Marine Pollution Bulletin, Vol. 31, Nos 4-12, pp. 464--470, 1995
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The Toxicity of Marine Sediments in Victoria Harbour, Hong Kong Y. S. WONG*, N. F. Y. TAMJ', P. S. LAU* and X. Z. XUE:~
*Research Centre~Biology Department, The Hong Kong University of Science and Technology, Hong Kong ~fDepartment of Biology and Chemistry, City University of Hong Kong, Hong Kong ~Environmental Science Research Centre, Xiamen University, People's Republic of China
When the toxicity of marine sediment in Hong Kong was evaluated, it was found that the seven sediments collected within Victoria Harbour were severely contaminated with heavy metals, at concentrations many times higher than those in sediments collected from outside the harbour. The highest metal content was recorded in site VSI4 (located near the airport runway and the industrialized area), with copper, zinc, lead and chromium values of 3789, 610, 138 and 601mgkg - z dry wt, respectively. This site also had the greatest alkaline phosphatase activities (15 fluorescent intensity unit g-1 wet wt), the largest number of total coliforms (910CFUg -1 wet vet) and sulphate-reducing bacteria (8.5 x 104 cells g - 1 wet wt), implying that site VS14 was also contaminated with organic matter and nutrients. Sediment bioassays, Microtox and algal tests, demonstrated that sediment elutriates obtained from site VS14 were of greatest toxicity. The EClo value in Microtox tests was 17% elutriate, and the 96-h ICso values using Skeletonema costatum and Dunaliella tertiolecta were 40 and 79% elutriate, respectively. No toxic effects were found in sediment samples collected from the control site outside Victoria Harbour. Significant correlations were found between the results of the algal toxicity test (using S. costatum) and the coliform count and metal content of the sediments. The Microtox test was less sensitive than the algal bioassay, and no sediment elutriate, even from the site mostly contaminated by heavy metals, caused more than 50% inhibition of the light-emitting activity of the bacteria. In this study, S. costatum (the diatom) provided a more sensitive and reliable test species than D. tertiolecta (the flagellate) in differentiating the toxicity of marine sediments.
Owing to the continual rise in human population density and the rapid industrial growth and land reclamation, large quantities of dredged sediments are generated in Hong Kong (EPD, 1992). Marine sediments (especially those from the inshore waters of urban centres such as Victoria Harbour, Hong Kong) are contaminated with both inorganic and organic chemicals, particularly heavy metals. Marine sediments act both as a sink and as a reservoir for 464
these persistent contaminants, influencing the fate and concentration of toxicants, and potentially damaging aquatic ecosystems (Chapman, 1988). Traditional techniques to predict the adverse effects of sediments involve analysis of the bulk chemical characteristics and contaminant concentrations of sediments and/or the use of field surveys to detect the presence or absence of certain types of organisms. Although these techniques provide useful information, it is difficult to extrapolate from such data to the likely impacts on living organisms, and the in situ toxicities of contaminant mixtures in sediments are often unknown. Sediment bioassays that measure the toxic effects of contaminated sediments on test organisms have been recently developed (Chapman, 1988; Burton & Scott, 1992), and a large variety of bioassays is becoming available (USEPA, 1990). Various types of organisms and several biological parameters have been used to evaluate the toxicity of sediments. Marine unicellular algae (phytoplankton, microalgae) are essential to the normal function of marine ecosystems, as they are the main primary producers that form the first link in food webs, oxygenate the water, and are important in cycling dissolved organic and inorganic substances (Walsh, 1988). Microalgae also show great sensitivity to environmental pollutants, and bioassays using organisms such as Skeletonema costatum (a diatom) provide information on sediment toxicity (Walsh, 1988). Acute toxicity tests employing bacteria (e.g. the Microtox assay) are rapid, sensitive and cost effective; thus, they are valuable as preliminary screening tests for determining the toxicity of contaminated sediments (Kwan, 1993). The use of sediment-based microbial activity such as alkaline phosphatase activity and total bacterial counts can enhance the understanding of the impact of aquatic toxicants, as microbial activities regulate the mineralization, co-metabolism (co-oxidation) and biodegradation of many toxicants (Burton & Lanza, 1985). The present study aims to 1. assess the toxicity of contaminated marine sediments collected from various locations in Hong Kong using these microorganisms; 2. compare the sensitivity and comparability of different bioassays; and 3. determine the relationships between the results of toxicity tests and the concentrations of heavy metals in sediments.
Volume 31/Numbers 4-12/April-December 1995
Materials and Methods Eight surface sediment samples (each from two replicates, taken approximately 50m apart) were collected. Seven samples (labelled as VS) were collected from within Victoria Harbour, a heavily urbanized and industrialized water body that receives organic and inorganic contaminants from surrounding domestic and industrial sources. One sample was collected from
outside the harbour (VM site) in the East Lamma Channel of Hong Kong, and this was used as the control (Fig. 1). Samples of sediments were collected using a Srnith-McIntyne grab and were transported to the laboratory and stored at 4°C before analyses. Most sediments collected were anaerobic and black in colour (Table 1). Concentrations of heavy metals and boron in these sediments were determined by digesting the samples with a mixture of H202 (30%), concentrated
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Fig. 1 Locations of the eight sediment collection sites around Victoria Harbour.
Marine Pollution Bulletin TABLE 1 General features of sediments collected in this study.
Water depth (m)
VSI VS2 VS3 VS4 VS6 VS8 VSI4 VM2
21.0 6.4 7.2 8.1 11.5 11.6 6.2 13.0
8.22 8.39 8.02 8.66 8.24 7.95 8.50 8.17
Fine mud, black, strong H2S smell Sand mud, black, strong H2S smell Fine mud, black, no smell Fine mud, black, no smell Fine mud, black, no smell Fine mud, grey, no smell Fine mud, black, strong H2S smell Fine mud, grey, no smell
HNO3, H F and HC104. Copper, zinc, cadmium, chromium, nickel, lead, manganese, iron, boron and aluminium in the digests were then analysed by ICP/ AES (model PE plasma 1000). The numbers of total aerobic heterotrophic bacteria, total coliform, faecal coliform, sulphate-reducing bacteria and yeasts in the sediments were defined by shaking 6 g of sediment samples (wet wt) with 200 ml sterilized seawater, followed by serial dilution. The diluted sediment suspension (1 ml) was transferred to 'Millipore Samplers' (MILLIPORE, Bedford, UK), for the measurement of total bacteria, coliform and yeast/ mould counts. The Millipore filters were then incubated for 48h at 35°C for total bacteria count, 24h at 30°C for total coliform bacteria, 24h at 44°C for faecal coliform and 48 h at 30°C for yeast/mould. The number of colonies formed on the Millipore filter at the end of incubation period was counted. Sulphate-reducing bacteria were enumerated using test tubes containing a sulphate-reducing medium, incubated at 30°C for 144 h. The number of positive tubes at the end of the incubation period was counted and the MPN values were recorded (APHA et al., 1992). The activity of alkaline phosphatase in sediments was determined by extracting 1 g of sediments (wet wt) with Tris buffer containing methylfluorescein phosphate (as substrate) at pH 9. The enzyme activity was measured in terms of fluorescent intensity, as described by Hill et al. (1968). The sediment extraction procedures described by Ankley et al. (1991) were used to prepare the sediment elutriates. One part of sediment was mixed with four parts of artificial seawater (prepared from commercially available salt, 'Instant Ocean') and the mixture was shaken and centrifuged. The aqueous fraction was decanted as the elutriate. The toxic effects of elutriates were assessed by the static 96-h algal bioassay following
the standard ASTM protocol (ASTM, 1990). Two marine microalgal species, S. costatum (a diatom) and Dunaliella tertiolecta (a flagellate), were used and the cell numbers were counted by haematocytometer. The IC50 value (the concentration of elutriate causing 50% inhibition of cell growth) was estimated by plotting the percentage of responses against the concentrations (log10 values) of elutriate. The toxicity of elutriate was also evaluated by the standard Microtox test employing a marine bacterial species, Photobacterium phosphoreum (Microtox ® model 205, Microbics Co.). The end-point is the 15-min ECs0, EC20 and EClo, i.e. the concentrations of elutriate causing 50, 20 and 10% inhibition of the light-emitting ability of the bacteria after 15rain exposure at 15°C, respectively (Casarini et al., 1991). The relationship between heavy metal concentrations, enzymatic activities, bacterial population sizes and bioassay results were calculated by Spearman rank correlation coefficients (Siegel, 1956).
Results and Discussion Table 2 shows that the sediment samples collected from Victoria Harbour (the VS sites) were heavily contaminated with Cu, Zn, Cr, Ni and Pb and their concentrations were many times higher than those collected from outside the harbour (VM2). Concentrations of Cd, Fe and A1 were similar among the eight sediment samples. The highest metal levels were recorded in sediments collected from site VS14, which is close to the airport runway and near the industrialized area of Kwun Tong. The site exhibiting the second highest metal levels was VS2, which also receives effluents from this industrialized area but has more flushing to dilute and disperse the contaminants. Sites VS6 and VS8, towards the west of Victoria Harbour (near Stonecutters Island), were relatively less contaminated than other sites within the Harbour (Table 2). This trend was similar to that reported by EPD (1992), showing that sediments near urbanized centres and industrialized areas are more polluted than those of open marine areas. It is considered that the discharge of industrial wastes by textile, printed-circuit-board and electroplating factories contribute significantly to the high concentrations of heavy metals in bed sediments (EPD, 1992). The heavy metal levels recorded in this study were higher than those reported in 1992 (EPD, 1992), indicating that the contamination of Victoria Harbour may be increasing.
TABLE 2 Heavy metal concentrations of surface sediments (mg k g - t dry wt).
VS1 VS2 VS3 VS4 VS6 VS8 VS14 VM2
312.6 921.9 353.5 633.7 176.5 63.3 3789.5 45.2
171.2 258.8 165.9 189.7 154.1 129.2 610.4 97.9
3.33 2.63 2.62 2.89 2.61 2.69 3.29 3.24
135.8 170.7 100.8 120.5 74.0 116.8 601.2 57.5
36.1 63.8 31.6 40.4 25.4 28.4 177.1 23.6
47.4 70.2 56.6 70.8 47.4 68.3 138.1 48.1
428.1 373.9 432.8 414.5 389.3 568.8 371.5 453.0
55.1 37.4 39.7 48.5 46.8 53.3 28.2 51.7
3.41 2.89 3.12 3.19 2.99 3.01 3.02 3.21
2.19 1.53 2.05 1.35 1.34 1.74 2.24 1.50
*% dry wt.
Volume 31/Numbers 4-12/April-December 1995 Organic pollutants and faecal matter were substantial in the sediments studied. The faecal pollution is due to the discharge o f large quantities of untreated or partially treated municipal sewage from the densely populated urban areas located on both side o f Victoria H a r b o u r (EPD, 1992). The numbers o f total coliform and faecal coliform bacteria in sediments collected from VS sites were much higher than those from the control (VM) site (Table 3). Among the seven sediment samples collected from Victoria Harbour, site VS14 exhibited the highest counts of total coliform bacteria and yeasts/moulds, the greatest alkaline phosphatase activity and the largest population size of sulphate-reducing bacteria (Table 3). These results suggest that site VS14 was severely contaminated with organic wastes and nutrients. Microtox tests on sediment elutriates all produced relatively non-toxic responses. The ECs0 values were either impossible to calculate or greater than 100% elutriate (Table 4). The relatively non-toxic responses were probably due to the fact that artificial seawater extracts only water-soluble toxicant(s) and these may not exert significant toxicity (Kwan, 1993). The EC20 and EC10 data show that the sediments o f sites VS14, VS2 and VS1 were more toxic than those of other locations (Table 4). Although the Microtox assay is a rapid and simple method for screening a large number of samples, the test was less sensitive under the conditions used here, compared to other bioassays such as algal tests. The microalgal bioassays revealed that elutriates prepared from VS14 sediments had the highest inhibitory effects on the cell division and cell growth o f both S. costatum and D. tertiolecta, followed
by VS4 (Fig. 2). Some inhibition o f S. costatum was also found in sediment elutriates prepared from site VS2. Based on the effects o f 100% elutriates on algal growth, the toxicity of four o f the eight sediments followed a descending order o f V S 1 4 > V S 2 > V S 4 > V S 1 (Fig. 3). Sediments collected from the other four sites (including the control site) did not exert any inhibitory effects on algal growth, and the elutriates stimulated cell division. When the two algal species were compared, S. costatum was more sensitive to the toxic effects o f the sediment elutriates than D. tertiolecta, with IC50 value at 38 and 87% elutriates for the most polluted sediments (VS14), respectively (Fig. 3, Table 4). Significant correlations were found between the results o f algal toxicity tests and heavy metal concentrations in sediments. Greater levels of cell growth inhibition by 100% sediment elutriates were recorded when higher concentrations o f Cu, Ni, Zn and Cr were measured (Table 5). The responses of S. costatum were also positively related to the total coliform count and the alkaline phosphatase activities (Table 5). The positive correlation between alkaline phosphatase activity and total content of Zn and Pb suggests that this enzymatic activity may be stimulated by heavy metals. Burton & Lanza (1985) reported that, although alkaline phosphatase activity represents a broad spectrum of microbial activity and has potential as a tool to detect the impacts o f toxicants in sediments, additional factors influencing microbial responses to toxicants also affect the toxicity. The presence of large quantities of organic matter and nutrients (particularly phosphorus as identified in this
Bacteria counts (number g-t wet wt) and enzymeactivities (fluorescentunit g-1 wet wt) of surface sedment samples. Sample VSI VS2 VS3 VS4 VS6 VS8 VS14 VM2
Total bacteria count 27 000 450 000 385 000 1800 000 480 000 540 000 390000 230 000
Total coliform count 670 700 720 360 265 215 910 <5
Faecal coliform count
SO4-reducing bacteria count
35 150 205 165 53 <5 182 <5
8300 55000 2300 36 000 75 600 10500 85000 25 700
33 700 360 <5 315 110 1210 400
Alkaline phosphatase 5.75 8.40 4.42 8.10 8.55 6.60 15.03 2.75
TABLE 4 Toxicity of surface sediment samples based on two bioassays: Microtox and algal tests.* Microtox
VS1 VS2 VS3 VS4 VS6 VS8 VS14 VM2
40.81 36. l 5 NT NT NT NT 16.91 51.21
79.23 74.26 NT NT NT NT 57.07 75.33
246.30t 254.23t NT NT NT NT 456.44t 145.69t
SM 60.95 SM NT SM SM 38.19 SM
NT NT SM NT SM SM 87.49 NT
*EClo, EC2oand ECso, elutdate concentrations causing 10, 20 and 50% inhibition of fight emission, respectively. tEC50 value was greater than the highest concentration (i.e. 100% elutriate) and the result was calculated from extrapolated data. NT, No toxicity was detected in any elutriate concentration. ICs0, elutriate concentration causing a 50% reduction of algal cell counts. SM, stimulating effect. 467
Marine Pollution Bulletin
(B) VS2 100
60 40 20 0 -20 ( 1.0
60 40 20 •
-40 -60 -80 1.2
80 60 40
20 o~ -20 -40 1.0
ol I 1.2
i ~'~04.----~ 1.4 1.6 1.8
(~.) v s s
100 75 50 25 0 -25( -50q -75 -100 -125
100 80 ~ o
4O 20 0
40 20 O, .0
Log e l u t r i a f l e c o n e .
Log e l u t r i a t e
Fig. 2 Responsesof microalgal ceils to sediment elutriates of different dilutions. A positive value indicates the % inhibition in cell growth, while a negativevalue represents the % increasein cell number, i.e. a stimulating effect(O, Skeletonema costatum; • , Dunaliella tertiolecta).
study) might affect the responses of microorganisms to toxic sediments. In summary, the present study shows that the sediments tested were only moderately toxic to nontoxic, except for those from site VS14. The growth and cell division o f the two microalgae (S. costatum and D. tertiolecta) were significantly inhibited by elutriates prepared from sediments collected from site VS14. Based on the concentrations of total heavy metals and the bacterial data, sediments collected from some sites, VS2 and VS14 in Victoria Harbour were heavily contaminated with toxic metals and organic matter. Chapman (1988) recommended that, in order to evaluate the toxicity o f sediments, integrated ap468
proaches such as the 'Sediment Quality Triad' and the 'Apparent Effects Threshold' should be adopted. By using as a battery of toxicity tests, biotic community indices, habitat evaluations and chemical characterization studies, accurate evaluations or predictions of ecosystem perturbations are more likely (Burton & Scott, 1992). Elutriates of all sediments in this study were non-toxic to a marine bacteria (P. phosphoreum) which was not comparable with the algal bioassays. Ahlf et al. (1989) pointed out that the comparability of bioassays is limited by the different sensitivities of test organisms. Complete agreement between different toxicity tests only occurs when sediments are either highly toxic or non-toxic; thus, for sediments which are
Volume 31/Numbers 4-12/April-December 1995 VS2
80 60 40
-60 -80 -100
Fig. 3 Effects of 100% sediment elutriates on algal growth. Positive
values indicate the % inhibition in algal growth, while negative values represent the % increases in cell number, i.e. a stimulating effect (1~, Skeletonema costatum; , Dunaliella tertiolecta).
o f low to m o d e r a t e toxicity, the observed differences between test results m a y be expected ( C h a p m a n , 1992)•
o o 0 0 ~ 0 o o ~
The authors would like to express sincere thanks to Dr H. Y. Yeung from the Environmental Protection Department, Hong Kong Government, for their assistance in collecting sediment samples, and the technicians in the Research Centre of the Hong Kong University of Science and Technology for their help in analytical work.
0 0 0 ~ 0 0 0 0 0 0 0 ~ 0 0 ~
Ahlf, W., Calmano, W., Erhard, J. & Forstner, U. (1989). Comparison of five bioassay techniques for assessing sediment-bound contaminants. Hydrobiologia 1~/189, 285-289. Ankley, G. T., Katko, A. & Arthur, J. W. (1991). Predicting the toxicity of bulk sediments to aquatic organisms with aqueous test fractions: pore water vs. elutriate. Environ. Toxicol. Chem. 10, 13591366. APHA-AWWA-WPCF (1992). Standard Methods for the Examination of Water and Wastewater. APHA-AWWA-WPCF, New York, USA. ASTM (1990). Standard guide for conducting static 96-h toxicity tests with microaigae. In Annual Book of A S T M Standards, Water and Environmental Technology, Vol. E1218-90. American Society for Testing and Materials, Philadelphia, PA, USA. Burton, G. A. & Lanza, G. R. (1985). Sediment microbial activity tests for the detection of toxic,an impacts. In Aquatic Toxicology and Hazard Assessment: Seventh Symposium (R. D. Cadwell, R. Purdy & R. C. Bahner, eds), pp. 214-228. American Society for Testing and Materials, Philadelphia, PA, USA. Burton Jr, G. A. & Scott, K. J, (1992). Sediment toxicity evaluations: their niche in ecological assessments. Environ• Sci. Technol. 26(l 1), 2068-2075. Casarini, D. C. P., Cunha, R. C. A., Sato, M. I. Z. & Sanchez, P. S. (1991). Evaluation of toxicity test procedure to define loading rates in a land treatment system. Wat. Sci. Technol. 24(12), 183-188. Chapman, P. M. (1988). Marine sediment toxicity tests. In Chemical and Biological Characterization of Sludges, Sediments, Dredged Spoils, and Drilling Muds, ASTM STP 976 (J. J. Lichtenberg, F. A. Winter, C. I. Weber & L. Fradkin, eds), pp. 391-402. American Society for Testing and Materials, Philadelphia, PA, USA. Chapman, P. M. (1992). Pollution status of North Sea sediments--an international integrative study. Mar. EcoL Progr. Set. 91, 313-322. EPD (1992). Environment Hong Kong 1992. Hong Kong Environmental Protection Department, Hong Kong Government Printer. Hill, H. D., Summer, G. K. & Waters, M. D. (1968). An automatic fluorometric assay for alkaline phosphatase using 3-O-methylfluoreseeiu phosphate. Anal. Biochem. 24, 9-17.
Marine Pollution Bulletin Kwan, K. K. (1993). Direct toxicity assessment of solid phase samples using the toxie-ehromotest kit. Environ. ToxicoL Wat. Qual. 8, 223230. Siegel, S. (1956). Non-parametric Statistics for the Behavioral Sciences. McGraw-Hill Kogakusha, Tokyo, Japan.
USEPA (1990). Managing Contaminated Sediments. Report EPA506-90/002, Sediment Oversight Technical Committee, US Environmental Protection Agency, Washington, DC, USA. Walsh, G. E. (1988). Prindples of toxicity testing with marine unicellular algae. Environ. Toxicol. Chem. 7, 979-987.