In Collaboration with the Netherlands Institute for Sea Research
URNAL
‘SEA ELSEVIER
Journal of Sea Research
OF RESEARCE
38 (1997) 35-45
Heavy metals in Diadema setosum (Echinodermata, Singapore coral reefs
Echinoidea) from
P. Flammang a,*, M. Warnau b, A. Temara b, D.J.W. Lane ‘, M. Jangoux a,b aLaboratoire de Biologie marine, UniversitP de Mons-Hainaut, 19 av. Maistriuu B-7000, Mom, Belgium b Laboratoire de Biologie marine (CP 160-15), Universite’Libre de Bruxelles, 50 av. ED. Roosevelt B-1050, Bruxelles, Belgium L’ School ofBiological Sciences, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119260, Singapore Received
13 January
1997; accepted
2 July 1997
Abstract Concentrations of Zn, Pb, Cd, Fe, Cr, Cu and Ti were measured in body compartments of the echinoid Diadema setosum and in the silt fraction of surface sediment from eight coral reefs in Singapore coastal waters. Metal concentrations in the echinoid varied with the body compartment analysed and with the site of sampling. Amongst the body compartments studied, Zn, Cd, Fe, Cr and Cu were preferentially accumulated in the gonads, whereas Pb and Ti were accumulated mainly in the calcified body compartments. The concentrations of most metals differed according to the reef sampled. A decreasing north-south gradient of metal concentrations was observed in D. setosum populations, correlating to the distance from Singapore. A similar gradient was observed for metal concentrations in sediment. It is suggested that D. setosum could be a valuable bioindicator for assessing heavy metal contamination in coral reef ecosystems of the Indo-West Pacific.
Keywords: heavy metals; coral reef ecosystem;
Diadema setosum; Singapore
1. Introduction Coral reef ecosystems are recognized as being particularly threatened by pollutants (Howard and Brown, 1984). In Singapore, almost all reefs are found around the Southern Islands, south of the mainland (Chou and Chia, 1991), where port and shipping facilities and the various petroleum industries are also centred (Ho, 1992). These industries are likely to release pollutants such as heavy metals and hydrocarbons into the marine environment (Howard and Brown, 1984; Augier et al., 1989). However, published data *Corresponding author:
[email protected]
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373434;
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1385-l 101/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIZ S1385-1 101(97)00033-6
of the concentrations of pollutants in the marine environment around Singapore are rather sparse. Recent studies have demonstrated that echinoids are valuable bioindicators of heavy metal contamination (Augier et al., 1989; Ablanedo et al., 1990; Warnau et al., 1995a,b,c,d) and can be used to ascertain spatial and temporal trends in heavy metal abundance and bioavailability. It has been shown that, in these organisms, metal concentrations differ according to whether the body compartment considered is calcified (body wall, spines, skeleton, Aristotle’s lantern) or non-calcified (digestive wall, gonads) (Warnau et al., 1995b,d). Any study using echinoids as bioindicators should thus investigate at least one compartment of each category.
36
P. Flammang et al. /Journal of Sea Research 38 (1997) 35-45
The aim of the present work was to assess the global status of heavy metal contamination in the Southern Islands area, using the echinoid Diadema setosum as a bioindicator species. Indeed, D. setosum is one of the most abundant and ubiquitous benthic species on Singapore coral reefs (Lim and Chou, 1988; Grignard et al., 1997). Among body compartments, one non-calcified (gonads) and three calcified compartments (body wall, spines and skeleton) were considered for metal analyses. For comparative assessment, heavy metal concentrations were also investigated in surface sediments. 2. Material and methods
2.1. Sampling and preparation
of samples
Diadema setosum was sampled in April 1994 from eight stations located to the south of the Singapore mainland (Fig. 1). At each station, 10 large echinoids (ambital diameter 2 70 mm; see Wamau et al., 1995d) and 5 samples of surface sediment (top 1 cm) were collected by SCUBA divers between 1 and 5 m depth. The stations were selected to approximately follow two perpendicular transect lines: Cyrene reefs (Station l), Pulau Semakau west side (Station 2), and Raffles Lighthouse (Station 3) from north to south; and Sultan Shoal Lighthouse (Station 4), Terumbu Pempang Laut (Station 5), Pulau Semakau east side (Station 6), Pulau Jong (Station 7), and St John’s Island (Station 8) from west to east (Fig. 1). After collection, the echinoids were kept in sea water from the sampling site and dissected directly upon arrival at the laboratory (i.e. never more than 5 h after collection). Three body compartments were separated: the gonads, the spines and the body wall (i.e. the integument without the spines). A fraction of each body wall sample (ca. 20%) was further cleansed of non-calcified tissues using a 0.1% (w/v) proteinase N (Serva) solution (Dubois and Jangoux, 1985), thus isolating a fourth compartment: the skeleton. A small portion of gonad from each echinoid was collected and a smear was made on a glass slide for microscopic examination to determine the sex. All samples were then dried at 100°C for 48 h, weighed (DW) and stored in hermetic polyethylene containers until analysis.
Sediment samples were dried at 100°C for 48 h and sieved. The silt fraction (~50 pm, Luoma, 1990) was collected and analysed for metals. 2.2. Metal analysis Samples were prepared and metals were analysed according to the methods described in Warnau et al. (1995b). Briefly, a known amount (usually 0.5 g DW) of each echinoid sample (gonads, spines, body wall and skeleton) was digested with 1 ml of 65% HNOs (Merck, p.a.) per g DW. This digestion was carried out successively at 20, 40, 60 and 80°C for 24, 24, 12 and 12h, respectively. Digests were diluted to 12 ml with milli-Q water (Millipore) and filtered on Whatman GF/A glass microfiber filters. For sediment samples, about 0.2 g DW of the silt fraction was digested with 2 ml HNOs using the same protocol, and diluted to 10 ml before filtration. The concentrations of zinc (Zn), lead (Pb), cadmium (Cd), iron (Fe), chromium (Cr), copper (Cu,) and titanium (Ti) were measured by atomic emission spectrometry using a Jobin-Yvon 38+ ICPS, taking into account matrix corrections. Accuracy of the metal analysis methodology was checked by processing a certified reference material, Myths edulis tissue (CRM no. 278, Community Bureau of Reference), along with the experimental samples (Table 1). 2.3. Data analysis Mean metal concentrations were computed taking into account all measurements, i.e. data below the detection limits were included as half the value of the detection limit (Black, 1991). Detection limits for Zn, Pb, Cd, Fe, Cr, Cu and Ti were 2, 14, 1, 4, 1, 2 and 1 pg of metal per litre of solution, respectively. Comparisons between heavy metal concentrations were performed using l- or 2-way analysis of variance (ANOVA) followed by the multiple comparison test of Tukey (Zar, 1984). The variability explained by each factor is derived from the sum of squares. Simple linear regressions were used to test the relationships between the concentrations of a given metal in different body compartments and between the concentrations of different metals in a given body compartment or in the sediment (Zar, 1984). The level of significance was set at a! = 0.05.
P. Flammang et al. /Journal of Sea Research 38 (1997) 35-45
SINGAPORE
0
Fig.
Table 1 Certified and measured Reference)
31
*,.a
1.Map of Singapore Mainland and Southern Islands, showing the eight sampling stations (l-8).
metal concentrations
Certified (mean value f 95% ci.) Measured (min. and max. n = 6) Values shown in parentheses
(kg
gg ’ DW) in the certified
reference
material
(CRM
278, Community
Bureau
of
Zn
Pb
Cd
Fe
Cr
cu
Ti
76 zk 2 77-82
1.91 kO.04 1.91-1.95
0.34 f 0.02 0.31-0.37
133&4 126-161
0.8 zk 0.08 0.87-1.04
9.60 zk 0.16 9.60-10.71
(2 f 0.2) 2.8-3.8
are not certified.
3. Results 3.1. Metal distribution in the body compartments Diadema setosum, and in sediment
of
The mean concentrations of the metals measured in the gonads, spines and body wall of D. setosum from the reefs sampled are presented in Table 2. Concentrations in the skeleton of D. setosum and in the silt fraction of surface sediment are presented
in Table 3. The mean metal concentrations in D. setosum ranged between 4 and 300 ug gg’ DW for Zn; 1 and 7 pg gg’ for Pb; 0.3 and 11 pg g-’ for Cd; 10 and 300 ug gg’ for Fe; 0.3 and 5 pg g-’ for Cr; 0.2 and 8 ug g-t for Cu; and 0.01 and 0.5 ug gg’ for Ti. Mean metal concentrations were a factor higher in the silt fraction of the sediment (up to 20 mg gg’ DW) than in the echinoids, except for Zn and Cd. The whole dataset for echinoid metal concentrations was analysed using 2-way ANOVA (Table 4).
B 14.69 k 2.33a A3.75 k 0.93ab Bl.16f0.46a A8l +=46a AB1.09 k 0.63a A0.66 + 0.27= A0.42 f 0.1 6a
‘12.33 f 1.71= A2.82 f 0.92b B0.96 f 0.3a *48 f 25a B0.65 f 0.25a AB0.49 f 0.44” A0.15 f 0.08 (l)b
84.35 ??0.8V A2.11 f 0.67b ‘0.36 f 0.06a B12&5a 80.39 f 0.09a *0.32 * 0.1 la AO. 14 It 0.05a
A205 f 10la ‘0.81 f 0.24a ‘410.15 f4.51a A228 f 18V A2.79 + 1.55b A3.97 f 0.42b A0.03 & 0.03 (4)b
3
line
A14.67 f 2.ga A2.67 f O.ga AB2.16*0.75” n45 f 21” B0.57 f 0.16a ‘0.58 f 0.44a A0.17 f 0.1 I (l)a
A4.64 f 3a AB2.1 f 0.35a B0.3 f 0.07a *20 f 6a *0.28 &0.22a 80.22 f 0.22” A0.03 f 0.03 (6)”
A 169 & 177a B 1.04 f 0.35a A5.86 f 5.98” A205 f 140ab A2.53 f 2.6gab A3.35 f 1.53ab A0.06 & 0.08 (4)a
4
*12.39f 1.12” A2.15 k 0.71= Bo.55 f 0.09a %4*41a Bl.11 jI0.95a B0.95 *0.56a A0.16 f 0.07”
*4.2 ik l.31a AB1.85 f OHa Bo.3 f 0.05a B20*8a *0.32 &0.09a no.31 f 0.14a AO. 11 k 0.05a
-
transect line
A290 f 303a B 1.04 f 0.43” A6.48 f 1.75a A285 f 229a A3.61 * 2. 12a A4.47 f 0.93a A0.03 & 0.06 (5)a
5
Stations along the west-east
f 177a ??0.29” k 3.5a k 186ab f l.llab f 0.45b It 0.21 (5)a
A12.83 f l.51a A2.48 k 0.67a A0.5 1 f O.Oga AB79 xt 49a AB0.74 f 0.25a 80.31 f 0.22a A0.18&0.12a
A5.67 *3.39a A2.55 5 0.5ga A0.25 * OIkta B33f lla B0.36 f 0.13” *0.3 1.rt 0.22a AO. 12 5 0.07a
Al75 B0.78 A4.49 A202 A2.13 A2.91 A0.13
6
coral reefs
‘13.83 & l.39a A2.85 f 0.7ga B0.63 k 0.26a A25 & 16a A0.5 f 0. 14a 80.54 * 0.43” AO.l7+0.14(l)a
‘6.51 f 2.77” A2.84 k 1 .05a B0.33 f 0.12” A25 f lOa A0.48 f 0.24” a0.33 f 0.13” Aa0.14*o.la
*307 k 369a ‘0.94 f 0.2ga A8.61 f 11.51a A88 f 77b Al .39 f 0.99b A4.76 =k 3.05” ‘0.02 + 0.03 (6)a
I
A15.14&2.18” AB2.6 f 0.95” A 1.32 k 0.42a A32 f- 17” AB0.48 f 0. 15a * 1.29 ??0.66a A0.07 * 0.07a
A4.02 & 1.38 A2.79 & 1 .2a A0.34 f 0.09” Al4f4a B0.33 f 0.23a B0.27f0.11a A0.09 f 0.06 (l)a
Al29 * 162a ‘1.34f0.66” A5.03 & 1.87” A120f90b A2 f 0.716 A4.37 f 1.1 l”b AO.Ol f 0.01 (8)a
8
Numbers in parentheses indicate the number of points under the detection limits. Differences between the mean concentrations are indicated by superscripts; means sharing the same superscript are not significantly different from each other (Jq&y>O.O5). Upper case superscripts indicate differences between body compartments (read vertically): lower case superscripts indicate differences between sampling stations (read horizontally).
C. Body wall Zn B14.56 + 2.4a Pb A4.24 k 1.3” Cd nl.l7*0.49” Fe B62 f 37a Cr *0.98 k 0.4a cu ‘1.65 f 1.28” Ti A0.41 * 0.199
0.14” 0.31” O.OV
1 14a 0.4a 2.96b 83” l.14b 0.83b 0.07 (6)b
k 3.19a f 0.81ab *0.14a 7a =k 0. la xk 0.84a & 0.06 (l)a
B7.12 A3.29 60.41 A28 f ‘0.48 A0.73 B0.13
B. Spines Zn B5.62 f Pb A4.33 f Cd *0.55 + Fe B19*7a Cr 80.54 It CU B0.67 f Ti A0.22 f
0.81a 0.92= 0. 16a
Al65 f a 1.22 f A6.76 * A153 f A2.75 f A4.07 f ‘0.05 f
2
A. Gonads Zn A229 L!=267’ Pb Bl.86f0.65a Cd ‘411.34*4.45a Fe A243 f 176a Cr A4.83 f 3.~57~ CU A7.73 & 7.1sa Ti A0.28 f 0.34 (2)a
1
transect
(Kg g-’ DW, *SD, n = 10) of heavy metals in the tissues of Diuderna setosum from Singapore
Stations along the north-south
Table 2 Mean concentrations
? h
g ;r, z cc 2 % 2
F
$
a f %
2 P
P 3n 00 % F
?u 2
line
64f 13b 36 zlz5b 0.81 f 0.25ab 13731 z!z 1 162b 114&74a 97 It 48= 3.84 & 1.53b
5.26 + 0.93a 5.29 zlz0.7gb 0.53 z!z0.08a 32 LII42a 2.14f 1.4” 0.64 zt 0.29b 0.32 I!=0.1”
3
0.195 0.039 0.18 0.783 0.704 0.01 0.052
0.001 0.042 0.036 0.65) and of Zn, Pb and Cd on the west-east transect line (p > 0.15). However, the geographical variations were generally of low amplitude. The Tukey multiple comparison test revealed significant differences between sites in the gonads but not in the spines or the body wall for Cd, Fe, Cr and Cu; in the spines and the body wall but not in the gonads for Pb; and in the gonads and the body wall but not in the spines for Ti (Table 2). In the skeleton, significant differences between metal concentrations measured in the different sampling stations were detected for Pb and Cu on the north-south transect line and for Cd, Fe, Cu and Ti on the west-east transect line (one-way ANOVA, Table 3). On the north-south transect line, station 1 was always the station where the highest concentrations of Pb, Cd, Cr, Cu and Ti were observed (Tables 2 and 3A), the concentrations of these metals decreasing towards station 3. On the west-east transect line, the geographical variations in metal concentrations did not follow such a clear gradient. However, it appeared that station 4 was the most contaminated by Cd; station 5 by Fe and Cu: and station 7 by Cr, Cu and Ti. Except for Cr and Cu, the metal concentrations measured in the silt fraction of surface sediment also varied between stations (one-way ANOVA, Ta-
I? Flammang et al. /Journal of Sea Research 38 (1997) 35-45
42 15 I
y=o.13x_1.41;
r’=0.5;
p=0.0001
0
.g
m
.:
I
.
0 /
/ 0
0
20 Pb
100
conce?i~atitions cg g -’ I$
Fig. 4. Relationship between Ti and Pb concentrations fraction of surface sediment, all stations together.
in the silt
ble 3B). On the north-south transect line, there were clear differences between the sampling stations: station 1 exhibited highest concentrations of Pb, Cd and Ti, whereas station 2 was the most contaminated by Zn and Fe. On the west-east transect line, on the other hand, such differences were of low amplitude; only for Fe was one station (station 7) significantly more contaminated than the others. Note that, like in the echinoids, there was a significant relationship between the concentrations of Pb and Ti in the different sediment samples (Fig. 4). 4. Discussion The present study aimed at establishing a baseline for heavy metal contamination in a coral reef species, the echinoid Diadema setosum, from the Southern Islands off Singapore. Both the body compartment analysed and the sampling station affected metal concentrations in the echinoid, although the former factor was the more important. All the metals studied were found to be selectively distributed among the body compartments of D. setosum. The highest concentrations of Zn, Cd, Fe, Cr and Cu were measured in the gonads. Zn, Fe, Cr and Cu are biologically essential elements (George, 1990), and the elevated concentrations recorded in the gonads could be related to the high metabolic activity of this organ (Hori et al., 1987). Although Cd is not an essential metal, its concentrations followed the same pattern, possibly because this metal
binds to similar ligands as those bound by Zn and Cu (Sorensen, 1991). It has been demonstrated that metallothioneins (which are present in echinoderms) are used in the storage of essential metals, but can also bind other metals (including Cd) with a strong affinity (Nemer et al., 1984; Hamer, 1986; Temara et al., 1997). The highest concentrations of Pb were measured in the spines, the body wall and the skeleton. Similar observations have been reported for several other echinoderm species (e.g. Lawrence et al., 1993; Warnau et al., 1995a,d; Temara et al., 1996). It is known that Pbf+ can either adsorb onto the surface of the carbonate skeleton (Temara et al., 1995) or replace Ca++ and/or Mg++ tons in the crystal lattice (Beeby, 1991; Sorensen, 1991). In the present study, concentrations of Ti were only rarely significantly different from one body compartment to another, but the highest concentrations were generally observed in the skeleton. Comprehensive data on heavy metal concentrations in the body compartments of echinoids are scarce in the literature, although data are available for Echinus esculentus from the Irish Sea, Paracentrotus lividus from the Mediterranean Sea, and Echinometra lucunter from the Gulf of Mexico (Table 5). The data presented in this table confirm that, whatever the species investigated, heavy metals are similarly distributed in the body compartments of echinoids. Concentrations of Zn, Fe and Cu are always higher (up to one order of magnitude) in the gonads than in the calcified compartments while concentrations of Pb are always higher in the calcified compartments. No significant difference was found between male and female individuals of D. setosum for any of the metals in any of the body compartments, and this differs from the situation in asteroids (Den Besten et al., 1991). The only recurrent significant relationships between the concentrations of two metals in the body compartments of D. setosum were found for Pb and Ti in the body wall, the spines and the skeleton. A similar relationship was also observed in the sediment samples. This proportionality between Pb and Ti concentrations in the echinoids and the sediments is probably an indication that these metals are derived from common sources.
l? Flammang
Table 5 Heavy metal concentrations Species
et al. /Journal
in the body compartments Body compartment
Oral body wall Aboral body wall Spines Gonads
Paracentrotus
Body wall
lividus
110 12 28 110
Gonads
Echinometra
Integument
lucunter
Diadema setosum
124 161
Integument
8.21 12.2 230 436
Body wall Spines Skeleton Gonads
Integument
3.84 5.21
Gonads
Gonads
Pb
