Hydrobiologia 381: 53-61, 1996. © 1996 Kluwer Academic Publishers. Printed in Belgiun. 53 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 fluminalis) 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 sta- bility in sediments and scarcity in natural environments (Saeki et al., 1993), Heavy metals introduced to aquatic environments by industrial, domestic and mining activities are ulti- mately absorbed by deposits and incorporated into sed- iments. Hence sediments are the most concentrated physical pool of metals in aquatic systems. Sediments may contribute significantly to con- centrations of metals in benthic invertebrates, either by absorption/adsorption from interstitial water or by direct ingestion (Clements, 1991; Gethardt, 1990).. (On the basis of controlled laboratory feeding exper- iments, one might anticipate the existence of a consis- tent relationship between metal accumulation in ben- thic 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 associ- ation with sediments, and the ability of certain species totolerate and accumulate metals, these organisms rep- resent 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 deter- mine heavy metal concentrations in surficial sediments from different sites of the Anzali wetland and compar- ison 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 mactobenthic 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 impor- tant 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 km?, There is a little indus- trial 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,‘igure 1. Map of Anzali wetland showing location of the six sampling sites.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?, 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 organ- ic matter determination). The other five were sieved in the field through a 200 jum nylon mesh. Macroben- thic 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 mol- luscs, a bottom dredge (with net bag of 5 mm mesh size) was used and specimens were stored as men- tioned 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 Merek, 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 tothe nearest | mg) were taken to a 100 ml kjeldahl flask, to which 10 ml diluted aqua regia (3:3:1 H,O:HCI:HNOs) ‘was added, covered with a watch glass and allowed to stand overnight at room temperature (c. 20°C). The fol- lowing day samples were digested to near dryness at ‘90°C on a hot plate. Cooled digested samples were fil- tered through Whatman No. | filter papers and collect- ed 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- 55 ume with DDDW. The solutions were analysed for metals in a Pye Unicam flame atomic absorption spec- trophotometer, 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. lin- eatus samples were also measured and separated into two size groups: < 15 mm and > 15 mm. Macroin- vertebrate 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 HNO:/HC10, (5:1) was applied and diluted to a maxi- ‘mum of S ml volume with 6% HNOs (Young & Harvey, 1991), Pooled samples were analysed (at least in trip- licate) on a Perkin Elmer model 503 atomic absorption spectrophotometer and a HGA72 graphite atomizer. Amounts of organic matter in sediments were deter- mined by loss of weight on ignition at 600°C (Holme & Mcintyre, 1984), To determine grain-size, sediments were split into fa sand (particles > 62 jzm) and a silty-clay fraction (particles < 62 um). The sand fraction was divided through a series of graded sieves (pore sizes 125, 250, 500, 1000 and 2000 um) (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 mea- sures) 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, 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 asthe interaction of these factors on concentrations of heavy metals in sediments ‘Source of variation a ss MS ratio P-value Main effets: ‘Sampling sites 5 88905 177810378 <001 Metals 3 1194262 3764753.9 802.14 <001 Seasons 29763 92.02.11 005 Interactions: Sampling sites » Metals 15 S0909409 3393.61 7.23 <0.01 Sampling sites « Seasons 15 7396.90 4931.33.08. >0.05 Metals « Seasons 9 7036353 781.7. «1.65 > 0.05 Residual 45 21120318 4693.40 Total 95 122775607 ‘A wwortailed 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-Smimov goodness of fit proce- dure it could be reasonably assumed that heavy metals contents of sediments and chironomid larvae (sampled from the same sites) were normally distributed, Pear- son’s product moment correlation coefficient were used toexamine the relationship between the two latter vari- ables (Rees, 1991). All statistical analyses were performed using SPSS- X (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) high- ly significant differences in accumulation of different heavy metals, (c) no significant differences could be detected in accumulation between seasons, (d) a sig- nificant 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 I (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 sam- pling sites are shown separately in Figure 3. Compar- ison between the results illustrated in this Figure and the grain-size distribution of the sediments (see Fig- ure 4), particularly percentage of fine fraction (grain- size < 63 pm) reflected no clear relationship. How- ever, 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 pri- marily 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), tis interesting to note that the pattern of Mn accu- mulation 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,7 uctdion diatance 10 8 20 2 sempling $$$} $$$} tee Li y 5 2 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 lat dendrogram compares the sampling sites with respect concentrations ofthe four metals contents in organic fraction of the wetland sediments (originated from decomposition of aquatic plants). Metal concentration in surficial sediments of fresh- water 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 deter- mination in three size groups were only obtained from sampling site 5 in winter. The Kruskal-Wallis test pre- sented 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 differ- ences 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 corre- lated with levels in sediments. Other work (e.g. Young & Harvey, 1991) has revealed that under conditions of high metal loading, metal contents of benthic inverte- brates may be related to metal concentrations in sed: iments. More often, however, concentrations in sedi- ‘ments 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 vvia contaminated food, and surface adsorption plays a dominant role in metal flux into organisms (Soechtig, 1990). This may account for the observed inverse rel58 100% 75% 50x. asx oO Figure 4. Grain size distribution of sediments from the sampling sites tionship. The importance of body surface as a passway of metals uptake by benthic invertebrates is also seen in some other investigations. For instance similar con- centrations of certain metals in live and dead Chirono- ‘mus sp., exposed to contaminated sediments have been reported (Clements, 1991). Sufficient bivalve molluscs (two species, M. lin- eatus and C. fluminalis) for heavy metal measure- ‘ment were gathered from sampling site 5 (in sum- ‘mer and autumn, respectively). Of 214 aquatic mollusc species occurring in central Asia, 56 have been used as bioindicators of environmental conditions (Izzatul- layev, 1993). C. fluminalis is among these. Biocon- centration factor values revealed that this species accu- mulates Cu, Zn, Mn and Pb at levels about 6888.9, 2950.0, 10.1 and 397.1 times greater than in ambient water, respectively. As previously mentioned, speci- mens of M. lineatus were separated to two size groups. Table 5 shows that in the case of Cu and Pb the null hypothesis can be rejected (P < 0.01). The mean concentration of Lead was higher in smaller molluscs while a reverse case was detected for Copper. The rel- atively higher level of Cu in the larger specimens may reflect their pumping rate, because the pumping rate of some filter-feeder species (e.g. Mytilus edulis) is posi- tively correlated with shell length (Bunt et al., 1993). But in the case of Lead, a reduction of body surface ratio in the larger specimens may be more effectivePRRCENTAGE 59 a8 30 2 1s ‘SPRING ‘Figure 5. Seasonal changes in the organic mater content of sediments sampled from the sampling sites. Table 2. Heavy metal concentrations (ug ¢~' dry weight) inthe surficial sediments (bulk) from various geographical regions and world average shale Geographical area Pb Cu Zn Mn Lake Teganuma, Japan(I) 309 654 2339 815.1 Lake Hjalmaren, Sweden (2) 4240190, ~ ‘Mean 52 Quebec and 56.1 200 1252 10100 Ontario lakes, Canada (3) Lake George, Canada(4) 21,5 75.0 1686.5 Lake Lumsden, Canada (4) 358 589 7169 Lake Plastic, Canada (4) = = 12.7 91.0 427.8 Lake Crosson, Canada (4) 129 535 1568 Lake Blue Chalk, Canada (4) - 83 1303 1091.7 Freshwater section of the Elbe estuary, Germany (5) Cauvery river, India (6) Bayou d'Inde, USA (7) 1673 2264 495.4 — 202 255 95.0 S407 119 585 St 2226 ‘Oker river, Germany (8) 4475.7 927.9 6323.1 = Ecker river, Germany (8) 86.5. 29.7 6939 ~ World average (9, 10) 2% 45 95 880 Anzali wetland Iran (11) 242-383 87.5 840.2 References: (I) Saeki & Okazaki, 1992; @) Dave, 1991; @) Rowan & Kalff; (4) Young & Harvey, 1991; (5) Heckman, 1990; (6) Vaithiyanathan etal; (7) Ramelow etal, 1991; (8) Soechtig, 1990, (9) Chapman, 1992; (10) Gibbs, 1993; (1) present study. than the increase in pumping rate. Accumulation of heavy metals by aquatic molluscs is influenced by sex, reproductive condition, temperature, salinity and con- centrations of other metals (Kinne, 1984), Table 3. Mean of heavy metal contents (ugg! dry weight) in the three size groups of chironomid larvae and results of Kruskal-Wallis and Tukey test assessing differences of heavy metals accumulations among size groups. Groups linked by lines are not significantly different from one another; n.refers to number of pooled samples analyzed (5-8 individuals per sample) 0-10 > 1018 > 15 Mel (n=5) (n=7) (n=8) H_ Povalue Cu 67.70 4018 41.91 488 > 0.102 Zn 10702 10151 2994798 0.010 Mn ~ 3835 25 182 > 0.102 Ph 18811633671 9.14 < 0.009 Table 4. Spearman correlation coefficients (r,) between ‘heavy metals concentrations in sediments (s) and chironomid larvae (¢) Asterisks indicate significance of , at P < 0.01 Ma(@ Cute) Poi) Za(@) Mn(s) 06123 0859508734 0.4352 Cus) 08818-05492 0.5702 -0.4945 Pbis) -07333 0.7473 0.9294" 05243 Zn(s) 0473002333 0.7832. 846" 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 5. Means metal concentrations (ug g~ dry weight SD) in two size groups of M.linearus and results of two tailed student’s ‘Hest examing the null hypothesis that there were no significant diferences between heavy metal contents in the two size groups ‘refers to the number of samples analyzed (4-6 individuals per samples) 15mm Meal (n=8) (n=6) t Mn O47 0.22 0.77 030 1.94 005 < P< 0.10 Zn 228.12479.98 297.80:458.02 0.26 0.80< P< 0.90 Cu 318941229 39.264 7.68 1204 < 0.01 Ph —«2826L SAB NAIL 76 1238 — < 0.01 P-value Table 6, Mean beavy metal concentrations (++SD) inthe investigated ‘macroinvertebrates from Anza wetland, n refers tothe total number cof analyzed individuals. All data in ug g~! dry weight ‘Taxon Mn Gu Zn Po Chironomidae 33° 499795139122 (14) (188) G64) 7.6) Tubifex rubifex 84-74 154319246 (46) G45) 634) (3) Mytilasterlineams 62-386 2629-43-93 23) 9) 69 67 Corbicula flaminalis 43° 248 33 S88 en 8%) @44) G9 of these metals for other investigated benthos w tubificid worms > chironomid larvae > C. fluminalis. Mn and Cu concentrations showed a different pattern in decreasing order, as follows: tubificid worms > chi- ronomid larvae > M. lineatus > C. fluminalis. With regards to above mentioned results, it can be concluded that in the case of Mn and Cu, detritivorous bbenthos 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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