Iranian Fisheries Research & Training Organization, Tehran, Iran. Received 21 ... by industrial, domestic and mining activities are ulti- mately absorbed by ...
53
Hydrobiologia 331 : 53-61, 1996 . © 1996 Kluwer Academic Publishers . Printed in Belgium.
Heavy metal concentrations in surficial sediments and benthic macroinvertebrates from Anzali wetland, Iran N. Pourang Iranian Fisheries Research & Training Organization, Tehran, Iran Received 21 September 1994; in revised form 16 April 1996 ; accepted 17 April 1996
Abstract Lead, Copper, Zinc and Manganese were measured in surficial sediments, chironomid larvae, tubificid worms and two species of bivalve molluscs (Mytilaster lineatus and Corbicula fiuminalis) from the Anzali wetland, Iran . No distinct relationship was observed between heavy metal levels and percentage fine fraction in sediments . The pattern of Mn accumulation was parallel to trends of organic matter variation . There were highly significant differences between sampling sites in contents of heavy metals but no significant differences between seasons . Significant differences were found in bioaccumulation of Lead and Zinc in three size categories of chironomid larvae . Lead was higher in smaller M. lineatus, while the reverse was observed for Copper .
Introduction Among aquatic pollutants, heavy metals are the most appropriate indicator of pollution, because of their stability in sediments and scarcity in natural environments (Saeki et al ., 1993) . Heavy metals introduced to aquatic environments by industrial, domestic and mining activities are ultimately absorbed by deposits and incorporated into sediments . Hence sediments are the most concentrated physical pool of metals in aquatic systems . Sediments may contribute significantly to concentrations of metals in benthic invertebrates, either by absorption/adsorption from interstitial water or by direct ingestion (Clements, 1991 ; Gerhardt, 1990) . On the basis of controlled laboratory feeding experiments, one might anticipate the existence of a consistent relationship between metal accumulation in benthic organisms and metal partitioning in the adjacent or host sediment . Metal concentrations found in benthos are affected by different geochemical and biological factors (Bodou & Ribeyre, 1989) . On the other hand, benthic invertebrates are often a major component the diet of many fish species . Because of their close association with sediments, and the ability of certain species to tolerate and accumulate metals, these organisms represent an important link in the transfer of contaminants to higher levels (Clements, 1991) .
In the present study, heavy metals in sediments and benthic fauna have been investigated from different point of view. The main objectives were : (a) to determine heavy metal concentrations in surficial sediments from different sites of the Anzali wetland and comparison with results from other geographical regions, (b) to assess relationship between heavy metal contents in sediments and benthic invertebrates, (c) to investigate the relation of body size to total body burdens of metals in macrobenthic organisms, (d) to study the influence of feeding habits on concentrations of metals in benthic invertebrates .
Materials and methods Study area This study was conducted between June 1993 and March 1994 in Anzali wetland, one of the most important water bodies in northern Iran (37°28'N, 49°25'W), connected to the Caspian sea . Its open water surface area is estimated at 58 km2 . There is a little industrial activity in the wetland's watershed. Only 10 .5 percent of the total population of the watershed works in industry. The wetland represents an internationally important wildlife reserve and sanctuary, listed under the Ramsar Convention (Olah, 1990 ; Holcik & Olah,
54
Figure 1 . Map of Anzali wetland showing location of the six sampling sites .
55 1992) . The location of our sampling sites is shown in Figure 1 . Sampling and storage Surficial sediments and benthic macroinvertebrates (tubificid worms and chironomid larvae) were sampled from six sites in various parts of the wetland using a standard Ekman grab with a sampling dimension of 231 cm 2 . The samples were taken four times (June, September, December 1993 and March 1994) . Each time 12 sub-samples were collected from each site . Seven of them were used for sediment analysis (five for heavy metal analysis and two for grain size and organic matter determination) . The other five were sieved in the field through a 200 ,urn nylon mesh. Macrobenthic organisms retained in the net were transferred to acid-washed polypropylene bags and stored on ice until delivered to the laboratory and then frozen at -20 ° C prior to heavy metal analysis . To collect bivalve molluscs, a bottom dredge (with net bag of 5 mm mesh size) was used and specimens were stored as mentioned above, before analysis . The surface layer of the sediment sub-samples in the undisturbed grabs was carefully removed with a plastic spatula and the remainder of grab contents (without broken shells and plant remains) stored at 4°C until testing . Analytical procedures Throughout analytical work, deionized double distilled water (DDDW) was used . All acids were Merck, with high purity . To minimize trace element contamination, all labware was washed with detergent and tapwater and was then soaked in 15% nitric acid for 25 h and followed by repeated rinsing in DDDW . To prepare sediment samples (as bulk) for heavy metal determination, samples were oven-dried at 65°C on glass dishes, homogenized with a pestle and mortar and each of weighted samples (approximately 1 g to the nearest 1 mg) were taken to a 100 ml kjeldahl flask, to which 10 ml diluted aquaregia (3 :3 :1 H20 :HC1 :HNO 3 ) was added, covered with a watch glass and allowed to stand overnight at room temperature (c . 20'C) . The following day samples were digested to near dryness at 90°C on a hot plate . Cooled digested samples were filtered through Whatman No . 1 filter papers and collected in 100 ml beakers . The filter papers was washed with about 20 ml of water and the contents of the beakers transferred to 50 ml volumetric flask, brought to vol-
ume with DDDW. The solutions were analysed for metals in a Pye Unicam flame atomic absorption spectrophotometer, Model SP9 . All analyses were analyses were undertaken in triplicate and mean values were calculated . A standard reference material (SRM) and two acid blanks were run with each batch of samples . The standard solutions were made from Merck stock solutions . Frozen invertebrate samples were thawed at room temperature . Chironomid larvae were divided into three size categories : 0-10 mm (small), 10-15 mm (medium), and > 15 mm (large) . Shell length of M. lineatus samples were also measured and separated into two size groups : < 15 mm and > 15 mm . Macroinvertebrate individuals were pooled to obtain at least 0 .1 g dry weight. Digestion was done in a similar way to that for sediments, except that a mixture of HNO3/HC104 (5 :1) was applied and diluted to a maximum of 5 ml volume with 6% HNO3 (Young & Harvey, 1991) . Pooled samples were analysed (at least in triplicate) on a Perkin Elmer model 503 atomic absorption spectrophotometer and a HGA72 graphite atomizer . Amounts of organic matter in sediments were determined by loss of weight on ignition at 600 ° C (Holme & McIntyre, 1984) . To determine grain-size, sediments were split into a sand (particles > 62 µm) and a silty-clay fraction (particles < 62 pm) . The sand fraction was divided through a series of graded sieves (pore sizes 125, 250, 500, 1000 and 2000 µm) (Holme & McIntyre, 1984) . Data analysis A three-way analysis of variance (ANOVA) (Sokal & Rohlf, 1981) was used to test for significant differences in heavy metal accumulation in sediments (6 sampling sites x 4 seasons x 4 metals). Each used datum was the mean of metal concentrations in five sub-samples . Hierarchical cluster analysis (using Euclidian measures) was used to group different sampling sites based on accumulation of each heavy metal in sediments (Ludwig & Reynolds, 1988 ; Green, 1979) . Since the pooled chironomid samples were few, a Kruskal-Wallis test (a non-parametric equivalent of one-way ANOVA) was conducted to determine whether the differences among concentrations of each heavy metal in the three size categories of chironomid larvae were significant . A nonparametric Tukey-type multiple comparison test was applied to determine the differences between the three size groups of chironomid larvae (Zar, 1984) .
56 Table 1 . Results of a three-way analysis of variance (ANOVA) test to examine the influ-
ence of sampling sites, metals and seasons as well as the interaction of these factors on concentrations of heavy metals in sediments Source of variation
df
SS
MS
F-ratio
P-value
Main effects : Sampling sites
5
88905
17781 .0
3 .78
< 0 .01
Metals Seasons
3 3
11294262 29763
3764753 .9 9921 .0
802 .14 2 .11
< 0 .01 > 0.05
Interactions: Sampling sites * Metals
15
509094.09
33939 .61
7 .23
< 0.01
Sampling sites * Seasons Metals * Seasons
15 9
73969 .90 70363 .53
4931 .33 7818 .17
1 .05 1 .66
> 0.05 > 0.05
Residual
45
211203 .18
4693 .40
Total
95
12277560 .7
A two-tailed Student's t-test was employed to examine the null hypothesis that concentrations of each heavy metal was the same (no statistical differences) in the two size groups of M. lineatus . Since on the basis of Kolmogorov-Smirnov goodness of fit procedure it could be reasonably assumed that heavy metals contents of sediments and chironomid larvae (sampled from the same sites) were normally distributed, Pearson's product moment correlation coefficient were used to examine the relationship between the two latter variables (Rees, 1991) . All statistical analyses were performed using SPSSX (version 3 .0, 1988) and Statgraphics (version 5 .0, 1991) . Bioconcentration factors (Bodou & Ribeyre, 1989 ; Heath, 1990) were calculated for bivalve molluscs in relation to ambient water (average concentrations of metals in different water sampling sites measured by Department of the Environment, unpublished data) .
Results and discussion In general, metal concentrations in the sediments from different sampling sites of the study area were in the following order: Mn > Zn > Cu > Pb. Based on Table 1, it can be concluded that : (a) there were highly significant differences between the sampling sites in contents of the four metals, (b) highly significant differences in accumulation of different heavy metals, (c) no significant differences could be detected in accumulation between seasons, (d) a significant difference between sampling sites .
Figure 2 shows dendrograms derived by average linkage clustering of the six sampling sites . Arbitrary dashed lines have been used . Comparison between sampling sites with respect to all four heavy metals shows at a distance about 8, three distinct clusters : I (sampling sites 2, 3, 4 and 6) and II (sampling site 5) and II (sampling site 1) . At a higher distance (about 14), clusters I and II fuse, forming a single cluster . The major differences are in clustering of sampling site 1 and the remaining ones . Mean concentrations of metals at different sampling sites are shown separately in Figure 3 . Comparison between the results illustrated in this Figure and the grain-size distribution of the sediments (see Figure 4), particularly percentage of fine fraction (grainsize < 63 um) reflected no clear relationship . However, in all cases, except for Mn, the lowest average levels of metals were measured in sediments from site 2 which contained the lowest percentage of fine fraction . This was unexpected, because trace metals (especially those originated from anthropogenic sources) are primarily associated with the fine fractions of surficial sediments, due to an increase in surface area and to the surface properties of clay minerals (Kinne, 1984 ; Sericano, 1982) . It is interesting to note that the pattern of Mn accumulation was mostly parallel to the trends in organic matter variation in the same sites (with the exception of site 2 ; Figure 5) . Mn concentration in the wetland macrophytes is relatively high (Pourang, unpublished results) . Hence, the similarity in the pattern of Mn and organic matter variation is probably due to high plant
57 tuolidian diatanoe 0 #ampting site# 2 3
5
10
15
20
25
J
Mn 5 1 ----------------------------------------------------------------------3 6 4 1 2 ------------------------------------------ ----------------------------
CU
5 6
Zn
4 1 3 ----------------------------------------------------------------------
3 6 5 Pb 2 4 1 ---------------------------------------------------------------------I _ 2 3 4 J 6 p 5 E 1 ------------------------------ ----------- ----------------------------
Figure 2 . Dendrograms for hierarchical cluster analysis of the six sampling sites based on heavy metal concentrations in sediments . The
vertical dashed lines show the arbitrary division lines for defining clusters . The last dendrogram compares the sampling sites with respect to concentrations of the four metals.
contents in organic fraction of the wetland sediments (originated from decomposition of aquatic plants) . Metal concentration in surficial sediments of freshwater bodies in various regions of the world (Table 2), it suggests that the levels found in this study present no danger to wetland ecological equilibrium . Sufficient chironomid larvae for heavy metal determination in three size groups were only obtained from sampling site 5 in winter. The Kruskal-Wallis test presented in Table 3 suggests that in the case of zinc and lead, the null hypothesis (heavy metal contents the same in the three size categories) can be rejected (a = 0.05) . However, there are no significant differences between the small and medium size categories (Tukey test) .
As shown in Table 4, only Lead and Zinc body burdens in chironomid larvae were significantly correlated with levels in sediments . Other work (e .g. Young & Harvey, 1991) has revealed that under conditions of high metal loading, metal contents of benthic invertebrates may be related to metal concentrations in sediments . More often, however, concentrations in sediments are poor predictors of metal concentrations in the associated fauna. An inverse relationship occurs between size and metal levels in the larvae (Table 4), probably due to reduction of body surface ratio in larger individuals . It appears that only 10 percent of metal accumulation in the larvae derived from uptake via contaminated food, and surface adsorption plays a dominant role in metal flux into organisms (Soechtig, 1990) . This may account for the observed inverse rela-
58
Figure 3. Mean heavy metal concentrations in sediments from the six sampling sites . Mn values are divided by 10 . r
r i
V4 ,
Zac~a o
Hilili!IhIIiIii
0 >2000 pro 1000-2000puM 500-1000pm l1 p~ 125-250pm ® 83-125).m ME 10-15
21 .5 35 .8
75 .0 1686 .5 58 .9 716.9
mm Metal (n = 5)
Lake Plastic, Canada (4)
12 .7
91 .0 1427 .8
Cu
67 .70
40 .18
Lake Crosson, Canada (4) Lake Blue Chalk, Canada (4)
12 .9 8 .3
53 .5 156 .8 130 .3 1091 .7 495 .4
Zn Mn
107 .02 3 .8
101 .51 3 .5
Pb
18 .81
16 .33
Lake George, Canada (4) Lake Lumsden, Canada (4)
Freshwater section of the Elbe estuary, Germany (5) Cauvery river, India (6) Bayou d'Inde, USA (7) Oker river, Germany (8) Ecker river, Germany (8) World average (9, 10) Anzali wetland, Iran (11)
-
-
167 .3 226 .4 20 .2 11 .9
25 .5 58 .5
95 .0 54 .1
4475 .7 927 .9 6323 .1 86 .5 29 .7 693 .9 20 24 .2
45 38 .3
95 87 .5
> 15
mm mm (n = 7) (n = 8)
H
P-value
41 .91 4 .88 > 0.102 29 .94 7 .98 0 .010 2 .5 1 .82 > 0.102 6 .71 9 .14 < 0 .009
540 .7 222.6
Table 4. Spearman correlation coefficients (rs) between
-
heavy metals concentrations in sediments (s) and chironomid larvae (c) . Asterisks indicate significance of r 5 at P < 0 .01 .
850 840 .2
References : (1) Saeki & Okazaki, 1992 ; (2) Dave, 1991 ; (3) Rowan & Kalff; (4) Young & Harvey, 1991 ; (5) Heckman, 1990 ; (6) Vaithiyanathan et al . ; (7) Ramelow et al., 1991 ; (8) Soechtig, 1990 ; (9) Chapman, 1992 ; (10) Gibbs, 1993 ; (11) present study .
than the increase in pumping rate . Accumulation of heavy metals by aquatic molluscs is influenced by sex, reproductive condition, temperature, salinity and concentrations of other metals (Kinne, 1984) .
Mn (c)
Cu (c)
Pb (c)
Cu (s) Pb (s)
-0 .6123 0 .8318 -0.7333
0 .8595 -0 .5492
0 .8734 -0 .5702
Zn (s)
-0.4730
Mn (s)
0 .7473 0 .2333
0.9294* 0 .7832
Zn (c) 0 .4352 -0 .4945 0 .5243 0 .8846*
Gerhardt (1990) has reported that the highest heavy metal levels in aquatic invertebrates are generally observed in filter feeders . The results presented in Table 6 indicate that the highest concentrations of Zn and Pb were measured in M. lineatus and the order
60 Table S . Means metal concentrations (µg g - 1 dry weight ±SD) in two size groups of M. lineatus and results of two tailed student's t-test examing the null hypothesis that there were no significant differences between heavy metal contents in the two size groups . n refers to the number of samples analyzed (4-6 individuals per samples) .
Metal Mn Zn Cu Pb
< 15 mm (n = 8)
> l5 mm (n = 6)
t
P-value
0 .47± 0.22 0 .77± 0.30 1 .94 0 .05 < P < 0 .10 228 .12±79 .98 297 .80±58 .02 0.26 0.80 < P < 0 .90 31 .89±12.29 39.26± 7 .68 12.04 < 0.01 28.26± 5 .88 20.41± 7 .6 12.38 < 0.01
Table 6. Mean heavy metal concentrations (±SD) in the investigated macroinvertebrates from Anzali wetland, n refers to the total number of analyzed individuals . All data in sg g - I dry weight Taxon
Mn
Cu
Chironomidae
3 .3 (1 .4)
49 .9 (18 .8)
Tubifex tubifex
Mytilaster lineatus
Corbicula fluminalis
Zn
Pb
n
79 .5
13 .9
122
(36 .4)
(7 .6)
8 .4
77 .4 (34.5)
154 .3 (63 .4)
19 .2 (8 .3)
46
(4 .6)
6 .2 (2 .3)
35 .6 (9 .9)
262 .9 (69 .0)
24 .3 (5 .7)
93
4 .3
24.8
53 .1
5 .8
(2 .1)
(8 .9)
(24 .4)
(1 .9)
8
of these metals for other investigated benthos were : tubificid worms > chironomid larvae > C. fluminalis . Mn and Cu concentrations showed a different pattern in decreasing order, as follows : tubificid worms > chironomid larvae > M. lineatus > C. fluminalis . With regards to above mentioned results, it can be concluded that in the case of Mn and Cu, detritivorous benthos showed higher metal concentrations than filter feeders whereas no definite trend was found for Zn and Pb .
Acknowledgements I am grateful to B . Riazi and Dr F. A . Moghadam for their useful guidance and to G . Minasian for his invaluable assistance in the laboratory . I also thank Dr B . Kiabi and D . Rostami for helpful comments on statistical analyses .
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