Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf

Marine Environmental Research xxx (2014) 1e10

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Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf A. Nasrolahi a, *, B.D. Smith b, M. Ehsanpour c, M. Afkhami c, P.S. Rainbow b a

Department of Marine Biology, Faculty of Biological Sciences, Shahid Beheshti University, G. C., Evin, 198 396 9411 Tehran, Iran Department of Life Sciences, Natural History Museum, Cromwell Road, London SW7 5BD, UK c Young Researchers and Elite Club, Islamic Azad University, Bandar Abbas Branch, P.O. Box 79159-1311, Bandar Abbas, Iran b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 May 2014 Received in revised form 8 July 2014 Accepted 11 July 2014 Available online xxx

The fouling barnacle Amphibalanus amphitrite is a cosmopolitan biomonitor of trace metal bioavailabilities, with an international comparative data set of body metal concentrations. Bioavailabilities of As, Cd, Cr, Cu, Fe, Mn, Pb, V and Zn to A. amphitrite were investigated at 19 sites along the Iranian coast of the understudied Persian Gulf. Commercial and fishing ports showed extremely high Cu bioavailabilities, associated with high Zn bioavailabilities, possibly from antifouling paints and procedures. V availability was raised at one port, perhaps associated with fuel leakage. Cd bioavailabilities were raised at sites near the Strait of Hormuz, perhaps affected by adjacent upwelling off Oman. The As data allow a reinterpretation of the typical range of accumulated As concentrations in A. amphitrite. The Persian Gulf data add a new region to the A. amphitrite database, confirming its importance in assessing the ecotoxicologically significant trace metal contamination of coastal waters across the world. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Trace metals Biomonitoring Barnacles Amphibalanus amphitrite Persian Gulf

1. Introduction The use of biomonitors provides direct evidence on differences in the local bioavailabilities of contaminants between sites, or at the same site over time (Phillips and Rainbow, 1994; Luoma and Rainbow, 2008). A biomonitor is defined here as an organism which accumulates chemical contaminants in its tissues, the accumulated concentration of which provides a relative measure of the total amount of contaminant taken up by all routes by that organism, integrated over a preceding time (Luoma and Rainbow, 2008). This accumulated concentration is, therefore, an integrated measure of the total amount of contaminant in bioavailable form to which that organism has been exposed over a period that can be defined for that organism, for example by biodynamic modelling (Luoma and Rainbow, 2008). Only bioavailable forms of a contaminant are taken up by an organism, and therefore have any potential to be of ecotoxicological significance. Thus, when studying contaminant pollution, biomonitoring is to be preferred to other physical measures of contaminant concentrations in the local environment, such as water or sediment concentrations, in that it provides measures of amounts of bioavailable contaminants

* Corresponding author. Tel.: þ98 21 29902704; fax: þ98 21 22431664. E-mail addresses: [email protected], [email protected] (A. Nasrolahi).

present, as opposed to total concentrations which potentially include non-bioavailable forms of the contaminant (Phillips and Rainbow, 1994; Luoma and Rainbow, 2008). In assessing the contamination by toxic metals in coastal waters, several biomonitors have been employed worldwide (Rainbow and Phillips, 1993; Rainbow, 2006). Prominent amongst these have been barnacles (Phillips and Rainbow, 1988; Rainbow and Blackmore, 2001; Rainbow et al., 2002, 2004a; Reis et al., 2011). Barnacles take up and subsequently strongly accumulate trace metals from solution and from the diet, consisting of plankton and suspended detritus filtered from the water column by the thoracic legs, apparently for the most part from the diet (Wang et al., 1999a,b; Rainbow and Wang, 2001). Thus accumulated metal concentrations in the bodies of barnacles reflect metal bioavailabilities in these two sources, but particularly in the detritus and plankton filtered by the barnacles. Because accumulated metal concentration data strictly apply only to the integrated metal sources available to the biomonitor analysed, a more thorough biomonitoring programme should ideally employ a suite of biomonitors to cover a range of potential metal sources in a habitat (Rainbow, 2006; Luoma and Rainbow, 2008). Although it is indeed preferable to use a large suite of biomonitors in a biomonitoring programme, it is usual for metal contamination in a habitat to be manifested in more than one component of the aquatic system. However, there are often local circumstances where a suite of

http://dx.doi.org/10.1016/j.marenvres.2014.07.008 0141-1136/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Nasrolahi, A., et al., Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.008

2

A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

potential biomonitors is not available. Under such circumstances, the analysis of even just one selected biomonitor will still show up that the habitat is contaminated with metal in a form that is bioavailable to at least some resident organisms. A biomonitor is particularly useful if it is cosmopolitan, thereby allowing comparisons of local metal bioavailabilities over a wide geographical range, encompassing a wide range of degrees of trace metal pollution (Rainbow and Phillips, 1993; Luoma and Rainbow, 2008). Such a cosmopolitan trace metal biomonitor is the ubiquitous fouling barnacle Amphibalanus amphitrite (previously known as Balanus amphitrite) (Luoma and Rainbow, 2008; Reis et al., 2011), used as a biomonitor across the world, for example in Australia (da Silva et al., 2005, 2009), Hong Kong (Phillips and Rainbow, 1988; Rainbow and Blackmore, 2001), India (Anil and Wagh, 1988), Croatia (Barbaro et al., 1978), Spain (Morillo et al., 2005, 2008), Brazil (Silva et al., 2006), and California, USA (Fialkowski and Newman, 1998). Correspondingly, a large database of accumulated metal concentrations is available for A. amphitrite, allowing the recognition of what is a typical accumulated metal concentration, or a high accumulated metal concentration that indicates an atypically high local bioavailability of the metal concerned (Luoma and Rainbow, 2008: Reis et al., 2011). This study extends the use of A. amphitrite as a trace metal biomonitor to the Persian Gulf. The Persian Gulf has been relatively understudied in terms of coastal pollution, in spite of being potentially one of the most polluted marine ecosystems in the world, with almost two-thirds of the world's proven oil reserves located there. Furthermore, the Persian Gulf has undergone considerable development, urbanization and industrialization, and local port areas are major sources of pollution (Bastami et al., 2013). The 19 sites chosen stretch the length of the Iranian coast of the Persian Gulf, and represent locations with different potential anthropogenic inputs of trace metals into the local coastal waters. Such sources include urban effluent discharge, as well as effluent from commercial and industrial concerns. Spillages from the oil industry into the sea can be characterised by high vanadium levels found in oil (Pearson and Green, 1993). Vessels, from large passenger and merchant ships to small fishing boats and pleasure craft, may release trace metals such as copper from antifouling paint (Woods Hole Oceanographic Institution, 1952). Sediments such as organically rich muds are typically associated with high concentrations of iron and manganese.

This Persian Gulf data set will add a new region and more data to the database of accumulated trace metal concentrations in the bodies of A. amphitrite. The data set will allow even better interpretation of the significance of trace metal contamination in coastal waters across the world, particularly in the IndoPacific e a region with increasing environmental problems in coastal waters as industrial development moves forward apace. 2. Materials and methods 2.1. Barnacle identification A. amphitrite is not the only barnacle occurring at the sites of interest, although it is much the predominant balanoid species occurring intertidally at the sites (Shahdadi et al., 2014). All barnacle identifications were confirmed by A Shahdadi, a taxonomic expert on Persian Gulf barnacles. 2.2. Sample sites Barnacles, A. amphitrite, were collected between 2 and 15 June 2013 from piers, rocks of artificial breakwaters, rocky shores or mangrove roots in the eulittoral zone of the 19 sites listed in Table 1 (Fig. 1). The sites chosen cover a wide range of potential anthropogenic input of trace metals into the local coastal waters. They include sites in commercial ports (sites 8, 9, 12, 19), a passenger vessel terminal (site 17), a port with pleasure craft (site 7), fishing ports (sites 2, 18, 19) and jetties for fishing boats (sites 5, 11, 15, 16), areas of urban discharge (sites 1, 6), and sites with no apparent anthropogenic effluent discharge close nearby (sites 4, 14). 2.3. Sample analysis Barnacles were removed from the substratum with a new stainless steel scraper, and placed in polythene bags to be transported to the laboratory in a cool box. 10 bodies were subsequently dissected out with new stainless steel instruments, each rinsed in distilled water and pooled in each of 10 replicate samples from each site. The body of a barnacle consists of the thorax with six pairs of filtering thoracic limbs, the residual abdomen and part of the head (the oral cone with mouthparts). Bodies are easily recognised and removed from the mantle cavity, leaving behind the remainder of

Table 1 The 19 sampling sites along the Iranian coast of the Persian Gulf, and dates of collection of barnacles. Site

Name

Latitude

Longitude

Date

Comment

1 2 3 4

Khour-e-Gorsuzan Posht Shahr Port Tavanir Bostanu

27 100 46.7700 N 27 100 13.5300 N 27 90 1.0200 N 27 40 26.6500 N

56 170 36.0300 E 56 150 46.0500 E 56 70 35.4000 E 55 590 27.1500 E

02.06.2013 02.06.2013 02.06.2013 02.06.2013

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Bandar-e-Khamir Bandar-e-Lengeh Nayband (Khour-e-Haleh) Nakhl Taghi Bandar-e-Kangan Bandar-e-Dayer Shif Island Bandar-e-Ganaveh Bandar-e-Emam Hassan Bandar-e-Deylam Hendijan Bandar-e-Emam Khomaini Qeshm (Zakeri port) Qeshm (Ramchah port) Hormuz Island

26 560 45.6500 N 26 320 31.5000 N 27 240 16.8000 N 27 290 26.1400 N 27 490 53.6900 N 27 490 50.5800 N 29 40 25.2400 N 29 330 36.5900 N 29 490 51.6100 N 30 30 26.8000 N 30 60 25.3800 N 30 280 30.1200 N 26 570 57.5600 N 26 530 56.4300 N 27 50 37.9700 N

55 350 54.4100 E 54 520 28.0200 E 52 380 42.9500 E 52 350 5.7700 E 52 30 24.5600 E 51 550 50.1300 E 50 520 19.8100 E 50 300 43.1000 E 50 150 32.0000 E 50 80 37.9000 E 49 460 24.3600 E 49 40 8.6200 E 56 160 22.1800 E 56 90 50.4500 E 56 260 43.5300 E

03.06.2013 04.06.2013 04.06.2013 05.06.2013 05.06.2013 05.06.2013 06.06.2013 06.06.2013 06.06.2013 07.06.2013 07.06.2013 07.06.2013 14.06.2013 14.06.2013 15.06.2013

Urban wastewater discharge Fishing port Electric power plant nearby Mangrove 4 km from commercial port and industrial complex Fisheries jetty near mangrove Open area with some urban discharge Port with pleasure craft Commercial port with fishing boats Commercial port with large ships Largest fishing port in Persian Gulf Fisheries jetty with iron uprights Commercial port Open rock shore in oil industry area Rocks outside commercial port Fisheries jetty with mud sedimentation Fisheries jetty with iron columns Large passenger vessel terminal Fishing port Commercial port with fishing

Please cite this article in press as: Nasrolahi, A., et al., Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.008

A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

the head which forms the mantle, base, shell plates and opercular plates. Any egg masses in the mantle cavity were ignored. Given their ease of identification, removal and therefore reproducibility, it is the bodies of barnacles that are usually analysed in biomonitoring studies (e.g. Phillips and Rainbow, 1988; Rainbow and Blackmore, 2001; Rainbow et al., 2004a). Barnacles were not kept in the laboratory in any effort to depurate the gut contents in order to avoid laboratory contamination. All the barnacle bodies, therefore, contain gut contents but these are considered to represent only a small proportion of total body metal content, given the characteristics of barnacles as particularly strong trace metal accumulators (Rainbow, 1987, 2002, 2007; Rainbow and Blackmore, 2001). The pooled bodies were dried in acid-washed pre-weighed plastic vials to constant weight at 60  C. The pooled samples were then acid-digested in acid-washed test tubes at 100  C (glassstoppered reflux) in Aristar grade (Merck) concentrated nitric acid. Each digest was made up to 10 ml with double-distilled water and analysed for nine trace metals, As, Cd, Cr, Cu, Fe, Mn, Pb, V and Zn, on a Vista-Pro CCD Simultaneous ICP-OES. Simultaneous comparative analyses of trace metals were made in blanks and the Standard Reference Material Lobster Hepatopancreas TORT-2 (Table 2). Agreement with the TORT-2 certified concentrations is considered satisfactory. All metal concentrations are quoted in terms of mg g1 dry weight.

2.4. Statistical analyses When comparing bioaccumulated concentrations of metals in invertebrates from different locations, it is necessary to consider any potential effect of size on body metal concentrations. Size will inevitably be correlated with age, but in the absence of an independent measure of age (but see Marsden et al., 2013), it is size that is used to allow for age differences. The power function y ¼ axb, where y is the metal concentration (mg g1), x is body dry weight (g), and a and b are constants, is an accepted model for barnacles

3

Table 2 Comparisons of mean measured trace metal concentrations (mg g1 dry weight) against certified concentrations in Standard Reference Material Lobster Hepatopancreas TORT-2 (National Research Council Canada) (±95% Confidence limits, n ¼ 3). Measured concentration As Cd Cr Cu Fe Mn Pb V Zn

22.1 23.4 1.20 95.9 104 12.0 0.99 1.56 165

± ± ± ± ± ± ± ± ±

0.5 0.14 0.37 2.4 16 0.7 0.30 0.40 11

Certified concentration 21.6 26.7 0.77 106 105 13.6 0.35 1.64 180

± ± ± ± ± ± ± ± ±

1.8 0.6 0.15 10 13 1.2 0.13 0.19 6

(Phillips and Rainbow, 1988; Rainbow and Blackmore, 2001; Rainbow et al., 2004a). In this case, x is the mean body weight of the ten pooled bodies in each sample. All data were transformed logarithmically (to the base ten) to render the variance homogeneous and to normalise the data, creating an additive data set with skews removed. Data were first analysed for significant regression coefficients (slopes) in the regressions of log body weight (x) against log metal concentration (y) in the data set for each metal at each site, and in the whole data set for each metal. For each of the 9 metals analysed, there was in fact a significant effect of size on accumulated body metal concentration in the whole data set and/or in at least one of the data sets for an individual site. It is, therefore, not meaningful to quote means or use Analysis of Variance to make comparisons between barnacle metal concentrations at different sites. Analysis of Covariance (ANCOVA) was therefore carried out in order to allow for the size effect. The data for each metal were next tested to see if the data set for any site showed a regression coefficient significantly different from those of the other sites. When this occurred, that data set was removed from any further statistical comparison. Analysis of the data for those sites for which the slopes of best-fit

Fig. 1. Persian Gulf coastal waters showing the 19 sites from which barnacles (Amphibalanus amphitrite) were collected. See Table 1 for details.

Please cite this article in press as: Nasrolahi, A., et al., Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.008

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 1730 13,900 5460 5180 1320 10,700 743 14,000 5790 19,500 19,500 6220 11,100 8580 3590 6760 3220 3760 3830 1220 7450 2770 975 785 5600 473 9980 3910 9470 6270 1500 5070 2630 780 3550 1260 2690 1730 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 7.32 6.62 6.95 7.55 4.01 3.25 3.70 2.81 6.92 30.7 6.54 5.64 12.3 4.49 12.5 3.23 8.20 4.74 10.2 0.96 3.77 2.81 1.27 1.09 1.52 1.95 1.23 3.36 13.5 2.99 1.35 3.57 1.94 3.53 0.89 4.68 1.92 3.85 2 5 3 3 8 10 2 10 6 1 10 6 8 3 4 4 2 8 10 18.2 7.43 2.65 6.60 2.79 13.7 3.11 7.19 8.46 3.88 6.72 2.59 8.77 10.1 8.08 8.33 3.72 5.14 3.35 6.59 2.61 2.11 1.90 0.83 3.55 2.33 2.22 3.16 3.88 2.75 2.42 2.33 3.65 4.00 4.96 3.03 2.15 1.76 10 10 10 10 10 10 10 10 10 10 10 9 10 10 10 9 10 10 10 42.4 30.6 36.4 94.2 28.1 27.4 18.9 13.2 36.9 26.3 113 32.6 110 37.5 66.1 29.0 43.5 34.2 56.9 15.9 16.3 13.5 19.1 9.67 11.4 11.0 7.69 22.3 10.9 47.1 7.95 43.7 12.2 23.3 5.48 22.9 14.5 34.6 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 9 10 10 10 1940 1420 1180 3160 1790 1310 1070 1270 2330 2590 2390 1790 2640 1390 3440 1570 2830 2260 2330 832 475 618 489 512 614 773 526 1520 644 993 448 1230 788 1390 475 1370 914 903 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 26.8 874 276 276 188 326 108 2150 321 2090 737 968 942 549 237 47.4 343 99.0 670 14.9 445 122 73.3 81.2 152 81.0 1190 199 395 263 238 366 187 95.2 16.3 122 59.5 255 3 7 10 8 10 4 10 8 10 5 10 10 10 5 9 2 10 9 10 6.54 5.45 21.9 10.4 6.84 4.44 5.04 3.75 11.4 5.65 20.0 8.27 15.9 6.74 12.2 8.27 8.20 5.84 7.62 5.10 1.45 2.11 1.73 1.53 2.54 2.81 1.31 4.20 2.43 7.74 1.68 5.26 4.00 4.29 1.65 3.65 2.30 3.33 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4.55 45.6 35.3 34.0 41.6 102 8.64 11.8 40.9 17.3 13.7 27.8 57.9 44.2 19.5 13.3 76.2 221 72.6 2.63 17.7 14.2 7.94 21.3 47.2 6.25 6.67 26.0 8.77 3.21 11.8 22.0 24.8 7.32 8.27 24.8 122 19.0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 108 160 155 184 128 327 235 198 290 260 183 368 292 239 183 76.2 178 164 179 0.00087 0.00206 0.00299 0.00257 0.00588 0.00145 0.00243 0.00359 0.00139 0.00130 0.00185 0.00243 0.00235 0.00100 0.00129 0.00120 0.00174 0.00235 0.00318

Max

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

20.7 95.9 60.0 36.8 92.5 182 168 128 212 121 73.5 217 125 115 91.9 18.0 86.0 93.3 103

Max Min

Zn

n Max Min

V

n Max Min

Pb

n Max Min

Mn

n Max Min

Fe

n Max Min

Cu

n Max Min

Cr

n Min

Max Cd

Min

n As

Table 3 summarises the metal concentration data for trace metals accumulated in the bodies of A. amphitrite from 19 sites along the Iranian coast of the Persian Gulf in June 2013. The accumulated concentrations of Cr and Pb were often below detection limits, and the concentrations reported summarise only the measured concentrations (Table 3). While Table 3 shows clear accumulated metal concentration differences between barnacles from different sites, these concentrations cannot be compared directly between sites without allowing for any effect of differences in size (body weight in this case) between samples from different sites. For every metal there was indeed a significant effect of size on accumulated body metal concentration in the whole data set and/or in at least one of the data sets for an individual site. For each metal this size effect was, therefore, allowed for by ANCOVA of best-fit double log regressions of individual body dry weight against accumulated metal concentrations for each site (Table 4). For every metal there was a significant a priori difference in metal concentrations across the sites compared, a prerequisite of this concentration comparison being a lack of significant difference across the regression coefficients of the regressions under comparison. Concentration data for all 19 sites could be compared for Fe, Mn and V, but slightly reduced numbers of sites were compared for As (17 sites), Cd (18) and Cu (17) as a result of elimination of data sets with significantly different regression coefficients (Table 4). The data set for Zn could be divided into two groups of 14 and 5 sites respectively for comparison, each group containing data sets with no significant differences between regression coefficients, although the regression coefficients in each group differed significantly from those of sites in the other group (Table 4). In the cases of Cr and Pb, individual site data sets with 3 or fewer replicates were excluded from ANCOVA analyses, leaving 17 and 12 sites compared for Cr and Pb respectively (Table 4). Table 4 presents a summary of the ANCOVA comparisons made for each metal, comparative accumulated metal concentrations being quoted as the estimated accumulated concentrations

Mean individual dry wt

3. Results

Site

regressions did not differ significantly, was continued to identify any differences in elevation of metal concentration/body dry weight regressions (double log) between sites. For every metal there was a significant a priori difference in accumulated metal concentration across all the sites (up to 19) included in the ANCOVA comparison. Subsequently Tukey's Honest Significant Difference (HSD) test was applied a posteriori, to identify which sites differed significantly from each other in accumulated metal concentrations in the barnacle bodies. Regression analyses and ANCOVA were carried out using STATISTICA (Statsoft). Because mean accumulated metal concentrations in the bodies of barnacles from different sites have no comparative meaning in the presence of size effects, even in just a subset of data sets for a metal, comparative accumulated metal concentrations are quoted as the estimated accumulated concentrations (with 95% confidence limits) in barnacle bodies of a standard dry weight, as calculated from each double log regression. The mean dry weight of all barnacle bodies analysed was 0.00221 g. The standard dry weight chosen for expression of data was, therefore, 0.002 g dry weight. The ordination technique Principal Components Analysis (PCA) was used finally to compare the similarity between sites, with respect to trace metal profiles for the 7 of the 9 metals for which there were full data sets of estimated metal concentrations for barnacle bodies of 0.002 g dry weight. CAP5 (Community Analysis Package, Pisces Conservation Ltd, Lymington SO41 8GN, UK) was used for ordination analyses (Henderson and Seaby, 2008).

n

A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10 Table 3 Amphibalanus amphitrite: Minimum and maximum concentrations (mg g1 dry weight) of trace metals in bodies (mean individual dry weight (g)) of up to 10 replicate samples, each of 10 pooled bodies, collected from the 19 sites listed in Table 1.

4

Please cite this article in press as: Nasrolahi, A., et al., Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.008

A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10 Table 4 Amphibalanus amphitrite: Comparison by ANCOVA of accumulated concentrations of As, Cd, Cr, Cu, Fe, Mn, Pb, V and Zn in the bodies of barnacles from the 19 sites listed in Table 1. Trace metal concentrations [M] are estimated concentrations (mg g1 dry wt) with 95% confidence limits (CL) in a barnacle body of 0.002 g dry weight [W] from each site, using the best-fit regression equation Log [M] ¼ Log a þ b Log [W]. Barnacles from any sites sharing a common letter in the ANCOVA column for a metal do not differ significantly in body concentrations of that metal. Asterisks indicate sites that could not be included in the ANCOVA analysis for that metal because of a significant difference in regression coefficient (b) in comparison with best-fit regressions from other sites. In the cases of Cr and Pb, samples from 2 sites (Cr) and 6 sites (Pb) had 3 or fewer replicates with measurable concentrations, and were excluded from the ANCOVA analysis. Cr and Pb concentrations quoted for these sites are means with standard errors, distinguished by parentheses. In the case of Zn, asterisks indicate sites that could not be included in the main ANCOVA analysis (letters A to I) because of a significant difference in regression coefficient (b) in comparison with best-fit regressions from other sites. The regression coefficients of these 5 sites did not, however, differ significantly between themselves, and these sites have been subjected to a separate ANCOVA analysis (letters W to Z). Site

As 6 12 9 7 13 10 8 14 19 18 15 5 2 11 17 3 4 1 16 Cd 18 6 17 19 13 9 14 5 2 3 4 12 10 16 15 8 11 7 1 Cr 11 13 15 9 14 3 19 4 17 12 18 7 10 6 2

[M] Estimate

95% CL

ANCOVA

Lower

Upper

276 253 212 211 211 273 149 131 169 138 133 137 125 119 108 112 96.3 110 10.5

198 212 174 182 182 225 81.8 60.2 116 111 96.4 90.3 109 99.2 86.0 79.2 85.5 9.39 0.90

384 305 257 229 246 333 272 287 248 172 184 208 144 143 136 157 109 1283 123

A A A A,B A,B,C A,B,C,D B,C,D,E B,C,D,E C,D,E D,E D,E E E E E * * F F

192 62.3 39.8 61.8 41.3 30.0 17.2 61.1 29.3 24.3 17.9 17.8 14.2 7.80 11.5 8.12 7.44 7.91 2.35

151 41.2 28.4 28.0 34.2 22.0 12.4 37.2 23.1 16.0 14.3 13.8 9.91 2.77 7.99 3.38 5.47 6.87 1.22

243 94.1 55.7 137 49.9 41.1 23.9 100 37.2 36.8 22.1 23.1 20.4 22.7 16.6 19.5 10.1 9.11 4.50

A B C C,D C,D C,D C,D D D E * E E,F,G F,G,H G,H,I H,I H,I I J

11.2 7.67 5.34 8.32 5.64 6.20 5.05 4.77 4.44 4.35 3.76 3.85 2.55 2.51 3.34

14.4 5.99 3.48 4.39 0.53 1.92 3.03 3.24 3.69 2.60 2.53 2.83 1.17 1.78 2.57

8.68 9.83 8.21 15.8 59.6 20.0 8.43 7.02 5.34 7.27 5.59 5.24 5.56 3.54 4.33

A A,B A,B A,B,C A,B,C,D B,C,D B,C,D B,C,D B,C,D B,C,D B,C,D B,C,D,E B,C,D,E B,C,D,E C,D,E

5

Table 4 (continued ) Site

5 8 1 16 Cu 8 10 2 13 11 19 12 9 6 17 3 15 5 4 14 7 18 1 16 Fe 15 17 9 13 11 19 18 1 4 12 14 10 5 7 16 6 8 3 2 Mn 11 13 19 15 4 17 1 9 14 3 18 2 12 10 6 5 7 16 8 Pb 6 16 8 11 9 2 15 13

[M] Estimate

95% CL Lower

Upper

ANCOVA

3.58 4.59 (5.71) (4.96)

0.56 0.79 (5.28) (1.65)

23.0 26.8 (6.14) (8.27)

D,E E (n ¼ 3) (n ¼ 2)

2195 1097 631 610 481 540 365 222 181 186 211 167 360 149 97.2 93.7 86.9 14.5 9.8

986 534 536 545 372 295 241 154 127 135 146 11 162 124 40.5 82.2 73.1 6.7 1.5

4884 2253 745 684 622 987 553 321 258 257 305 250 798 180 233 107 103 31.3 65.4

A B B * B,C C,D C,D D,E E,F E,F,G F,G,H G,H,I H,I * * I,J J K K

1707 1707 1695 1679 1719 2123 1334 644 1307 1147 1317 934 1041 837 752 643 1044 983 839

1075 1417 1154 1414 1399 1297 908 246 973 669 707 469 194 659 13.3 468 357 714 681

2709 2057 2489 1993 2112 3473 1960 1688 1757 1966 2454 1859 5599 1064 42,618 883 3054 1352 1033

A A,B A,B,C A,B,C,D A,B,C,D,E A,B,C,D,E,F A,B,C,D,E,F,G B,C,D,E,F,G C,D,E,F,G C,D,E,F,G D,E,F,G E,F,G F,G F,G F,G F,G G G G

69.8 62.1 34.7 29.5 39.7 27.6 13.8 23.2 36.7 30.0 21.1 21.0 19.7 15.7 12.9 17.8 14.5 18.9 11.6

58.8 49.0 25.5 17.5 29.8 23.0 4.4 16.4 11.7 15.6 14.2 17.2 11.5 9.47 8.91 4.03 11.5 0.08 5.62

82.9 78.7 47.2 49.9 53.0 33.2 42.8 32.8 115 57.9 31.5 25.6 33.8 26.0 18.8 78.6 18.4 4223 23.9

A A A,B B,C B,C,D B,C,D,E B,C,D,E,F B,C,D,E,F,G C,D,E,F,G C,D,E,F,G D,E,F,G D,E,F,G E,F,G,H F,G,H,I F,G,H,I G,H,I G,H,I H,I I

4.07 1.65 8.79 4.51 2.08 4.29 4.02 4.35

2.26 0.01 1.78 3.70 0.28 2.46 0.79 3.59

7.35 268 43.5 5.49 15.5 7.51 20.4 5.28

A A,B A,B,C A,B,C A,B,C A,B,C,D A,B,C,D B,C,D (continued on next page)

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A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

Table 4 (continued ) Site

[M] Estimate

95% CL

ANCOVA

Lower

Upper

12 18 19 5 1 14 4 10 17 7 V 10 19 13 15 17 2 9 3 11 12 18 7 4 14 1 16 5 6 8 Zn 11 8 10 2 13 6 16 9 14 18 3 12 19 4 17 1 15 5 7

3.11 3.92 2.17 4.88 (12.4) (7.69) (4.53) (3.88) (3.37) (2.41)

1.86 2.83 1.40 1.71 (6.59) (5.66) (3.15)

5.20 5.43 3.35 13.9 (18.2) (9.72) (5.91)

(3.03) (2.25)

(3.71) (2.57)

B,C,D C,D D E (n ¼ 2) (n ¼ 3) (n ¼ 3) (n ¼ 1) (n ¼ 2) (n ¼ 3)

24.0 10.4 6.34 4.94 4.85 4.88 5.44 5.60 4.60 3.17 3.14 2.75 3.16 2.48 1.36 7.51 2.28 1.68 2.27

15.1 6.42 5.12 2.79 4.16 4.44 3.22 3.71 3.77 1.96 2.19 2.10 2.41 1.07 0.08 0.94 0.37 1.11 0.69

38.1 16.8 7.86 8.74 5.64 5.36 9.19 8.46 5.62 5.13 4.51 3.62 4.13 5.73 23.9 59.9 13.9 3.62 7.44

A B B B B B,C B,C,D B,C,D,E B,C,D,E C,D,E,F C,D,E,F D,E,F,G E,F,G E,F,G E,F,G F,G F,G F,G G

11,900 13,800 14,200 10,200 9700 6330 3500 4660 1050 3310 4480 2540 3460 2460 1780 1090 1830 1910 575

9210 7950 9150 8660 8250 4760 1140 3390 342 2900 3590 1740 2070 2020 1390 663 1030 1360 470

15,500 24,000 22,100 11,900 11,400 8420 10,700 6400 3250 3770 5590 3700 5770 3000 2270 1780 3260 2670 702

A A A,B A,B *W B,C C,D D *X E *X E,F E,.F *Y F,G G,H H *Z I

(with 95% confidence limits) in barnacle bodies of a standard 0.002 g dry weight. Data for Cr and Pb excluded from ANCOVA comparisons are included for completeness, expressed as mean concentrations with standard errors (Table 4). Barnacles from any sites sharing a common letter in the ANCOVA column for a metal do not differ significantly a posteriori in body concentrations of that metal. In Table 4, sites have been ordered firstly in terms of the a posteriori ANCOVA groups to which they belong (as denoted by a single letter), and secondly within those groups in terms of the estimated body metal concentration of a body of 0.002 g dry weight. It needs to be remembered, however, that it is regression lines that are being compared. These lines will cross (even in the absence of significant differences between slopes), and estimated metal concentrations for different body weights would probably give different site orders. Secondly, according to how the chosen standardised weight falls in the real data set of body weights analysed for a given site, the 95% confidence limits of the estimated concentrations might be narrow or broad. For these two reasons a

posteriori ANCOVA site groupings will not always follow the strict rank order of the weight standardised metal concentrations quoted (Table 4). As is clear from Table 4, there was a significant difference in accumulated concentrations of each metal across the sites compared, with a gradient of metal concentrations in the bodies of barnacles across the sites. The barnacles from six sites (6, 12, 9, 7, 13 and 10) accumulated the highest arsenic concentrations (ANCOVA group A) (Table 4). Thereafter sites followed a gradient of accumulated As concentrations in the barnacle bodies, down to the lowest concentrations recorded from sites 1 and 16. Cadmium concentrations were extraordinarily high in the bodies of barnacles collected from site 19, significantly above those measured at all other sites. Cadmium concentrations at site 6 were lower than at site 19, but still significantly raised above all other accumulated Cd concentrations (Table 4). Barnacles from site 1 had very low accumulated Cd body concentrations, significantly below concentrations measured at all other sites (Table 4). Chromium concentrations in the bodies of the barnacles lacked the large inter-site variation seen for Cd, and body Cr concentrations at all but 4 of the 17 sites compared did not differ significantly (ANCOVA group B) (Table 4). In the case of copper, barnacles from site 8 contained remarkably high accumulated concentrations, significantly higher than all other accumulated Cu concentrations measured in the bodies of barnacles from the other sites (Table 4). Cu concentrations were also significantly raised in barnacles from sites 10, 2 and 11, while Cu concentrations in barnacles from site 13 were similarly high but not amenable to ANCOVA analysis (Table 4). Accumulated copper concentrations in the barnacle bodies followed a long gradient with the lowest concentrations recorded for barnacles from sites 1 and 16 (Table 4). Iron concentrations in the barnacle bodies varied significantly across all sites, but the highest estimated concentrations for a site are only about twofold higher than the lowest (Table 4). Manganese concentrations in barnacle bodies at sites 11 and 13 were significantly raised above all others, except those at site 19 which in turn did not differ significantly from those at 5 further sites (Table 4). Lead concentrations in the barnacle bodies showed relatively little (but still significant) variation across the 12 sites compared by ANCOVA, with concentration data for 8 of these sites showing no significant difference (ANCOVA group B, Table 4). Vanadium concentrations in barnacle bodies from site 10 were significantly raised above those at all other sites, which followed a gradient down to the lowest accumulated concentration at site 8. Zinc concentrations in the barnacle bodies analysed ranged approximately tenfold (Tables 3 and 4). ANCOVA comparisons were made on two separate groupings of sites, the significantly lowest accumulated body Zn concentrations being found at site 7. Principal Components Analysis (PCA) was used to compare the similarity between sites with respect to trace metal profiles for the 7 of the 9 metals for which there were full data sets of estimated metal concentrations for barnacle bodies of 0.002 g dry weight (Fig. 2). Groupings of sites containing barnacles with similar profiles of accumulated trace metals are distinguishable (Fig. 2). Eigenvectors for Zn, Cu, V and As behave similarly, causing sites 10 and 8 to be separable from the other sites along Principal Axis 1 (Fig. 2). Eigenvectors for Fe and Mn are similar drivers of the separation of sites 11, 13 and 19 along Principal Axis 2 (Fig. 2). Sites 15, 17, 4, 14 and 18 are clumped, and the Eigenvector for Cd appears to be a factor in their separation (Fig. 2). The two Principal Axes shown together account for 57% of the variance in the data.

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A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

7

Table 5 Amphibalanus amphitrite: Accumulated concentrations of 8 trace metals (mg g1 dry weight), categorised as ‘typical’ or ‘high’ when possible, in the bodies of the barnacle A. amphitrite. The low end of the typical range is indicative of uncontaminated conditions; the high end is indicative of concentrations representative of moderate contamination on a regional scale. The ‘high’ concentrations are indicative of atypically raised bioavailability of that metal in the local habitat (After Phillips and Rainbow, 1988; Rainbow and Blackmore, 2001; Luoma and Rainbow, 2008; Morillo and Usero, 2008; Rainbow and Luoma, 2011a,b). Also shown for comparison are the ranges across the sites of estimated metal concentrations in 0.002 g dry weight barnacle bodies, summarised from Table 4.

Amphibalanus amphitrite Typical High Persian Gulf Range of weight-corrected metal concentrations

As

Cd

Cr

Cu

Fe

Mn

Pb

Zn

7e75 200e460

0.8e10 20e170

1e10 20e40

52e100 500e9400

300e2000 3000e6000

10e300 400e900

0.4e10 35e40

2700e10,000 12,000e50,000

11e276

2.4e192

2.5e11.2

10e2195

643e2123

12e70

2.2e8.8

575e14,200

4. Discussion The accumulated trace metal concentration in the body of a barnacle is an integrated measure of the total metal taken up by that barnacle from solution and diet over a preceding period. It is appropriate then to refer to high accumulated body concentrations of a trace metal as indicative of high ambient bioavailabilities (summed from both sources) to that barnacle over that period. Barnacles are typically very strong accumulators of trace metals (Rainbow, 1987, 2002, 2007; Luoma and Rainbow, 2008), and the period concerned is very long, probably of the order of months or even more than a year for those trace metals investigated. Half lives of metals accumulated in the body of A. amphitrite may vary with the site of origin and exposure pre-history (Rainbow et al., 2003, 2004b). Nevertheless estimated half lives of metals accumulated in the body of these barnacles are of the order of 165e654 days for zinc, 89e187 days for cadmium, and 30e396 days for silver (Rainbow et al., 2003, 2004b; Rainbow and Luoma, 2011c). The half life of chromium accumulated in the body of another barnacle, Austrominius modestus, is 126 days (Rainbow and Wang, 2001).

Table 5 is a summary of literature data on accumulated concentrations of eight trace metals in the bodies of A. amphitrite from coastal sites across the world. There are no equivalent data available for vanadium. Typical accumulated concentrations are those usually found, and range from body concentrations found in uncontaminated conditions to those concentrations more usually measured, given the presence of some trace metal contamination in most coastal waters analysed (Luoma and Rainbow, 2008). High bioaccumulated concentrations listed in Table 5 are rarely found, and indicate atypically very high local bioavailabilities of the trace metals concerned. Included in Table 5 for comparison are the ranges across the sites of the estimated metal concentrations in 0.002 g dry weight barnacle bodies. Arsenic concentrations measured in the bodies of A. amphitrite from at least six of the sites on the Iranian coast of the Persian Gulf (Tables 3 and 4) can be considered high (Table 5), and barnacles at 12 of the remaining 13 sites had body concentrations still above the typical range defined in Table 5. Barnacles from these 18 of the 19 Persian Gulf sites had accumulated As concentrations higher than similarly estimated As concentrations in 0.0045 g dry weight

Fig. 2. Principal Components Analysis of profiles for the metals As, Cd, Cu, Fe, Mn, V and Zn for Amphibalanus amphitrite. The numbering of sites is as in Table 1.

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A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

bodies of A. amphitrite from 11 of the 12 Hong Kong coastal waters sites investigated by Rainbow and Blackmore (2001). These latter data were used to define the upper limit of 75 mg As g1 in the typical range in Table 5. While arsenic does occur in oil, it is unlikely that spilled oil is the cause of raised arsenic bioavailability at the large number of sites concerned, given that the bioavailability of vanadium, a more classic indicator of oil pollution (Pearson and Green, 1993), is not generally raised at so many sites (see later). While a source of arsenic from oil contamination cannot be ruled out, it does appear unlikely that there are high arsenic bioavailabilities of international significance at so many of the sites investigated here, and the typical range deduced from the literature (Table 5) may need redefinition in the light of these Persian Gulf data. A relevant factor here may be growth dilution, or rather the lack of growth dilution, in the barnacle data presented here. Growth dilution is a reduction in accumulated metal concentration (mg g1) caused by the rate of growth outpacing the rate of accumulation of metal content (mg). The average dry weight (0.00221 g) of the bodies of the Persian Gulf barnacles, rounded down to 0.002 g in the estimated accumulated metal concentrations, was less than half the dry weight (0.0045 g) used in the estimates of the accumulated trace metal concentrations in the Hong Kong barnacles. The accumulated As contents (the total amounts of As accumulated), as opposed to accumulated As concentrations, would therefore be similar in the two sets of data, indicating that most of the Persian Gulf barnacles were not exposed to high As bioavailabilities. Even so, As bioavailabilities do appear to be raised above typical at sites 6, 12 and 9, and perhaps at sites 7, 13 and 10 (Table 4). While sites 12 and 13, and sites 9 and 10, are independently adjacent to each other, perhaps suggesting a separate common source of raised As bioavailability to each pair of sites, there does not seem to be a unifying feature of potential anthropogenic input of As (Table 1) across all the six sites picked out. No Persian Gulf barnacles reached the high estimated As body concentration (457 mg As g1) recorded by Rainbow and Blackmore (2001) at the top Hong Kong site (Chai Wan Kok), a site receiving industrial effluent with clearly exceptionally raised As bioavailability. Cadmium concentrations in the bodies of the Persian Gulf barnacles (Tables 3 and 4) were similarly high on an international scale (Table 5), and, as for arsenic, the low dry weight, with an associated absence of growth dilution, may again be a factor at many of the sites. Nevertheless, the Cd concentration (192 mg Cd g1, Table 4) estimated in the bodies of barnacles from site 18 is the highest ever recorded for A. amphitrite (Table 5). The Cd concentrations in barnacle bodies at sites 6 and 17 (Table 4) are also high on an international scale (Table 5), and none of these three high accumulated concentrations can be explained away by the low dry weights of the barnacles concerned. Cd concentrations can be high in mined phosphate fertilisers (Taylor, 1997), and Cd bioavailabilities can be raised in run off from agricultural land heavily treated with such fertilisers. No such situation appears to be the case here, although, at site 6, there is some very small run off from an aquaculture site that may employ fertilisers. It is interesting that sites 17, 18 and 19 (occupying 3 of the top 4 ranking positions for Cd in Table 4) are close to the Strait of Hormuz at the eastern end of the Persian Gulf. These sites may be affected by the entry into the Gulf of phosphate-enriched waters derived from upwelling in the Indian Ocean off Oman (Brewer and Dryssen, 1985). These waters can be expected to also have raised levels of associated dissolved cadmium, regenerated in the deeper oceanic waters subsequently rising to the surface, as seen off California (Luoma and Rainbow, 2008). Chromium concentrations accumulated in the bodies of the Persian Gulf barnacles (Tables 3 and 4) appear typical of coastal

waters on an international scale (Table 5). There is no evidence for atypically raised Cr bioavailability at any site. The situation is very different for copper. Cu concentrations in the bodies of barnacles from site 8 (Tables 3 and 4) are extremely high on an international scale, and those from sites 10, 2, 11 and 13 (Table 4) are also very high (Table 5). Copper bioavailabilities to barnacles also appear raised above the typical at sites 19, 12 and 9 (Tables 4 and 5), even after allowance for any effect of low body weight. Body copper concentrations estimated for 0.002 g barnacle bodies at sites 1 and 16, appear very low as a result of the chosen standardised weight falling outside the body weight range analysed at these two sites, thereby adding to the imprecision of the estimate and the wide associate confidence limits (Tables 3 and 4). The high accumulated Cu concentration (estimated 2195 mg Cu g1) at site 8 (Tables 3 and 4) is particularly remarkable, approximating to body concentrations in A. amphitrite at sites directly affected by copper mining effluent (3720e9430 mg Cu g1 in the Huelva estuary, Spain e Morillo and Usero, 2008) or by industrial effluent from an electroplating works (3472 mg Cu g1 at Chai Wan Kok, Hong Kong e Phillips and Rainbow, 1988). Of the sites with high Cu bioavailabilities, sites 8, 10, 2, 19, 12 and 9 are all significant commercial and/or fishing ports (Table 1). Cu is a key ingredient leaching from antifouling paints, and this is a possible source of high local Cu bioavailability. Nevertheless the Cu bioavailability at site 8 does seem extraordinarily high to be explained by this source, and site 11, also high in Cu bioavailability, lacks a high concentration of boats that might carry copper-based antifouling paint (Table 1). Data for iron and manganese are included in Table 5, although these two metals are of low ecotoxicological relevance in coastal waters (Luoma and Rainbow, 2008). For both metals, the accumulated body concentrations measured here fall in the typical range (Tables 4 and 5), with no suggestion of atypically raised iron or manganese bioavailabilities of anthropogenic origin at any Persian Gulf site. The occurrence of high accumulated concentrations of iron and manganese is typically associated with raised sediment loadings in the water column. The situation for lead is similar to that for chromium. Accumulated Pb concentrations in the Persian Gulf barnacles (Tables 3 and 4) are typical of coastal waters on an international scale (Table 5). There is no evidence for atypically raised Pb bioavailability at any site. Interpretation of the vanadium data is hampered by a lack of comparative information. It is clear, nevertheless, that vanadium bioavailability to the barnacles is raised at site 10 (Table 4). It is, however, difficult to conclude that there are atypically raised V bioavailabilities elsewhere, given the overlaps in bioaccumulated V concentrations in barnacles from the other 18 sites (Table 4). High V bioavailability can often be interpreted to result from the presence of oil or petroleum in the water, given the high V content of many oils (Pearson and Green, 1993). Site 10 is located in the largest fishing port in the Persian Gulf (Table 1). It is possible that there has been much fuel leakage or spillage into the local seawater, but this might also have been expected at many others of the commercial and fishing ports sampled (Table 1). The bioaccumulated concentrations of zinc in the Persian Gulf barnacles (Table 4) indicate variability in zinc bioavailability to the barnacles across the 19 sites. Zn concentrations in the barnacle bodies were particularly raised at sites 11, 8, 10 and 2 (Table 4), with no apparent common zinc source linking them (Fig. 1). These bioaccumulated concentrations can be considered at the top end, and just beyond, of typical coastal bioavailabilities of Zn, without suggesting atypically strong zinc contamination (Table 5). The Zn concentrations in the bodies of the barnacles collected from site 7, on the other hand, were anomalously low (Table 5). It is difficult to

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A. Nasrolahi et al. / Marine Environmental Research xxx (2014) 1e10

suggest any consistent strong candidate zinc sources to account for the pattern of zinc bioavailabilities observed. The bioavailability of a dissolved metal to a coastal invertebrate can be affected by physicochemical factors other than dissolved metal concentration, although the latter is a dominant factor (Phillips and Rainbow, 1988). Low salinity, for example, raises the bioavailabilities of dissolved cadmium and zinc, by reducing inorganic (particularly chloride) complexation of the free metal ion, which is considered to model the dissolved trace metal form available for uptake across the cell membrane of permeable external surfaces (Campbell, 1995; Luoma and Rainbow, 2008). A high concentration of dissolved organic matter (DOM) may also increase organic complexation of dissolved metal ions and reduce dissolved bioavailability (Luoma and Rainbow, 2008). There are no major river systems emptying into estuaries to cause reduced salinity along the coast studied, so it is unlikely that variation in salinity is an important factor affecting comparative bioavailabilities in this study. Increased DOM may also be associated with estuarine systems, but additionally with sewage effluent from large cities. There was, however, no pattern here to suggest that this might have been a significant factor. It needs to be remembered that biomonitoring provides an accurate record of local bioavailabilities after the effect of any physicochemical factor, but it is useful to be aware of potential causes of raised or lowered dissolved bioavailabilities, other than just dissolved metal concentration. When considering the data from the viewpoint of the different sites for which full metal data sets were available, PCA (Fig. 2) was able to distinguish different groupings, discernible to some extent in comparisons of the ANCOVA site rankings for the different metals (Table 4). Sites 10 and 8, and to a lesser extent site 2, featured high on the ANCOVA site rankings for both Cu and Zn (Table 4), and these sites could be separated from other sites along PCA Principal Axis 1, in association with Eigenvectors for Cu and Zn (Fig. 2). PCA showed Eigenvectors for V and As in the same direction along PCA Axis 1. Site 10 certainly also showed up with high V bioavailability in the ANCOVA site ranking for V (Table 4). However, sites 10, 8 and 2 were not in particularly high rank positions in the As ANCOVA site ranking (Table 4). Thus it can be concluded that it is the bioavailabilities of Cu and Zn to the barnacles that cause the association of sites 10 and 8 (Fig. 2). Both sites are in busy ports (Table 1). It is possible that the Cu is derived from leaching from Cu-based antifouling paints (Woods Hole Oceanographic Institution, 1952), while Zn may be released from sacrificial anodes designed to reduce fouling on ships or port installations. Sites 11, 13 and 19 are characterised by high rank positions in the ANCOVA lists for Fe and Mn (Table 4). These sites were correspondingly identifiable as a grouping along PCA Principal Axis 2, in association with Eigenvectors for Fe and Mn (Fig. 2). It is possible that these sites shared high suspended sediment loadings in the water column. Sites 15, 17, 4, 14 and 18 were clumped by PCA, apparently in association with the relatively short Eigenvector for Cd (Fig. 2). Of these sites, site 18 did indeed top the ANCOVA site ranking for Cd, but the other sites were not particularly prominent in this ranking (Table 4). It would not appear, therefore, that raised Cd bioavailability is the main agent behind the clumping of these sites by PCA. Sites 1, 16 and 17 generally appeared low in ANCOVA site rankings for the metals analysed (Table 4), and these sites did group together in the PCA analysis, not associated with any metal Eigenvector (Fig. 2). It can be concluded, then, that these sites were relatively uncontaminated by the metals investigated. There is a large comparative literature confirming the suitability of barnacles as biomonitors of trace metal pollution in coastal waters (Luoma and Rainbow, 2008; Reis et al., 2011). Barnacles are

9

particularly appropriate in this regard because they are sessile and accumulate many trace metals to very high body concentrations (Luoma and Rainbow, 2008). Furthermore there is a good literature now available on the biodynamic modelling of the accumulation of many trace metals in barnacles, including A. amphitrite, providing insight into the half times of accumulated body concentrations, the effects of growth rate on accumulated concentrations, and the relative importance of diet and solution as metal sources (Wang et al., 1999a,b; Rainbow and Wang, 2001; Rainbow et al., 2003; da Silva et al., 2009; Rainbow and Luoma, 2011b,c). Additionally, the use of regression analysis and ANCOVA allows comparisons to be made between populations of barnacles from sites with different growth rates. There are other well used trace metal biomonitors in coastal waters in addition to barnacles e not least mussels like Mytilus edulis or Perna viridis, which have wide occurrence globally, high soft tissue weights for analysis, but lower accumulated metal concentrations (Luoma and Rainbow, 2008). An ideal biomonitoring programme would include a suite of such biomonitors if available locally. This Persian Gulf data set adds a new region to the growing database of accumulated trace metal concentrations in the bodies of A. amphitrite, confirming its status as a cosmopolitan biomonitor for trace metals of significant value in assessing the potentially ecotoxicologically significant trace metal contamination of coastal waters across the world. Acknowledgements The authors are very grateful to A Shahdadi who confirmed all barnacle identifications. References Anil, A.C., Wagh, A.B., 1988. Accumulation of copper and zinc by Balanus amphitrite in a tropical estuary. Mar. Pollut. Bull. 19, 177e180. Barbaro, A., Francescon, A., Polo, B., Bilio, M., 1978. Balanus amphitrite (Cirripedia: Thoracica) e a potential indicator of fluoride, copper, lead, chromium and mercury in North Adriatic Lagoons. Mar. Biol. 46, 247e257. Bastami, K.D., Afkhami, M., Ehsanpour, M., Khazaali, A., Mohammadizadeh, M., Haghparast, S., Soltani, F., Zanjani, S.A., Farzaneh, G.N., Pourzare, R., 2013. Polycyclic aromatic hydrocarbons in the coastal water, surface sediment and mullet Liza klunzingeri from northern part of Hormuz strait (Persian Gulf). Mar. Pollut. Bull. 76, 411e416. Brewer, P.G., Dryssen, D., 1985. Chemical oceanography of the Persian Gulf. Prog. Oceanogr. 14, 188e197. Campbell, P.G.C., 1995. Interaction between trace metals and aquatic organisms: a critique of the free-ion activity model. In: Tessier, A., Turner, D.R. (Eds.), Metal Speciation and Aquatic Systems. Wiley, New York, pp. 45e102. da Silva, E.T., Ridd, M., Klumpp, D.W., 2005. Can body burdens in the barnacle Balanus amphitrite indicate seasonal variation in cadmium concentrations? Estuar. Coast. Shelf Sci. 65, 159e171. da Silva, E.T., Klumpp, D.W., Ridd, M., 2009. The barnacle Balanus amphitrite as a bioindicator for Cd: development and application of a simulation model. Estuar. Coast. Shelf Sci. 82, 171e179. Fialkowski, W., Newman, W.A., 1998. A pilot study of heavy metal accumulation in a barnacle from the Salton Sea, southern California. Mar. Pollut. Bull. 36, 138e143. Henderson, P., Seaby, R., 2008. A Practical Handbook for Multivariate Methods. Pisces Conservation Ltd, Lymington, Hants, UK. Luoma, S.N., Rainbow, P.S., 2008. Metal Contamination in Aquatic Environments: Science and Lateral Management. Cambridge University Press, Cambridge, UK. Marsden, I.D., Smith, B.D., Rainbow, P.S., 2013. Effects of environmental and physiological variables on the accumulated concentrations of trace metals in the New Zealand cockle Austrovenus stutchburyi. Sci. Total Environ. 470e471, 324e339. Morillo, J., Usero, J., 2008. Trace metal bioavailability in the waters of two different habitats in Spain: Huelva estuary and Algeciras Bay. Ecotoxicol. Environ. Saf. 71, 851e859. Morillo, J., Usero, J., Bakouri, H.E., 2008. Biomonitoring of heavy metals in the coastal waters of two industrialised bays in southern Spain using the barnacle Balanus amphitrite. Chem. Speciat. Bioavailab. 20, 227e237. Morillo, J., Usero, J., Gracia, I., 2005. Biomonitoring of trace metals in a minepolluted estuarine system (Spain). Chemosphere 58, 1421e1430. Pearson, C.D., Green, J.B., 1993. Vanadium and nickel complexes in petroleum resid acid, base, and nickel fractions. Energy Fuels 7, 338e346.

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Phillips, D.J.H., Rainbow, P.S., 1988. Barnacles and mussels as biomonitors of trace elements: a comparative study. Mar. Ecol. Prog. Ser. 49, 83e93. Phillips, D.J.H., Rainbow, P.S., 1994. Biomonitoring of Trace Aquatic Contaminants, second ed. Chapman and Hall, London. Rainbow, P.S., 1987. Heavy metals in barnacles. In: Southward, A.J. (Ed.), Barnacle Biology. A. A. Balkema, Rotterdam, pp. 405e417. Rainbow, P.S., 2002. Trace metal concentrations in aquatic invertebrates: why and so what? Environ. Pollut. 120, 497e507. Rainbow, P.S., 2006. Biomonitoring of trace metals in estuarine and marine environments. Australas. J. Ecotoxicol. 12, 107e122. Rainbow, P.S., 2007. Trace metal bioaccumulation: models, metabolic availability and toxicity. Environ. Int. 33, 576e582. Rainbow, P.S., Blackmore, G., 2001. Barnacles as biomonitors of trace metal bioavailabilities in Hong Kong coastal waters: changes in space and time. Mar. Environ. Res. 51, 441e463. Rainbow, P.S., Luoma, S.N., 2011a. Biodynamic parameters of the accumulation of toxic metals, detoxification, and the acquisition of metal tolerance. In: Amiardo, M. (Eds.), Tolerance to Environmental ConTriquet, C., Rainbow, P.S., Rome taminants. CRC Press, Boca Raton, FL, USA, pp. 127e151. Rainbow, P.S., Luoma, S.N., 2011b. Trace metals in aquatic invertebrates. In: Beyer, W.N., Meador, J.P. (Eds.), Environmental Contaminants in Biota: Interpreting Tissue Concentrations. Taylor and Francis Books, Boca Raton, FL, USA, pp. 231e252. Rainbow, P.S., Luoma, S.N., 2011c. Metal toxicity, uptake and bioaccumulation in aquatic invertebrates e modelling zinc in crustaceans. Aquat. Toxicol. 105, 455e465. Rainbow, P.S., Phillips, D.J.H., 1993. Cosmopolitan biomonitors of trace metals. Mar. Pollut. Bull. 26, 593e601. Rainbow, P.S., Wang, W.-X., 2001. Comparative assimilation of Cr, Cr, Se, and Zn by the barnacle Elminius modestus from phytoplankton and zooplankton diets. Mar. Ecol. Prog. Ser. 218, 239e248.

Rainbow, P.S., Blackmore, G., Wang, W.-X., 2003. Effects of previous field exposure history on the uptake of trace metals from water and food by the barnacle Balanus amphitrite. Mar. Ecol. Prog. Ser. 259, 201e213. Rainbow, P.S., Fialkowski, W., Wolowicz, M., Smith, B.D., Sokolowski, A., 2004a. Geographical and seasonal variation of trace metal bioavailabilities in the Gulf of Gdansk, Poland using mussels (Mytilus trossulus) and barnacles (Balanus improvisus) as biomonitors. Mar. Biol. 144, 271e286. Rainbow, P.S., Ng, T.Y.-T., Shi, D., Wang, W.-X., 2004b. Acute dietary pre-exposure and trace metal bioavailability to the barnacle Balanus amphitrite. J. Exp. Mar. Biol. Ecol. 311, 315e337. Rainbow, P.S., Smith, B.D., Lau, S.S., 2002. Biomonitoring of trace metal availabilities in the Thames estuary using a suite of littoral biomonitors. J. Mar. Biol. Assoc. U.K. 82, 793e799. Reis, P.A., Salgado, M.A., Vasconcelas, V., 2011. Barnacles as biomonitors of metal contamination in coastal waters. Estuar. Coast. Shelf Sci. 93, 269e278. Shahdadi, A., Sari, A., Naderloo, R., 2014. A checklist of the barnacles (Crustacea: Cirripedia: Thoracica) of the Persian Gulf and Gulf of Oman with nine new records. Zootaxa 3784, 201e223. Silva, C.A.R., Smith, B.D., Rainbow, P.S., 2006. Comparative biomonitors of coastal trace metal contamination in tropical South America (N. Brazil). Mar. Environ. Res. 61, 439e455. Taylor, M.D., 1997. Accumulation of cadmium derived from fertilisers in New Zealand soils. Sci. Total Environ. 208, 123e126. Wang, W.-X., Qiu, J.-W., Qian, P.-Y., 1999a. The trophic transfer of Cd, Cr, and Se in the barnacle Balanus amphitrite from planktonic food. Mar. Ecol. Prog. Ser. 187, 191e201. Wang, W.-X., Qiu, J.-W., Qian, P.-Y., 1999b. Significance of trophic transfer in predicting the high concentration of zinc in barnacles. Environ. Sci. Technol. 33, 2905e2909. Woods Hole Oceanographic Institution, 1952. Marine Fouling and its Prevention. WHO Contribution No. 580. U.S. Naval Institute, Annapolis, Maryland, USA.

Please cite this article in press as: Nasrolahi, A., et al., Biomonitoring of trace metal bioavailabilities to the barnacle Amphibalanus amphitrite along the Iranian coast of the Persian Gulf, Marine Environmental Research (2014), http://dx.doi.org/10.1016/j.marenvres.2014.07.008