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Benthic marine diatom deformities associated with contaminated sediments in Hong Kong
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Pergamon
Environment International, Vol. 24, No. 7, pp. 749-759, 1998 Copyright O1998 Elsevier Science Ltd Printed in the USA. All rights reserved 0160-4120/98 $19.00+.00
PH S0160-4120(98)00060-9
BENTHIC MARINE DIATOM DEFORMITIES ASSOCIATED WITH CONTAMINATED SEDIMENTS IN HONG KONG Mike D. Dickman Ecology and Biodiversity Dept., The University of Hong Kong, Hong Kong, China
E1 9710-209 M (Received 31 October 1997; accepted 21 May 1998)
Three thousand benthic diatoms were examined at each of twelve sites to determinethe frequency of diatom deformities. Two basic types of diatom deformities were observed: 1) diatoms with deformed cell wall morphology; and 2) diatoms with deformedvalve wall ornamentation.Only six deformed diatoms were observed. Deformed individuals ofFragilaria capucina and Achnanthes hauckiana displayed external cell wall deformities,while deformedindividualsof Diatoma vulgate displayed valve walls with a wavy pattern of longitudinal lines in which the valve ornamentation, rather than the valve cell wall, was abnormal.The deformityfrequencyofNavicula rhyncocephala, Achnanthes hauckiana, Fragilaria capucina, and Diatoma vulgare was significantly lower at unpolluted sites than at polluted sites based on Fisher exact probability estimates. Cadmium reached 1.1 mg/kg dry wt. of sediment, while other levels were chromium (42 mg/kg of sediment), copper (440 mg/kg), nickel (31 mg/kg), lead (130 mg/kg), and zinc (450 mg/kg of sediment) atthe most polluted site. There was a significant correlation (P < 0.05, r~ = 0.652) between sediment toxicity, as reflected by the Microtox test, and benthic diatom diversity. The most polluted site exhibited the highest Microtox acute toxicity levels, the lowest diversity, and moderate diatom deformity levels. It was concluded that abnormal cell morphology of diatoms may be a valid indicator of ecosystem health. However, because the amount of time and effort required to identify a statistically meaningful number of deformed diatoms is so great, diatom deformity frequency is unlikely to become a useful tool unless an automated system of assessing diatom deformity f r e q u e n c y is d e v e l o p e d . 01998 Elsevier Science Ltd
INTRODUCTION There is growing acceptance o f the view that chemical measurements alone are poor gauges o f the biological impacts o f pollutants. As a consequence, there is an increasing shift away from the strict reliance on chemical measurements to assess the health of ecosystems. Biological communities appear to provide a direct means o f observing the impact of contaminants. The bioavailability o f heavy metals, once they have bound with nonionic organic compounds, colloids, or clays, is extremely difficult to assess from chemical data alone. The present study o f the relationship be-
tween contaminated sediments and deformity frequencies of benthic diatoms was an attempt to determine if these microscopic plants might serve as biological indicators o f heavy metal bioavailability. The sediments o f coastal areas around Hong Keng, such as Victoria Harbour and Tolo Harbour, are contaminated with a variety o f heavy metals and persistent organic compounds (Enviro-Chem Engineering and Laboratory Co. Ltd. 1994; EPD 1994). A number o f studies have been conducted to assess sediment toxicity in Hong Kong, but assessments o f chemical 749
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M. Dickman
Tubing Handpump wiLh one-way valve
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Clear plastic sleeve
Fig. 1. A schematic illustration of the shallow water epipelic suction sampler.
bioavailability have proved difficult. The chemical form of a pollutant determines its potential bioavailability to marine organisms (Burgess and Scott 1992). Bioavailability is reduced in the presence of sedimentbound nonionic organic compounds or colloidal matter. Heavy metal toxicity is also decreased in the presence of organic ligands of sulfide precipitates (Landrum et al. 1987). Ecological conditions in coastal waters near Hong Kong are a concern to its citizens and scientists alike because there are a number of toxic chemicals present that have been linked to reproductive, metabolic, neurologic, and behavioural abnormalities (Wu 1988; Hong 1993). The elevated level of heavy metals in Tolo Harbour (EPD 1994) was the main reason this study was undertaken in this part of Hong Kong. Lam et al. (1987) concluded that the assimilative capacity of Tolo Harbour was limited due to its enclosed characteristics, weak currents, vertical salinity, and thermal stratification. The copper, chromium, and zinc in Tolo Harbour are all elevated above the U.S. Environmental Protection Agency (U.S. EPA) guidelines (Lam et al. 1987). Benthic diatom deformities have been correlated with the presence of genotoxic and teratogenic chemicals in aquatic sediments in a number of countries (Andresen and Tuchman 1991; Barber and Carter 1981; Dickman et al. 1992; Yang and Duthie 1993). William et al. (1980) described the effect of 11 heavy metals on the morphology of Thalassiosira aestivalis.
Diatoms as indicator organisms
Diatoms are single-celled algae which are classified by the shape and ornamentation of their silica cell walls. These cell walls preserve extremely well and, as a result, diatoms can be efficiently and permanently identified and archived. Cell shape and ornamentation in natural diatom populations vary only slightly within a species, and deformities in diatom morphology from unpolluted waters are rare. METHODS
In the present study, 12 sites were examined to determine their diatom diversity, sediment toxicity as indicated by the Microtox test, heavy metal concentrations of the sediments, and frequency of diatom deformities. An Ekman grab sampler was used to sample the sediments for heavy metals and Microtox determinations. A suction sampler (Fig. 1) was used for quantitative samples of sediment diatoms. Because sand grains in the Ekman grab samples obscured the diatoms, making their accurate enumeration far more time consuming, a method was developed that removed little sand in sampling the epipelic diatoms from the surface layer of sandy substrates. This epipelic diatom suction sampler (Fig. 1) was used to sample the diatoms from all sites except sites 3 and 4, which were too deep for the suction hose to reach the bottom. An Ekman dredge sampler was used at two of the twelve sites which were too deep to employ the suction
Benthic marine diatom deformities
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Fig. 2. The upper two insets indicate the location of Ping Chau in Mirs Bay relative to the location of the six Tolo Harbour sample sites. The latitude of Ping Chau is 114 ° 25' and its longitude is 22 ° 35'. The location of Cape d'Aguilar is indicated by the dot at the end of the cape in the upper inset. The inset at the far right of the upper portion of Fig. 2 provides a general idea of Hong Kong's location in the South China Sea.
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sampler. The Ekman dredge penetrated the sediment to a depth of about 4 cm ± 1 cm depending on the amount of sediment consolidation. Three replicates were taken from each of the twelve sites. Each site was sampled once during the period January 1994 to February 1995. Sample sites 1-4 are located in the Tai Mei Tuk Typhoon Shelter in Tolo Harbour near Sha Lan (lower inset, Fig. 2). Sample site 5 is located near the Tai Po Industrial Estates. Site 6 is located at the mouth of a small stream that enters Tolo Harbour between Tai Mei Tuk and the northern end of the Plover Cove Reservoir. Ping Chau is located at 114 ° 25' north latitude and 22 ° 35' east longitude (upper inset, Fig. 2). The sample site at Cape d'Aguilar is indicated by the dot at the end of the cape in the upper inset. There are six sites in Tolo Harbour, three sites at Ping Chau, and three sites at Cape d'Aguilar on Hong Kong Island. The Ping Chau and Cape d'Aguilar sites were selected because of their low levels of sediment heavy metals. One thousand diatom frustules were counted for each of the three replicates at each of the twelve sites (36 000 diatoms were counted in all). A Horiba portable water quality analyzer was used by the author to measure the specific conductivity of the water at the mud-water interface at each sample site. Values are reported as the meanbased on three replicates at each site. In addition, dissolved oxygen, temperature, and salinity were measured (n=3 per site) with the same unit. Total dissolved solids and oxidation-reduction potential (ORP) for the mud-water interface were measured according to the Standard Methods (APHA 1975).
Heavy metal analyses The Cd, Cr, Cu, Fe, Ni, Pb, and Zn concentrations in the sediment were determined by the AWT Science and Environment Consulting Co. (1995) using atomic absorption spectrometry on nitric acid digested samples. All glassware was acid-washed after first soaking it in Alconox solution for 24 h. After the acid washing, theglassware was rinsed in three separate washes of deionized water. The dried sediment was next dissolved in high purity grade nitric acid. To each test tube, 8 mL of 70% nitric acid (Fisher Scientific, Trace Metal Grade) were added. The test tubes were covered with glass condensers and the samples were digested at room temperature for 12 h. Digestion was continued for 8 h more at 120 C. Digests were cooled to room temperature and brought to 50 mL with Nanopure deionized water. Two sample blanks were analyzed
M. Dickman
together with each sample batch. Concentrations of metals in blanks were below detection limits in all analyses. A spiked blank was analyzed during each analysis to ensure day-to-day reproducibility. Each standard and sample was measured in duplicate and the sample was re-analyzed if the relative standard deviation of the two measurements was higher than 10%. Standard reference materials from the National Institute of Standards and Technology and the National Research Council of Canada were digested and analyzed with each sample batch. The results of metal concentrations always fell within 1 SD of certified values.
Biological analyses The biological assessment of the contaminants present in the sediment was carried out by screening for both acute toxicity (Hong Kong Environmental Protection Dept.) and the frequency of diatom deformities (Ecotoxicology Laboratory, The University of Hong Kong). The samples from each of the 12 sites were coded to avoid unconscious bias. After 36 000 diatoms had been counted and the number of individuals of each species recorded, the number of deformed diatoms on each slide was noted and the slides were then decoded.
Toxicity testing The method chosen for sediment toxicity screening was the non-extractive Microtox Solid Phase Test (Microbics Corp. 1992). This is a bacterial inhibition bioassay, using a 50% reduction in the bioluminescenee of a colony of Photobacteriumphosphoreum as the criterion for calculating an ECso value. The ECs0 is defined as the median effective concentration and represents the time it takes for a 50% reduction in light intensity of the bioluminescent bacteria. The wet sediments collected from each site were centrifuged and the resulting pellet was made into an aqueous suspension before being serially diluted. The effective concentration (ECs0), which is the concentration of the sediment sample causing a 50% decrease in bioluminescence measured after 25 min total contact time, was then determined (Microbics Corp. 1992). The Microtox test is a rapid and simple method for comparing and ranking the relative toxicity of sediment samples taken from different locations. A detailed discussion of the physiology of Photobacterium phosphoreum, the influence of test conditions on the final toxicity values, and the experimental procedure of
Benthicmarinediatomdeformities
this bioassay were provided by Krebs (1983). Unlike methods which require solvent extracts or elutriates to be prepared from the sediments, the Microtox Solid Phase Test allows the Photobacterium phosphoreum bacteria to come into direct contact with the sediment. The bacteria are then separated using a filter column and analyzed using the Microtox Toxicity Analyzer Model M 500 (Beckman 1982).
Diatom deformity testing A Pasteur Pipette was used to transfer 5 mL of the homogenized sediment-water mixture to an Utermohl sedimentation tube which was observed using an Olympus Research compound microscope (Model BX 50, equipped with a photoautomat). At each site, a total of 1000 diatoms were counted for each of the 3 samples taken from that site. Diatoms were counted at 400 times magnification and identified at 1000 times magnification. Identifications were based on Cheng et al. (1993), Germain (1981), Jin et al. (1986), and Patrick and Reimer (1966; 1975). Deformities were photographed using an Olympus Research microscope equipped with a photoautomat. A total of 36 000 diatoms were examined for deformities.
Site descriptions Site 1: The sediments at this site were a brownishyellow colour and were collected using an Ekman grab sampler. Amphipods, crabs, and hermit crabs were found among the collected sediments (water depth was about 1.2 m). Site 2: The sediments at this site were observed to be relatively inorganic. Filamentous green algae, primarily Enteromorpha, were collected along with the sediment (water depth was about 0.9 m). Sediments were collected using the epipelic suction sampler (Fig. 2). Site 3: Water depth was about 0.75 m. Numerous benthic invertebrates were found at this site and sediments were collected using the epipelic suction sampler (Fig. 2). Site 4: Water depth was about 3 m. The sediment was black and gave off a strong hydrogen sulphide smell. No invertebrates were found. Sediment at site 4 was collected using an Ekman grab sampler. Site 5: Water depth was 0.8 m. The area was near the Tai Po Industrial Estate storm drain which was a double storm drain out-fall located about 500 m north of the Motorola Building. Sediments were collected at this and all remaining sites using an epipelic suction sampler.
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Site 6: Water depth was less than 0.75 m. The sediment was quite coarse grained, brownish-yellow sand. Site 7 was located about 300 m northwest of the ferry dock on Ping Chau Island in about 0.1 m of water. The sediments at this site were coarse grained sands which were relatively free of organic matter except for some filamentous green algae. Site 8 was located about 300 m southeast of the ferry dock on Ping Chau Island (Fig. 2). The water depth at this site was about 0.1 m. The sandy sediments at site 8 were slightly finer than those at site 7 and filamentous green algae was also present. Site 9 was located about 1.3 km southeast of the ferry dock on Ping Chau Island (Fig. 2). The water depth at this site was about 0.8 m. The sandy sediments at site 9 were quite fine and a flocculant organic layer was present at the sand-water interface. Sites 10, 11, and 12 were located at Cape d'Aguilar in shallow water (0.1 - 0.4 m). No filamentous green algae were present at these sites. All three of these sites (Fig. 2) had coarse sandy sediments. Samples were collected at low tide to make sediment sample conditions as uniform as possible. Salinity, conductivity, dissolved oxygen, ORP, pH, water depth, temperature, and total dissolved solids were measured at each site using a YSI Model 6000 Environmental Monitoring System (Campbell Scientific Inc., Logan,
UT, USA). RESULTS
With the exception of site 4, where the sediments were anaerobic and had an ORP of- 154 (Tables 1 and 2), the diatoms were active when viewed live under the microscope. Secchi transparencies were nearly always less than 1.5 m and the compensation depth was generally less than 3.5 m. A list of the diatom species found at each site was compiled but is not included because of its large size. This list is available from the author.
Deformed diatoms As shown in Fig. 3, normal cells of Achnanthes hauckiana Grun. (Jin et al. 1985) were easily distinguished by their bilateral symmetry (left), whereas deformed cells (right) lacked symmetry. Normal (upper inse0 and deformed Diatoma species (Fig. 4) were recognizable because deformed individuals displayed a wavy pattern of longitudinal lines in which the valve ornamentation rather than the valve cell wall was the critical trait. Deformed individuals of Fragilaria
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Table 1. Water chemistry data for mud-water interface samples collected at sites 1-6. Parameter
Units
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
Conductivity Total dissolved solids Salinity Dissolved oxygen ORP pH Temperature Depth
(mS) (mg/kg) (g/kg) (% Sat.) (mV)
50.1 32.5 32.7 79 85 7.55 27.2 0.9
50.0 32.6 32.7 48.6 84 7.51 27.2 0.7
48.9 32.5 32.7 92 73 7.49 27.3 0.6
49.2 31.8 31.9 42.9 -154 7.61 27.1 1.3
49.6 31.7 31.8 37 33 7.59 27.2 1.2
49.8 31.9 26.0 84.1 37 7.55 27.3 0.7
(°C) (m)
Table 2. Water chemistry data for the mud-water interface for samples collected at sites 7-12. Parameter
Units
Site 7
Site 8
Site 9
Site 10
Site 11
Site 12
Conductivity Total dissolved solids Salinity Dissolved oxygen ORP pH Temperature Depth
(mS) (mg/kg) (g/kg) (% Sat.) (mV)
50.1 32.5 32.7 90.4 77 7.48 27.3 0.1
50.0 32.6 32.7 98.6 79 7.37 27.2 0.1
50.9 32.5 32.7 92.0 73 7.56 27.3 0.8
50.2 32.8 32.9 92.1 81 7.96 27.0 0.3
50.6 32.7 32.8 90.7 83 7.46 27.3 0.4
50.8 32.9 32.7 91.3 79 7.66 27.3 0.1
(°C) (m)
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Fig. 3. Achnanthes hauckiana Grun. collected from site 6 where it occurred with deformities (left side) and without deformities (right side). The three normal valves ofA. hauckiana depicted on the right are modified from Jin et al. (1986). The solid line represents a distance of 15/~m (left side) and 20 ~zm, (right side).
Benthic marine diatom deformities
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Fig. 5. Fragilaria capucina from site 6 with the with the top valve representing a normal individual and the two lower valves representing deformed valves from site 6. The solid line represents a distance of 20 lam (top) and 35 ~tm (bottom two halves).
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Fig. 4. Three deformed individuals of Diatoma collected from site 6 (top). Each display a slightly different pattern of abnormal valve ornamentation. The three normal individuals (n) are modified from Jin et al. (1986). The solid line represents a distance of 45 ~m.
capucina (Fig. 5) displayed a lack of longitudinal symmetry. Both it and .4chnanthes hauckiana Grun. illustrated external cell wall deformities while Diatoma (Fig. 4) represented abnormal cell wall ornamentation deformities. Achnanthes was also studied by Schmid
(1985), who exposed various species of this genus to a variety of osmoregulators and drugs and was able to induce the formation of a triradiate form (Round et al. 1990). Sediment toxicity, as indicated by the Microtox test, was examined at each of the study sites (Table 3) to determine if a relationship between sediment mutagenicity and diatom deformity frequency existed. Diatom diversity and species richness atthe 12 study sites were also calculated (Table 3). Diatom diversity is (H) where H = -~(Pi Log2 Pi) and pi = the number of diatoms in the ith species divided by the total number of diatoms counted in the sample. The results were calculated on a count of 3000 diatom frustules per site. The number of deformed diatoms observed at each site ranged from none at sites 1, 2, 7-12, one at sites 3, 4, and 5, to three at site 6 (Table 3). A total of 3000 diatom frustules were counted at each site, 1000 for each of the 3 replicate slides. The concentrations of seven elements (Table 4)were analyzed to determine whether sites that exhibited high levels of heavy metals also exhibited elevated Microtox and diatom deformity levels. Copper, chromium, zinc, nickel, and lead concentrations exceeded U.S. EPA guidelines at sites 4 and 5 (EPD 1994). Similar findings were reported by Lam et al. (1987) for copper, chromium, and zinc in Tolo Harbour, and were all elevated above the U.S. EPA guidelines.
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Table 3. Results of the sediment microtoxicity tests. Sample site number*
ECs0(%) (g/kg)**
Descending toxicity ranking***
Number of deformities****
Diversity (H)*****
Species richness (#/3000)
Site 1 5.5931 6 0 3.19 20 Site 2 3.2105 5 0 3.65 26 Site 3 2.6446 4 1 3.22 18 Site 4 0.1455 2 1 3.14 15 Site 5 0.0328 1 1 2.70 15 Site 6 1.5846 3 3 3.09 17 Site 7 5.6241 7 0 3.74 42 Site 8 5.8844 10 0 3.77 39 Site 9 5.7558 8 0 3.61 41 Site 10 5.8964 11 0 3.59 38 Site 11 5.8978 12 0 3.45 40 Site 12 5.8793 9 0 3.65 35 * Site locations are provided in Fig. 1. Sites 7-12 were controls. * * ECs0-Effective concentration causing a 50% decrease in bioluminescence (g/kg wet wt). * * * 1 represents sediments with the highest acute toxicity based on Microtox Solid Phase test. * * * * The number of deformed benthic diatoms per thousand observed. ***** The total number of benthic diatom taxa observed in a count of 3000 diatom frustules. Table 4. The concentrations of seven common sediment contaminants in mg/kg. Contaminant*
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
7-12
Cadmium < 0.5 < 0.5 < 0.5 0.75 1.1 < 0.5 <0.5-0.8 Chromium 2.0 3.1 2.3 32 42 2.2 < 2-3.7 Copper 16 25 30 260 440 20 24-35 Iron 14 8.1 1.0 19 27 5.3 5-8.6 Nickel 1.8 2.2 1.8 18 31 2.8 < 1-2.4 Lead 39 34 26 110 130 22 25-38 Zinc 31 35 34 330 450 32 30-36 * Sediment contaminant determinations were carried out by the EPD, Wanchai, Hong Kong.
Site 5 (Tai Po Industrial Estate discharge area) had the highest M i c r o t o x toxicity levels (Table 3) and the highest levels o f h e a v y metals (Table 4). Site 6 w a s associated with the illegal d u m p i n g o f acidified wastes f r o m a o n e - m a n operation involving the reclamation o f preci6us metals f r o m discarded c o m p u t e r parts and other electronic equipment. Site 6 had the highest dia t o m d e f o r m i t y levels (one per thousand) vs. one per three thousand at sites 3, 4, and 5. This site, which is a river m o u t h site, exhibited the lowest salinities (Table 1; 26 parts per thousand) and, thus, o s m o t i c factors m a y h a v e p l a y e d a role in d e f o r m i t y induction at this site. The sediments collected at site 6 were coarse ( m e a n particle size o f 0.2-0.3 c m ) by comparison to s a m p l e s f r o m the other sites. It is well doc u m e n t e d that the bulk o f the total metals m e a s u r e d in
sediment tends to be associated with the finer particles, which h a v e a greater surface area ( H o n g 1993). It is possible that o r g a n i s m s collected at site 6, including the diatoms, were e x p o s e d to m e t a l c o n t a m i n a n t s in the pore water or in the w a t e r c o l u m n on a regular basis, but that the metals did not absorb or a c c u m u l a t e in these coarser sediments. In order to determine whether or not there were significant differences between clean and contaminated sediment sites in the n u m b e r o f diatom deformities, the Fisher Exact Probability test w a s used (Fisher and Yates 1953). A total o f six deformities w e r e o b s e r v e d at the Tolo H a r b o u r c o n t a m i n a t e d sediment sites, while no deformities were o b s e r v e d at the "control" sites. W h a t is the probability that all six d e f o r m i t i e s would occur b y chance alone for c o n t a m i n a t e d sites while
Benthic marine diatom deformities
none occurred at the clean sites? According to the Fisher Exact Probability test, the exact probability of this occurring by chance alone is 0.01562. Thus, there were less than two chances in a hundred that all six deformed diatoms would have been observed at the contaminated sites and none at the clean sites. Because all three slides were coded, these observations were not affected by subjective bias during the counting of the samples. DISCUSSION Contaminated sediments
The presence of hazardous pollutants in water and sediments has resulted in some of today's most troublesome environmental problems. Many contaminants tend to accumulate, not only in the sediment, but also in living organisms, and these accumulations are often linked to such things as morphological deformities, reproductive and immune system failures, and population collapse (Dickman et al. 1989; William et al. 1980; Yu 1994; Dickman and Rygiel 1996). There was a significant correlation (P < 0.05, rs = 0.652; Siegel 1956) between sediment toxicity as reflected by the Microtox test and benthic diatom diversity (Table 3). Site 5 displayed the highest heavy metal concentrations, the lowest diversity, and the highest Microtox acute toxicity levels. Site 6, by contrast, had some of the lowest heavy metal concentrations, intermediate Microtox and diversity levels, and high deformity frequencies relative to the other five Tolo Harbour sites (Tables 3 and 4). It is possible that mutagenic or teratogenic substances other than heavy metals may have been present at site 6 or that sporadic discharges of heavy metals that reacted with the developing diatoms and then washed off the creek sediments into the sea were responsible for the elevated deformity frequencies at this site. It is also true that, because site 6 is a freshwater site, the pH of the water is lower than at the other sites which are all marine sediments. Heavy metals are more bioavailable at lower pH than they are in the sea water sites where pH is high (Krebs 1983). The Microtox test for acute toxicity was correlated (P < 0.05) with diatom species richness as assessed by the Spearman Rank Correlation test (Siegel 1956). The deformity frequencies and Microtox tests were not significantly correlated. The heavy metal levels of the sediments were not correlated with the Microtox tests. It is possible that
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substances other than heavy metals were the primary toxic chemicals, but since no tests for toxic organic compounds in the sediments was carried out, it is not possible to critically evaluate this possibility. Diatoms as continuous monitors
Because benthic diatoms remain in place for a number of months, they act as continuous monitors during that period (Buikeman and Herricks 1978; Dickman et al. 1980). This is a decided advantage to using diatoms rather than Microtox bacteria in situations where toxic and/or mutagenic discharges are infrequent (i.e., sporadic). Diatom deformities
Patterns in diatom valves are more variable than one might initially guess. If thousands of individuals of the same species are observed, all but one or two will look identical. Andrews and Stoelzel (1984) collected the rare variants for the species Perissonoe cruciata, which displayed what they considered to be abnormal pore patterns (Figs. 6-8) and compared them to what they considered normal variants of that species (Figs. 9-12). The ability to distinguish between normal and abnormal variants requires the skill of an individual who has taken the time to carefully examine hundreds of individuals of the same species. Until methods of automating the reliable enumeration of diatom deformities have been developed, using diatom deformity frequencies to infer the condition of a marine site cannot be justified in terms of the time and effort required to reach statistically significant sample sizes. Species diversity and species richness levels at the 12 sites were closely correlated with one another and inversely correlated with sediment toxicity as reflected by the Microtox results. The r, for the inverse relationship between Microtox toxicity and species richness was 0.809, which was significant at the 0.01 level of probability (Spearman Rank Correlation test, Siegel 1956). The Microtox sediment toxicity levels were inversely correlated with species richness (P < 0.05). The metals, present at mg/L concentrations, were divided into three groups on the basis of their toxic effects (Braeck and Jensen 1976). In the first group, the presence of either Cu, Zn, or Ge increased the chain length and the normal separation of cells failed to occur. In the second group, Hg, Cd, and Pb, there was some disruption of cell division and elongated or bent cells occurred. In the third group, Cr, Ni, Se, and Sb were without effect at or below 1 mg/L.
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F o r d e f o r m i t i e s to occur, h e a v y m e t a l concentrations m u s t be high e n o u g h to cause the formation o f a b n o r m a l frustules, but low enough to allow at least for one division ( P i c k e t t - H e a p s et al. 1990). This range o f concentration is often quite narrow (Braeck and Jensen 1976). For e x a m p l e , m o r p h o l o g i c a l aberrations occur in Thalassiosira p s e u d o n a n a o v e r a narrow range o f Cu 2+ ion activities, (10 .8.3 to 10 .8.6 M) (Braeck and Jensen 1976). A b n o r m a l cell m o r p h o l o g y o b s e r v e d in s a m p l e s f r o m nature m u s t be used with caution as an indicator o f h e a v y m e t a l pollution because the abnormalities can also be caused b y a variety o f other factors including nutrient limitation (William et al. 1980). T h e station with the highest h e a v y metal concentrations (Station 5) displayed the highest sediment toxicity as indicated b y the solid phase M i c r o t o x test (Table 3). T h e highest n u m b e r o f d e f o r m e d diatoms occurred at site 6 w h e r e h e a v y m e t a l concentrations w e r e e l e v a t e d a b o v e the control sites but w e r e not as high as those o b s e r v e d at sites 4 and 5. The data presented in this p a p e r do not permit an u n e q u i v o c a l conclusion about the use o f diatom def o r m i t y frequencies as a m e a n s o f assessing the mutagenic/teratogenic i m p a c t o f anthropogenic substances on a m a r i n e benthic diatom c o m m u n i t y . In addition, the large n u m b e r o f d i a t o m s that m u s t be evaluated for deformities m a k e s this m e t h o d e x t r e m e l y labour- and time-intensive, and f e w scientists are likely to adopt it as a standard screening tool for contaminated sediments.
Acknowledgment--I am grateful to Prof. John Hodgkiss and Mr. Tom Glenwright for reading over the manuscript, to Philippe Jaouen, Ivan Tsoi, Gina Sequeira, and Alfred Lai of the University of Hong Kong for their assistance in taking and analyzing the samples, and to Ann Neller and the staff of the Chemical Waste and Ecotoxicology Laboratories of the Hong Kong Environmental Protection Department for their technical assistance. I am also grateful to the Director of the Environmental Protection Department of Hong Kong for permission to cite the EPD heavy metal data in this paper. Funds to support this research were provided by a grant to the author (UHK-CRCG #337/023/0003).
REFERENCES Andresen, N.A.; Tuchman, M.L. Anomalous diatom populations in Lake Michigan and Lake Huron in 1983. J. Great Lakes Res. 17: 144-149; 1991. Andrews, G.W.; Stoelzel, V.A. Morphology and evolutionary significance of Perissonoe, a new marine diatom genus. In: Mann, D.G., ed. Proc. seventh intematinal diatom symposium. Koenigstein: Otto Koeltz Science Publishers; 1984. APHA (American Public Health Association). Standard methods for the examination of water and waste water. APHA, Washington, DC; 1975. AWT Science and Environment Consulting Co., West Ryde, New South Wales, Australia; 1995. Barber, H.G.; Carter, J.R. Observations on some deformities found in British diatoms. Microscopy 34: 214-226; 1981. Beckman Scientific Co. Microtox system operating manual. Carlsbad, CA: Beckman Instruments, Inc., 1982. Braeck, G.S.; Jensen, A. Heavy metal tolerance of marine phytoplankton. The combined effects of copper and zinc ions on cultures of four common species. J. Exp. Mar. Biol. Ecol. 25: 3750; 1976. Buikeman, A.L. Jr.; Herricks, E.E. Effects of pollution on freshwater invertebrates. J. Water Pollut. Cont. Fed. 50: 1637-1648; 1978.
Benthic marine diatom deformities
Burgess, R.M.; Scott, K.J. The significance of in-place contaminated marine sediments on the water column: Processes and effects. In: Burton, A.G. Jr., ed. Sediment toxicity assessment. Boca Raton, FL: Lewis Publishers; 1992. Cheng, H.; Gao, Y.; Liu, S. Nanodiatoms from Fujian Coast. ISBN 7-5027-2974-7/Q.92. Beijing: China Ocean Press; 1993: 91. Dickman, M.D.; Smol, J.; Steele, P. The impact of industrial shock loading on selected biocoenoses in the lower Welland River, Ontario. Water Pollut. Res. Canada 15: 17-31; 1980. Dickman, M.; Que, L.; Matthews, B. Teratogens in the Niagara River watershed as reflected by Chironomid (Diptera: Chironomidae) labial plate deformities. Water Pollut. Res. J. Canada 24: 47-79; 1989. Dickman, M.D.; Yang, J.R.; Brindle, I.D. Impacts of heavy metals on higher aquatic plant, diatom and benthic invertebrate communities in the Niagara River watershed near Welland, Ontario. Water Pollut. Res. J. Canada 25: 131-159; 1992. Dickman, M.D.; Rygiel, G. Chironomid larval deformity frequencies, mortality and diversity in heavy-metal contaminated sediments of a Canadian riverine wetland. Environ. Int. 22: 693703; 1996. Enviro-Chem Engineering and Laboratory Co. Ltd. Report on marine sediment analyses in Victoria Harbour. Doc. Ref. 93/MS110-R 1. Hong Kong: Enviro-Chem Engineering and Laboratory Co. Ltd., 95-97 Des Voeux Road West; 1994. EPD (Environmental Protection Department). Marine water quality in Hong Kong. Hong Kong: Monitoring Section, Waste and Water Services Group, EPD; 1994: 236. Fisher, R.A.; Yates, F. Statistical tables for biological, agricultural and medical research. London: Oliver and Boyd; 1953: 125. Germain, J. Flora des diatomees d'eaux douce et saumatre. Paris, France: Societe Nouvelle des Editions Boubee; 1981: 444. Hong, H. Victoria Harbour contaminated sediment study report. Publication 93-03. Hong Kong: The Hong Kong University of Science and Technology Research Centre; 1993: 89. Jin, D.; Cheng, Z.; Lin, J.; Liu, S. Marine benthic diatoms in China. Vol. 1. Beijing, China: China Ocean Press; 1985: 313. Jin, D.; Cheng, Z.; Lin, J.; Liu, S. Marine benthic diatoms in China. Vol. 2. Beijing, China: China Ocean Press; 1986: 527. Krebs, F. Toxicity testing using freeze dried luminescent bacteria. Gew~serschutz, Wasser, Abwasser 63: 173-230; 1983. Lam, C.W.Y.; Kong, M.F.; Lui, P.H. Marine water quality in Hong Kong. Hong Kong: EPD, Marine Section, Water Quality Group, Hong Kong Government; 1987: 109.
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Landrum, P.F,; Nihart, S.R.; Eadie, B.J.; Herche, L.R. Reduction in bioavallability of organic contaminants to the amphipod Pontoporeia hoyi by dissolved organic matter of sediment interstitial waters. Environ. Toxicol. Chem. 6:11-20; 1987. Microbics Corporation. Microtox manual: A toxicity testing handbook. Vol. 3. Carlsbad, CA: Microbics Corporation; 1992. Patrick, R.; Reimer, C. The diatoms of the United States. Vol I. Monograph of the Academy of Natural Sciences of Philadelphia. Philadelphia, PA: Academy of Natural Sciences of Philadelphia; 1966. Patrick, R.; Reimer, C. The diatoms of the United States. Vol II. Monograph of the Academy of Natural Sciences of Philadelphia. Philadelphia, PA: Academy of Natural Sciences of Philadelphia; 1975.
Pickett-Heaps, J.; Schmid, A.-M.M.; Edgar, L.A. The cell biology of diatom valve formation. In: Progress in phycological research. Vol. 7. Biopress Ltd.; 1990. Round, F.E.; Crawford, R.M.; Mann, D.G. The diatoms. Cambridge, UK: Cambridge University Press; 1990: 747. 1990. Schmid, A.-M.M. Wall morphogenesis in Thalassiosira eccentrica: Comparison of auxospore formation and the effect of MTinhibitors. In: Mann, D.G., ed. Proc. seventh intemational diatom symposium. Koenigstein: O. Koeltz; 1984: 47-70. Schmid, A.-M.M. Centronella reichelti Voigt - A very unusual diatom in the surface sediments of the grabensee. In: Danielopol, D.; Schmid, R.; Schultze, E. Contributions to the paleolimnology of the Trumer Lakes (Salzburg) and the Lakes Mondsee. Attersee and Traunsee (Upper Austria). Limnologisches Institut. Osterreichische Akademie der Wissenschaften; 1985: 65-78. Siegel, S. Nonparametric statistics for the behavioral sciences. New York, NY: McGraw-Hill Book Co. Inc.; 1956:312. William, H.T.; Hollibaugh, J.T.; Seibert, D.L.R. Effects of heavy metals on the morphology of some marine phytoplankton. Phycologia 19(3): 202-209; 1980. Wu, R.S.S. Marine pollution in Hong Kong: a review. Asian Mar. Biol. 5:11-23; 1988. Yang, J.R.; Duthie, H.C. Morphology and ultrastructure of teratological forms of the diatoms Stephanodiscus niagare and S. parvus (Bacillariophyceae) from Hamilton Harbour (Lake Ontario, Canada). Hydrobiologia 269/270: 57-66; 1993. Yu, D. Pollution solutions. Vol. 3. Kowloon, Hong Kong: Private Sector Committee Environment Centre, 83 Tat Chee Avenue; 1994.
Pergamon
Environment International, Vol. 24, No. 7, pp. 749-759, 1998 Copyright O1998 Elsevier Science Ltd Printed in the USA. All rights reserved 0160-4120/98 $19.00+.00
PH S0160-4120(98)00060-9
BENTHIC MARINE DIATOM DEFORMITIES ASSOCIATED WITH CONTAMINATED SEDIMENTS IN HONG KONG Mike D. Dickman Ecology and Biodiversity Dept., The University of Hong Kong, Hong Kong, China
E1 9710-209 M (Received 31 October 1997; accepted 21 May 1998)
Three thousand benthic diatoms were examined at each of twelve sites to determinethe frequency of diatom deformities. Two basic types of diatom deformities were observed: 1) diatoms with deformed cell wall morphology; and 2) diatoms with deformedvalve wall ornamentation.Only six deformed diatoms were observed. Deformed individuals ofFragilaria capucina and Achnanthes hauckiana displayed external cell wall deformities,while deformedindividualsof Diatoma vulgate displayed valve walls with a wavy pattern of longitudinal lines in which the valve ornamentation, rather than the valve cell wall, was abnormal.The deformityfrequencyofNavicula rhyncocephala, Achnanthes hauckiana, Fragilaria capucina, and Diatoma vulgare was significantly lower at unpolluted sites than at polluted sites based on Fisher exact probability estimates. Cadmium reached 1.1 mg/kg dry wt. of sediment, while other levels were chromium (42 mg/kg of sediment), copper (440 mg/kg), nickel (31 mg/kg), lead (130 mg/kg), and zinc (450 mg/kg of sediment) atthe most polluted site. There was a significant correlation (P < 0.05, r~ = 0.652) between sediment toxicity, as reflected by the Microtox test, and benthic diatom diversity. The most polluted site exhibited the highest Microtox acute toxicity levels, the lowest diversity, and moderate diatom deformity levels. It was concluded that abnormal cell morphology of diatoms may be a valid indicator of ecosystem health. However, because the amount of time and effort required to identify a statistically meaningful number of deformed diatoms is so great, diatom deformity frequency is unlikely to become a useful tool unless an automated system of assessing diatom deformity f r e q u e n c y is d e v e l o p e d . 01998 Elsevier Science Ltd
INTRODUCTION There is growing acceptance o f the view that chemical measurements alone are poor gauges o f the biological impacts o f pollutants. As a consequence, there is an increasing shift away from the strict reliance on chemical measurements to assess the health of ecosystems. Biological communities appear to provide a direct means o f observing the impact of contaminants. The bioavailability o f heavy metals, once they have bound with nonionic organic compounds, colloids, or clays, is extremely difficult to assess from chemical data alone. The present study o f the relationship be-
tween contaminated sediments and deformity frequencies of benthic diatoms was an attempt to determine if these microscopic plants might serve as biological indicators o f heavy metal bioavailability. The sediments o f coastal areas around Hong Keng, such as Victoria Harbour and Tolo Harbour, are contaminated with a variety o f heavy metals and persistent organic compounds (Enviro-Chem Engineering and Laboratory Co. Ltd. 1994; EPD 1994). A number o f studies have been conducted to assess sediment toxicity in Hong Kong, but assessments o f chemical 749
750
M. Dickman
Tubing Handpump wiLh one-way valve
JI [
~
Lead
Sieve p l a t e ~ Removable
Clear plastic sleeve
Fig. 1. A schematic illustration of the shallow water epipelic suction sampler.
bioavailability have proved difficult. The chemical form of a pollutant determines its potential bioavailability to marine organisms (Burgess and Scott 1992). Bioavailability is reduced in the presence of sedimentbound nonionic organic compounds or colloidal matter. Heavy metal toxicity is also decreased in the presence of organic ligands of sulfide precipitates (Landrum et al. 1987). Ecological conditions in coastal waters near Hong Kong are a concern to its citizens and scientists alike because there are a number of toxic chemicals present that have been linked to reproductive, metabolic, neurologic, and behavioural abnormalities (Wu 1988; Hong 1993). The elevated level of heavy metals in Tolo Harbour (EPD 1994) was the main reason this study was undertaken in this part of Hong Kong. Lam et al. (1987) concluded that the assimilative capacity of Tolo Harbour was limited due to its enclosed characteristics, weak currents, vertical salinity, and thermal stratification. The copper, chromium, and zinc in Tolo Harbour are all elevated above the U.S. Environmental Protection Agency (U.S. EPA) guidelines (Lam et al. 1987). Benthic diatom deformities have been correlated with the presence of genotoxic and teratogenic chemicals in aquatic sediments in a number of countries (Andresen and Tuchman 1991; Barber and Carter 1981; Dickman et al. 1992; Yang and Duthie 1993). William et al. (1980) described the effect of 11 heavy metals on the morphology of Thalassiosira aestivalis.
Diatoms as indicator organisms
Diatoms are single-celled algae which are classified by the shape and ornamentation of their silica cell walls. These cell walls preserve extremely well and, as a result, diatoms can be efficiently and permanently identified and archived. Cell shape and ornamentation in natural diatom populations vary only slightly within a species, and deformities in diatom morphology from unpolluted waters are rare. METHODS
In the present study, 12 sites were examined to determine their diatom diversity, sediment toxicity as indicated by the Microtox test, heavy metal concentrations of the sediments, and frequency of diatom deformities. An Ekman grab sampler was used to sample the sediments for heavy metals and Microtox determinations. A suction sampler (Fig. 1) was used for quantitative samples of sediment diatoms. Because sand grains in the Ekman grab samples obscured the diatoms, making their accurate enumeration far more time consuming, a method was developed that removed little sand in sampling the epipelic diatoms from the surface layer of sandy substrates. This epipelic diatom suction sampler (Fig. 1) was used to sample the diatoms from all sites except sites 3 and 4, which were too deep for the suction hose to reach the bottom. An Ekman dredge sampler was used at two of the twelve sites which were too deep to employ the suction
Benthic marine diatom deformities
751
N
'1'olo I..I~-bour
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Cape d'Aguil~r
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YN T~'I'S~
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Fig. 2. The upper two insets indicate the location of Ping Chau in Mirs Bay relative to the location of the six Tolo Harbour sample sites. The latitude of Ping Chau is 114 ° 25' and its longitude is 22 ° 35'. The location of Cape d'Aguilar is indicated by the dot at the end of the cape in the upper inset. The inset at the far right of the upper portion of Fig. 2 provides a general idea of Hong Kong's location in the South China Sea.
752
sampler. The Ekman dredge penetrated the sediment to a depth of about 4 cm ± 1 cm depending on the amount of sediment consolidation. Three replicates were taken from each of the twelve sites. Each site was sampled once during the period January 1994 to February 1995. Sample sites 1-4 are located in the Tai Mei Tuk Typhoon Shelter in Tolo Harbour near Sha Lan (lower inset, Fig. 2). Sample site 5 is located near the Tai Po Industrial Estates. Site 6 is located at the mouth of a small stream that enters Tolo Harbour between Tai Mei Tuk and the northern end of the Plover Cove Reservoir. Ping Chau is located at 114 ° 25' north latitude and 22 ° 35' east longitude (upper inset, Fig. 2). The sample site at Cape d'Aguilar is indicated by the dot at the end of the cape in the upper inset. There are six sites in Tolo Harbour, three sites at Ping Chau, and three sites at Cape d'Aguilar on Hong Kong Island. The Ping Chau and Cape d'Aguilar sites were selected because of their low levels of sediment heavy metals. One thousand diatom frustules were counted for each of the three replicates at each of the twelve sites (36 000 diatoms were counted in all). A Horiba portable water quality analyzer was used by the author to measure the specific conductivity of the water at the mud-water interface at each sample site. Values are reported as the meanbased on three replicates at each site. In addition, dissolved oxygen, temperature, and salinity were measured (n=3 per site) with the same unit. Total dissolved solids and oxidation-reduction potential (ORP) for the mud-water interface were measured according to the Standard Methods (APHA 1975).
Heavy metal analyses The Cd, Cr, Cu, Fe, Ni, Pb, and Zn concentrations in the sediment were determined by the AWT Science and Environment Consulting Co. (1995) using atomic absorption spectrometry on nitric acid digested samples. All glassware was acid-washed after first soaking it in Alconox solution for 24 h. After the acid washing, theglassware was rinsed in three separate washes of deionized water. The dried sediment was next dissolved in high purity grade nitric acid. To each test tube, 8 mL of 70% nitric acid (Fisher Scientific, Trace Metal Grade) were added. The test tubes were covered with glass condensers and the samples were digested at room temperature for 12 h. Digestion was continued for 8 h more at 120 C. Digests were cooled to room temperature and brought to 50 mL with Nanopure deionized water. Two sample blanks were analyzed
M. Dickman
together with each sample batch. Concentrations of metals in blanks were below detection limits in all analyses. A spiked blank was analyzed during each analysis to ensure day-to-day reproducibility. Each standard and sample was measured in duplicate and the sample was re-analyzed if the relative standard deviation of the two measurements was higher than 10%. Standard reference materials from the National Institute of Standards and Technology and the National Research Council of Canada were digested and analyzed with each sample batch. The results of metal concentrations always fell within 1 SD of certified values.
Biological analyses The biological assessment of the contaminants present in the sediment was carried out by screening for both acute toxicity (Hong Kong Environmental Protection Dept.) and the frequency of diatom deformities (Ecotoxicology Laboratory, The University of Hong Kong). The samples from each of the 12 sites were coded to avoid unconscious bias. After 36 000 diatoms had been counted and the number of individuals of each species recorded, the number of deformed diatoms on each slide was noted and the slides were then decoded.
Toxicity testing The method chosen for sediment toxicity screening was the non-extractive Microtox Solid Phase Test (Microbics Corp. 1992). This is a bacterial inhibition bioassay, using a 50% reduction in the bioluminescenee of a colony of Photobacteriumphosphoreum as the criterion for calculating an ECso value. The ECs0 is defined as the median effective concentration and represents the time it takes for a 50% reduction in light intensity of the bioluminescent bacteria. The wet sediments collected from each site were centrifuged and the resulting pellet was made into an aqueous suspension before being serially diluted. The effective concentration (ECs0), which is the concentration of the sediment sample causing a 50% decrease in bioluminescence measured after 25 min total contact time, was then determined (Microbics Corp. 1992). The Microtox test is a rapid and simple method for comparing and ranking the relative toxicity of sediment samples taken from different locations. A detailed discussion of the physiology of Photobacterium phosphoreum, the influence of test conditions on the final toxicity values, and the experimental procedure of
Benthicmarinediatomdeformities
this bioassay were provided by Krebs (1983). Unlike methods which require solvent extracts or elutriates to be prepared from the sediments, the Microtox Solid Phase Test allows the Photobacterium phosphoreum bacteria to come into direct contact with the sediment. The bacteria are then separated using a filter column and analyzed using the Microtox Toxicity Analyzer Model M 500 (Beckman 1982).
Diatom deformity testing A Pasteur Pipette was used to transfer 5 mL of the homogenized sediment-water mixture to an Utermohl sedimentation tube which was observed using an Olympus Research compound microscope (Model BX 50, equipped with a photoautomat). At each site, a total of 1000 diatoms were counted for each of the 3 samples taken from that site. Diatoms were counted at 400 times magnification and identified at 1000 times magnification. Identifications were based on Cheng et al. (1993), Germain (1981), Jin et al. (1986), and Patrick and Reimer (1966; 1975). Deformities were photographed using an Olympus Research microscope equipped with a photoautomat. A total of 36 000 diatoms were examined for deformities.
Site descriptions Site 1: The sediments at this site were a brownishyellow colour and were collected using an Ekman grab sampler. Amphipods, crabs, and hermit crabs were found among the collected sediments (water depth was about 1.2 m). Site 2: The sediments at this site were observed to be relatively inorganic. Filamentous green algae, primarily Enteromorpha, were collected along with the sediment (water depth was about 0.9 m). Sediments were collected using the epipelic suction sampler (Fig. 2). Site 3: Water depth was about 0.75 m. Numerous benthic invertebrates were found at this site and sediments were collected using the epipelic suction sampler (Fig. 2). Site 4: Water depth was about 3 m. The sediment was black and gave off a strong hydrogen sulphide smell. No invertebrates were found. Sediment at site 4 was collected using an Ekman grab sampler. Site 5: Water depth was 0.8 m. The area was near the Tai Po Industrial Estate storm drain which was a double storm drain out-fall located about 500 m north of the Motorola Building. Sediments were collected at this and all remaining sites using an epipelic suction sampler.
753
Site 6: Water depth was less than 0.75 m. The sediment was quite coarse grained, brownish-yellow sand. Site 7 was located about 300 m northwest of the ferry dock on Ping Chau Island in about 0.1 m of water. The sediments at this site were coarse grained sands which were relatively free of organic matter except for some filamentous green algae. Site 8 was located about 300 m southeast of the ferry dock on Ping Chau Island (Fig. 2). The water depth at this site was about 0.1 m. The sandy sediments at site 8 were slightly finer than those at site 7 and filamentous green algae was also present. Site 9 was located about 1.3 km southeast of the ferry dock on Ping Chau Island (Fig. 2). The water depth at this site was about 0.8 m. The sandy sediments at site 9 were quite fine and a flocculant organic layer was present at the sand-water interface. Sites 10, 11, and 12 were located at Cape d'Aguilar in shallow water (0.1 - 0.4 m). No filamentous green algae were present at these sites. All three of these sites (Fig. 2) had coarse sandy sediments. Samples were collected at low tide to make sediment sample conditions as uniform as possible. Salinity, conductivity, dissolved oxygen, ORP, pH, water depth, temperature, and total dissolved solids were measured at each site using a YSI Model 6000 Environmental Monitoring System (Campbell Scientific Inc., Logan,
UT, USA). RESULTS
With the exception of site 4, where the sediments were anaerobic and had an ORP of- 154 (Tables 1 and 2), the diatoms were active when viewed live under the microscope. Secchi transparencies were nearly always less than 1.5 m and the compensation depth was generally less than 3.5 m. A list of the diatom species found at each site was compiled but is not included because of its large size. This list is available from the author.
Deformed diatoms As shown in Fig. 3, normal cells of Achnanthes hauckiana Grun. (Jin et al. 1985) were easily distinguished by their bilateral symmetry (left), whereas deformed cells (right) lacked symmetry. Normal (upper inse0 and deformed Diatoma species (Fig. 4) were recognizable because deformed individuals displayed a wavy pattern of longitudinal lines in which the valve ornamentation rather than the valve cell wall was the critical trait. Deformed individuals of Fragilaria
754
M. Dickman
Table 1. Water chemistry data for mud-water interface samples collected at sites 1-6. Parameter
Units
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
Conductivity Total dissolved solids Salinity Dissolved oxygen ORP pH Temperature Depth
(mS) (mg/kg) (g/kg) (% Sat.) (mV)
50.1 32.5 32.7 79 85 7.55 27.2 0.9
50.0 32.6 32.7 48.6 84 7.51 27.2 0.7
48.9 32.5 32.7 92 73 7.49 27.3 0.6
49.2 31.8 31.9 42.9 -154 7.61 27.1 1.3
49.6 31.7 31.8 37 33 7.59 27.2 1.2
49.8 31.9 26.0 84.1 37 7.55 27.3 0.7
(°C) (m)
Table 2. Water chemistry data for the mud-water interface for samples collected at sites 7-12. Parameter
Units
Site 7
Site 8
Site 9
Site 10
Site 11
Site 12
Conductivity Total dissolved solids Salinity Dissolved oxygen ORP pH Temperature Depth
(mS) (mg/kg) (g/kg) (% Sat.) (mV)
50.1 32.5 32.7 90.4 77 7.48 27.3 0.1
50.0 32.6 32.7 98.6 79 7.37 27.2 0.1
50.9 32.5 32.7 92.0 73 7.56 27.3 0.8
50.2 32.8 32.9 92.1 81 7.96 27.0 0.3
50.6 32.7 32.8 90.7 83 7.46 27.3 0.4
50.8 32.9 32.7 91.3 79 7.66 27.3 0.1
(°C) (m)
.X~.z
!
!
Fig. 3. Achnanthes hauckiana Grun. collected from site 6 where it occurred with deformities (left side) and without deformities (right side). The three normal valves ofA. hauckiana depicted on the right are modified from Jin et al. (1986). The solid line represents a distance of 15/~m (left side) and 20 ~zm, (right side).
Benthic marine diatom deformities
755
~____a_tl_ltlir'll¢l~'l'ltili~-,.~ I -~
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Fig. 5. Fragilaria capucina from site 6 with the with the top valve representing a normal individual and the two lower valves representing deformed valves from site 6. The solid line represents a distance of 20 lam (top) and 35 ~tm (bottom two halves).
11
n
Fig. 4. Three deformed individuals of Diatoma collected from site 6 (top). Each display a slightly different pattern of abnormal valve ornamentation. The three normal individuals (n) are modified from Jin et al. (1986). The solid line represents a distance of 45 ~m.
capucina (Fig. 5) displayed a lack of longitudinal symmetry. Both it and .4chnanthes hauckiana Grun. illustrated external cell wall deformities while Diatoma (Fig. 4) represented abnormal cell wall ornamentation deformities. Achnanthes was also studied by Schmid
(1985), who exposed various species of this genus to a variety of osmoregulators and drugs and was able to induce the formation of a triradiate form (Round et al. 1990). Sediment toxicity, as indicated by the Microtox test, was examined at each of the study sites (Table 3) to determine if a relationship between sediment mutagenicity and diatom deformity frequency existed. Diatom diversity and species richness atthe 12 study sites were also calculated (Table 3). Diatom diversity is (H) where H = -~(Pi Log2 Pi) and pi = the number of diatoms in the ith species divided by the total number of diatoms counted in the sample. The results were calculated on a count of 3000 diatom frustules per site. The number of deformed diatoms observed at each site ranged from none at sites 1, 2, 7-12, one at sites 3, 4, and 5, to three at site 6 (Table 3). A total of 3000 diatom frustules were counted at each site, 1000 for each of the 3 replicate slides. The concentrations of seven elements (Table 4)were analyzed to determine whether sites that exhibited high levels of heavy metals also exhibited elevated Microtox and diatom deformity levels. Copper, chromium, zinc, nickel, and lead concentrations exceeded U.S. EPA guidelines at sites 4 and 5 (EPD 1994). Similar findings were reported by Lam et al. (1987) for copper, chromium, and zinc in Tolo Harbour, and were all elevated above the U.S. EPA guidelines.
756
M. Dickman
Table 3. Results of the sediment microtoxicity tests. Sample site number*
ECs0(%) (g/kg)**
Descending toxicity ranking***
Number of deformities****
Diversity (H)*****
Species richness (#/3000)
Site 1 5.5931 6 0 3.19 20 Site 2 3.2105 5 0 3.65 26 Site 3 2.6446 4 1 3.22 18 Site 4 0.1455 2 1 3.14 15 Site 5 0.0328 1 1 2.70 15 Site 6 1.5846 3 3 3.09 17 Site 7 5.6241 7 0 3.74 42 Site 8 5.8844 10 0 3.77 39 Site 9 5.7558 8 0 3.61 41 Site 10 5.8964 11 0 3.59 38 Site 11 5.8978 12 0 3.45 40 Site 12 5.8793 9 0 3.65 35 * Site locations are provided in Fig. 1. Sites 7-12 were controls. * * ECs0-Effective concentration causing a 50% decrease in bioluminescence (g/kg wet wt). * * * 1 represents sediments with the highest acute toxicity based on Microtox Solid Phase test. * * * * The number of deformed benthic diatoms per thousand observed. ***** The total number of benthic diatom taxa observed in a count of 3000 diatom frustules. Table 4. The concentrations of seven common sediment contaminants in mg/kg. Contaminant*
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
7-12
Cadmium < 0.5 < 0.5 < 0.5 0.75 1.1 < 0.5 <0.5-0.8 Chromium 2.0 3.1 2.3 32 42 2.2 < 2-3.7 Copper 16 25 30 260 440 20 24-35 Iron 14 8.1 1.0 19 27 5.3 5-8.6 Nickel 1.8 2.2 1.8 18 31 2.8 < 1-2.4 Lead 39 34 26 110 130 22 25-38 Zinc 31 35 34 330 450 32 30-36 * Sediment contaminant determinations were carried out by the EPD, Wanchai, Hong Kong.
Site 5 (Tai Po Industrial Estate discharge area) had the highest M i c r o t o x toxicity levels (Table 3) and the highest levels o f h e a v y metals (Table 4). Site 6 w a s associated with the illegal d u m p i n g o f acidified wastes f r o m a o n e - m a n operation involving the reclamation o f preci6us metals f r o m discarded c o m p u t e r parts and other electronic equipment. Site 6 had the highest dia t o m d e f o r m i t y levels (one per thousand) vs. one per three thousand at sites 3, 4, and 5. This site, which is a river m o u t h site, exhibited the lowest salinities (Table 1; 26 parts per thousand) and, thus, o s m o t i c factors m a y h a v e p l a y e d a role in d e f o r m i t y induction at this site. The sediments collected at site 6 were coarse ( m e a n particle size o f 0.2-0.3 c m ) by comparison to s a m p l e s f r o m the other sites. It is well doc u m e n t e d that the bulk o f the total metals m e a s u r e d in
sediment tends to be associated with the finer particles, which h a v e a greater surface area ( H o n g 1993). It is possible that o r g a n i s m s collected at site 6, including the diatoms, were e x p o s e d to m e t a l c o n t a m i n a n t s in the pore water or in the w a t e r c o l u m n on a regular basis, but that the metals did not absorb or a c c u m u l a t e in these coarser sediments. In order to determine whether or not there were significant differences between clean and contaminated sediment sites in the n u m b e r o f diatom deformities, the Fisher Exact Probability test w a s used (Fisher and Yates 1953). A total o f six deformities w e r e o b s e r v e d at the Tolo H a r b o u r c o n t a m i n a t e d sediment sites, while no deformities were o b s e r v e d at the "control" sites. W h a t is the probability that all six d e f o r m i t i e s would occur b y chance alone for c o n t a m i n a t e d sites while
Benthic marine diatom deformities
none occurred at the clean sites? According to the Fisher Exact Probability test, the exact probability of this occurring by chance alone is 0.01562. Thus, there were less than two chances in a hundred that all six deformed diatoms would have been observed at the contaminated sites and none at the clean sites. Because all three slides were coded, these observations were not affected by subjective bias during the counting of the samples. DISCUSSION Contaminated sediments
The presence of hazardous pollutants in water and sediments has resulted in some of today's most troublesome environmental problems. Many contaminants tend to accumulate, not only in the sediment, but also in living organisms, and these accumulations are often linked to such things as morphological deformities, reproductive and immune system failures, and population collapse (Dickman et al. 1989; William et al. 1980; Yu 1994; Dickman and Rygiel 1996). There was a significant correlation (P < 0.05, rs = 0.652; Siegel 1956) between sediment toxicity as reflected by the Microtox test and benthic diatom diversity (Table 3). Site 5 displayed the highest heavy metal concentrations, the lowest diversity, and the highest Microtox acute toxicity levels. Site 6, by contrast, had some of the lowest heavy metal concentrations, intermediate Microtox and diversity levels, and high deformity frequencies relative to the other five Tolo Harbour sites (Tables 3 and 4). It is possible that mutagenic or teratogenic substances other than heavy metals may have been present at site 6 or that sporadic discharges of heavy metals that reacted with the developing diatoms and then washed off the creek sediments into the sea were responsible for the elevated deformity frequencies at this site. It is also true that, because site 6 is a freshwater site, the pH of the water is lower than at the other sites which are all marine sediments. Heavy metals are more bioavailable at lower pH than they are in the sea water sites where pH is high (Krebs 1983). The Microtox test for acute toxicity was correlated (P < 0.05) with diatom species richness as assessed by the Spearman Rank Correlation test (Siegel 1956). The deformity frequencies and Microtox tests were not significantly correlated. The heavy metal levels of the sediments were not correlated with the Microtox tests. It is possible that
757
substances other than heavy metals were the primary toxic chemicals, but since no tests for toxic organic compounds in the sediments was carried out, it is not possible to critically evaluate this possibility. Diatoms as continuous monitors
Because benthic diatoms remain in place for a number of months, they act as continuous monitors during that period (Buikeman and Herricks 1978; Dickman et al. 1980). This is a decided advantage to using diatoms rather than Microtox bacteria in situations where toxic and/or mutagenic discharges are infrequent (i.e., sporadic). Diatom deformities
Patterns in diatom valves are more variable than one might initially guess. If thousands of individuals of the same species are observed, all but one or two will look identical. Andrews and Stoelzel (1984) collected the rare variants for the species Perissonoe cruciata, which displayed what they considered to be abnormal pore patterns (Figs. 6-8) and compared them to what they considered normal variants of that species (Figs. 9-12). The ability to distinguish between normal and abnormal variants requires the skill of an individual who has taken the time to carefully examine hundreds of individuals of the same species. Until methods of automating the reliable enumeration of diatom deformities have been developed, using diatom deformity frequencies to infer the condition of a marine site cannot be justified in terms of the time and effort required to reach statistically significant sample sizes. Species diversity and species richness levels at the 12 sites were closely correlated with one another and inversely correlated with sediment toxicity as reflected by the Microtox results. The r, for the inverse relationship between Microtox toxicity and species richness was 0.809, which was significant at the 0.01 level of probability (Spearman Rank Correlation test, Siegel 1956). The Microtox sediment toxicity levels were inversely correlated with species richness (P < 0.05). The metals, present at mg/L concentrations, were divided into three groups on the basis of their toxic effects (Braeck and Jensen 1976). In the first group, the presence of either Cu, Zn, or Ge increased the chain length and the normal separation of cells failed to occur. In the second group, Hg, Cd, and Pb, there was some disruption of cell division and elongated or bent cells occurred. In the third group, Cr, Ni, Se, and Sb were without effect at or below 1 mg/L.
758
M. Diekman
,~':e'.* ,,,nk~. - - - .'7".~ o
6
8
7
Figs. 6-8. Three deformed individuals ofPerissonoe cruciata (modified from Andrews and Stoelzel 1984).
•t , . . . .,~ ~
~*a " e ' t l 4
40
•
9
~.,~ ~i',.t.w-~' 10
,IP
oee•.
4
~""f""~,'~ " ~
•
4o
•
s--
11
~. . . . " " " : " ~a,#
at
12
Figs. 9-12. Four normal variants ofPerissonoe cruciata (modified from Andrews and Stoelzel 1984).
F o r d e f o r m i t i e s to occur, h e a v y m e t a l concentrations m u s t be high e n o u g h to cause the formation o f a b n o r m a l frustules, but low enough to allow at least for one division ( P i c k e t t - H e a p s et al. 1990). This range o f concentration is often quite narrow (Braeck and Jensen 1976). For e x a m p l e , m o r p h o l o g i c a l aberrations occur in Thalassiosira p s e u d o n a n a o v e r a narrow range o f Cu 2+ ion activities, (10 .8.3 to 10 .8.6 M) (Braeck and Jensen 1976). A b n o r m a l cell m o r p h o l o g y o b s e r v e d in s a m p l e s f r o m nature m u s t be used with caution as an indicator o f h e a v y m e t a l pollution because the abnormalities can also be caused b y a variety o f other factors including nutrient limitation (William et al. 1980). T h e station with the highest h e a v y metal concentrations (Station 5) displayed the highest sediment toxicity as indicated b y the solid phase M i c r o t o x test (Table 3). T h e highest n u m b e r o f d e f o r m e d diatoms occurred at site 6 w h e r e h e a v y m e t a l concentrations w e r e e l e v a t e d a b o v e the control sites but w e r e not as high as those o b s e r v e d at sites 4 and 5. The data presented in this p a p e r do not permit an u n e q u i v o c a l conclusion about the use o f diatom def o r m i t y frequencies as a m e a n s o f assessing the mutagenic/teratogenic i m p a c t o f anthropogenic substances on a m a r i n e benthic diatom c o m m u n i t y . In addition, the large n u m b e r o f d i a t o m s that m u s t be evaluated for deformities m a k e s this m e t h o d e x t r e m e l y labour- and time-intensive, and f e w scientists are likely to adopt it as a standard screening tool for contaminated sediments.
Acknowledgment--I am grateful to Prof. John Hodgkiss and Mr. Tom Glenwright for reading over the manuscript, to Philippe Jaouen, Ivan Tsoi, Gina Sequeira, and Alfred Lai of the University of Hong Kong for their assistance in taking and analyzing the samples, and to Ann Neller and the staff of the Chemical Waste and Ecotoxicology Laboratories of the Hong Kong Environmental Protection Department for their technical assistance. I am also grateful to the Director of the Environmental Protection Department of Hong Kong for permission to cite the EPD heavy metal data in this paper. Funds to support this research were provided by a grant to the author (UHK-CRCG #337/023/0003).
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Benthic marine diatom deformities
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