ni (10.6± 67.0 mg kgÿ1), Cr (18.3±85.4 mg kgÿ1), Cu (26.8± 449 mg kgÿ1), Pb (168±1529 mg kgÿ1) and Zn (238± 1220 mg kgÿ1) in the river sediments (Fig. 2). The highest contents of Cu, Zn, Ni, Cd, and Cr were recorded in Fo Tan, with the values of 449, 1220, 67.0, 4.03 and 85.4 mg kgÿ1 (dry weight), respectively. The concentrations of Pb in Tai Wo and Fo Tan (1529 and 1387 mg kgÿ1 dry weight, respectively) were ®ve times higher than those obtained from other sites. Statistical comparisons between river sediments and ®sh pond sediment showed that the levels of heavy metals (except Cd) found in Fo Tan, Tai Wai, Siu Lek Yuen and Tai Wo were signi®cantly higher (p < 0.05) than those found in the ®sh pond. As to Tai Po, signi®cant dierences of Pb, Ni and Cr were also found when compared to the ®sh pond. As to Cd, Fo Tan and Tai Wo contained relatively high concentrations (4.03 and 3.87 mg kgÿ1, respectively), Tai Wai and Siu Lek Yuen had moderately levels (2.41 and 2.77 mg kgÿ1, respectively), and Tai Po had the lowest level (1.67 mg kgÿ1). On average, the degree of metal
3333
contamination in sediment followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po>Mai Po. In all the river sediment samples, only Cu was observed in the water-soluble phase. The greatest percentage (55±70%) of Cu was associated with organic bound fraction. On average, the percentage of Cu associated with dierent fractions in the seven sediments was in the order of: organic>residual >carbonate>Fe±Mn oxide>exchangeable>watersoluble. Zinc was concentrated in the residual (23± 45%), Fe±Mn oxide (24±38%) and carbonate (21± 36%) fractions whereas Pb and Cr were mainly associated with the residual fraction (62±87% for Pb and 79±93% for Cr). Nickel was found in all the fractions except the water-soluble, and mainly associated with residual (27±63%) and carbonate (15± 25%) fractions. Cadmium was not found in Fe±Mn oxide and organic fractions (Fig. 3). The results of heavy metal contents in dierent organs of tilapia collected from dierent sites are shown in Table 1. It was noted that the concentrations of Pb, Cd and Cr in the muscle were well below the limit of Public Health Regulation in Hong Kong (Hong Kong Government, 1989), whereas no limits are provided for Cu, Zn and Ni. However, gill, skin and viscera contained much higher levels of metals than muscle. Gill accumulated higher contents of Pb, Ni and Cd than other organs. Signi®cantly higher concentrations (p < 0.05) of Pb and Cd were found in ®sh gill collected from Tai Wai (Pb: 12.6 mg gÿ1 and Cd: 1.85 mg gÿ1) and Fo Tan (Pb: 11.0 mg gÿ1 and Cd: 1.76 mg gÿ1) than those collected from the ®sh pond (Pb: 6.40 mg gÿ1 and Cd: 1.30 mg gÿ1). Zinc tended to accumulate in the skin of tilapia and samples collected from Fo Tan and Tai Wai had signi®cantly higher (p < 0.05) concentrations than the ®sh pond, with the values of 266 and 250 mg gÿ1, respectively. A high content of Cu was found in viscera. Tilapia collected from Fo Tan contained the highest Cu content of 168 mg gÿ1, followed by Tai Wai of 86.4 mg gÿ1 and Mai Po of 4.93 mg gÿ1. Chromium distributed approximately equally in skin and muscle.
DISCUSSION
Heavy metal concentrations in sediments In general, relatively high concentrations of Cu, Zn and Pb were obtained in all the river sediments. The magnitude of heavy metal concentrations in river sediments was Zn>Pb>Cu>Cr, Ni>Cd. The results matched the regular monitoring results of river water quality by EPD of Hong Kong Government (EPD, 1995b). Sediments collected from Fo Tan were mostly polluted by Cu, Zn, Ni, Cd and Cr because it is located next to an industrial estate (Fig. 1). Fo Tan and Tai Wo contained sig-
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H. Y. Zhou et al.
Fig. 2. Total heavy metal concentrations of sediments. Columns with the same letters are not signi®cant dierent according to Duncan's multiple range test (p < 0.05). FT: Fo Tan; TWai: Tai Wai; SLY: Siu Lek Yuen; TWo: Tai Wo; TP: Tai Po and MP: Mai Po.
ni®cantly higher (p < 0.05) concentrations of Pb (®ve times higher than other sampling sites). Copper, Zn, Pb, Ni, Cd, and Cr are commonly used in textile dyes, electroplating, galvanizing, battery manufacture, and plastic fabrication. The cor-
relations between any two pair of Cu, Zn, Pb, Ni, Cd and Cr are shown in Table 2. Close correlations (p < 0.01) were found for Zn±Cu, Cd±Cu, Cd±Zn, Cr±Cu, Cr±Zn, Cr±Cd, Ni±Cu, Ni±Zn, Ni±Cr, Pb± Zn, Pb±Cd and Pb±Cr in the river sediments,
Metal concentrations in sediments and tilapia
Fig. 3. Percentage of metals in each chemical fraction of the river sediments. ex: exchangeable; carb: carbonate; oxide: Fe±Mn oxide; org: organic; res: residual; FT: Fo Tan; TWai: Tai Wai; SLY: Siu Lek Yuen; TWo: Tai Wo; TP: Tai Po and MP: Mai Po.
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H. Y. Zhou et al.
Table 1. Metal contents in dierent organs of tilapia (mg gÿ1, dry weight). Means with the same superscript in the same row are not signi®cant dierent according to Duncan's multiple range test (p < 0.05)
Organ
Metal
Fo Tan
Tai Wai a
Mai Po b
Viscera
Cu Zn Pb Ni Cd Cr moisture (%)
168 260 30.6 24.8 c 0.77 20.03 b 0.72 20.14 b 0.39 20.06 b 0.09 20.01 c 60 24
86.42 4.2 96.0 25.4 a 7.7621.77 a 10.1 21.6 a 0.5720.03 a 1.0020.05 a 65 29
4.93 20.94 c 48.82 8.0 b 5.20 21.14 a 1.27 20.20 b 0.31 20.06 b 0.39 20.18 b 46 24
Gill
Cu Zn Pb Ni Cd Cr moisture (%)
3.86 20.20 b 77.8 26.6 a 11.0 21.2 a 3.322 0.42 a 1.762 0.13 a 0.162 0.08 a 77 21
7.2420.58 a 74.2211.1 a 12.6 21.5 a 4.0420.54 a 1.8520.22 a 0.1420.04 a 76 24
3.62 20.33 b 87.2 28.0 a 6.40 20.53 b 2.51 20.29 b 1.30 20.13 b 0.11 20.03 a 75 22
Skin
Cu Zn Pb Ni Cd Cr moisture (%)
3.042 0.82 a 266 231 a 1.642 0.42 a 1.322 0.19 a 0.312 0.04 a 0.512 0.06 a 72 21
2.9120.12 a 250 218 a 2.2620.16 a 1.5720.14 a 0.3620.00 a 0.3220.02 a 70 21
2.56 20.07 a 124 2 18 b 2.19 20.53 a 0.82 20.06 b 0.35 20.06 a 0.39 20.18 a 71 25
Cu Zn Pb Ni Cd Cr moisture (%)
2.092 0.56 a 22.42 1.5 a 1.532 0.55 a 0.71 20.13 b 0.542 0.19 a 0.552 0.19 a 81 21
2.4920.50 a 23.426.13 a 1.9320.28 a 0.8220.04 a 0.502 0.07 a,b 0.442 0.02 a,b 802 1
1.99 20.16 a 26.7 23.1 a 1.38 20.22 a 0.62 20.05 c 0.34 20.02 b 0.30 20.04 b 78 22
Public Health Regulation (wet weight) (Hong Kong Government, 1989)
ÿ ÿ 6.0 ÿ 2.0 1.0
±: no data.
suggested that these metals were derived from a common origin. Metal contents in sludge is a good indicator of metal usage in local industries. Dramatic increases of Cu, Pb and Cd were observed in sludge samples collected from Shatin Sewage Treatment Plant near Fo Tan from July 1992 to June 1994 (Pun et al., 1995). When compared to Mai Po, which is a relative unpolluted area, metal contents in river sediments of Fo Tan were signi®cantly (p < 0.05) higher in general (Fig. 2). So far, little information is available concerning heavy metal contamination in sediments of inland water systems in Hong Kong. The EPD report (EPD, 1995b) indicated that Zn was the most abundant in the river water, followed by Cu and Pb, and the least was Cd and Cr while Ni was not recorded. The results of the present study were in line with the report that Shing Mun River was the most pol-
Table 2. Pearson correlation matrix showing the relationships between metal concentrations of the sediments Cd Ni Pb Zn Cu
0.94* Zn
0.69* 0.56 Pb
0.39 0.86* 0.92* Ni
0.50 0.91* 0.78* 0.63* Cd
0.73* 0.93* 0.62* 0.97* 0.98* Cr
*Indicates the correlation coecient was signi®cant at 0.01 probability level using one-tailed test, n = 15.
luted followed by Lam Tsuen River and Tai Po River, and metal contamination in river sediments followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po. When compared to other Asian countries (Kurokawa and Tatsukawa, 1990; Prudente et al., 1994; Baruah et al., 1996), the heavy metal contents obtained in the present study were rather high (Table 3). Furthermore, the Pb levels in sediments of Shing Mun River were two times higher than those obtained from Pasig River, Philippines (Prudente et al., 1994) where the place was considered to be heavily polluted by rapid industrialization and increasing population. Sequential extraction provides data on the mode of metals occurrence and the potential bioavailability in the sediments. Levels in the residual fraction should be considered as the background value for the elements in the sediments (Tessier et al., 1979). The water-soluble, exchangeable and carbonate fractions are considered to be moderately available in the environment (Ma and Rao, 1997). In the present study, only Cu was found in the water-soluble fraction. The sum of these three extractions for Cu, Zn, Pb, Ni, Cd and Cr were 4.6±6.9%, 21±36%, 1.5±12%, 22±38%, 55±100% and 1.6±11%, respectively. Copper was present in all fractions, but it was primary associated in the organic fraction (55±
Metal concentrations in sediments and tilapia
3337
Table 3. Comparison of heavy metal concentrations (mg kgÿ1, dry weight) in sediments collected from Shing Mun River, Lam Tsuen River and Tai Po River with published data in other countries Location Shing Mun River (Hong Kong) Lam Tsuen and Tai Po River (Hong Kong) Duyen Hai and Ho Chi Min (Vietnam) Jhanji River (India) Pasig River (Philippines) Marikina River (Philippines)
Cu
Zn
Pb
Ni
Cd
Cr
53.2±449 26.8±69.6
461±1220 238±593
246±1387 169±1529
16.0±67.0 10.6±17.0
2.4±4.0 01.7±3.9
15±25
68±106
7.1±1.1
31±57
0.06±0.10
ÿ
28±70 110±189 28±79
32±288 236±1560 74±169
8.7±87 66±137 18±31
17.5±101 17.1±27.3 10.5±21.7
ÿ 2.03±15.2 1.47±2.59
28.5±399 ÿ ÿ
32.4±85.4 This study 18.3±37.1 This study Kurokawa and Tatsukawa (1990) Baruah et al. (1996) Prudente et al. (1994) Prudente et al. (1994)
ÿ: no data.
70%). Copper can easily form complexes with organic compounds because of the high formation constants of organic-Cu compounds which makes it rather stable in the environment (Stumm and Morgan, 1981). In aquatic systems, the distribution of Cu is markedly aected by natural organic matter such as humic materials and amino acid. The predominance of Cu in organic phase has also been observed in freshwater and marine sediments (Baruah et al., 1996; Chen et al., 1996; Wong, 1996). Zinc was mainly associated with carbonate (21± 36%), Fe±Mn oxide (24±38%) and residual (23± 45%) fractions (Fig. 3). Calcium carbonate is a strong absorbent to form complexes with Zn as double salts CaCO3ZnCO3 in the sediments (Ramos et al., 1994). This coprecipitation phenomena with carbonate may be an important elimination mechanism for Zn when organic matter and hydrous Fe oxides are less abundant in the aquatic system (Forstner and Wittmann, 1979). As to Pb and Cr, residual fraction was the predominant form (62±87% for Pb and 79±93% for Cr) in the sediment. Cr contents were not detected in the Fe±Mn oxide fraction in all sediments, while Cr in the organic fraction was only present in the sediments collected from Fo Tan, Tai Wai and Tai Wo (Fig. 3). Fe±Mn oxide was another important fraction for Pb, although it only accounted for 8±22% of the total Pb. Like Zn, Pb also formed stable complexes with Fe±Mn oxide, and these compounds were even more stable than those of Zn (Ramos et al., 1994). The concentration of Cd was the lowest when compared to other elements monitored. It mainly existed in exchangeable (22±29%) and carbonate (34±61%) fractions (Fig. 3). Cadmium is not found in the organic fraction for low adsorption constant and labile complexion with organic matter (Baron et al., 1990). The pattern of Ni distribution was dierent in dierent sediments. In the sediments collected from Siu Lek Yuen, Tai Wo and Tai Po, the residual fraction was by far the most important, ranging from 40±63% of total Ni. Signi®cantly higher concentrations of Ni were found in carbonate and Fe± Mn oxide fractions in the sediments collected from
Fo Tan and Tai Wai. These two sediments also contained much higher (p < 0.05) concentrations of Ni (67.0 mg kgÿ1 for Fo Tan and 43.1 mg kgÿ1 for Tai Wai) than other sites. Heavy metal concentrations in dierent organs of tilapia When comparing metal accumulation in tilapia living in dierent locations, tilapia collected from the heavily polluted Shing Mun River had signi®cantly higher heavy metal concentration (p < 0.05) than those collected from the Mai Po ®sh farm. In general, muscle contained a smaller amount of metals than skin, gill and viscera. Dierent metals seemed to accumulate in dierent organs. Higher levels of Pb, Ni and Cd were found in gill, while higher contents of Zn were recorded in skin, and Cu in viscera. Gill as the ®rst target for pollutants in water, absorption across the gill (Heath, 1987; Sorensen, 1991) seemed to be the major pathway of Pb, Ni and Cd entering tilapia. The relatively high contents of heavy metals found in viscera may be due to the fact that most of the heavy metals are accumulated in the liver and kidney after ingestion (Badsha and Goldspink, 1982), or excessive metals in the diets are not absorbed but remained in the gut and intestine. On the other hand, ®sh have dierent routes for possible excretion of heavy metals when exposed in heavy metal contaminated water bodies, these include gill, bile (via feces), kidney and skin (Sorensen, 1991). All these factors may contribute to higher levels of heavy metals in viscera, skin and gill than in muscle. Greatest contents of Cu were accumulated in viscera of all tilapia samples. It has been noted that ®sh liver had the highest concentration factor (the ratio of the concentration of a chemical inside an organ to the environmental concentration) of Cu for almost any exposure time (Heath, 1987; Ghazaly et al., 1992). Although liver, kidney and gill are vital organs in maintaining Zn to a considerable extent (Chen et al., 1987; Hilmy et al., 1987), skin showed the highest accumulation of Zn in the present study, followed by gill, viscera, and muscle. Similar results were obtained by Gupta and Sharma (1994) and
3338
H. Y. Zhou et al.
Seymore et al. (1996), who studied Zn accumulation in ®sh. Although Zn is an essential element for ®sh, excessive Zn will be toxic causing mortality, growth retardation and tissue alterations (Sorensen, 1991). Relationships between dierent chemical fractions of metals in sediments and their accumulation in ®sh Relationships of metals in each fraction (except water-soluble fraction) of sediment with metal con-
tents in dierent parts of ®sh were studied using Pearson correlation matrix (Table 4). The accumulations of Cu in viscera were closely related (p < 0.05) to the concentrations of this metal in each fraction, while Zn concentrations in skin were closely related to carbonate, Fe±Mn oxide, organic and residual fractions of sediments. Lead contents in all ®sh organs analyzed were closely related (p < 0.05) to the exchangeable fractions of sediments. No signi®cant correlations were found
Table 4. Pearson correlation matrix showing the relationship of metals between tilapia and each fraction of sediment
Cu
Zn
Pb
Ni
Cd
Cr
Gill
Skin
Viscera
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.043 ÿ0.047 0.422 0.403 ÿ0.164 0.395 ÿ0.262 ÿ0.143
1 0.322 0.073 ÿ0.141 ÿ0.014 ÿ0.252 ÿ0.029 ÿ0.125
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 ÿ0.629* ÿ0.162 0.599* 0.110 ÿ0.377 ÿ0.484 ÿ0.373 ÿ0.508
1 ÿ0.043 ÿ0.492 0.261 0.810** 0.865** 0.845** 0.897**
1 0.066 0.380 ÿ0.593* ÿ0.496 ÿ0.515 ÿ0.390
1 0.010 ÿ0.453 ÿ0.507 ÿ0.488 ÿ0.553
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.901** 0.838** 0.904** 0.746** 0.252 ÿ0.019 0.022 ÿ0.167
1 0.921** 0.986** 0.765** ÿ0.090 ÿ0.313 ÿ0.227 ÿ0.369
1 0.913** 0.786** ÿ0.014 ÿ0.488 ÿ0.479 ÿ0.624*
1 0.805** ÿ0.018 ÿ0.239 ÿ0.170 ÿ0.313
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.757** 0.517 0.818** 0.406 0.245 0.319 0.050 0.481
1 0.347 0.887** 0.528 0.212 0.273 0.152 0.474
1 0.473 ÿ0.245 0.103 0.210 ÿ0.447 0.339
1 0.244 ÿ0.025 0.042 ÿ0.121 0.288
1 0.409 0.734** 0.915** 0.652* 0.882** 0.786**
Muscle
1 0.476 0.311 0.623* 0.168 0.152
Exchangeable
Carbonate
Fe±Mn oxide
1 0.810** 0.960** 0.739** 0.757**
1 0.758** 0.985** 0.931**
1 0.662* 0.681*
1 0.958**
1
1 0.055 ÿ0.022 0.202 0.173
1 0.980** 0.985** 0.964**
1 0.957** 0.970**
1 0.970**
1
1 0.070 ÿ0.203 ÿ0.172 ÿ0.261
1 0.649* 0.491 0.339
1 0.967** 0.926**
1 0.964**
1
1 0.991** 0.817** 0.916**
1 0.757** 0.945**
1 0.660*
1
ÿ ÿ ÿ
ÿ ÿ
1
ÿ ÿ ÿ
1 0.732**
1
1 0.578* 0.561 0.732** 0.566
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 ÿ0.128 0.393 0.510 0.319 0.516 ÿ ÿ ÿ0.229
1 0.740** ÿ0.529 ÿ0.402 ÿ0.531 ÿ ÿ 0.616*
1 ÿ0.120 ÿ0.185 ÿ0.248 ÿ ÿ 0.405
1 0.524 0.603* ÿ ÿ ÿ0.199
1 0.916** ÿ ÿ ÿ0.058
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.040 ÿ0.014 0.300 ÿ0.139 0.040 ÿ 0.250 0.278
1 ÿ0.231 0.054 ÿ0.527 ÿ0.264 ÿ ÿ0.204 0.278
1 ÿ0.393 0.912** 0.150 ÿ ÿ0.007 ÿ0.540
1 ÿ0.178 0.739** ÿ 0.783** 0.729**
1 0.365 ÿ 0.180 ÿ0.476
1 ÿ ÿ ÿ0.253
1 ÿ 0.921** 0.558
Organic Residual
* and ** indicate the correlation coecients were signi®cant at 0.05 and 0.01 probability levels, using two-tailed test, n = 9. ÿ: no data.
Metal concentrations in sediments and tilapia
between the contents of Ni and Cd in ®sh organs and sediments. As to Cr, signi®cant correlations were noted in Cr contents in muscle with Cr contents in carbonate, organic and residual fractions. Cu, Zn and Pb were more abundant than Ni, Cd and Cr both in sediment and river water, correlations of Cu, Zn and Pb concentrations between tilapia and sediments were thus more evident than those of Ni, Cd and Cr. The eect of heavy metals on aquatic organisms is controlled by the concentrations and chemical forms of the metals in water and sediment. Theoretically, free metal ion is the most bioavailable form of the element, and the concentration of the free metal ion varies signi®cantly with pH and organic substances (Sorensen, 1991; Rainbow, 1995). It is believed that complexation of metals by organic substances reduces metal bioavailability and its potential toxicity. Assuming that bioavailability is related to solubility, the six extraction fractions followed the decreasing solubility order of: watersoluble>exchangeable>carbonate bound>Fe±Mn oxide bound>organic bound>residual (Ma and Rao, 1997), then metals bioavailability decreased in the same order. Metals in the residual fraction and associated with silicates would not expect to release under natural condition. Whatever the precise uptake mechanisms, most heavy metals can be accumulated by aquatic biota and pose toxic eect at high trophic levels including human beings. CONCLUSION
Sediments collected from inland river system had much higher concentrations of heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) than those collected from the ®sh pond. The magnitude of heavy metal contents in sediments were Zn>Pb>Cu>Cr, Ni>Cd. Tilapia living in heavily polluted Shing Mun River contained a substantial amount of heavy metals when compared to those collected from the commercial ®sh pond. It was indicated that dierent metals had dierent accumulation targets in ®sh body (Cu in viscera, Zn in skin, Pb, Ni, and Cd in gill, Cr in skin and muscle). Signi®cant correlations between dierent chemical fractions of metals (exchangeable, carbonate, Fe± Mn oxide, organic and residual) in river sediments and their accumulation in ®sh organs (Cu in viscera, Zn in skin, Pb in all organs, and Cr in muscle) were obtained. Sequential extraction of metals from the sediment may re¯ect their chemical species as well as their adsorbed fraction. However, their bioavailability will re¯ect not only their solubilities but also the presence or absence of chelating compounds in water phase, structure of foodchains, and other parameters such as pH and redox potential. AcknowledgementsÐThe authors would like to thank Dr Y. Liang, Mr K. W. Chan and Miss F. Y. K. Lee for
3339
technical assistance. Financial support from the Industry Department, Hong Kong Government is gratefully acknowledged.
REFERENCES
Allen S. E., Grimshaw H. M., Parkinson J. A. and Quarmby C. (1989) Chemical Analysis of Ecological Materials, 2nd edn. Blackwell Scienti®c Publications, Oxford, U.K. AOAC (1990) Ocial Methods of Analysis, 15th edn. Association of Ocial Analytical Chemist, Washington, D.C. Badsha K. S. and Goldspink C. R. (1982) Preliminary observations on the heavy metal content of four species of freshwater ®sh in N.W. England. J. Fish. Biol. 21, 251± 267. Baron J., Legret M. and Astruc M. (1990) Study of interactions between heavy metals and sewage sludge: Determination of stability constants and complexation capacities of complexes formed with Cu and Cd. Environ. Technol. 11, 151±162. Baruah N. K., Kotoky P., Bhattacharya K. G. and Borah G. C. (1996) Metal speciation in Jhanji river sediments. Sci. Tot. Environ. 193, 1±12. Chen Y., Cai F. L., Qiu M. H. and Xu P. A. (1987) The behavior of 65 Zn in arti®cial marine microhabitat. Acta. Oceanol. 6, 444±451. Chen W., Tan S. K. and Tay J. H. (1996) Distribution, fractional composition and release of sediment-bound heavy metals in tropical reservoirs. Wat. Air Soil Pollut. 92, 273±287. Environmental Protection Department (EPD) (1995a) Environment in Hong Kong. Government Printer, Hong Kong. Environmental Protection Department (EPD) (1995b) River Water Quality in Hong Kong: Results from the EPD River Water Monitoring Program for 1994. Government Printer, Hong Kong. Forstner U. and Wittmann G. T. W. (1979) Metal Pollution in the Aquatic Environment. Springer-Verlag, Berlin. Ghazaly K. S., El-Mofty M. M. and Attia S. I. (1992) Eects of locality and tissue type on heavy metal levels of some commercial ®shes from Alexandria Ð Egypt. Bull. Natl. Inst. Oceanogr. Fish. Egypt. 18, 43±52. Gupta A. K. and Sharma S. K. (1994) Bioaccumulation of zinc in Cirrhinus mrigala (Hamilton) ®ngerlings during short-term static bioassay. J. Environ. Biol. 15, 231±237. Heath A. G. (1987) Water Pollution and Fish Physiology. CRC Press, Boca Raton, FL. Hilmy A. M., El-Domiaty N. A., Daabees A. Y. and Abdel-Latife H. A. (1987) Toxicity in tilapia zilli and Clarias lazera (Pisces) induced by zinc seasonally. Comp. Biochem. Physiol. C. 86, 263±265. Hong Kong Government (1989) Public Health and Municipal Services Ordinance, Ch. 132. Food Adulteration (Metallic Contamination) Resolution, Hong Kong. Huet M. (1994) Text Book of Fish Culture: Breeding and Cultivation of Fish, 2nd edn. University Press, Cambridge. Kurokawa A. and Tatsukawa R. (1990) Distribution of heavy metals in soils of Vellar River estuary, South India. In Proc. Ann. Symp. of the Soc. of Environ. Sci., Japan, pp. 152. Toranomon, Tokyo. Ma L. Q. and Rao G. N. (1997) Chemical fraction of cadmium, copper, nickel and zinc in contaminated soils. J. Environ. Qual. 26, 259±264. Miller W. P. and McFee W. W. (1983) Distribution of cadmium, zinc, copper and lead in soils of industrial northwestern Indiana. J. Environ. Qual. 12, 29±33.
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Morton B. (1989) Pollution of the coastal waters of Hong Kong. Mar. Pollut. Bull. 20, 310±318. Phillips D. J. H. (1989) Trace metals and organochlorines in the coastal waters of Hong Kong. Mar. Pollut. Bull. 20, 319±327. Prudente M. S., Ichihashi H. and Tatsukawa R. (1994) Heavy metal concentrations in sediments from Manila Bay, Philippines and in¯owing rivers. Environ. Pollut. 86, 83±88. Pun K. C., Cheung R. Y. H. and Wong M. H. (1995) Characterization of sewage sludge and toxicity evaluation with microalgae. Mar. Pollut. Bull. 31, 394±401. Rainbow P. S. (1995) Physiology, physiochemistry and metal uptake Ð a crustacean perspective. Mar. Pollut. Bull. 31, 55±59. Ramos L., Hernandez L. M. and Gonzalez M. J. (1994) Sequential fraction of copper, lead, cadmium and zinc in soils from or near Donana National Park. J. Environ. Qual. 23, 50±57. Rauret G., Rubio R., Lopez-Sanchez J. F. and Casassas E. (1989) Speci®c procedure for metal solid speciation in heavily polluted river sediments. Int. J. Environ. Anal. Chem. 35, 89±100. Seymore T., Dupreez H. H. and Vanvuren J. H. J. (1996) Concentrations of zinc in Barbus marequensis from the lower Olifants River Mpumalanga. South Africa Hydrobiol. 332, 141±150. Sorensen E. M. B. (1991) Metal Poisoning in Fish. CRC Press, Boca Raton, FL. Stover R. C., Sommers L. E. and Silviera D. J. (1976) Evaluation of metal in wastewater sludge. J. Wat. Pollut. Control Fed. 48, 2165±2175.
Stumm W. and Morgan J. J. (1981) Aquatic Chemistry: An Introduction Emphasizing Chemical Equilibria in Natural Waters, 2nd edn. Wiley, New York. Tessier A., Campbell P. G. C. and Bisson M. (1979) Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 51, 844±851. Watanabe W. O., Ellingson L. J. and Wicklund R. I. (1987) The eects of salinity on growth, food consumption and conversion in Juvenile, Monosex Mal Florida Red tilapia. In The 2nd Int. Symp. on tilapia in Aquacult, ed. R. S. V. Dullin, T. Bhukaswas, K. Tonguthai and J. L. Maclean. ICLARM, Thailand. Williams M. T. (1994) The Green Dragon: Hong Kong's Living Environment. Green Dragon Publication, Hong Kong. Wong J. W. C. (1996) Heavy metal and nutrient contamination of marine sediments in Hong Kong. Toxicol. Environ. Chem. 53, 175±189. Wong M. H. and Chiu S. T. (1993) Feasibility studies on the use of sewage sludge as supplementary feed for rearing tilapia, I. Trace metal contents of the treated ®sh. Environ. Technol. 14, 1155±1162. Wong M. H., Hung K. M. and Chiu S. T. (1996) Sludgegrowth algae for culturing aquatic organisms. Part II. Sludge-growth algae as foods for aquatic organism. Environ. Manage. 20, 375±384. Wong Y. S., Tam N. F. Y., Lau P. S. and Xue X. Z. (1995) The toxicity of marine sediments in Victoria Harbour, Hong Kong. Mar. Pollut. Bull. 31, 464±470.
Wat. Res. Vol. 32, No. 11, pp. 3331±3340, 1998 # 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0043-1354/98 $19.00 + 0.00
METAL CONCENTRATIONS IN SEDIMENTS AND TILAPIA COLLECTED FROM INLAND WATERS OF HONG KONG M H. Y. ZHOU1, R. Y. H. CHEUNG2, K. M. CHAN3 and M. H. WONG1**
Institute for Natural Resources and Waste Management, and Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China; 2Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, PR China and 3Department of Biochemistry, Chinese University of Hong Kong, Shatin, Hong Kong, PR China 1
(First received November 1997; accepted in revised form February 1998) AbstractÐThis paper presents data on the concentrations of six heavy metals, copper (Cu), zinc (Zn), lead (Pb), nickel (Ni), cadmium (Cd) and chromium (Cr), in sediments and ®sh sampled from inland waters of Hong Kong. Sediments and tilapia (Tilapia mossambica) were collected from Shing Mun River (at Fo Tan, Tai Wai and Siu Lek Yuen), Lam Tsuen River (at Tai Wo) and Tai Po River (at Tai Po), as well as from a commercial ®sh pond (at Mai Po) for comparison. The total concentrations of the six metals in river sediments were signi®cantly (p < 0.05) higher than those collected from the ®sh pond. The highest metal concentrations of Cu, Zn, Ni, Cd and Cr were recorded in the sediment of Fo Tan at Shing Mun River, with the values of 449, 1220, 67.0, 4.03 and 85.4 mg kgÿ1 (dry weight), respectively. On average, the total metal contamination in sediments followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po>Mai Po. Dierent metals seemed to accumulate in dierent organs of ®sh (muscle, gill, skin and viscera). A substantial amount of heavy metals was observed in tilapia which lived in contaminated environment (Fo Tan and Tai Wai), when compared to those collected from the ®sh pond. Tilapia collected from Fo Tan contained the highest concentrations of Cu (168 mg gÿ1) and Zn (266 mg gÿ1). Signi®cant relationships (p < 0.05) between metals in dierent chemical fractions (exchangeable, carbonate, Fe±Mn oxide, organic and residual) of sediments and metals in ®sh organs (Cu in viscera, Zn in skin, Pb in all organs and Cr in muscle) were found. # 1998 Elsevier Science Ltd. All rights reserved Key wordsÐcopper, zinc, nickel, lead, cadmium, chromium, river sediment, tilapia
INTRODUCTION
Hong Kong is one of the most densely populated cities in the world. It supports over 6 million people with a total area of only 1068 km2. More than half of the population (3.7 million) resided on the shores of Victoria Harbor and the Kowloon Peninsula (north of Hong Kong Island) and the rest is scattered around the New Territories in several satellite cities such as Sha Tin and Tai Po (Williams, 1994). The main source of water pollution comes from the domestic sewage of the inhabitants and euents discharged by various industries. The untreated inorganic and organic waste running into the aquatic systems contributed to the degradation of the inland (EPD, 1995a,b) and coastal water systems (Morton, 1989; Phillips, 1989). The coastal water and sediments are signi®cantly contaminated by trace metals (Morton, 1989; Phillips, 1989; Wong et al., 1995; Wong, 1996), while inland water systems are facing the same problem (EPD, 1995a). More than two-third of the rivers are grossly polluted (EPD, 1995b). Monitoring of the water qual*Author to whom all correspondence should be addressed.
ity has been undertaken regularly by the Environmental Protection Department (EPD) of the Hong Kong Government. Since the enactment of the waste disposal regulations in the late 1980s, improvement in water quality in some rivers is evident. At present, a lot of eort has been put into tackling pollution at the source and improving the water quality of the 12 priority rivers including Shing Mun River, Lam Tsuen River and Tai Po River. However, there is a lack of data on the heavy metals in river sediment and in living organisms concerning the inland water systems of Hong Kong. In this project, sediments and ®sh (Tilapia mossambica) were collected from three major waterways namely Shing Mun River, Lam Tsuen River and Tai Po River of the New Territories, Hong Kong. The heavy metal contents in dierent geochemical phases of the sediments and in dierent parts of the ®sh were investigated. It was hoped that the obtained data could serve as a baseline to evaluate the metal levels in the river sediments and their eects on associated biological organisms. The analysis of the total metal concentrations only provides information of metal enrichment of
3331
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H. Y. Zhou et al.
the sediment, but not direct information on the biological eects of the metals. This is due to the fact that dierent chemical forms of a metal in the sediment will determine its behavior in the environment. On the contrary, sequential extraction which provides information on the speciation of metals can give detailed information concerning the mode of occurrence, metal mobility and bioavailability (Tessier et al., 1979). More than ten dierent sequential extraction procedures have been developed to study the geochemical phase of metals in aquatic sediments, soils, road dust and sludge (e.g. Tessier et al., 1979; Stover et al., 1976; Miller and McFee, 1983). Among these methods, the one proposed by Tessier et al. (1979) becomes a widely used method. The problems such as limited selectivity of extractants and overlap between the fractions can be avoided by careful control of the parameter in each step (Rauret et al., 1989). Tilapia are omnivores which have a diversi®ed food spectrum (Huet, 1994). Various kinds of food including crustaceans, debris, vascular plants, microalgae and mud could be found in the gut of tilapia (Wong et al., 1996). They can survive at adverse environmental conditions because their resistance to disease is strong, their respiratory demands are slight so that they can tolerate low oxygen and high ammonia levels. Even some freshwater tilapia are able to survive and grow over a wide range of salinities (Watanabe et al., 1987). It has been noted that heavy metals were accumulated in tilapia after they were fed with metal contaminated sludge (Wong and Chiu, 1993). They are commonly found in ®sh ponds and streams of Hong Kong and, therefore, they were chosen to study the eect of environmental impact on this widely distributed organism. The objectives of the present study were: (1) to identify and compare dierent forms of heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the sediments of Shing Mun River, Lam Tsuen River and Tai Po River, using sequential extraction to fractionate the metals contained in sediments into six groups: water-soluble, exchangeable, carbonate, Fe± Mn oxide, organic and residual fractions, (2) to determine whether the ®sh which live in heavily contaminated Shing Mun River would contain a substantial amount of these metals. Sediments and ®sh were also collected from a commercial ®sh pond at Mai Po for comparison. MATERIALS AND METHODS
Sediment and ®sh sampling A total of six sediments (each composed of three replicates) were collected using a box sediment grab. Five samples were collected from Shing Mun River (at Fo Tan, Tai Wai and Siu Lek Yuen), Lam Tsuen River (at Tai Wo) and Tai Po River (at Tai Po) (Fig. 1). Another sediment was collected from a commercial ®sh pond at Mai Po. All the river sediments except that from the ®sh pond
Fig. 1. Map of sampling locations. Sampling sites: (1) Tai Wai, (2) Fo Tan, (3) Siu Lek Yuen, (4) Tai Po, (5) Tai Wo and (6) Mai Po. Other places: (A) Fo Tan industrial estate, (B) Sha Tin sewage treatment plant and (C) Tai Po industrial estate. were black in color and with strong disgusting odour. They were sealed in polyethylene bags embedded in ice during transportation to the laboratory. They were then freeze-dried and passed through a 1 mm size sieve to separate the stones, leaves and dead invertebrates. The sediments were then ground into powder using a mortar and a pestle to ensure the particle size was less than 100 mesh. Tilapia were also collected from Shing Mun River (at Fo Tan and Tai Wai) and the ®sh pond (at Mai Po). Three tilapia were collected from each site and transported to the laboratory in an ice compartment. They were then divided into gill, skin, muscle and viscera and the freezedried samples were stored at 48C. The moisture contents of the ®sh tissue were determined according to weight loss before and after freeze-drying. Analysis of total metal contents 0.1 g sediment was digested using 5 ml conc. nitric acid at 1358C for 4 h. 2 ml hydrogenperoxide (30%) and conc. perchloric acid were then added and the temperature was maintained at 1508C until the liquor was clear and the particles turned white or gray. This digestion method is precisely an extraction rather than a total digestion method. However, it gave good recoveries (90210%) for all the six heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the standard reference material (NBS 2704 river sediment, National Bureau of Standards (now National Institute of Standards and Technology, NIST), U.S. Department of Commerce, Gaithersberg, MD: less than 100 mesh in size). 2 g ®sh samples were predigested with 5 ml conc. nitric acid at room temperature overnight, and then at 1358C until the liquor was clear (AOAC, 1990). A standard reference material (NBS 1566a oyster tissue, U.S. Department of Commerce National Bureau of Standards, Gaithersberg, MD 20899) was also used to verify the accuracy of metal determination. The recovery rates for all
Metal concentrations in sediments and tilapia heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) in the standard were within 90210%. The analyses were triplicated for each sediment and ®sh sample. All the digested liquors were ®ltered through an Advantec 5C ®lter paper and diluted to 25 ml with distilled water. They were stored in acid-rinsed polyethylene bottles at 48C prior to analysis. The heavy metal concentrations (Cu, Zn, Pb, Ni, Cd and Cr) were determined using an atomic absorption spectrophotometer (Allen et al., 1989). Chemical fractionation of metals in sediments The extraction scheme adopted (Tessier et al., 1979) in the present project separated the metals into water-soluble, exchangeable, carbonate, Fe±Mn oxide, organic and residual fractions, respectively. 1 g of the freeze-dried sediment was sequentially extracted with 15 ml deionized water, 8 ml 1 M magnesium chloride, 8 ml 1 M sodium acetate (adjusted to pH 5 with acetic acid), 5 ml 0.04 M hydroxylammonium chloride in 25% acetic acid, 5 ml 30% hydrogenperoxide (adjusted to pH 2 with nitric acid) and 5 ml mixture of conc. nitric acid and perchloric acid (2:1, v/v). After each successive extraction, the solution was centrifuged at 14 000 rpm for 20 min and then ®ltered through an Advantec 5C ®lter paper. The remaining residue was washed with deionized water before further extraction. The heavy metals were analyzed using the method described above. The metal forms that are extracted by deionized water, magnesium chloride, sodium acetate, hydroxylammonium chloride, hydrogenperoxide and mixture of acids are, respectively, water-soluble, exchangeable, carbonate, Fe±Mn oxide, organic and residual bound phases. Statistical analysis An ANOVA test followed by Duncan's multiple range test were used to determine any signi®cant dierences (at p < 0.05) in terms of metal concentrations of ®sh and sediment between dierent sampling sites
RESULTS
Among all the metals monitored, Cd was the lowest (1.67±4.03 mg kgÿ1), followed by Ni (10.6± 67.0 mg kgÿ1), Cr (18.3±85.4 mg kgÿ1), Cu (26.8± 449 mg kgÿ1), Pb (168±1529 mg kgÿ1) and Zn (238± 1220 mg kgÿ1) in the river sediments (Fig. 2). The highest contents of Cu, Zn, Ni, Cd, and Cr were recorded in Fo Tan, with the values of 449, 1220, 67.0, 4.03 and 85.4 mg kgÿ1 (dry weight), respectively. The concentrations of Pb in Tai Wo and Fo Tan (1529 and 1387 mg kgÿ1 dry weight, respectively) were ®ve times higher than those obtained from other sites. Statistical comparisons between river sediments and ®sh pond sediment showed that the levels of heavy metals (except Cd) found in Fo Tan, Tai Wai, Siu Lek Yuen and Tai Wo were signi®cantly higher (p < 0.05) than those found in the ®sh pond. As to Tai Po, signi®cant dierences of Pb, Ni and Cr were also found when compared to the ®sh pond. As to Cd, Fo Tan and Tai Wo contained relatively high concentrations (4.03 and 3.87 mg kgÿ1, respectively), Tai Wai and Siu Lek Yuen had moderately levels (2.41 and 2.77 mg kgÿ1, respectively), and Tai Po had the lowest level (1.67 mg kgÿ1). On average, the degree of metal
3333
contamination in sediment followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po>Mai Po. In all the river sediment samples, only Cu was observed in the water-soluble phase. The greatest percentage (55±70%) of Cu was associated with organic bound fraction. On average, the percentage of Cu associated with dierent fractions in the seven sediments was in the order of: organic>residual >carbonate>Fe±Mn oxide>exchangeable>watersoluble. Zinc was concentrated in the residual (23± 45%), Fe±Mn oxide (24±38%) and carbonate (21± 36%) fractions whereas Pb and Cr were mainly associated with the residual fraction (62±87% for Pb and 79±93% for Cr). Nickel was found in all the fractions except the water-soluble, and mainly associated with residual (27±63%) and carbonate (15± 25%) fractions. Cadmium was not found in Fe±Mn oxide and organic fractions (Fig. 3). The results of heavy metal contents in dierent organs of tilapia collected from dierent sites are shown in Table 1. It was noted that the concentrations of Pb, Cd and Cr in the muscle were well below the limit of Public Health Regulation in Hong Kong (Hong Kong Government, 1989), whereas no limits are provided for Cu, Zn and Ni. However, gill, skin and viscera contained much higher levels of metals than muscle. Gill accumulated higher contents of Pb, Ni and Cd than other organs. Signi®cantly higher concentrations (p < 0.05) of Pb and Cd were found in ®sh gill collected from Tai Wai (Pb: 12.6 mg gÿ1 and Cd: 1.85 mg gÿ1) and Fo Tan (Pb: 11.0 mg gÿ1 and Cd: 1.76 mg gÿ1) than those collected from the ®sh pond (Pb: 6.40 mg gÿ1 and Cd: 1.30 mg gÿ1). Zinc tended to accumulate in the skin of tilapia and samples collected from Fo Tan and Tai Wai had signi®cantly higher (p < 0.05) concentrations than the ®sh pond, with the values of 266 and 250 mg gÿ1, respectively. A high content of Cu was found in viscera. Tilapia collected from Fo Tan contained the highest Cu content of 168 mg gÿ1, followed by Tai Wai of 86.4 mg gÿ1 and Mai Po of 4.93 mg gÿ1. Chromium distributed approximately equally in skin and muscle.
DISCUSSION
Heavy metal concentrations in sediments In general, relatively high concentrations of Cu, Zn and Pb were obtained in all the river sediments. The magnitude of heavy metal concentrations in river sediments was Zn>Pb>Cu>Cr, Ni>Cd. The results matched the regular monitoring results of river water quality by EPD of Hong Kong Government (EPD, 1995b). Sediments collected from Fo Tan were mostly polluted by Cu, Zn, Ni, Cd and Cr because it is located next to an industrial estate (Fig. 1). Fo Tan and Tai Wo contained sig-
3334
H. Y. Zhou et al.
Fig. 2. Total heavy metal concentrations of sediments. Columns with the same letters are not signi®cant dierent according to Duncan's multiple range test (p < 0.05). FT: Fo Tan; TWai: Tai Wai; SLY: Siu Lek Yuen; TWo: Tai Wo; TP: Tai Po and MP: Mai Po.
ni®cantly higher (p < 0.05) concentrations of Pb (®ve times higher than other sampling sites). Copper, Zn, Pb, Ni, Cd, and Cr are commonly used in textile dyes, electroplating, galvanizing, battery manufacture, and plastic fabrication. The cor-
relations between any two pair of Cu, Zn, Pb, Ni, Cd and Cr are shown in Table 2. Close correlations (p < 0.01) were found for Zn±Cu, Cd±Cu, Cd±Zn, Cr±Cu, Cr±Zn, Cr±Cd, Ni±Cu, Ni±Zn, Ni±Cr, Pb± Zn, Pb±Cd and Pb±Cr in the river sediments,
Metal concentrations in sediments and tilapia
Fig. 3. Percentage of metals in each chemical fraction of the river sediments. ex: exchangeable; carb: carbonate; oxide: Fe±Mn oxide; org: organic; res: residual; FT: Fo Tan; TWai: Tai Wai; SLY: Siu Lek Yuen; TWo: Tai Wo; TP: Tai Po and MP: Mai Po.
3335
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H. Y. Zhou et al.
Table 1. Metal contents in dierent organs of tilapia (mg gÿ1, dry weight). Means with the same superscript in the same row are not signi®cant dierent according to Duncan's multiple range test (p < 0.05)
Organ
Metal
Fo Tan
Tai Wai a
Mai Po b
Viscera
Cu Zn Pb Ni Cd Cr moisture (%)
168 260 30.6 24.8 c 0.77 20.03 b 0.72 20.14 b 0.39 20.06 b 0.09 20.01 c 60 24
86.42 4.2 96.0 25.4 a 7.7621.77 a 10.1 21.6 a 0.5720.03 a 1.0020.05 a 65 29
4.93 20.94 c 48.82 8.0 b 5.20 21.14 a 1.27 20.20 b 0.31 20.06 b 0.39 20.18 b 46 24
Gill
Cu Zn Pb Ni Cd Cr moisture (%)
3.86 20.20 b 77.8 26.6 a 11.0 21.2 a 3.322 0.42 a 1.762 0.13 a 0.162 0.08 a 77 21
7.2420.58 a 74.2211.1 a 12.6 21.5 a 4.0420.54 a 1.8520.22 a 0.1420.04 a 76 24
3.62 20.33 b 87.2 28.0 a 6.40 20.53 b 2.51 20.29 b 1.30 20.13 b 0.11 20.03 a 75 22
Skin
Cu Zn Pb Ni Cd Cr moisture (%)
3.042 0.82 a 266 231 a 1.642 0.42 a 1.322 0.19 a 0.312 0.04 a 0.512 0.06 a 72 21
2.9120.12 a 250 218 a 2.2620.16 a 1.5720.14 a 0.3620.00 a 0.3220.02 a 70 21
2.56 20.07 a 124 2 18 b 2.19 20.53 a 0.82 20.06 b 0.35 20.06 a 0.39 20.18 a 71 25
Cu Zn Pb Ni Cd Cr moisture (%)
2.092 0.56 a 22.42 1.5 a 1.532 0.55 a 0.71 20.13 b 0.542 0.19 a 0.552 0.19 a 81 21
2.4920.50 a 23.426.13 a 1.9320.28 a 0.8220.04 a 0.502 0.07 a,b 0.442 0.02 a,b 802 1
1.99 20.16 a 26.7 23.1 a 1.38 20.22 a 0.62 20.05 c 0.34 20.02 b 0.30 20.04 b 78 22
Public Health Regulation (wet weight) (Hong Kong Government, 1989)
ÿ ÿ 6.0 ÿ 2.0 1.0
±: no data.
suggested that these metals were derived from a common origin. Metal contents in sludge is a good indicator of metal usage in local industries. Dramatic increases of Cu, Pb and Cd were observed in sludge samples collected from Shatin Sewage Treatment Plant near Fo Tan from July 1992 to June 1994 (Pun et al., 1995). When compared to Mai Po, which is a relative unpolluted area, metal contents in river sediments of Fo Tan were signi®cantly (p < 0.05) higher in general (Fig. 2). So far, little information is available concerning heavy metal contamination in sediments of inland water systems in Hong Kong. The EPD report (EPD, 1995b) indicated that Zn was the most abundant in the river water, followed by Cu and Pb, and the least was Cd and Cr while Ni was not recorded. The results of the present study were in line with the report that Shing Mun River was the most pol-
Table 2. Pearson correlation matrix showing the relationships between metal concentrations of the sediments Cd Ni Pb Zn Cu
0.94* Zn
0.69* 0.56 Pb
0.39 0.86* 0.92* Ni
0.50 0.91* 0.78* 0.63* Cd
0.73* 0.93* 0.62* 0.97* 0.98* Cr
*Indicates the correlation coecient was signi®cant at 0.01 probability level using one-tailed test, n = 15.
luted followed by Lam Tsuen River and Tai Po River, and metal contamination in river sediments followed the order of Fo Tan>Tai Wai>Tai Wo>Siu Lek Yuen>Tai Po. When compared to other Asian countries (Kurokawa and Tatsukawa, 1990; Prudente et al., 1994; Baruah et al., 1996), the heavy metal contents obtained in the present study were rather high (Table 3). Furthermore, the Pb levels in sediments of Shing Mun River were two times higher than those obtained from Pasig River, Philippines (Prudente et al., 1994) where the place was considered to be heavily polluted by rapid industrialization and increasing population. Sequential extraction provides data on the mode of metals occurrence and the potential bioavailability in the sediments. Levels in the residual fraction should be considered as the background value for the elements in the sediments (Tessier et al., 1979). The water-soluble, exchangeable and carbonate fractions are considered to be moderately available in the environment (Ma and Rao, 1997). In the present study, only Cu was found in the water-soluble fraction. The sum of these three extractions for Cu, Zn, Pb, Ni, Cd and Cr were 4.6±6.9%, 21±36%, 1.5±12%, 22±38%, 55±100% and 1.6±11%, respectively. Copper was present in all fractions, but it was primary associated in the organic fraction (55±
Metal concentrations in sediments and tilapia
3337
Table 3. Comparison of heavy metal concentrations (mg kgÿ1, dry weight) in sediments collected from Shing Mun River, Lam Tsuen River and Tai Po River with published data in other countries Location Shing Mun River (Hong Kong) Lam Tsuen and Tai Po River (Hong Kong) Duyen Hai and Ho Chi Min (Vietnam) Jhanji River (India) Pasig River (Philippines) Marikina River (Philippines)
Cu
Zn
Pb
Ni
Cd
Cr
53.2±449 26.8±69.6
461±1220 238±593
246±1387 169±1529
16.0±67.0 10.6±17.0
2.4±4.0 01.7±3.9
15±25
68±106
7.1±1.1
31±57
0.06±0.10
ÿ
28±70 110±189 28±79
32±288 236±1560 74±169
8.7±87 66±137 18±31
17.5±101 17.1±27.3 10.5±21.7
ÿ 2.03±15.2 1.47±2.59
28.5±399 ÿ ÿ
32.4±85.4 This study 18.3±37.1 This study Kurokawa and Tatsukawa (1990) Baruah et al. (1996) Prudente et al. (1994) Prudente et al. (1994)
ÿ: no data.
70%). Copper can easily form complexes with organic compounds because of the high formation constants of organic-Cu compounds which makes it rather stable in the environment (Stumm and Morgan, 1981). In aquatic systems, the distribution of Cu is markedly aected by natural organic matter such as humic materials and amino acid. The predominance of Cu in organic phase has also been observed in freshwater and marine sediments (Baruah et al., 1996; Chen et al., 1996; Wong, 1996). Zinc was mainly associated with carbonate (21± 36%), Fe±Mn oxide (24±38%) and residual (23± 45%) fractions (Fig. 3). Calcium carbonate is a strong absorbent to form complexes with Zn as double salts CaCO3ZnCO3 in the sediments (Ramos et al., 1994). This coprecipitation phenomena with carbonate may be an important elimination mechanism for Zn when organic matter and hydrous Fe oxides are less abundant in the aquatic system (Forstner and Wittmann, 1979). As to Pb and Cr, residual fraction was the predominant form (62±87% for Pb and 79±93% for Cr) in the sediment. Cr contents were not detected in the Fe±Mn oxide fraction in all sediments, while Cr in the organic fraction was only present in the sediments collected from Fo Tan, Tai Wai and Tai Wo (Fig. 3). Fe±Mn oxide was another important fraction for Pb, although it only accounted for 8±22% of the total Pb. Like Zn, Pb also formed stable complexes with Fe±Mn oxide, and these compounds were even more stable than those of Zn (Ramos et al., 1994). The concentration of Cd was the lowest when compared to other elements monitored. It mainly existed in exchangeable (22±29%) and carbonate (34±61%) fractions (Fig. 3). Cadmium is not found in the organic fraction for low adsorption constant and labile complexion with organic matter (Baron et al., 1990). The pattern of Ni distribution was dierent in dierent sediments. In the sediments collected from Siu Lek Yuen, Tai Wo and Tai Po, the residual fraction was by far the most important, ranging from 40±63% of total Ni. Signi®cantly higher concentrations of Ni were found in carbonate and Fe± Mn oxide fractions in the sediments collected from
Fo Tan and Tai Wai. These two sediments also contained much higher (p < 0.05) concentrations of Ni (67.0 mg kgÿ1 for Fo Tan and 43.1 mg kgÿ1 for Tai Wai) than other sites. Heavy metal concentrations in dierent organs of tilapia When comparing metal accumulation in tilapia living in dierent locations, tilapia collected from the heavily polluted Shing Mun River had signi®cantly higher heavy metal concentration (p < 0.05) than those collected from the Mai Po ®sh farm. In general, muscle contained a smaller amount of metals than skin, gill and viscera. Dierent metals seemed to accumulate in dierent organs. Higher levels of Pb, Ni and Cd were found in gill, while higher contents of Zn were recorded in skin, and Cu in viscera. Gill as the ®rst target for pollutants in water, absorption across the gill (Heath, 1987; Sorensen, 1991) seemed to be the major pathway of Pb, Ni and Cd entering tilapia. The relatively high contents of heavy metals found in viscera may be due to the fact that most of the heavy metals are accumulated in the liver and kidney after ingestion (Badsha and Goldspink, 1982), or excessive metals in the diets are not absorbed but remained in the gut and intestine. On the other hand, ®sh have dierent routes for possible excretion of heavy metals when exposed in heavy metal contaminated water bodies, these include gill, bile (via feces), kidney and skin (Sorensen, 1991). All these factors may contribute to higher levels of heavy metals in viscera, skin and gill than in muscle. Greatest contents of Cu were accumulated in viscera of all tilapia samples. It has been noted that ®sh liver had the highest concentration factor (the ratio of the concentration of a chemical inside an organ to the environmental concentration) of Cu for almost any exposure time (Heath, 1987; Ghazaly et al., 1992). Although liver, kidney and gill are vital organs in maintaining Zn to a considerable extent (Chen et al., 1987; Hilmy et al., 1987), skin showed the highest accumulation of Zn in the present study, followed by gill, viscera, and muscle. Similar results were obtained by Gupta and Sharma (1994) and
3338
H. Y. Zhou et al.
Seymore et al. (1996), who studied Zn accumulation in ®sh. Although Zn is an essential element for ®sh, excessive Zn will be toxic causing mortality, growth retardation and tissue alterations (Sorensen, 1991). Relationships between dierent chemical fractions of metals in sediments and their accumulation in ®sh Relationships of metals in each fraction (except water-soluble fraction) of sediment with metal con-
tents in dierent parts of ®sh were studied using Pearson correlation matrix (Table 4). The accumulations of Cu in viscera were closely related (p < 0.05) to the concentrations of this metal in each fraction, while Zn concentrations in skin were closely related to carbonate, Fe±Mn oxide, organic and residual fractions of sediments. Lead contents in all ®sh organs analyzed were closely related (p < 0.05) to the exchangeable fractions of sediments. No signi®cant correlations were found
Table 4. Pearson correlation matrix showing the relationship of metals between tilapia and each fraction of sediment
Cu
Zn
Pb
Ni
Cd
Cr
Gill
Skin
Viscera
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.043 ÿ0.047 0.422 0.403 ÿ0.164 0.395 ÿ0.262 ÿ0.143
1 0.322 0.073 ÿ0.141 ÿ0.014 ÿ0.252 ÿ0.029 ÿ0.125
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 ÿ0.629* ÿ0.162 0.599* 0.110 ÿ0.377 ÿ0.484 ÿ0.373 ÿ0.508
1 ÿ0.043 ÿ0.492 0.261 0.810** 0.865** 0.845** 0.897**
1 0.066 0.380 ÿ0.593* ÿ0.496 ÿ0.515 ÿ0.390
1 0.010 ÿ0.453 ÿ0.507 ÿ0.488 ÿ0.553
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.901** 0.838** 0.904** 0.746** 0.252 ÿ0.019 0.022 ÿ0.167
1 0.921** 0.986** 0.765** ÿ0.090 ÿ0.313 ÿ0.227 ÿ0.369
1 0.913** 0.786** ÿ0.014 ÿ0.488 ÿ0.479 ÿ0.624*
1 0.805** ÿ0.018 ÿ0.239 ÿ0.170 ÿ0.313
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.757** 0.517 0.818** 0.406 0.245 0.319 0.050 0.481
1 0.347 0.887** 0.528 0.212 0.273 0.152 0.474
1 0.473 ÿ0.245 0.103 0.210 ÿ0.447 0.339
1 0.244 ÿ0.025 0.042 ÿ0.121 0.288
1 0.409 0.734** 0.915** 0.652* 0.882** 0.786**
Muscle
1 0.476 0.311 0.623* 0.168 0.152
Exchangeable
Carbonate
Fe±Mn oxide
1 0.810** 0.960** 0.739** 0.757**
1 0.758** 0.985** 0.931**
1 0.662* 0.681*
1 0.958**
1
1 0.055 ÿ0.022 0.202 0.173
1 0.980** 0.985** 0.964**
1 0.957** 0.970**
1 0.970**
1
1 0.070 ÿ0.203 ÿ0.172 ÿ0.261
1 0.649* 0.491 0.339
1 0.967** 0.926**
1 0.964**
1
1 0.991** 0.817** 0.916**
1 0.757** 0.945**
1 0.660*
1
ÿ ÿ ÿ
ÿ ÿ
1
ÿ ÿ ÿ
1 0.732**
1
1 0.578* 0.561 0.732** 0.566
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 ÿ0.128 0.393 0.510 0.319 0.516 ÿ ÿ ÿ0.229
1 0.740** ÿ0.529 ÿ0.402 ÿ0.531 ÿ ÿ 0.616*
1 ÿ0.120 ÿ0.185 ÿ0.248 ÿ ÿ 0.405
1 0.524 0.603* ÿ ÿ ÿ0.199
1 0.916** ÿ ÿ ÿ0.058
gill skin viscera muscle exchangeable carbonate Fe±Mn oxide organic residual
1 0.040 ÿ0.014 0.300 ÿ0.139 0.040 ÿ 0.250 0.278
1 ÿ0.231 0.054 ÿ0.527 ÿ0.264 ÿ ÿ0.204 0.278
1 ÿ0.393 0.912** 0.150 ÿ ÿ0.007 ÿ0.540
1 ÿ0.178 0.739** ÿ 0.783** 0.729**
1 0.365 ÿ 0.180 ÿ0.476
1 ÿ ÿ ÿ0.253
1 ÿ 0.921** 0.558
Organic Residual
* and ** indicate the correlation coecients were signi®cant at 0.05 and 0.01 probability levels, using two-tailed test, n = 9. ÿ: no data.
Metal concentrations in sediments and tilapia
between the contents of Ni and Cd in ®sh organs and sediments. As to Cr, signi®cant correlations were noted in Cr contents in muscle with Cr contents in carbonate, organic and residual fractions. Cu, Zn and Pb were more abundant than Ni, Cd and Cr both in sediment and river water, correlations of Cu, Zn and Pb concentrations between tilapia and sediments were thus more evident than those of Ni, Cd and Cr. The eect of heavy metals on aquatic organisms is controlled by the concentrations and chemical forms of the metals in water and sediment. Theoretically, free metal ion is the most bioavailable form of the element, and the concentration of the free metal ion varies signi®cantly with pH and organic substances (Sorensen, 1991; Rainbow, 1995). It is believed that complexation of metals by organic substances reduces metal bioavailability and its potential toxicity. Assuming that bioavailability is related to solubility, the six extraction fractions followed the decreasing solubility order of: watersoluble>exchangeable>carbonate bound>Fe±Mn oxide bound>organic bound>residual (Ma and Rao, 1997), then metals bioavailability decreased in the same order. Metals in the residual fraction and associated with silicates would not expect to release under natural condition. Whatever the precise uptake mechanisms, most heavy metals can be accumulated by aquatic biota and pose toxic eect at high trophic levels including human beings. CONCLUSION
Sediments collected from inland river system had much higher concentrations of heavy metals (Cu, Zn, Pb, Ni, Cd and Cr) than those collected from the ®sh pond. The magnitude of heavy metal contents in sediments were Zn>Pb>Cu>Cr, Ni>Cd. Tilapia living in heavily polluted Shing Mun River contained a substantial amount of heavy metals when compared to those collected from the commercial ®sh pond. It was indicated that dierent metals had dierent accumulation targets in ®sh body (Cu in viscera, Zn in skin, Pb, Ni, and Cd in gill, Cr in skin and muscle). Signi®cant correlations between dierent chemical fractions of metals (exchangeable, carbonate, Fe± Mn oxide, organic and residual) in river sediments and their accumulation in ®sh organs (Cu in viscera, Zn in skin, Pb in all organs, and Cr in muscle) were obtained. Sequential extraction of metals from the sediment may re¯ect their chemical species as well as their adsorbed fraction. However, their bioavailability will re¯ect not only their solubilities but also the presence or absence of chelating compounds in water phase, structure of foodchains, and other parameters such as pH and redox potential. AcknowledgementsÐThe authors would like to thank Dr Y. Liang, Mr K. W. Chan and Miss F. Y. K. Lee for
3339
technical assistance. Financial support from the Industry Department, Hong Kong Government is gratefully acknowledged.
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