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RELATIONSHIP BETWEEN COPPER SPECIATION IN
SEDIMENTS A N D B I O A C C U M U L A T I O N BY M A R I N E
BIVALVES OF T A I W A N
Bor-Cheng Han, a Woei-Lih Jeng, b Tsu-Chang H u n g b
& Ming-Yi
Wen C
aSchool of Public Health, Taipei Medical College, Taipei, Taiwan, Republic of China bInstitute of Oceanography, National Taiwan University, Taipei, Taiwan, Republic of China
CSchool of Public Health, Tulane University, New Orleans, USA.
(Received 25 October 1994; accepted 7 April 1995)
Abstract
This paper evaluates the relationships between copper species in sediments and accumulation by the purple clam
(Hiatula diphos) and venus clam (Gomphina aeguilat- era) collected from the fieM and culture (aquaculture)
ponds in the polluted coastal area of Lukang, Taiwan. Sediment was sampled along with the molluscs, including oysters (Crassostrea gigas), purple clams (Hiatula diphos), rock-shells (Thais clavigera), venus clams (Gomphina aeguilatera), and hard clams (Meretrix lusoria), from two unique environments of Lukang dur-
ing the period from August 1993 to July 1994.
The data indicate that the total copper concentrations in sediments from culture ponds (185 ixg g-l) was higher than those of the field (44.0 p~g g t). Copper species in sediments were analyzed by a sequential leaching tech- nique. Results show that concentrations of various copper species in the sediments are in the range of 1.14 + 0.59 to 13.2 + 22.4 p~g g-i and 0.36 + 0.24 to 133 + 36.7 ~zg g 1 for the two environments, respectively. Also the exchange-
able copper in sediment from culture ponds was 15 times higher than that from the field. In addition, the sum of exchangeable and copper carbonates had the highest per- centages of copper in both the pond sediment (86.6 %) and the field sediment (50.7 %).
Maximum copper concentrations (309 + 35.1 txg gJ) in oysters were much higher than those in the other ben- thic organisms by about 4-127 times. Similarly, the data also showed that copper concentrations in Thais clavigera were 12-32 times higher than those in other
benthic organisms. Copper concentrations in various ben- thic organisms differed significantly (p < 0.05) from that in Thais clavigera. This capacity makes Thais clavigera
a potential candidate for monitoring copper in marine sediments. In terms of copper species, the best correla- tion was generally obtained between copper carbonates in sediments and copper concentrations in Hiatula diphos
(r = 0.886*). A strong multiple regression correlation (p < 0"05, r 2 = 0.7894) also indicates that the copper carbonates may dominate as the available form of copper to Hiatula diphos from various environments in the
Lukang coastal area under natural physicochemical con- ditions.
Key words: Copper speciatiorr, sediments, accumula- tion, bivalves.
35
INTRODUCTION
Taiwan is a modern industrialized island country with a population of about 21 million and a strong economy. Societal demand for protein increases when the stan- dard of living rises. Oysters (Crassostrea gigas) and other molluscs (e.g. purple clams, Hiatula diphos) are the most popular seafood in Taiwan. However, due to dense population and rapid industrial expansion, the estuarine and coastal environments of Taiwan are suffer- ing ever-increasing impact from all kinds of human activities (Hung, 1988; Han & Hung, 1990; Hung & Han, 1992; Han et al., 1994a; Jeng & H a n , 1994).
We monitored for heavy metals in the coastal envi- ronment of Taiwan and found that heavy metal pollu- tion is the most serious and one of the most studied marine environmental problems in Taiwan. Moreover, most current health risks associated with seafood safety originate in the environment; for example, some alarm has been expressed concerning possible health implica- tions of copper residues in oysters (Han et al., 1994a). Because of the incident of 'green oyster' in the Chart- ing coastal area ( H a n & Hung 1989, 1990), the green oysters, collected from the Erhjin Chi estuary in January
1989, gave copper concentration of 4401 + 79 p.g gl (Han & Hung, 1989, 1990; Han et al., 1994a). The area around the Erhjin Chi estuary is especially affected by large discharges of heavy metals from acid cleaning of metal scrap on the riverbank. Besides, the purple clam
(Hiatula diphos) incident was attributed to an aquacul- ture source resulting from polluted groundwater which caused abundant localized blooms of dinoflagellates
(Alexandrium tamarense) when conditions were optimal (so-called 'red tides'). Dinoflagellates containing para- plytic shellfish poisoning (PSP) were eaten by purple clams which accumulated the toxins becoming themselves toxic to human consumers. Consumption of contami- nated purple clams caused the death of one person and illness of six others during 1986 (Chang & Hong, 1986).
inquiry because of its importance in the understanding of the fate and effects of metals in the environment. As these different trace metal forms will generally exhibit different physical and chemical properties, measure- ment of the total metal concentration provides little indication of a metal's potential interaction with the biotic and abiotic components present in the aquatic environment (Tessier & Campbell, 1991). Relationship between speciation, bioavailability, and toxicity of met- als to aquatic organisms has been clearly demonstrated under experimental conditions (Zamuda & Sunda,
1982; O'Donnel et al., 1985; Hunt, 1987). Han et al.
(1993) described the accumulation and depuration pro- cesses of metals in green oysters and blue mussels from Taiwanese waters. Furthermore, a multiple regression equation was developed on the basis of the results of field experiments relating copper accumulation by green oysters and various copper species in natural sea- water. There has been some investigation exploring the role of acid volatile sulfide (AVS) in determining bioavailability and toxicity to benthic organisms (Carl- son et al., 1991; Ankley et al., 1991,1993).
In effect, a major fraction of the trace metals intro- duced into the aquatic environment is found to be associated with the bottom sediments, where they con- stitute a potential danger for both autotrophic and heterotrophic benthic organisms. (Tessier & Campbell, 1991). Marine molluscs have a large capacity for the accumulation of metals such as Cu, Zn and Cd, and have therefore commonly been suggested as biomoni- tors of metal contamination (Phillips, 1990). In this context, Tessier and Campbell (1991) reported that there is much circumstantial evidence that speciation in sediments had an important influence on metal uptake, but there was a pressing need for further research. Fur- thermore, there is no information on the relationships between metal speciation in sediments and bioaccumu- lation by the benthic organisms of Taiwan. Especially, it is interesting to understand the bioaccumulation of various copper species by different benthic organisms under the same environmental conditions.
Therefore, this study presents the distribution of copper species in sediment collected from the Lukang culture (aquaculture) area of central Taiwan. The paper also evaluates the relationships between copper species
in sediments and accumulation by purple clams (Hiat-
ula diphos) and venus clams (Gomphina aeguilatera) col- lected from field and culture ponds of coastal Lukang. Finally, the purpose of this work is to better under- stand different copper species as indicators of metal bioavailability in the sediments of Taiwan.
MATERIALS AND METHODS
Five species of marine invertebrates including oysters
(Crassostrea gigas), purple clams (Hiatula diphos),
rock-shells (Thais clavigera), venus clams (Gomphina
aeguilatera) and hard clams (Meretrix lusoria) were sampled from the same culture areas of Lukang during the period from August 1993 to July 1994. Oysters are
a filter feeder; purple clams, venus clams, and hard clams are infaunal deposit feeders; and rock-shells are a carnivorous feeder.
Sediment was collected along with the molluscs and sieved (< 63 /zm mesh) to obtain the mud fraction. Accurately, 0.200 g of the dried and homogenized sedi- ment was placed in a 50 ml capped polypropylene centrifuge tube and was sequentially treated with different reagents as shown in Fig. 1 (Pal et al., 1993; Han et al.,
1994b).
Field and pond-harvested molluscs were returned to the laboratory, individually scrubbed, shucked, and flesh placed in tared acid-washed Teflon beakers for weighing to obtain individual wet values. All biological samples were digested with a mixture of nitric and sulfuric acids (1/1, v/v), and the supernatant analyzed for copper by atomic absorption spectrophotometry (Hung et al., 1982; H a n & Hung, 1990).
Determination of metals was performed with a Hitachi Zeeman FAAS (model Z-8000 with an autosampler). NBS oyster tissue standard (NBS 1566, USA) was also used as a reference. The results generated were, in most cases, in good agreement with certified values. The influ- ence of copper concentrations upon copper accumulation from various biological samples were statistically exam- ined by t-test, analysis of variance (ANOVA), Shette's test, and multiple regression analysis (SAS, 1988). RESULTS AND DISCUSSION
Copper concentrations in sediments and molluscs
Concentrations of copper in sediments are so much higher than those in the overlying water that even a minute fraction may represent an important source for uptake, especially in benthic filter-feeding and burrow-
ing organisms (Bryan et al., 1985; Han et al., 1994a).
J SedLment samples I
I IM Ammonium Acetate, 15ml, pH=7, 30 rnins [ Exchangeable phase [
IM Sodium Acetate, 2Oral, pH=5. 5hrs
I C~bo~,esph~ ]
I M Sodium Acetate+O.25M Hydroxylamine Hydrnchloride, 20ml, pH=5, overnight
[ Easd,v reducible Fe & Mn oxides phase
25% Acetic Acid +0.25M Hydroxylamine Hydrochlotide, 25ml, 6 hrs ] Resistant cr'/stalline Mn oxides phase [
(a). Add 3 ml of0.01M HNO3+5 ml of 30e./, H202 heat to 85°C for 2 hrs.
(b). Add 2 ml of0.01N HNO~+3 ml of 30% H202 heat to 85*(: for 3 hrs.
(c) Cool and add l0 ml of IM Ammonium Acetate (oH=2). shake ovem ght.
[ Residual organic matter and sulphides phase ] Residual digest HF/HNO~ (1 : !) I LaRic held material phase ]
Fig. 1. Flow chart showing the sediment sequential extraction scheme (after Pal et al., 1993; Han et al., 1994b).
Table I. Concentration of copper species (/~g g-l) in sediments collected from two different environments. Mean values are presented with standard deviations and high and low values in parentheses
Copper Phase Number Culture environments
species of samples Field Pond Exchangeable phase 1 6 9.04 + 14-1 (0.20-35.8) Carbonates phase 1I 6 13-2 _+ 22.4 ( 1-05-63.2)
Easily reducible Fe and II1 6 1.14 + 0.59
Mn oxides phase (0.20-2.08)
Resistant crystalline Mn IV 6 9.03 + 2.07
oxide phase (7.15-13.1)
Organic matter and V 6 6.92 + 2.19
sulphides phase (4.26-9-89)
Lattice held material VI 6 4.61 + 1-47
phase (2.00-7.00) Total 44.0 133 + 36.7 (0.20-604) 27.2 + 54.2 (0.20-137) 0-36 + 0.24 (0.20-0.78) 9.58 + 2.85 (3-90-11.7) 10.8 + 6-10 (4.72-17.0) 4.00 + 1.41 (2.00-5.50) 185 ~,4o
Table l shows the average concentrations of copper species in sediments collected from two different envi- ronments in Lukang. The above data indicate that total copper concentrations in sediments from culture ponds (185/~g g-~) were higher than those from the field (44.0 /~g g i). From visual observation, culture pond sedi- ments are muddy, and field sediments are sandy. It is possible that the higher copper concentrations reflect finer grain size in sediments. In general, with sediments the finer the particle size, the greater the concentration of metals observed (Hung et al., 1993).
Results of the sediment sequential extractions show that the concentrations o f various copper species are in the range o f 1.14 _ 0.59 to 13.2 +_ 22.4 /~g g~ and 0.36 _ 0.24 to 133 _ 36.7 p,g g~ for the two culture environments, respectively (Table 1). The highest copper concentration of 133 _ 36-7 /~g g l found in the exchangeable phase was obtained from the culture pond sediment. This result was 15 times higher than exchangeable copper from the field. In addition, the
I00 [~]I 9o D n 80 • I n 70 • IV 3 6 0 [ ] v 50 [] vI 30 20 10 0 Field Pond
Fig. 2. The percentages of various copper species in sediments collected from two different culture environments (I) exchange- able phase; (II) carbonates phase; (III) easily reducible Fe and Mn oxides phase; (IV) resistant crystalline Mn oxides phase; (V) organic matter and sulphides phase; (VI) lattice held
material phase.
sum of phases I and II had higher percentages of cop- per both in culture pond (86.6%) and field sediments (50.5%) (Fig. 2). This suggests that the Lukang coastal environment is influenced by the local anthropogenic input of chemicals, especially copper. In general, the binding capacity o f a sediment determines its trace metal capacity. Sediments with high percentages of smaller grain size fractions (e.g. clay or silt) and with high percentages of iron and manganese can scavenge trace metals very effectively (Timmermans, 1993).
Indicator organisms should be good accumulators of metals and their tissue concentrations must reflect metal availability. For this reason, organisms having an ability to regulate metals are clearly unsuitable (Bryan et al., 1985). Figure 3 shows the copper concentrations in var- ious marine molluscs (soft) collected for this study. Maximum copper concentrations (309 _+ 35.1 /~g g 1
dry wt) in oysters (Crassostrea gigas) were much higher
than those of other benthic organisms by about 4 to 127 times (Fig. 3). At the same time, it can be seen that
•
-
~.~
A**
* Collected from culture ponds ** Collected from the field
B* C** D** E** F*
Organisms
G**
Fig. 3. The average concentration of copper in various mol- luscs (soft) collected from the Lukang coastal area of Taiwan. (A) Crassostrea gigas; (B) Hiatula diphos; (C) Hiatula diphos; (D) Thais clavigera; (E) Gomphina aeguilatera; (F) Meretrix
the range of copper concentrations in the oysters did differ significantly (p < 0.05) from other organisms. This suggests the ability of oysters to concentrate copper is much larger than that of other species. In other words, the oysters can accumulate copper from surrounding water 4-127 times more efficiently than other species. However, copper concentrations in oysters were significantly correlated (at the level of p < 0.05) with the concentration of particulate copper; this sug- gests that the food pathway from surrounding water may dominate the accumulation of copper by oysters (Han & Hung, 1990).
Figure 3 also shows copper concentrations in various benthic organisms collected from culture ponds and the field. The results reveal that relatively high copper
concentrations (77-7 + 15.2 /~g g-l, dry wt) in Thais
clavigera were found as compared with those in other benthic organisms (range from 2.43 + 0.21 to 6.30 +
1.62 ~g g-l, dry wt). Copper concentrations in Thais
clavigera were 12-32 times higher than those in other benthic organisms which was significant (p < 0.05).
The No Observed Effect Concentration for accumu- lation (NOEC accumulation) can be defined as the highest copper concentration in the sediment which did not result in a significant (p < 0-05) increase of the cop-
per concentration in the organisms (Kraak et al., 1993);
therefore, suggesting that Hiatula diphos, Gomphina
aeguilatera and Meretrix lusoria were able to regulate (i.e. low efficiency in accumulation) their internal cop-
per concentration. Conversely, Thais clavigera has a
high capacity for accumulating copper at t h e same location (i.e. under the same physico--chemical condi-
tions). This capacity makes Thais clavigera a potential
candidate for monitoring copper in marine sediments. On the other hand, relatively high copper concentra-
tions were obtained for Hiatula diphos and Meretrix
lusoria (4.98 + 1.20 and 3.72 + 0.42/xg g-i, dry wt, respec- tively) sampled from culture ponds as compared with those from the field (3.21 + 0.45 and 2.43 + 0.21 /xg g-l, dry wt, respectively). The total copper~ concentrations in sediments from two different environments differed greatly, that is 44-0 and 185/~g g-l, respectively (Table
1). However, copper concentrations in Hiatula diphos
and Meretrix lusoria collected from culture ponds did not differ significantly (p < 0.05) from those collected
from the field. This also confirmed that Hiatula diphos
and Meretrix lusoria were able to regulate their internal concentrations. In other words, copper concentrations in Hiatula diphos and Meretrix lusoria are not affected by copper concentrations in sediment.
Relationship between copper species and bioaecumulation
Bioavailable metals are defined according to Campbell
et al. (1988) as biological available chemical states that can be taken up by an organism and can react with its metabolic machinery. High trace metal concentrations in one type of sediment can be quite harmless for organisms, whereas low concentrations in another type can be very available and result in a considerable accu- mulation (Timmermans, 1993). Currently, a simple
relationship between external trace metal concentra- tions in sediment and internal concentrations in organ- isms is absent in Taiwan. Correlations of accumulation of various copper species have been calculated for
Hiatula diphos and Gomphina aeguilatera (Table 2). The best correlations were generally obtained between cop-
per carbonates and copper concentrations in Hiatula
diphos (r = 0.886). Additionally, significant regression correlations have also been found for resistant crys- talline Mn oxides (phase IV) and copper concentration in Hiatula diphos (r = 0-798) and for total copper in
sediments and copper concentration in Hiatula diphos
(r : 0.804). The present result might suggest that copper carbonates gave better information about copper bioavailability than did either the total concentration in the sediment or any one of a range of other extractants
(Table 2). Although Hiatula diphos is able to regulate
copper (low efficiency in accumulation), this result indi- cates that at low copper body burden, there still exists some degree of correlation between copper species in
sediment and copper concentrations in Hiatula diphos.
In a laboratory study of copper uptake from natural
copper-spiked sediments by Tubificid worms, Diks and
Allen (1983) also found a good correlation between accumulation and a dilute acid extractable fraction (which is similar to our carbonates phase). Our finding
supports the conclusion of Tessier et al. (1984) that
iron oxides and organic matter have a protective effect
on the accumulation of copper by Hiatula diphos and
indicate a similar role for carbonate.
The multiple regression correlation coefficient (p < 0.05, r 2 = 0.7894) was also obtained between the copper con-
centration of Hiatula diphos studied and the concentra-
tions of various copper species in the sediments. From these results, the copper carbonates may dominate as a
copper source to Hiatula diphos from several locations
along the Lukang coastal area under natural physico- chemical conditions. Furthermore, a multiple regres- sion equation was developed based on the results of
field experiments relating copper accumulation by Hiat-
ula diphos and the various copper species in natural sediments. This equation can be represented as follows:
Y = 0.991 + 0.0059X~* + 0.0150X2 (r 2 = 0.7984)
where Y is the copper concentration in Hiatula diphos;
Table 2. Relationships between copper accumulation by Hiatula
diphos and various copper species
Copper species Regression coefficient
Exchangeable phase -0.490
Carbonates phase 0.886 a
Easily reducible Fe and Mn oxides phase 0.175
Resistant crystalline Mn oxides phase 0.798 a
Organic matter and sulphides phase 0.748
Lattice held material phase 0.117
Total copper 0.804 a
X~ is the copper carbonates concentrations in the sediments; X2 is the lattice held material copper concen- trations in the sediments; and * means significant at p < 0.05.
This demonstrates that the chemical characteristics of the sediment affect the extent o f copper accumula- tion. Furthermore, it was the copper carbonates (phase II) and lattice held material (phase VI) which over- whelmingly gave the best prediction of accumulation in our studies. So, if the concentration o f various copper species has been measured in natural sediment, these copper species can be used to estimate the copper con-
centration in Hiatula diphos. Unfortunately, reliable
correlation between the concentrations of various cop-
per species in the sediments and Hiatula diphos m a y
not prove useful to monitoring requirements as Hiatula
diphos has the ability o f regulating copper which makes it clearly unsuitable for monitoring purposes.
A C K N O W L E D G E M E N T S
The authors would like to thank Dr Ming-Shiou Jeng of the Institute of Zoology, Academia Sinica, Republic of China, for providing chemical analyses and assistance in the collection of marine organisms for this study. The authors are grateful to one anonymous reviewer for constructive comments and suggestions. This study was supported by a grant from the National Science Council, Republic of China (NSC 83-0421-B-038-003-Z).
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