Biomonitoring of trace metals in a mine-polluted estuarine system (Spain)

Chemosphere 58 (2005) 1421–1430 www.elsevier.com/locate/chemosphere

Biomonitoring of trace metals in a mine-polluted estuarine system (Spain) Jose´ Morillo *, Jose´ Usero, Ignacio Gracia Department of Chemical and Environmental Engineering, University of Seville, Camino de los Descubrimientos s/n, 41092 Seville, Spain Received 4 December 2003; received in revised form 3 August 2004; accepted 29 September 2004

Abstract In this paper, we examine metal concentrations in the water and in the crustacean Balanus balanoides from the Huelva estuary, one of the most polluted estuaries in Europe. Metal levels in waters are very high, especially those of Zn, Fe and Cu. Zn presents the highest concentrations, with a mean value of 690 lg l 1 in 2001 and 301 lg l 1 in 2002. As the water flows down through the estuary toward the sea, the metal concentrations drop sharply and the pH rises. The metal concentrations in balanoides tissues are, in general, very high, undoubtedly due to the high metal pollution of the water where it lives. Metal concentrations in Balanus balanoides tissues behave similarly to those in the water, reaching maximums in the upper part of the estuary and diminishing as we approach the sea. The element that reaches the highest levels in Balanus balanoides is Zn, with a mean value of 54.6 g kg 1 in 2001 and 29.9 g kg 1 in 2002, followed by Cu and Fe. There is a significant correlation (p < 0.01) for concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Balanus balanoides relative to their concentration in waters. Barnacles showed a great capacity to accumulate metals, especially Zn, Cu and Fe. Based on the results obtained, we can conclude that Balanus balanoides is a good tool for monitoring trace metals in the Huelva estuary. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Biomonitoring; Metal pollution; Barnacles; Balanus balanoides; Huelva estuary; Tinto River; Odiel River

1. Introduction There is currently a great deal of interest in using living organisms as pollution biomonitors in aquatic ecosystems, given that the method that has been used traditionally—chemical analysis of the water—does not provide information on the bioavailability of metals present in the environment. Furthermore, the metal concentrations in water often lie near or below the detection

*

Corresponding author. Tel.: +34 954 487 276. E-mail address: [email protected] (J. Morillo).

limit of instruments and they fluctuate drastically, depending on water flow and intermittence of discharge (Rainbow, 1995). Biomonitors have been defined as species that accumulate trace pollutants in their tissues, responding essentially to the fraction in the environment that is of direct ecotoxicological relevance, i.e. the bioavailable chemical forms (Ruelas-Inzunza and Pa´ez-Osuna, 2000). The organisms most commonly used for biomonitoring are bivalve molluscs. Mussels of the genus Mytilus are popular biomonitors and have been used extensively in Mussel Watch programs both in the US and Europe (Rainbow et al., 2000). However, the use of bivalve

0045-6535/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.09.093

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molluscs is not always possible since these organisms do not survive in extreme conditions, e.g. in a badly polluted environment (Paulson et al., 2003). Thus, in highly polluted areas, it is necessary to use other organisms as biomonitors. In this study, we examine the suitability of the barnacle Balanus balanoides as a biomonitor of spatial and temporal variations in the bioavailability of trace metals in the Huelva estuary; because of the pollution, it is not possible to find bivalves in a large part of this estuary. Among crustaceans, barnacles appear to be the best biomonitors (Rainbow, 1995; Pa´ez-Osuna et al., 1999) and are being used increasingly as such, particularly in the Indo-Pacific (Rainbow et al., 2000). Barnacles are sessile and exist in most salinities and shore types and with varying degrees of exposure to wave action (Blackmore, 1999). Balanus balanoides lives attached to surfaces found in intertidal areas, like rocks, piles, piers, etc. where it can be easily captured at low tide.

2. Materials and methods 2.1. Study area On their way to the sea, the Tinto and Odiel rivers join to form the Padre Santo Canal, which drains into the Gulf of Cadiz. Taken together, the estuaries of both rivers and the canal form the Huelva estuary (Fig. 1). The Tinto and Odiel rivers run through a metalliferous mining area in the Iberian Pyrite Belt. The waters of both rivers are acidic and contain large amounts of metals from erosion and mining activity. The water flow in these rivers is directly related to rainfall in the catchment area, which is highly variable on a seasonal and annual scale (Braungardt et al., 2003). The estuary is relatively well mixed due to the high tidal amplitude of 3 m (Elbaz-Poulichet and Dupuy, 1999). The Huelva estuary is one of the most industrialised areas of southern Spain and, consequently, receives the discharge of industrial and urban waste. 2.2. Collection and analysis of barnacles Balanus balanoides was sampled at 11 stations (Fig. 1) along the Huelva estuary: two stations were located on the Tinto estuary (T1, T2), three on the Odiel estuary (O1, O2 and O3) and six in the common section where both estuaries join, in the Padre Santo Canal (C1–C6). Two sampling exercises were carried out, one in 2001 (18 and 19 October) and the other in 2002 (14 and 15 October). Sixty organisms of the same size (10–13 mm rostrocarinal axis) were selected and collected from rocks and piers at each station in the intertidal area and transported in closed, refrigerated containers. In the labora-

Fig. 1. Study area indicating the location of the sampling sites.

tory, the soft parts were separated and pooled to prepare a composite sample per site. Each sample was weighed and then dried to constant weight at 60 °C and weighed again. We noted that among the stations studied there were no significant differences in the mean dry weight of individuals of the 60 pooled bodies, which means that the effect of body size on accumulated trace metals can be ruled out. The samples were digested in an automatic microwave digestion system because of the advantages of this technique, which include speed of digestion and less possibility of contamination during the process (Sures et al., 1995). A portion (approximately 0.25 g) of the dry, finely powdered solid tissue was accurately weighed in a dry, pre-cleaned Teflon digestion vessel. 5 ml of 65% HNO3 were added to each vessel. The vessels were sealed and placed in the microwave chamber at 300 W for 5 min and at 600 W for another 5 min. The vessels were allowed to cool for a few minutes before carefully adding 1 ml of 30% H2O2, and digestion continued for a further 5 min at 300 W and 5 min at 600 W. The contents of each microwave vessel were allowed to cool before transferring carefully into 50-ml volumetric flasks and diluted to volume with deionised water. The digestions were performed in batches of twelve samples (the microwave has a rotating 12-position sample carousel).

J. Morillo et al. / Chemosphere 58 (2005) 1421–1430

Following acid digestion, all the samples were analysed for eight elements by atomic absorption spectrometry (AAS). A Perkin-Elmer 4110 ZL atomic absorption spectrometer with longitudinal Zeeman background correction equipped with a THGA graphite furnace (transverse heated graphite atomiser) and AS-70 autosampler was used to determine Cd and Ni. Cu, Fe, Mn and Zn were determined using an air–acetylene flame (PerkinElmer 2380 with a double beam and deuterium background corrector). The working parameters and matrix modifiers employed were those recommended by the unitÕs manual. The hydride-generation technique was used to determine As, employing a Perkin-Elmer MSH-10 connected to a Perkin-Elmer 2380 spectrophotometer. An aliquot (1–10 ml) of the resulting analyte solution was added to 5 ml of 35% HCl and 5 ml of a solution containing 5% potassium iodide and 5% ascorbic acid in a PE flask for pre-reduction of As (V). Heating to 50 °C for 1 h ensured complete reaction to As (III). The vessels were then filled to 25 ml, and the final solutions were analysed for As by hydride generation, using a solution of 3% NaBH4 in 1% NaOH under the conditions recommended in the unitÕs manual. The cold-vapour technique was used for the analysis of Hg, employing a Perkin-Elmer MSH-10 connected to a Perkin-Elmer 2380 spectrophotometer. The solution was analysed using sodium borohydride as reducing agent (3% NaBH4 in 1% NaOH) in the conditions recommended in the unitÕs manual. All the analyses were performed within the laboratoryÕs updated rigorous quality control system (International Standard Organization ISO/IEC 17025, 1999), which guarantees the reliability of all the results. The accuracy of the metal-testing results was evaluated with both a certified reference material (CRM 278R, Community Bureau of Reference) and matrix spikes. The test results with the reference material showed that the accuracy was satisfactory. The recovery percentages were 89% for As, 93% for Hg, 96% for Mn, 101% for Cd, 102% for Zn and 105% for Cu. One sample and one acid blank in every batch digestion were spiked with a known amount of metal spike standard (added to the microwave vessel prior to digestion). The analysis of spike recoveries showed that the sample digestion was complete and matrix interference did not occur. The matrix spike recoveries for As, Cd, Cu, Fe, Mn, Hg, Ni and

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Zn were usually in the range of 92–104% (Table 1). These results met our laboratoryÕs acceptance criteria for results, which require that the percent recovery for a matrix spike must fall between 90% and 110%. Precision was verified by analysing a replicate sample in every batch digestion. Most of the replicates agreed within 10% for the relative percent difference and were therefore considered satisfactory. The metal pollution sources were evaluated analysing one acid blank in every batch. The results of the blanks were always below the methodÕs detection limit (Table 1). The detection limits were calculated using the procedure recommended by the EPA (US Environmental Protection Agency, Method 1631, 1999). This procedure involves spiking seven replicate aliquots of reference matrix or the sample matrix with the analytes of interest at a concentration within one to five times the estimated detection limit. The seven aliquots were carried through the entire analytical process, and the standard deviation of the seven replicate determinations was calculated. The standard deviation was multiplied by 3.14 (the StudentÕs t value at 6 degrees of freedom) to give the MDL (method detection limit). 2.3. Water collection and analysis Water samples were taken eight times in 2001 and 2002 (once every three months) from the same sampling sites as the organisms using 1-l acid-leached polyethylene bottles. All the samples were taken at low tide with a tidal coefficient of 0.65. The recommendations in ‘‘Standard Methods for the Examination of Water and Wastewater’’ (APHA, 1998) were the basis for collecting and storing the samples. The analyses were made using filtered samples. The samples were filtered at the time of collection using a pre-conditioned vacuum filtering device and passed through a pre-washed ungridded membrane filter with a pore diameter of 0.45 lm (Whatman, cellulose nitrate). The filter and filter device were pre-conditioned in 1 M HNO3 and rinsed with deionised water. The metal analyses were carried out by means of atomic absorption spectrophotometry (AAS) using a double-beam Perkin-Elmer 2380 AAS with deuterium background correction. With the exception of As and Hg, the metals were analysed with two procedures contained in ‘‘Standard Methods for the Examination of Water and Wastewater’’ (APHA, 1998): 3111C

Table 1 Method detection limit (MDL) and spike recoveries in analysis of metals in barnacles MDL (mg kg 1) Spiked additions (% recovered*) *

Mean ± standard deviation.

As

Cd

Cu

Fe

Mn

Hg

Ni

Zn

0.4 92 ± 2

0.1 98 ± 4

1.5 101 ± 3

2 102 ± 5

1.0 101 ± 4

0.1 93 ± 2

0.2 96 ± 2

5 97 ± 4

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of the CRM were spiked with a known amount of metal spike standard, and one spike was analysed with the 3111C method and other with 3111B (APHA, 1998). The CRM and one spike were also analysed according to ISO 11969 for As and EN 1483 for Hg. The metal recoveries were usually in the range of 94–102%, indicating that accuracy was acceptable (Table 2). The accuracy of the results for pH and salinity was evaluated with a certified reference material (Radiometer Analytical). Precision was verified by replicate analysis. Values within 10% were obtained for the relative percent difference and were therefore considered acceptable. The sources of metal pollution were evaluated by analysing blanks, and the results were always below the methodsÕ detection limits (Table 2).

(extraction air–acetylene flame method) and 3111B (direct air–acetylene flame method). The former (3111C) was used to measure metals present at low concentrations and the latter to measure high metal concentrations (above the linear range of the first method). The hydride-generation technique was used to determine As, employing a Perkin-Elmer MSH-10 connected to a Perkin-Elmer 2380 spectrophotometer. The procedure described in ISO standard 11969 (International Standards Organization ISO 11969, 1996) was used for preparing and analysing the samples. The cold-vapour technique was used to test for Hg, employing a Perkin-Elmer MSH-10 connected to a Perkin-Elmer 2380 spectrophotometer. The procedure described in European Standard EN 1483 (European Standard EN 1483, 1997) was followed, using a solution of 3% NaBH4 in 1% NaOH as a reducing agent. Finally, pH and salinity were measured in situ using field equipment (ORION model 230-A and WTW model LF330, respectively). Our laboratory is accredited by the Spanish National Accreditation Association (accreditation no. 248/LE499, www.enac.es) to determine pH, test for metals in water and take samples of surface water. This organisation has signed multilateral mutual recognition agreements with numerous international accreditation bodies, such as European Cooperation for Accreditation (EA), International Laboratories Accreditation Cooperation (ILAC) and the International Accreditation Forum (IAF). Accreditation is a formal recognition that a laboratory is qualified to carry out specific calibrations or tests, or specific types of calibrations or tests. The accuracy of the results for metals was evaluated with both a certified reference material (CASS-3, National Research Council, Canada) and matrix spikes. The certified reference material (CRM) was analysed with the 3111C method (APHA, 1998). Two aliquots

2.4. Reagents The nitric acid and hydrogen peroxide used for barnacle digestion were Suprapur (Merck). All other reagents used for analysing barnacles and water were of analytical reagent grade (Merck). Stock solutions (Merck) of 1000 mg l 1 of the different elements analysed were used to prepare the calibration standards and spike solutions. Working mercury standard solutions were prepared just before use by appropriately diluting the stock standard solution with a stabilising solution consisting of potassium permanganate/nitric acid/sodium chloride hydroxylamine hydrochloride. The reducing solution used for hydride generation and mercury analysis (NaBH4 in NaOH solution) was obtained by dissolving NaOH pellets (for analysis, Merck) and NaBH4 (for analysis, Merck) in deionised water. This solution was freshly prepared prior to use. The deionised water used for dilutions and reagents was obtained with a Millipore-Q system. All glassware was soaked in nitric acid and rinsed with deionised water before use.

Table 2 Method detection limit (MDL) and results for analysis of certified reference water Cd 1

Cu **

MDL (lg l )

Fe

**

Mn **

Ni

**

Zn **

**

Asa

Hgb

0.3 15*

0.8 60*

0.8 90*

0.9 30*

0.8 130*

0.6 15*

0.5

0.1

CASS-3 Certified (lg l 1) Measured (lg l 1)

0.030 <0.3**

0.517 <0.8**

1.26 1.2**

2.51 2.5**

0.386 <0.8**

1.24 1.2**

1.09 1.0

n.a.c <0.1

Spike 1 of CASS-3 (% recoveredd )

99 ± 4**

102 ± 3**

97 ± 6**

95 ± 4**

101 ± 5**

98 ± 6**

94 ± 3

96 ± 4

d

Spike 2 of CASS-3 (% recovered ) * **

Method 3111B. Method 3111C. a ISO 11969. b EN 1483. c n.a.: Not available. d Mean ± standard deviation.

*

98 ± 2

*

101 ± 3

*

100 ± 2

*

98 ± 3

*

97 ± 4

*

102 ± 3

J. Morillo et al. / Chemosphere 58 (2005) 1421–1430

3. Results and discussion 3.1. Metal concentrations in waters The metal concentrations in the waters of the Huelva estuary (Tables 3 and 4) are very high as compared to other European estuaries (Nolting et al., 1999; Power et al., 1999; Monbet, 2004). This is not surprising, considering that the water of the Tinto and Odiel rivers, which flow into it, run through an area known for its mining activities throughout historical times. Mining activities have left a large quantity of tailings, from which metal-rich acidic leachate and eroded materials are washed into the rivers (Van Geen et al., 1997). As a result of all this, the waters of these rivers are acidic and have very high metal concentrations. The most polluted parts of the estuary are the Tinto and Odiel estuaries, especially the Tinto, where maxima for all metals were found (point T1). The lowest pH values were also found at this point: 6.9 in 2001 and 6.3 in 2002. This result makes sense if we take into account that mining activity has been more intense in the Tinto River catchment (Braungardt et al., 2003). As we go downstream along the two estuaries and along the Padre

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Santo Canal towards the sea, there is a great decrease in the metal levels due to seawater which, apart from the dilution effect (seawater has a low metal content), causes precipitation of large amounts of metals into the sediments as a result of the increased pH and salinity of the water. Tables 3 and 4 show the important role played by the waterÕs pH and salinity and how the metal concentrations decrease as pH and salinity increase. At point C6, the point nearest the sea, we found the highest pH values (8.1 in 2001 and 8.2 in 2002), the greatest salinity (33.5& in 2001 and 32.9& in 2002) and the lowest metal concentrations. It is worth noting that there were significant correlations between the pH levels and concentrations of all the metals studied, except for As, with a confidence level of p < 0.01. The correlations between salinity and the metal concentrations were significant, with a confidence level of p < 0.05, except for As and Fe. The correlation coefficients obtained were negative because an increase in pH and salinity causes a decrease in the metal concentrations. In the case of As, the concentrations downstream in the Odiel estuary (point O3) were greater than upstream (point O1); this may be due to the input of As from industrial plants located downstream from this point.

Table 3 Mean values for pH, salinity (&) and concentrations (lg l 1) of As, Cd and Cu in Huelva estuary waters pH

Salinity

As

Cd

Cu

Year 2001 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6

6.9 ± 0.9 7.2 ± 0.8 7.1 ± 0.6 7.3 ± 0.6 7.5 ± 0.5 7.7 ± 0.4 7.7 ± 0.4 7.8 ± 0.5 7.8 ± 0.4 8.0 ± 0.3 8.1 ± 0.1

28.1 ± 5.3 30.8 ± 3.4 26.1 ± 4.6 29.5 ± 4.5 30.9 ± 3.5 31.2 ± 3.1 31.7 ± 2.8 32.1 ± 2.5 32.3 ± 2.4 33.0 ± 2.1 33.5 ± 1.8

29 ± 5.0 7.9 ± 4.9 4.2 ± 5.1 6.3 ± 5.1 8.7 ± 4.8 11 ± 6 10 ± 6 11 ± 6 8.4 ± 5.1 7.4 ± 3.0 5.2 ± 2.0

14 ± 4.2 7.9 ± 3.8 8.4 ± 2.4 5.3 ± 2.0 2.8 ± 2.0 3.6 ± 2.2 3.6 ± 2.7 3.6 ± 2.1 2.9 ± 2.4 1.5 ± 1.9 1.0 ± 1.3

479 ± 300 210 ± 173 363 ± 237 112 ± 110 46 ± 57 62 ± 79 57 ± 71 82 ± 101 65 ± 91 34 ± 39 16 ± 14

Mean

7.6 ± 0.5

30.8 ± 3.3

9.9 ± 4.9

5.0 ± 2.5

139 ± 116

Year 2002 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6

6.3 ± 1.1 7.5 ± 0.7 7.4 ± 0.5 7.5 ± 0.5 7.7 ± 0.4 7.9 ± 0.2 8.0 ± 0.2 8.0 ± 0.2 8.0 ± 0.1 8.1 ± 0.1 8.2 ± 0.1

25.0 ± 11 28.7 ± 7.3 23.9 ± 10 26.6 ± 9.9 29.1 ± 7.3 29.6 ± 6.9 30.1 ± 6.7 30.6 ± 6.0 30.9 ± 5.9 31.7 ± 5.1 32.9 ± 3.7

40 ± 10 10 ± 7.3 9.0 ± 7.9 7.8 ± 6.3 12 ± 9 15 ± 5 14 ± 4 13 ± 4 13 ± 4 12 ± 3 7.0 ± 1.2

9.8 ± 4.3 5.4 ± 3.5 5.2 ± 1.8 3.8 ± 1.7 3.4 ± 2.0 2.2 ± 0.8 2.1 ± 0.7 2.1 ± 1.0 1.5 ± 0.5 1.2 ± 0.5 0.7 ± 0.4

240 ± 235 115 ± 169 60 ± 22 52 ± 28 40 ± 26 24 ± 9 21 ± 4 23 ± 6 17 ± 4 13 ± 2 9.0 ± 3.2

Mean

7.7 ± 0.4

29.0 ± 7.3

14 ± 5.5

3.4 ± 1.6

56 ± 46

Mean ± standard deviation (4 sampling exercises per year).

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Table 4 Mean concentrations of Fe, Mn, Hg, Ni and Zn (lg l 1) in Huelva estuary waters

Year 2001 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6 Mean Year 2002 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6 Mean

Fe

Mn

Hg

Ni

Zn

368 ± 220 94 ± 44 115 ± 109 58 ± 43 24 ± 19 38 ± 17 38 ± 22 61 ± 30 51 ± 32 48 ± 24 36 ± 12

1114 ± 600 365 ± 228 1013 ± 528 534 ± 230 242 ± 259 152 ± 150 153 ± 187 147 ± 195 141 ± 196 99 ± 157 60 ± 105

<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

18 ± 13 7.5 ± 6.0 17 ± 12 8.2 ± 4.2 3.1 ± 3.6 4.6 ± 2.7 4.1 ± 3.4 4.0 ± 3.3 4.0 ± 3.2 2.5 ± 1.9 1.5 ± 1.0

2250 ± 2000 935 ± 826 1816 ± 1104 640 ± 540 330 ± 340 338 ± 418 342 ± 438 358 ± 463 291 ± 383 184 ± 252 106 ± 143

85 ± 52

365 ± 258

<0.1

6.8 ± 4.9

690 ± 628

257 ± 104 66 ± 75 103 ± 65 73 ± 46 68 ± 56 71 ± 61 39 ± 24 72 ± 49 33 ± 31 31 ± 15 40 ± 13

670 ± 325 250 ± 232 364 ± 216 210 ± 196 170 ± 187 55 ± 31 52 ± 24 51 ± 39 35 ± 29 28 ± 25 8±7

<0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1

23 ± 15 9.8 ± 14 9.0 ± 7.0 6.3 ± 6.2 5.4 ± 4.6 3.0 ± 2.1 2.0 ± 0.9 1.9 ± 0.9 1.3 ± 0.5 1.4 ± 0.9 1.0 ± 0.6

1125 ± 469 512 ± 462 540 ± 299 320 ± 310 282 ± 276 112 ± 73 112 ± 80 126 ± 97 86 ± 59 53 ± 30 38 ± 16

78 ± 49

172 ± 119

<0.1

5.8 ± 4.8

301 ± 197

Mean ± standard deviation (4 sampling exercises per year).

We found that important changes take place in metal levels in samples taken throughout each year; the concentrations of most metals in the samples obtained in autumn and winter are higher than in spring and summer (see Table 5), which can be explained by weather conditions. In summer the oxidation of pyritic materials as a result of bacterial activity produces a large number of highly soluble metal-rich Fe sulphates and ochre deposits. In autumn and winter rains dissolve these soluble materials and carry them to the rivers, which causes a pronounced increase in the metal levels in the Tinto and Odiel River waters. However, a prolonged period of rain in winter causes a decrease in metal concentrations since the levels of both rivers rise notably, diluting the metals. What is more, the amount of highly soluble material that forms during the dry period also decreases. The metal with the highest concentration is Zn, which reached mean values of 690 lg l 1 in 2001 and 301 lg l 1 in 2002. This is not surprising, given that the pyritic minerals in this area have a high concentration of Zn (Van Geen et al., 1997). The Zn levels in other estuaries in Europe are considerably lower than those found in Huelva; for example, in a study carried out by Power et al. (1999) in the Thames estuary, there

Table 5 Mean metal concentrations (lg l 1) in the four seasons (2001 and 2002) Season

Winter Spring Summer Autumn

Element As

Cd

Cu

Fe

Mn

Ni

Zn

6.8 13 16 12

5.4 3.5 3.4 4.3

140 57 87 105

84 55 88 98

397 170 240 267

8.7 3.6 6.0 6.9

692 270 492 526

was a mean Zn concentration of 29 lg l 1 and Chiffoleau et al. (1999) reported a maximum value of 10 lg l 1 for Zn in the Seine estuary. The metals that present the highest values apart from Zn are Fe, Cu and Mn. The minerals of the Iberian Pyrite Belt are the main source of Fe and Cu, while Mn (which presents a low concentration in pyritic materials) probably comes from leaching of the ground by the acidic waters generated in the oxidation of pyritic materials. That Fe is not the most plentiful element in the estuary waters, as one might expect given that it is the most abundant metal in the minerals of the area (Van

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3.2. Metal concentrations in Balanus balanoides tissues

Geen et al., 1997), is probably because a good deal of it precipitates at below-neutral pH, i.e. before reaching the estuary. Iron precipitation has been observed at low pH in other mine-polluted systems (Bigham et al., 1990). The mean concentrations of As (9.9 lg l 1 in 2001 and 14 lg l 1 in 2002) are high compared to those obtained in other estuaries that also flow into the Gulf of Cadiz, like the Guadiana, which contains 2.2 lg l 1 of As, and the Guadalquivir, with 1.8 lg l 1 (Usero et al., 2000). The mean concentrations of Cd (5.0 lg l 1 in 2001 and 3.4 lg l 1 in 2002) are also greater than those reported by Usero et al. (2000) in nearby estuaries and in other European estuaries, where the Cd values are less than 0.5 lg l 1 (Power et al., 1999). For Ni, we found mean concentrations (6.8 lg l 1 in 2001 and 5.8 lg l 1 in 2002) similar to those from other estuaries in the Gulf of Cadiz, such as the Guadalquivir (4.2 lg l 1) and the Guadalete (4.0 lg l 1) (Usero et al., 2000). By contrast, Hg levels were always lower than the detection limit of the analytical method used (0.1 lg l 1). Such concentrations for Hg are normal in the estuaries that empty into the Gulf of Cadiz. Based on what we have seen here, we can conclude that there is no significant Ni or Hg pollution in the Huelva estuary.

The metal concentrations in balanoides tissues (Table 6) are, in general, very high in comparison to those measured in other barnacle species (Blackmore, 2001; Pa´ez-Osuna et al., 1999; Rainbow and Blackmore, 2001), undoubtedly due to the high metal pollution of the water where it lives. It is important to note that metal concentrations in Balanus balanoides tissues behave similarly to those in waters. They show the highest values in the two estuaries, especially the Tinto, and decrease as they flow downstream, through Padre Santo Canal and toward the sea. Zn is the element that presents the highest mean concentrations (54.6 g kg 1 in 2001 and 29.9 g kg 1 in 2002). Walker et al. (1975) identified metal-rich granules around the mid-gut of Balanus balanoides and showed that this species has the ability to accumulate large quantities of Zn. A high level of Zn in comparison with other elements is typical in barnacles (Fialkowsky and Newman, 1998; Pa´ez-Osuna et al., 1999) and in other living organisms habitually used as pollution biomonitors (Castro et al., 1999; Jeng et al., 2000; Wong et al., 2000).

Table 6 Metal concentrations (mg kg 1, dry mass) in Balanus balanoides soft parts As

Cd

Cu

Fe

Mn

Hg

Ni

Zn 101 000 43 000 98 500 48 000 48 400 46 900 46 200 72 000 33 800 33 700 28 800

Year 2001 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6

78 48 73 56 36 35 52 60 74 58 42

330 160 293 168 153 158 140 152 99 76 40

21 800 8250 20 300 11 200 10 000 11 000 11 200 15 300 11 600 7750 6240

12 400 5300 10 200 5300 2800 2450 3160 5100 5560 2650 2320

816 545 680 304 153 166 170 269 433 148 135

6.5 1.2 5.2 2.0 1.9 1.6 2.0 1.2 2.0 1.6 1.0

15 9.1 16 8 5.9 7.6 9.4 12 14 6.8 5.3

Mean

56

161

12 200

5200

347

2.4

10

54 600

124 58 153 62 43 46 73 78 104 81 59

231 96 153 84 72 110 84 94 60 49 24

14 000 8640 9430 6680 5290 5500 6840 7180 5800 3100 2496

13 700 4770 11 200 5830 2520 2940 3480 5610 5000 2900 2020

452 416 390 152 108 115 85 108 96 98 88

4.6 1.1 3.1 1.0 0.95 0.96 1.6 0.84 1.8 1.4 0.82

18 8.2 12 6.5 4.7 6.1 6.6 8.4 12 4.8 4.1

70 700 27 200 39 400 28 800 19 360 23 450 27 720 43 200 20 280 16 850 11 520

80

96

6810

5450

192

1.7

8.3

29 900

Year 2002 T1 T2 O1 O2 O3 C1 C2 C3 C4 C5 C6 Mean

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In decreasing order, Zn concentrations were followed by Cu (12 200 mg kg 1 in 2001 and 6810 mg kg 1 in 2002) and Fe (5200 mg kg 1 in 2001 and 5450 mg kg 1 in 2002). In other barnacle studies, the Cu and Fe levels are also lower than the Zn levels. In Balanus amphitrite captured in a polluted area on the coast of Xiamen, China, Blackmore et al. (1998) found a Zn concentration of 10 000 mg kg 1, followed by Fe (3126 mg kg 1) and Cu (2204 mg kg 1). In Mission Bay (Salton Sea, California) the barnacle used by Fialkowsky and Newman (1998) had a mean concentration of Zn (37 900 mg kg 1), far greater than that of Cu (3750 mg kg 1) and Fe (720 mg kg 1), similar to what we found in this study. The rest of the metals studied in the Balanus balanoides pooled samples present mean concentrations considerably lower than those observed for Zn, Cu and Fe. If we list the remaining metals in decreasing order of mean concentrations, the first is Mn, which has mean values (347 mg kg 1 in 2001 and 192 mg kg 1 in 2002) similar to those obtained by Rainbow et al. (2000) in Balanus improvisus in the Gulf of Gdansk (Baltic Sea), with values between 187 and 307 mg kg 1. Next is Cd, which reaches mean levels in the Huelva estuary (161 mg kg 1 in 2001 and 96 in 2002) that are notably higher than those found by Rainbow et al. (2000), between 18.7 and 23.6 mg kg 1. Then comes As, with mean concentrations of 56 mg kg 1 in 2001 and 80 mg kg 1 in 2002. The Ni concentrations (10 mg kg 1 in 2001 and 8.3 mg kg 1 in 2002) were the lowest in the Balanus balanoides tissues, along with Hg (2.4 mg kg 1 in 2001 and 1.7 mg kg 1 in 2002). These values are comparable to those obtained in other studies done using barnacles (Fialkowsky and Newman, 1998; Pa´ez-Osuna et al., 1999; Rainbow et al., 2000). 3.3. Bioconcentrations of metals To evaluate the efficiency of metal accumulation in Balanus balanoides soft parts, we calculated the accumulation factor, defined as the ratio of the metal concentration in the organism to that in the water (Fig. 2). The metals that this organism accumulates in the greatest amounts in its body are Cu, with an average factor of 88 in 2001 and 122 in 2002, and Zn, with a factor of 79 in 2001 and 99 in 2002. The accumula-

1000 88 122

100

79 99

61 71

32 28 5.6 5.7

10 1

1.0 1.1

1.5 1.4

Mn

Ni

0.1 As

Cd

Cu

Fe 2001

Zn

2002

Fig. 2. Mean values of the accumulation factors (metal concentration in Balanus balanoides/metal concentration in water).

tion factors for Fe (61 in 2001 and 71 in 2002) and Cd (32 in 2001 and 28 in 2002) are also high. Ni and Mn are the metals that Balanus balanoides accumulates least (with values near one), which may by due to lesser bioavailability of these metals as compared to the rest. If we compare the two years studied, in 2002 the accumulation factors were greater for all of the metals except Cd and Ni. 3.4. Metal correlation between organisms and water There is a significant correlation (p < 0.01) for concentrations of Cd, Cu, Fe, Mn, Ni and Zn in Balanus balanoides relative to their concentrations in water (Table 7). The correlation coefficient obtained for As was positive but it had a low confidence level (p > 0.05). The correlation coefficient was not calculated for Hg since this metal presents concentrations in the water below the detection limit for the method of analysis employed. It is interesting to note that, using Balanus balanoides, we can conclude that the areas with the highest levels of Hg are the Tinto and Odiel estuaries, particularly the Tinto estuary (Table 6). It would not have been possible to reach this conclusion had the analysis been based on the waters themselves, since the values fall below the detection limit, as indicated above. Fig. 3 shows the relation between metal concentrations in Balanus balanoides and water based on a linear regression model for the year 2002 (the results for 2001

Table 7 Correlation coefficients between metal concentrations in water and in Balanus balanoides tissues Year

Element As

2001 2002 *

p < 0.01.

0.378 0.370

Cd

Cu *

0.911 0.906*

Fe *

0.836 0.881*

Mn *

0.862 0.854*

Ni *

0.874 0.875*

Zn *

0.742 0.771*

0.877* 0.840*

J. Morillo et al. / Chemosphere 58 (2005) 1421–1430 Cd B. balanoides

1429

Cu B. balanoides

250

16000

200

12000 y = 41 x + 4540

150 8000

y = 19 x + 30

100 4000

50

0

0 0

5

10

0

15

100

Cd water

200

300

Cu water

Fe B. balanoides

Mn B. balanoides 600

16000

500 12000

400 y = 50 x + 1557

300

8000

y = 0.65 x + 80.47

200 4000 100 0

0 0

100

200

300

0

200

400

600

800

Mn water

Fe water

Zn B. balanoides

Ni B. balanoides 20

80000

15

60000 y = 0.50 x + 5.41

y = 42 x + 17123

10

40000

5

20000

0

0 0

5

10

15

20

25

0

500

1000

Fig. 3. Linear regression between metal concentrations in Balanus balanoides tissues (mg kg

are similar). The metals with the greatest regression line slopes are Fe, Zn, Cu and Cd, with values of 50, 42, 41 and 19, respectively. The lowest slopes are obtained for Mn, with a value of 0.65, and Ni, with 0.50. If one metal has a greater slope than another then, given the same increase in their concentrations in the water, there will be a greater increase of that metal in Balanus balanoides. We should note that the metals with the greatest slopes (Fe, Zn, Cu and Cd) are those that have the highest accumulation factors (see Fig. 2). Based on the results of this study, we can conclude that Balanus balanoides is a good tool for monitoring trace metals. It is also very useful in areas like the Huelva estuary where, because of their high pollution levels, it is difficult to find other organisms that can be used as biomonitors.

1

1500

dry mass) and in waters (lg l 1).

Acknowledgments This research was supported by the Environmental Council of the Junta de Andalucı´a (the Andalusian governing body), Spain. References APHA (American Public Health Association), AWWA (American Water Works Association), WEF (Water Environment Federation), 1998. Standard methods for the examination of water and wastewater. 20th ed., Washington, DC. Bigham, J.M., Schwertman, U., Carlson, L., Murad, E., 1990. A poorly crystallized oxyhydroxysulfate of iron formed by bacterial oxidation of Fe(II) in acid mine waters. Geochim. Cosmochim. Acta 54, 2743–2758.

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