The Science of the Total Environment, 120 (1992) 185-204 Elsevier Science Publishers B.V., Amsterdam

185

Clams as pollution bioindicators in Kuwait's marine environment: metal accumulation and depuration M a z i n A. Z o r b a , P.G. J a c o b , A m a l A1-Bloushi and R e e m AI-Nafisi Environmental Sciences Department, Environmental and Earth Sciences Division, Kuwait Institute .for Scientific Research, P.O. Box 24885, 13109 Safat, Kuwait

(Received November 6th, 1990; accepted March 18th, 1991) ABSTRACT This study has developed and verified clam transplantation for marine pollution monitoring in Kuwait and has assessed its reliability for monitoring pollution by heavy metals through laboratory and field transplantation experiments. In field experiments, live specimens of the clam Circenita callipyga were transplanted, either suspended in seawater or buried in sand, to 11 coastal sites in Kuwait; subsamples of transplanted clams were recovered at intervals over a 6-month period. In laboratory experiments, heavy metal accumulation for 36 days and depuration (body's release of pollutants) for 60 days in small and big clams were investigated. Clams subsampled in laboratory and field experiments were analyzed for Hg, Cu, V, Cd and Pb. Results showed the ability of clams to survive under the transplantation conditions. Accumulation of biofouling materials was a problem at only two transplantation sites and was overcome by periodic cleaning. Statistical analysis of laboratory experimental data showed significant accumulation of all test metals and significant depuration of Hg, Cu, V and Cd. Rates of metal accumulation and depuration differed in relation to clam size class. Field experiments indicated statistically significant increases in Hg and Cu concentrations in the transplanted clams at most stations, no change in Cd concentrations and an increase or, occasionally, a decrease in V and Pb concentrations. Key words." clams; bioindicators; Kuwait

INTRODUCTION D u r i n g the last decade, c o n c e r n has g r o w n o v e r e n v i r o n m e n t a l water quality a n d p o l l u t i o n in b o t h heavily industrialized and developing nations. T h e detection a n d m o n i t o r i n g o f e n v i r o n m e n t a l c o n t a m i n a n t s , such as h a l o g e n a t e d h y d r o c a r b o n s , h e a v y metals and p e t r o l e u m h y d r o c a r b o n comp o u n d s in a q u a t i c ecosystems, have p r o m p t e d n u m e r o u s field and l a b o r a t o r y investigations, including the use o f bioindicators. Organisms used as bioindicators in m o n i t o r i n g p r o g r a m s include p h y t o p l a n k t o n , macroalgae, annelides, e c h i n o d e r m s , crustaceans, molluscs, m a r i n e finfish and mammals. O f

186

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these, bivalve molluscs have been the most often employed in preliminary programs (Davies and Pirie, 1978; Cooper et al., 1982; Goldberg et al., 1983). In temperate regions, Mytilus spp. and Crassostrea spp. have been widely used with considerable success (De Wolf, 1975; Bernhard, 1978; Goldberg et al, 1983). In tropical and subtropical regions, however, bioindicator studies have lagged. It has been suggested that in Southeast Asia, the best candidate for bioindicator surveys is the green-lipped mussel Perna viridis (Phillips, 1985); the accumulation of heavy metals by this species has been studied by several authors (D'Silva and Kureishy, 1978; Sivalingam and Bhaskaran, 1980; Rosell, 1985). Data are also available for heavy metal accumulation in the mussel Perna canaliculus from New Zealand (Nielsen, 1974) and in the oyster Saccostrea glomerata in Hong Kong (Phillips, 1979). In pollution monitoring studies, sampling a bioindicator species from locations where natural populations are found may omit certain locations of interest where natural populations are absent; in addition, a bivalve's spatial distribution is sometimes inadequate for intensive studies of polluted areas. In such cases, the use of a transplantation technique may be valuable. This situation exists in Kuwait's coastal environment. The spatial distribution of the clam Circenita callipyga, which was found to satisfy the prerequisites for bioindicator organisms (Anderlini et al., 1982), has undergone drastic change with several natural populations having been destroyed in recent years as a result of intensive coastal construction works. C. callipyga is a large (up to 50 mm) solid species, rounded, with a pointed umbos. Shell color is variable, usually brownish orange with outer radial streaks of purple. In its natural habitat, C. callipyga is found buried in sand in the medium to high tidal zone. Populations usually live in protected zones or areas of low-energy waves. This study developed, verified and assessed the reliability of clam transplantation as a technique for monitoring pollution by heavy metals. Appropriate field experiments were conducted, and their results were augmented by appropriate accumulation and depuration (body's release of pollutants) experiments. MATERIALS AND METHODS

Clam specimen collection A large intertidal population of C. callipyga at Fahaheel beach ( - 4 0 km south of Kuwait City, Fig. 1) was used as a stock supply for the laboratory accumulation and depuration and field transplantation experiments. Different size clams (shell length 10-50 mm) were abundant at this site during the study period.

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Stainless steel garden forks were used to collect clams buried in beach sand; only live clams with closed shells were collected. The clams were washed with seawater from the site to remove adhering materials, they were kept in clean nylon bags and transported directly to the laboratory. Some clams were transplanted to other locations and some clams were acclimated to laboratory conditions for 1 week before being used in laboratory experiments. Prior to analyses, clams in both groups were kept overnight in aerated clean seawater for defecation and then deep-frozen.

Experimentation Laboratory accumulation-depuration experiment Laboratory experiments were conducted under static conditions using unfiltered seawater; clams were held in mesh bags in an aerated 90-liter (30 x 30 x 100 cm) plexiglass tank. The seawater used was collected weekly from a relatively clean site at AI-Salmiya beach at high tide and was stored prior to use in polyethylene containers in the laboratory. During the experimental period, seawater was changed every 3 days and no food was added. Tank seawater salinity, pH and temperature were measured daily and their ranges were 36-41°k~, 7.6-8.4 and 14.0-22.4°C, respectively. The accumulation-depuration experiment involved 100 large clams (shell length 36-38 mm) and 200 small clams (shell length 22-24 mm) placed in separate mesh bags in the same tank. The two groups were treated exactly the same in order to study differences in response to metal-polluted seawater. Throughout the experiment, a ratio of 5 large to 10 small clams per liter seawater was maintained within the experimental tank; the volume of seawater was adjusted as clams were removed for analysis. The experiment consisted of two stages. In the accumulation stage, the tank seawater was spiked with five metals (Cd, Cu, Hg, Pb and V) in dissolved form. An appropriate amount of stock solution containing 20 #g ml -l Cd, 100 ~g ml -l Cu, 20/zg m1-1 Hg, 200/~g m1-1 Pb and 200/~g m1-1 V was added to the seawater to maintain metal levels at 10/zg 1-~ Cd, 50/zg 1-~ Cu, 10 tzg 1-1 Hg, 100 tzg 1-~ Pb and 100 ttg 1-l V. Spiking was performed whenever the tank seawater was changed, i.e., every 3 days. Separate subsamplings of 10 large and 20 small clams were carried out 0, 6, 12, 24 and 36 days after the start of the experiment. In the depuration stage, the contaminated specimens were exposed to clean (i.e., unspiked) seawater to study the elimination of body-accumulated metals. Subsamples of 10 large and 20 small clams each were taken 6, 12, 24, 36 and 60 days after the start of this stage. A control group of 40 clams was kept in a separate tank of seawater during the entire laboratory experimental period. Subsamplings of 10 specimens were carried out 0, 12, 36 and 72 days after the start of the period.

CLAMS AS POLLUTION BIOINDICATORS 1N KUWAIT

189

Occasionally, samples of unspiked and spiked seawater were analyzed for metal concentrations by atomic absorption spectroscopy (AAS) after a preconcentration step using Chelex 100 (Kingston et al., 1978). In the unspiked seawater, the concentrations of total (dissolved + particulate) Hg, Cu, V, Cd and Pb averaged 0.04, 2.4, 3.0, 0.11 and 1.9/~g 1-~, respectively; the dissolved form represented 80-90% of the total. In spiked seawater, the percentage recovery averaged 92% for Hg, 108% for Cu and Cd, 88% for V and 95% for Pb.

Field transplantation experiments Medium-size clams (shell length 30-35 mm) from Fahaheel, were transplanted to 11 stations in Kuwait coastal areas (Fig. 1). Sufficient clams were transplanted to each site such that at least 10 specimens could be recovered on each sampling occasion. Two transplantation methods were used. In the first method, clams were suspended in seawater and, thus, were exposed to pollutants in the water column only. Clams were placed in double-layered nylon mesh bags (20 × 40 cm) inside boxes (10 x 20 × 30 cm) of galvanized netting with 1-cm square openings. This method was applied at six stations (IS, 2S, 3S, 4S, 5S and 6S). In the second method, clams were kept buried in sand and, thus, were exposed to pollutants from different environmental media, particularly sediments and interstitial water. Clams were placed in plastic cages (20 x 30 x 40 cm); each cage was filled with sand from the same site; the top cage was covered with a perforated plastic cover; and the cages were then buried in sand at the respective sites, The cage and cover openings were of suitable dimensions to prevent clam loss but permit interstitial water exchange. This method was applied at five stations (1B, 2B1, 2B2, 3B and 4B). The transplantation experiments started on different dates in September and early October, 1987. Clams transplanted by suspension in seawater (stations 1S, 2S, 3S, 4S, 5S and 6S) were usually resampled 1, 2, 4 and 6 months after transplantation; and clams buried in sand (stations IB, 2B1, 2B2, 3B and 4B) were usually resampled 1 and 6 months after transplantation. However, additional samples were retrieved from stations 2S, 5S and 2B1; and the experiments at stations 1B, 3B and 5S were terminated 4-5 months after transplantation, because of site problems.

Analytical techniques Shells of half-thawed clams were opened, excess water in the mantle cavity was allowed to drain for about 10 min and all soft tissue was removed from the shell. The tissue wet weight and the shell length for individual clams were recorded. Soft tissue of individuals was freeze-dried separately at 40°C for about 48 h, and the dry weights were recorded. Tissue dry/wet weight ratios

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M.A. ZORBA El" AL.

for the laboratory and field experiment specimens averaged 0.126 and 0.160, respectively. Freeze-dried tissues of individuals in each subsample were powdered and composited using a porcelain mortar and pestle. For Hg analysis, 0.2-0.5 g of dried tissue powder was treated overnight with H2SOdHNO3 acid mixture (2:1) and then digested for 1 h at 65°C; digestion was completed by oxidation with a 5% KMnO4 solution and a 5% K2S208 solution. Excess KMnO4 was reduced with a 12% NaC1hydroxylammonium chloride solution, and the volume was raised to 50 ml with deionized water. For the other metals (Cd, Cu, Pb and V), 0.2-0.5 g of dried tissue was treated with HNO3/HC104 acid mixture (3:1) for 2 h at 95°C. The digest volumes were reduced by evaporation, and then heated with 1 ml of HNO3 for 30 min. Volume reduction and HNO3 treatment were then performed twice. Volume was then raised to 25 or 50 ml with deionized water, depending on the tissue weight digested. Metal analysis was carried out using a Perkin-Elmer 5000 Atomic Absorption Spectrophotometer equipped with a graphite furnace for Cu, V, Cd and Pb determination and using the cold vapor/amalgam technique for Hg determination. Standard addition was applied in all analyses.

Quality assurance Necessary precautions were taken during sampling and sample preparation to avoid contamination by the tested metals. Reagent blanks processed regularly with each 20 samples showed no traces of contamination. Replicate analysis of a standard reference material from the U.S. National Bureau of Standards (oyster tissue NBS No. 1566) showed good accuracy, with all results comparable with certified values. Triplicate analysis of clam samples in the accumulation-depuration experiments also showed good precision (average coefficients of variation were 7.1% for Hg, 5.8% for Cu, 7.6% for V, 10.2% for Cd and 11.5% for Pb). RESULTS

Survival of transplanted clams Clams transplanted suspended in seawater or buried in sand survived under the experimental conditions for the entire 6 months of field transplantation experiments. About 12% of the total transplanted clams were either lost or died during the field experiment, an acceptable rate for such work. The mortality of transplanted clams at some sites was attributed to nonpollution causes. The growth of nuisance biofoulants on outer shell surfaces was encountered in clams suspended in seawater but not in those buried in

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sand. Fouling material growth was high at Stations 1S and 4S compared with other transplantation sites, where such growth was minor or non-existent. The most common biofoulants on the experimental cages were barnacles, polychaete tubes, sponges and acedians.

Laboratory accumulation-depuration experiment The results of Hg, Cu, V, Cd and Pb analyses of the control group and two size-classes of clams in the accumulation-depuration experiment are presented in Fig. 2. Hg, Cu and Cd concentrations in the large and small clams increased steadily during the accumulation stage and had not reached equilibrium by the end of the 36-day period. Maximum concentrations for the large and small clams were Hg 8.56 and 18.8 #g g-l, Cu 85 and 140 #g g-l, and Cd 5.37 and 6.13/zg g-l, respectively. On the other hand, V and Pb concentrations reached equilibrium after 24 days of exposure. Maximum concentrations in the large and smaller clams were V 2.51 and 2.85/~g g-l, and Pb 8.3 and 10.4/~g g-~, respectively. The test metals generally were accumulated faster by the small than by the larger clams. Large and small clam metal-release patterns during depuration differed (Fig. 2). Metal concentrations in clam tissue generally decreased at the beginning. Cu concentrations in the large and small clams decreased until it reached 17 and 41 #g g-~, respectively, after the 60-day depuration period. Hg concentration decreased gradually in the small clams, but remained at 6-7 #g g-~ in the large clams throughout depuration. V and Cd concentrations decreased sharply in the first 12 days of depuration. Pb release was minor during depuration, and differences between the large and small clams narrowed with time. At the end of depuration, concentrations of all tested metals were above concentrations at 0 time (at the start of accumulation).

Field transplantation experiments Clam analysis results for Hg, Cu, V, Cd and Pb in transplantation experiments showed that soft tissue metal concentrations in transplanted clams varied with time at some locations (Fig. 3).

Mercury Hg levels in clams transplanted to Shuwaikh Port stations (2S, 2B1 and 2B2) and to Kuwait City stations (3S and 3B) increased over time in a similar pattern: accumulation was fast during the first month after transplantation and subsequently more gradual. Concentrations in clams transplanted suspended in seawater (stations 2S and 3S) were higher than in those transplanted buried in sand (stations 2B1, 2B2 and 3B). Maximum concen-

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trations at the end of transplantation were 0.39, 0.33, 0.26, 0.30 and 0.19 #g g-~, recorded at stations 2S, 2B1, 2B2, 3S and 3B, respectively. Minor increases were observed at stations 4S, 4B, 5S and 6S; no change was observed at stations 1S and lB.

Copper Except at stations 1S and 1B, Cu concentrations in transplanted clams increased with time, but to different degrees at different stations. Higher concentrations were observed in clams transplanted suspended in seawater, particularly at the Kuwait City stations (3S and 3B) and Shuwaikh Port stations (2S, 2B1 and 2B2). Clams subsampled from station 2S showed higher maximum concentrations and higher fluctuations in their body Cu concentrations than those subsampled at other sites. Maximum concentration at station 2S (12.5 #g g-~) was reached the second month after transplantation, whereas maximum concentrations at other sites was reached at the end of the experimental period (6.7, 9.8, 11.7, 7.1, 9.4, 9.7, 5.8 and 7.1 /zg g-i at stations 2B, 2B2, 3S, 3B, 4S, 4B, 5S and 6S, respectively). Vanadium Change in V concentration with time was minor at most stations. Concentrations were unchanged at station 6S, fluctuated in the Shuwaikh Port stations (2S and 2B1), and increased gradually at stations 1S and lB. However, at stations 3S and 5S, at the end of the accumulation period, concentrations were four to six times the initial concentrations. Maximum concentrations observed were 1.75 and 2.67/zg g-~ at stations 3S and 5S, respectively. Cadmium Cd concentrations at most stations were unchanged in the transplantation period. However, at station 3S concentrations increased through the second month after transplantation and then decreased; at station 3B, clams transplanted buried in sand showed less elevated Cd levels; and at station 5S, concentrations were higher than those at station 6S. Average Cd concentrations at the different stations generally varied within a narrow range (0.18 and 0.32 #g g-l). Lead Changes in Pb concentrations differed among stations. The concentration decreased at stations 1B, 2S and 3S; increased at stations 1B, 2B2 and 3B; and fluctuated sharply at Shuaiba Port (station 5S) and Shuwaikh Port (station 2B1). Average concentrations at the different sites ranged between 1.23 and 2.91 #g g-~.

CLAMS AS POLLUTION BIOINDICATORS IN KUWAIT

195

DISCUSSION AND CONCLUSIONS

Suitability of clam transplantation Clam transplantation was found to be successful in terms of natural mortality and loss rates. The two tested transplantation methods were found to be applicable and may serve different purposes. In the first method, in which clams were suspended in seawater, they were exposed to dissolved and suspended pollutants present in the water column. In the second method, in which clams were buried in sand as is their natural occurrence, they were affected by pollutants from sediments and land-based sources. Pollutant forms in the two transplantation media are different due to the presence of different physico-chemical conditions; hence, the forms have different accessibility to the organism. Several studies on transplanted bioindicators have been conducted successfully in different regions of the world using cage/buoy systems (Young et al., 1976; Davies and Pirie, 1978 and 1980; Johnson and Lack, 1985). Transplantation has certain advantages over wild-population sampling, since it permits experimentation on certain age or sex classes not otherwise available at the test site, testing for a certain season, and calculation of contaminant concentration changes (Ritz et al., 1982). On the other hand, problems are encountered with transplantation. The major one encountered in this study was the introduction of biofoulant species that affects the ecological balance in the immediate vicinity of the transplantation. The loss of cage/buoy systems is a potentially serious problem at many sites and was considered during this study. High temperature could affect clams transplanted buried in sand, especially if wave action uncovers them and exposes them to direct sunlight.

Heavy metal accumulation and depuration patterns & clams Clams, like other bivalves, show certain heavy metal accumulation and depuration patterns. These usually are studied in laboratory tests under controlled metal concentration and environmental conditions (temperature, salinity, pH). In this study, the accumulation of mixed Hg, Cu, V, Cd and Pb concentrations were examined in the laboratory under normal temperature and salinity. The significance of metal concentration variation in each group of clams was assessed by an analysis of variance (ANOVA) test. During accumulation, all metals showed significant change with exposure time in both large and small clams (P < 0.001; Table 1). During depuration, only Pb exhibited

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TABLE 1

Inter-group ANOVA of heavy metal concentrations in clams in accumulation-depuration experiment Stage/group

ANOVA a

Metal Hg

Cu

V

Cd

Pb

Accumulation

Large

DF F P > F

4,8 464.36 0.0001

4,10 2,313.39 0.0001

4,10 43.75 0.0001

4,10 122.58 0.0001

4,10 34.78 0.0001

Small

DF F P > F

4,8 218.65 0.0001

4,7 724.18 0.0001

4,7 175.54 0.0001

4,10 86.54 0.0001

4,10 32.65 0.0001

DF F P > F

5,12 14.98 0.0001

5,12 133.94 0.0001

5,12 60.70 0.0001

5,12 37.21 0.0001

5,12 0.48 NS

DF F P > F

5,11 18.22 0.0001

5,9 390.01 0.0001

5,9 20.49 0.0001

5,12 9.08 0.0001

4,10 2.74 NS

DF F P > F

3,4 62.91 0.0008

3,7 2.40 NS

3,8 0.54 NS

3,8 0.54 NS

3,8 11.19 0.0031

Depuration Large

Small

Control

aAnalysis of variance parameters: vl, v2 degrees of freedom (DF); F value (F) and significance probability associated with the F statistic (P > F); NS, not significant (P > 0.01).

insignificant change in both size classes of clams. In the control group of clams, no significant change was observed for Cu, V and Cd concentrations, but minor increases occurred in Hg and Pb concentrations. When a linear regression model was assumed for the obtained data, levels of all metals during accumulation showed significant linear increases with exposure time in large and small clams (P < 0.001; Table 2). During depuration, however, levels of some metals did not significantly correlate with depuration time. Hg depuration in large, but not small, clams did not follow a linear pattern, while Pb depuration did not follow a specific pattern in either size class. Accumulation rates (/zg g-I day-l), i.e. the regression coefficients in Table 2, were 2-10-times higher than depuration rates for all metals that showed

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197

TABLE 2 Linear regression analysis of heavy metal concentrations in clams with exposure period in accumulation-depuration experiment Stage/Group

Regression a

Metal Hg

Cu

V

Cd

Pb

0.961 0.877 0.212 13 0.0001

0.989 4.194 2.167 15 0.0001

0.556 1.080 0.030 15 0.0314

0.928 0.215 0.124 15 0.0001

0.808 4.226 0.130 15 0.0003

0.993 0.063 0.532 13 0.0001

0.994 3.511 3.947 12 0.0001

0.737 1.100 0.052 12 0.0062

0.973 0.170 0.155 15 0.0001

0.897 3.956 0.216 15 0.0001

-0.288 7.65 -0.116 18 NS

-0.938 110.44 -1.044 18 0.0001

-0.715 1.74 -0.012 18 0.0009

-0.857 6.18 -0.045 18 0.0001

0.095 7.79 0.004 18 NS

-0.941 22.96 -0.116 17 0.0001

-0.994 203.44 -1.694 15 0.0001

-0.691 2.18 -0.014 15 0.0044

-0.711 6.42 -0.027 18 0.0009

-0.355 10.20 -0.022 15 NS

Accumulation Large

r a

b n

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r a

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Depuration Large

r a

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r a

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a Linear regression parameters: correlation coefficient (r), intercept (a), regression coefficient (b), number of observations (n) and significance probability of correlation (P > I r 1). NS, not significant (P > 0.05).

linear relationships in both experimental stages (Table 3). In addition, accumulation rates in small clams were consistently higher than those in large clams (about twice for Hg, Cu, V and Pb). Depuration rates were also higher in small clams, except that Cd showed the reverse relationship. Hg depuration rates for large clams and Pb rates for large and small clams were, on average, slow. For metals showing a significantly linear relationship during depuration, recovery times were estimated based on the regression coefficient parameters

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M.A. Z O R B A ET AL.

TABLE 3 Estimated recovery times for accumulated metals in two size classes of clam I~,e class

Metal

Accumulation rate (#g g-I day-t)

Depuration rate (/zg g-I day-l)

Recovery time a (days)

Large

Hg Cu V Cd Pb

0.212 2.167 0.030 0.124 O.130

0.016 1.044 0.012 0.045 0.004

b 102 112 127 b

Small

Hg Cu V Cd Pb

0.532 3.947 0.052 0.155 0.216

0.116 1.694 0.014 0.027 0.022

183 117 121 221 b

a Time required to reach initial concentration, calculated from regression equation parameters a and b in Table 2. bValue not considered due to insignificant correlation coefficient (P > 0.05).

(parameters a and b in Table 2), assuming that their concentrations would decrease to their levels at time 0 of the experiment. Estimated recovery times for the two clam size-classes are shown in Table 3. Despite the higher metal depuration rates of small clams, their recovery times were longer than those of the big clams. This was possible because the small clams had higher accumulation rates than the large clams and, hence, could attain higher concentrations of heavy metals by the end of accumulation. The linear accumulation pattern of the five test metals in clams in this study is similar to accumulation patterns reported for other bivalves (George and Coombs, 1977; D'Silva and Kureishy, 1978; Ritz et al., 1982); e.g. the mussel Mytilus galloprovincialis was found by Majori and Petronio (1973) to reach an equilibrium concentration following an initial linear accumulation phase. In the present study, clams reached equilibrium concentrations for V and Pb after 24 days of exposure to 100 #g 1-1 for each metal; concentrations of the other metals (Hg, Cu and Cd) did not reach equilibrium during the 36 days of exposure, probably because they were applied at lower concentrations than were Pb and V. This accords with other studies that indicate metal accumulation rates to be dependent on external concentration (SchulzBaldes, 1974; George and Coombs, 1977; D'Silva and Kurieshy, 1978; Kumaraguru and Ramamoorthi, 1979; Ritz et al., 1982).

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CLAMS AS POLLUTION BIOINDICATORS IN KUWAIT

TABLE 4 Inter-station ANOVA of heavy metal concentrations in clams of field transplantation experiments Station NOVA a

Metal Hg

Cu

V

Cd

Pb

1S

DF F P > F

4, 10 1.22 NS

4, 10 12.22 0.0002

4, 13 3.50 NS

4, 9 7.46 0.0020

4, 14 7.77 0.0041

2S

DF F P > F

5, 14 144.63 0.0001

5, 16 109.75 0.0001

5, 16 12.09 0.0001

5, 16 1.38 NS

5, 16 1.09 NS

3S

DF F P > F

4, 9 39.97 0.0001

4, 13 55.89 0.0001

4, 13 38.07 0.0001

4, 14 4.97 0.0105

4, 14 65.96 0.0001

4S

DF F P > F

4, 7 10.62 0.0043

4, 13 21.77 0.0001

4, 9 30.04 0.0001

4, 14 2.05 NS

4, 14 12.05 0.0002

5S

DF F P > F

4, 12 4.57 0.0179

4, 13 4.66 0.0148

4, 13 135.28 0.0001

4, 14 2.71 NS

4, 14 5.30 0.0082

6S

DF F P > F

4, 7 15.08 0.0015

4, 12 34.85 0.0001

4, 8 2.23 NS

4, 14 0.66 NS

3, 11 6.05 0.0109

1B

DF F P > F

2,5 13.41 0.0100

2,7 0.20 NS

2,6 0.41 NS

2,8 1.94 NS

2,8 10.06 0.0065

2BI

DF F P > F

5, 14 78.33 0.0001

5, 16 21.26 0.0001

5, 15 3.73 0.0214

5, 17 0.99 NS

5, 17 35.25 0.0001

2B2

DF F P > F

2, 6 175.56 0.0001

2, 7 33.38 0.0003

2, 3 1916.38 0.0001

2, 8 2.08 NS

2, 8 18.54 0.0010

3B

DF F P > F

2, 5 198.83 0.0001

2, 7 71.28 0.0001

2, 7 7.60 0.0176

2, 8 28.32 0.0002

2, 8 6.02 0.0254

4B

DF F P > F

2, 5 254.70 0.0001

2, 7 30.48 0.0004

2, 5 120.58 0.0001

2, 8 0.08 NS

2, 8 33.99 0.0001

a Analysis of variance parameters: vl, v2 degrees of freedom (DF), F value (F), and significance probability associated with the F statistic (P > F); NS, not significant (P > 0.05).

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Ritz et al. (1982) reported a decrease in accumulation and depuration rates for Cu, Cd and Pb with an increase in size of the mussel Mytilus edulis. This relationship had been obtained with Cd by Schutz-Baldes (1974), but Harris et al. (1979) found the inverse relationship in the same species. With the clam C. callipyga, the present experimental results showed greater accumulation and depuration rates for Hg, Cu, V and Cd in small clams than in large clams, and the reverse relationship for Cd (Table 3). The difference in response between bivalve species and sizes may be due to a physiological effect causing differences in metabolic, feeding or filtration rates by species and age. Metal accumulation by different organs of bivalves, though not examined in this study, was important in total body accumulation and depuration rates. Data from other authors show that most or all bivalve species accumulate the highest concentrations of metals in the kidney and digestive glands (NRC, 1980).

Assessment of heavy metal pollution by transplanted clams Bioindicators take up pollutants from the ambient water and food. Pollutant concentrations in their tissues, or sometimes changes in these concentrations, provide a time-integrated measure of pollutant bioavailability rather than of pollutant abundance. The bioindicator clam in this study was transplanted in two media: water column and sediment. The bioavailable concentrations of metals in the two media may be different; and consequently, the accumulation pattern will also be different. The significance of variations in heavy metal concentrations at each transplantation station was tested with an ANOVA involving data obtained from all subsamples (Table 4). Variations in Hg, Cu and V concentrations were significant (P < 0.05) at all stations except the Doha stations (IS and 1B). Variations in V concentration were insignificant at station 6S. Cd concentrations varied significantly during transplantation at only three stations (IS, 3S and 3B), all in Kuwait Bay. Pb concentrations in all subsamples were significantly different (P < 0.05), except at subsamples collected from station 2S. Results of the linear regression analysis of metal concentrations with time are shown in Table 5. The transplanted clams at the different stations can be classified into three groups according to the pattern of concentration change (increase or decrease). In the first group, levels showed significant linear increases with exposure time (P < 0.05). About 50% of the 'metalstation' combinations belonged to this group: Hg at stations 2S, 3S, 2B1 and 2B2; Cu at stations 3S, 4S, 5S, 6S, 2B1, 2B2 and 4B; V at stations IS, 3S and 4S; Cd at station 3B; and Pb at stations IS, 3S, 4S, 1B, 2B2, and 3B.

CLAMSAS POLLUTIONBIOINDICATORSIN KUWAIT

201

TABLE 5 Linear regression analysis of heavy metal concentrations in clams with t r a n s p l a n t a t i o n period in the field experiments Station

1S

Regression a

2S

3S

r a b n P>

Cu

V

Cd

Pb

r [

-0.536 0.047 0.000 15 0.0393

-0.412 5.951 -0.007 18 NS

0.756 0.149 0.003 14 0.0018

0.536 0.249 0.001 19 0.0179

-0.827 1.682 -0.007 15 0.0001

r l

0.873 0.133 0.002 20 0.0001

0.345 8.232 0.012 22 NS

-0.394 0.546 0.001 22 NS

-0.177 0.299 0.000 22 NS

-0.460 1.720 -0.002 22 0.0314

r I

0.878 0.102 0.001 14 0.0001

0.922 5.831 0.034 18 0.0001

0.812 0.297 0.007 18 0.0001

-0.152 0.361 0.000 19 NS

-0.685 2.490 -0.011 19 0.0012

r [

0.194 0.074 0.000 12 NS

0.897 4.804 0.027 18 0.0001

0.917 0.322 0.005 14 0.0001

0.287 0.212 0.000 19 NS

0.817 1.544 0.009 19 0.0001

r J

0.332 0.069 0.000 17 NS

0.711 4.782 0.008 18 0.0009

0.535 0.725 0.011 18 0.0222

0.536 0.274 0.001 19 0.0179

-0.359 2.563 -0.007 19 NS

r [

0.849 0.058 0.000 12 0.0005

0.910 4.508 0.012 17 0.0001

0.005 0.333 0.000 13 NS

0.077 0.203 0.000 19 NS

-0.174 1.311 0.000 15 NS

r[

0.001 0.057 0.000 8 NS

0.140 4.987 0.001 10 NS

-0.065 0.352 0.000 9 NS

0.540 0.236 0.000 11 NS

0.835 1.859 0.005 11 0.0014

0.914 0.127 0.001

0.901 4.671 0.012

-0.507 0.547 -0.002

-0.352 0.216 0.000

-0.358 1.770 0.005

r a b n P>

4S

Hg r a b n P>

r a

b n P> 5S

r a b n P>

6S

IB

r a b n P> r

a b n

P> 2B1

r a

b

Metal

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M.A. Z O R B A ET AL.

TABLE 5

(continued)

Station Regression a

Hg

Cu

V

Cd

Pb

20 0.0001

22 0.0001

21 0.0190

23 NS

23 NS

r

0.831 0.116 0.001 9 0.0055 0.654 0.112 0.001 8 NS

0.951 4.974 0.025 10 0.0001 0.382 6.260 0.008 10 NS

0.793 0.564 0.003 6 NS 0.244 0.435 0.001 10 NS

0.425 0.245 0.000 11 NS 0.935 0.123 0.001 11 0.0001

0.816 2.296 0.009 11 0.0022 0.774 1.115 0.006 11 0.0052

r

0.960 0.042 0.000 8 0.0002

0.946 4.73 0.026 10 0.0001

0.618 0.650 0.002 8 NS

0.138 0.225 0.000 11 NS

-0.679 2.75 -0.009 11 0.0215

n

P> 2B2

r

r a b n

3B

P> r a

r

b n

P> 4B

r a b n

P>

Metal

aLinear regression parameters: correlation coefficient (r), intercept (a), regression coefficient (b), n u m b e r of observations (n) and significance probability of correlation ( P > I r I); NS, not significant ( P > 0.05).

Here, the corresponding metal concentrations in the transplantation environment were higher than the background concentrations in the native (collection site) environment, and the bioindicator clam averaged out this elevated concentration. In the second group, concentrations did not show significant linear increases but varied during transplantation period. The 'metal-station' combinations in this group were Cu at station 2S; V at station 5S; Cd at station 3S; and Pb at stations 2B1, 4B and 5S. Here, the concentrations at these stations fluctuated probably because of the presence of nearby pollution sources coupled with a high ambient dilution capacity; again, the bioindicator clam averaged out intermittently high concentrations in the ambient environment during the transplantation period. The third group exhibited significant linear changes in concentrations but, on average, attained < 0.001 t~g g-i day-l accumulation or depuration rate; e.g. Hg at stations 6S and 4B, where the Hg accumulation rate was equal to the depuration rate. Metal accumulation patterns were evident in the transplanted clams at almost all stations, depuration patterns were observed only for Pb at stations

CLAMS AS POLLUTION BIOINDICATORS IN KUWAIT

203

IS, 2S, 3S and 4B and V at station 2B1. The highest accumulation rates (i.e., regression coefficient, Table 5) were recorded as follows: Hg at station 2S (0.002 #g g-~ day-l); Cu at station 3S (0.034 #g g-i day-l); V at station 3S (0.007/~g g-1 day-l); Cd at station 3B (0.001/~g g-i day-l); and Pb at station 4S (0.009/~g g-~ day-l). The highest depuration rate was observed for Pb at station 3S (0.011 /~g g-i day-l). In conclusion, the field transplantation experiment results showed that Khiran (station 6S) and D o h a (stations 1S and 1B) are relatively clean areas with respect to metal pollution. The other stations are more polluted with Cu; the Shuwaikh Port area (stations 2S, 2B1 and 2B2) and Kuwait City seafront (stations 3S and 3B) are more polluted with Hg; the Shuaiba Port area (station 5S) is more polluted with V; and the D o h a beach area (station 1B), near two major power desalination plants, and the Kuwait City seafront area (stations 2B2, 3B) are more polluted with Pb. So far, however, metal pollution in Kuwait's marine environment is not a serious problem, but clam transplantation could be o f use in cases of occasional contamination from the land-based sources, which is possible at any time. ACKNOWLEDGEMENTS The authors are grateful to Dr. D. A1-Ajmi, Environmental and Earth Sciences Division Director and to Dr. S. AI-Muzaini, Environmental Science Department Manager for their encouragement and support during this study. Thanks are also due to Mr. O. Samhan for his critical review of the manuscript. REFERENCES Anderlini, V.C., O.S. Mohamrnad, M.A. Zorba and N.A. Omar, 1982. Assessment of trace metal and biological pollution in the marine environment of Kuwait. Kuwait Institute for Scientific Research, Report No. KISR605, Kuwait. Bernhard, M., 1978. Heavy metals and chlorinated hydrocarbons in the Mediterranean. Ocean Manage., 3: 253-313. Cooper, R.J., D. Langlois and J. Olley, 1982. Heavy metals in Tasmania shellfish. 1-Monitoring heavy metal contamination in the Derwent Estuary: Use of oysters and mussels. J. Appl. Toxicol., 2: 99-109. Davies, I.M. and J.M. Pirie, 1978. The mussel Mytilus edulis as a bio-assay organism for mercury in seawater. Mar. Pollut. Bull., 9: 128-132. Davies, I.M. and J.M. Pirie, 1980. Evaluation of a 'Mussel Watch' program for heavy metals in Scottish coastal waters. Mar. Biol., 57: 87-93. De Wolf, P., 1975. Mercury content of mussels from West European coasts. Mar. Pollut. Bull., 6: 61-63. D'Silva, C. and T.W. Kureishy, 1978. Experimental studies on the accumulation of copper and zinc in the green mussel. Mar. Pollut. Bull., 9: 187-190.

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George, S.G. and T.L. Coombs, 1977. The effect of chelating agents on the uptake and accumulation of cadmium by Mytilus edulis. Mar. Biol., 39: 261-268. Goldberg, E.D., M. Kaide, V. Hodge, A.R. Flegal and J. Martin, 1983. U.S. 'Mussel Watch' 1977-1978: Results of trace metals and radionuclides. Estuarine Coast. Shelf Sci., 16: 69-93. Harris, J.E., G.J. Fabris, P.S. Statham and F. Taufik, 1979. Biogeochemistry of selected heavy metals in western part, Victoria, and use of invertebrates as indicators with emphasis on Mytilus edulis planulatus. Aust. J. Mar. Freshwater Res., 30:159-178. Johnson, D. and T.J. Lack, 1985. Some responses of transplanted Mytilus edulis to metalenriched sediments and sewage sludge. Mar. Environ. Res., 17: 277-280. Kingston, H.M., I.L. Barnes, T.J. Brady and T.C. Rains, 1978. Separation of eight transition elements from alkali and alkali earth elements in estuarine and seawater with chelating resin and their determination by graphite furnace atomic absorption spectrometry. Anal. Chem., 50: 2064-2070. Kumaraguru, A.K. and K. Ramamoorthi, 1979. Accumulation of copper in certain bivalves of Vellore Estuary, Porto Navo, S. India in natural and experimental conditions. Estuarine Coast. Mar. Sci., 9: 467-475. Majori, L. and F. Petranio, 1973. Marine pollution by heavy metals and their accumulation by biological indicators. Rev. Int. Oceanogr. Med., 31: 50-90. Nielsen, S.A., 1974. Vertical concentration gradients of heavy metals in cultured mussels. N. Z. J. Mar. Freshwater Res., 8: 631-636. NRC, 1980. The International Mussel Watch. Commission on Natural Resources and National Research Council. Washington, D.C National Academy of Sciences. Phillips, D.J.H., 1979. The rock oyster Saccostrea glomerata, as an indicator of trace metals in Hong Kong. Mar. Biol., 53: 353-360. Phillips, D.J.H., 1985. Organochlorines and trace metals in green-lipped mussels Perna viridis from Hong Kong waters: A test of indicator ability. Mar. Biol. Prog. Ser., 21: 251-258. Ritz, D.A., R. Swain and N.G. Elliott, 1982. Use of the mussel Mytilus edulis planulatus in monitoring heavy metal levels in seawater. Aust. J. Mar. Freshwater Res., 33: 419-506. Rosell, N.C., 1985. Uptake and depuration of mercury in green mussel Perna viridis. Philipp. J. Sci., 114: 1-29. Schulz-Baldes, M., 1974. Lead uptake from seawater and food, and lead loss in common mussel, Mytilus edulis. Mar. Biol., 25: 177-193. Sivalingam, P.M. and B. Bhaskaran, 1980. Experimental insight of trace metal environmental pollution problems in mussel farming. Aquaculture, 20: 291-303. Young, D.R., T.C. Heesen and D.J. McDermott, 1976. An offshore biomonitoring system for chlorinated hydrocarbons. Mar. Pollut. Bull., 7: 156-159.