Ecotoxicology and Environmental Safety 51, 28}34 (2002) Environmental Research, Section B doi:10.1006/eesa.2001.2114, available online at http://www.idealibrary.

com on

Toxicity of Bauxite Manufacturing By-products in Sea Urchin Embryos

Giovanni Pagano,* Sureyya Merii,- Antonella De Biase,* Mario laccarino,* Domenico Petruzzelli,? K Olcay Tunay,- and Michel WarnauA K
*Istituto Nazionale per lo Studio e la Cura dei Tumori, Fondazione Pascale, I-80131 Naples, Italy; -Istanbul Technical University, Environmental Engineering Department, Maslak 80600 Istanbul, Turkey; ?Department of Civil and Environmental Engineering, Polytechnic University of Bari, 1-70125 Bari, Italy; and AInternational Atomic Energy Agency, Marine Environment Laboratory, MC-98012 Monaco Received January 18, 2001

INTRODUCTION

By-products from a bauxite manufacturing plant located in Seydisehir, Turkey, were investigated for their composition and : any toxicity to sea urchin embryogenesis. Samples from three other bauxite plants located in France, Greece, and Italy were simultaneously tested for toxicity in sea urchin embryos. Samples included sludge and solid residues in the plant and sediment and water columns from two holding ponds (red sludge or cryolite residues). Samples were analyzed for their inorganic content by inductively coupled plasma optical emission spectroscopy (ICP-OES). Analyses were carried out either following strong acid extraction or after release of soluble components from seawater-suspended pellets. Toxicity was tested by sea urchin bioassays, to evaluate the following endpoints: (a) acute and/or developmental toxicity, (b) changes in fertilization success, and (c) transmissible damage from sperm to o4spring. The results revealed the following: (1) inorganic analysis, following strong acid extraction, showed a prevalence of Al and Fe; (2) seawater release of soluble contaminants was con5ned to Fe and Mn, whereas Al levels were not changed by suspending increasing sample amounts in seawater; (3) the most severe toxicity to sea urchin embryos was exerted by a 2% water column from the red sludge holding pond and by soil and sludge collected near the plant reactor; (4) sludge supernatant was the most toxic sample to sperm and o4spring. The data showed a prevailing association of free Fe (and possibly Mn) levels with Seydisehir sample toxicity. The water column of the red sludge holding:pond showed both excess levels of free Al and high pH, thus suggesting a combined e4ect. The di4erences in sample toxicity in the Seydisehir plant compared with other bauxite manufacturing plants: suggest a possible variable toxicity as related to bauxite 2002 ore composition and/or manufacturing processes.
Elsevier Science

Key Words: bauxite; sludge; solid residues; sea urchins; toxicity test system.

To whom correspondence may be addressed. Fax: (#39)-081-2296625. E-mail: gbpagano@tin.it. 28

0147-6513/02 $35.00 2002 Elsevier Science All rights reserved.

Bauxite manufacture involves ore processing leading to alumina (AlO }xHO), which is then submitted to an electrolytic process using molten cryolite (NaAlF) and leading to metallic aluminum production (Hudson, 1987). These processes involve the production of a number of waste materials, including sludge and solid residues both from primary bauxite manufacturing and from the electrolytic process. The sludge, termed &&red sludge,'' contains large amounts of aluminum and iron and lower amounts of other metals depending on bauxite ore composition (Hudson, 1987). Apart from these processes, the need for high electric power associates aluminumproducing facilities with power plants that may contribute to the overall environmental impact, especially in the case of coal-fueled plants. Therefore, aluminum-producing facilities may be involved in multifaceted events of environmental pollution, related both to the di!erent by-products disposed of and to the site of disposal which may a!ect marine coastal or inland dumping areas. Previous investigations have focused on bauxite manufacturing sludge, tested as plant e%uent (Trie! et al., 1995; His et al., 1996), or as solid residues, or as marine sediment from a coastal disposal site (unpublished data). Our previous "ndings on bauxite sludge toxicity were attributed to aluminum and iron being present in sludge at high nominal concentrations. (Trie! et al., 1995; Pagano et al., in press). Other reports also focused on Al(III)- and Fe(III)-associated toxicity, either as complex mixtures or as Al(III) or Fe(III) salts (Pagano et al., 1996). Together, the evidence provided by the previous studies pointed to developmental, reproductive, and cytogenetic toxicity in sea urchin and oyster early development induced by Al- and/or Fe-containing complex mixtures (Trie! et al., 1995; His et al., 1996; Pagano et al., 1989, 1996).

BAUXITE BY-PRODUCTS IN SEA URCHINS

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FIG. 1.

Location of the Seydisehir bauxite factory in Turkey.

The present study was carried out on the bauxite manufacturing plant in Seydisehir (southwest Turkey) (Fig. 1), as ' a part of a more extensive study of bauxite facilities located in four countries. A series of specimens were collected from bauxite sludge, soil samples collected at the facilities, and water and sediment from two disposal sites (&&holding ponds''), receiving either red sludge or cryolite process byproducts. The results provided further evidence for the varied toxicity of the by-products investigated (from severe e!ects to lack of toxicity), depending on the nature and quantity of free inorganics released from these complex mixtures, and providing further hypotheses about the relevance of the composition of bauxite ores as related to by-product-associated toxicity.
MATERIALS AND METHODS

either a brush or a shovel, respectively. The sludge sample from a red sludge process tank (SS3) was ejected at a temperature of &803C, and was collected in an iron jar. After approximately 0.5 h, the sludge was transferred into 150-mL polystyrene containers. Water (SS4) and red sludge (SS5) samples were collected from the red sludge holding pond. Moreover, a ground sample from the beach of the red sludge holding pond (SS6) was collected to evaluate the e!ect of solid residue weathering. One year later, red sludge samples from both the process tank (SS3bis) and red sludge holding pond (SS5bis) were collected for con"rmation of the chemical analysis and toxicity results. The samples were stocked in 150-mL polystyrene containers, at the laboratory, "ltered through a 1-mm sieve, then dried at 603C for 72 h. Other samples were collected at the cryolite holding pond (SS7 and SS8). Water and wet samples were tested within 1 month of collection. Dry samples were stocked in the dark at room temperature. Chemical Analysis Overall metal content in each sludge sample was analyzed by destructive determinations after complete dissolution by

Red Sludge and Solid Residues Red sludge is produced as a by-product in the bauxite manufacturing process, as shown in Fig. 2. Red sludge is discharged to a "rst holding pond by steel pipes after being mixed with water withdrawn from holding ponds. In recent years a second holding pond has been used mainly to dispose of by-products from the cryolite process with a high concentration of #uoride ('200 mg L\) in the water column. Solid residues derive from ground deposition of dried red sludge at and near plant facilities, or consist of either sediment in the red sludge holding pond or soil located at the pond beach. Samples were taken from some selected locations in the Seydisehir facilities and at the holding ponds. For compara' tive purposes, solid residues from three other bauxite plants located in Aghios, Nikolaos, Greece, Gardanne, France, and Portovesme, Italy, were tested. Sampling Collection and Storage Soil samples (SS1 and SS2) were collected from surface layers of the ground close to the thickener, by means of
FIG. 2. Bayer process #owsheet and sludge holding ponds.

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PAGANO ET AL.

microwave disaggregation and chemical attack. Speci"cally, 0.1 g (dry wt) of each sample was contacted with 15 mL of a mixture of concentrated strong acids [HNO (5 mL), HCl (3 mL), and HF (7 mL)], in a sealed polytetra#uorethylene (PTF) vessel (bomb). After insertion of the bomb into the microwave digestion system (MDS2100S from CEM, Matthew, NC), the latter was operated at 60% of its maximum power for 15 min. The homogeneous digested solution was directly injected into the ICP-OES system for metal analysis (Tessier et al., 1979; APHA/AWWA/WEF, 1992). To evaluate seawater release of inorganics, samples were dried to constant weight and soaked in seawater (100 mL) in a Jar Test System (F.6/S from Velp Scienti"c, Cambridge, UK), stirring continuously (100 rpm) for 24 h. The supernatant solutions, after "ltration on 0.45- m polycarbonate "lter, were analyzed for content of seawater-released metals by ICP-OES on a Perkin Elmer Optima 3000 System (Norwalk, CT). Sea Urchins Sea urchins from the species Sphaerechinus granularis were used; gametes were obtained and embryo cultures were run as described previously (Pagano et al., 1986, 1993). Controls throughout experiments were conducted as untreated negative controls ("ltered seawater, FSW) and 2.5;10\ M CdSO as a positive control (Pagano et al., 1982, 1986). Test samples were suspended in FSW at concentrations ranging from 0.1 to 2% (dry w/v). Exposure of embryos (&20}30 embryos/mL) occurred throughout development from zygote (10 min after fertilization) up to the pluteus larval stage (72 h after fertilization). This procedure allows for direct contact throughout cleavage up to hatching (approximately 10 h after fertilization). Sperm bioassays were conducted on sperm cell suspensions by standard exposure of a 0.2% suspension of &&dry'' sperm pellet for 10 min. During exposure, test pellets were allowed to settle and 0.5% supernatant sperm were used to inseminate untreated egg suspensions (50}100 eggs/mL). Changes in the fertilization success of exposed sperm were determined by scoring the percentage of fertilized eggs in fresh cleaving embryos (1}3 h postfertilization). All experiments were run at least in quadruplicate. Observations of larvae were performed on living plutei (n"100 for each replicate) immobilized in 10\ M chromium sulfate

(Pagano et al., 1983, 1986). The following outcomes were evaluated: (i) retarded (R) plutei [4 size vs normal (N)  plutei]; (ii) pathologic (P1) malformed plutei; (iii) pathologic embryos (P2) that were unable to di!erentiate up to the pluteus larval stage; and (iv) dead (D) embryos/larvae [scored as dead plutei (D1) or early dead embryos (D2)]. All observations were carried out double-blind by trained readers, each evaluating a complete set of readings. Statistical Analysis The outcomes were evaluated statistically using the  and G procedures. To carry out several simultaneous comparisons, Dunnett's, Tukey's, and Bonferroni's tests were used (Whorthon 1985, Zar 1996). Prior to the tests, data were arcsin-transformed, using the correction of FreemanTukey (1950) described by Zar (1996). Data analysis was carried out using the Statistica 6.0 software. The level of signi"cance for statistical data was always set at "0.05.
RESULTS

Chemical Analyses A preliminary inorganic analysis was carried out on two selected soil samples collected at the alumina plant (SS1) and near the reactor (SS2). The samples were submitted to strong-acid extraction, and the results, shown in Table 1, pointed to the prevailing content of Al and Fe, 36 g kg\ (Fe) and 203 g kg\ (Al) in sample SS1, and 79 and 128 g kg\, respectively, in sample SS2. The other metals showed levels ranking as follows: Mn'Cr:Pb:Zn: Ni'As (Table 1). The sample set was then analyzed for the release of soluble inorganics following a 24-h suspension in seawater of two aliquots 0.5 and 2 g (dry wt) in 100 mL seawater. As shown in Table 2, the highest Al(III) level was reached by the water column sample (SS4) from the red sludge holding pond, 1775 g L\; as for the other samples, the levels of Al(III) measured in seawater ranged from 40 to 86 g L\, yet there was no detectable change for the any given sample, regardless of whether it was suspended in 0.5 or 2 g/100 mL seawater. In the case of Fe(III) seawater release, the highest levels were reached by samples SS3, SS2, and SS1, and a shift in Fe(III) release as a function of suspended aliquot was displayed by samples SS1 and SS5. Regarding the other inorganics measured, only Mn showed substantial levels in the sediment sample from the red sludge

TABLE 1 Levels of Inorganics (mg/kg) in Seydisehir Soil Samples as Detected following Strong-Acid Extraction :
Sample SS1. Alumina plant SS2, Near red sludge reactor Al 203,234 128,691 Fe 36,070 79,478 Mn 462.69 258.37 Zn 231.59 156.50 Cr 132.34 253.44 Pb 93.03 156.50 Ni 85.07 156.25 As 50.50 82.43

BAUXITE BY-PRODUCTS IN SEA URCHINS

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TABLE 2 Concentrations ( g/L) of Some Selected Inorganics from Seydisehir Samples following Seawater Extraction at Di4erent Pellet Levels?
Sample Alumina plant SS1. Soil, alumina plant) SS2. Soil, near reactor SS3. Red sludge pellet Red sludge holding pond SS4. Water column SS5. Sediment pellet SS6. Beach pellet g/100 mL (dry wt) Al ( "396.152) Fe ( "238.204) Mn ( "257.610)

0.5 2 0.5 2 0.5 2 2 0.5 2 0.5 2

72 75 60 61 44 43 1775 82 86 39 40

43 134 148 243 326 327 7 62 141 95 97

5 17 15 67 (0.4 0.4 (0.4 57 125 (0.4 2.1

?The following elements were invariably below detection limits ( g/L): (Cr(4, Ni(3, Pb(30). Levels of 6$1 g/L Cu and 3$1 g/L Zn were measured and attributed to background seawater Cu and Zn concentrations.

holding pond (SS5), up to 125 g L\, and in the soil sample close to the reactor (SS2), 67 g L\, following suspension of a 2-g pellet in 100 mL seawater. The other elements analyzed for showed levels below detection limits (Cr(4, Ni(3, Pb(30), or, in the case of Cu and Zn, measured levels were not changed by increasing amounts of sample; suspended and, thus, were attributed to background seawater levels (Table 2). In conclusion, the samples only released increasing and substantial amounts of Fe(III), and only two samples (SS2 and SS5) released increasing Mn(II) levels. A particular case was the water column sample (SS4)

from the red sludge holding pond, which showed exceedingly high Al(III) levels and a high pH (:12). Sea Urchin Bioassays When S. granularis embryos were reared in the samples from the Seydisehir plant, the most severe toxicity was ' exerted by the 2% dilution of the SS4 sample (water column from red sludge holding pond), with 100% early embryonic mortality (D2), as shown in Table 3. Another sample displaying signi"cant toxicity (P(0.005) was the water

TABLE 3 Developmental Defects Re6ecting Larval Retardation (R), Larval Malformation (P1), Developmental Arrest (P2), and Early Embryonic Mortality Before Hatching (D2), in S. granularis Larvae Reared in Samples from a Selection of Sites at the Facilities and at a Dumping Site (99Sludge Lake::) of the Bauxite Manufacturing Plant in Seydisehir, Turkey? :
Treatment schedule Blank Alumina plant 0.5% soil, alumina plant (SS1) 0.5% soil, near reactor (SS2) 0.5% red sludge (pellet) (SS3) 2% red sludge (supernatant) (SS3) Red sludge holding pond 1% water column (SS4) 2% water column (SS4) 0.5% lake sediment pellet (SS5) 2% lake sediment pore water (SS5) 0.5% beach sediment pellet (SS6) 2% beach sediment pore water (SS6) ?Quadruplicate experiment. R 6.2$2.9 4.5$2.3 9.0$4.6 4.8$1.8 13.3$3.6 P1 3.7$0.8 6.8$0.9 29.5$ 10.9 6.3$1.8 25.8$8.8 P2 2.3$0.4 1.8$1.4 15.5$2.1 0.5$0.5 19.8$7.7 D2 0.5$0.2 0.0$0.0 0.0$0.0 0.0$0.0 0.0$0.0

7.8$6.1 0.0$0.0 26.8 $18.6 3.5$2.0 4.3$1.0 3.3$0.8

8.3$4.4 0.0$0.0 11.5$2.2 6.3$2.4 6.0$1.6 5.0$0.7

3.0$0.4 0.0$0.0 3.0$1.6 4.0$1.6 1.0$0.7 2.5$1.0

0.0$0.0 100.0$ 0.0 0.0$0.0 0.0$0.0 0.0$0.0 0.0$0.0

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PAGANO ET AL.

TABLE 4 Developmental Defects in S. granularis Larvae Reared in a Selection of Samples (0.5% dry wt/vol) from the Bauxite Manufacturing Facilities in Seydisehir (TR), Gardanne (F), : Aghios Nikolaos (GR), and Portovesme (IT)?
Treatment Schedule Blank Cd(SO ) 2.5;10\ M  TR-SS1 TR-SS2 F-10 F-12 F-20 F-21 GR-604 GR-719 IT-011 IT-522 IT-489 R 5.3$1.0 0.0$0.0 11.0$2.8 8.6$2.5 10.8$3.1 16.0$3.1 2.6$1.6 11.0$2.4 11.0$3.0 16.4$3.4 0.0$0.0 0.0$0.0 8.0$2.9 P1 5.7$1.1 0.0$0.0 13.2$4.4 46.8$11.6 50.0$10.8 34.2$8.3 49.0$12.9 11.3$3.7 42.4$11.3 32.2$8.9 0.0$0.0 3.0$3.0 14.8$5.3 P2 1.7$0.5 100.0$0.0 14.6$12.0 22.2$14.7 5.6$0.7 8.8$5.1 39.6$12.1 4.8$3.3 3.6$0.8 6.0$1.5 100.0$0.0 97.0$3.0 0.6$0.4

spermiotoxicity (FR"38%) and a!ecting o!spring quality (:78% P1#P2) (Table 5). Other data, not shown in the present report, include the following: (a) con"rmation of the relative toxicities of Seydisehir samples (SS3bis and SS5bis) collected 1 year later (June '1999); (b) the lack of any signi"cant cytogenetic e!ects; and (c) the lack of any e!ects from either the water column or sediment from samples collected at the cryolite holding pond (the dumping site of the electrolytic process).
DISCUSSION

Chemical Analyses Two sets of data have been obtained by analyzing Seydisehir samples either following strong-acid extraction ' or following a 24-h extraction in seawater. The former procedure leads to disruption of crystalline or amorphic solid structures, thus providing exhaustive information on the chemical composition of a complex mixture as for example, in the present study, bauxite manufacturing byproducts. The data reported in Table 1 represent an example of this kind of information which, however, may conceal the environmental availability of a number of

?Quadruplicate experiment.

component (SS3 supernatant) of the red sludge, with increased malformations (P1), prelarval arrest (P2), and larval retardation (R). As for solid residues (soil and pellet samples), the only one that resulted in developmental toxicity was the soil sample collected near the reactor (SS2), with '50% developmental defects (as R#P1#P2). Another quadruplicate experiments was carried out to compare the relative toxicities associated with a series of solid residue samples from four bauxite plants in Seydisehir ' (TR), Gardanne (F), Aghios Nikolaos (GR), and Portovesme (IT). Samples were selected according to previous evidence for varied degrees of toxicity at di!erent locations of the above facilities, and S. granularis embryos were reared in 0.5% of the dry pellet. Table 4 reports the results of the comparative test with the solid residue samples from the four bauxite plants. Among the Seydisehir samples tested ' the relatively high toxicity of SS2 resulting in 47% malformed larvae (P1) and 22% prelarval arrest (P2) was con"rmed, whereas the SS1 sample was con"rmed to be less toxic. Also con"rmed were the toxicity data for the samples collected at the other facilities either previously (F, May 1996; GR, January 1998) or contemporaneously (IT, May 1998). This con"rmation held true for both the French and the Italian sample subsets, with the highest toxicities displayed by samples F-20 (:90% P1#P2) and IT-011 (100% P2), compared with relatively non-toxic samples, such as F-21 and IT-489. As for the samples from Aghios Nicolas (GR), their toxicities were intermediate and did not di!er signi"cantly from each other (Table 4). When S. granularis sperm were suspended for 10 min in seawater with Seydisehir samples, the supernatant from red ' sludge (SS3) was the only sample both exerting signi"cant

TABLE 5 Fertilization Rate (FR) and O4spring Quality (P1, P2) in S. granularis Larvae following Exposure of Sperm to Some Selected Samples from the Facilities and at the 99Sludge Lake:: of the Bauxite Manufacturing Plant in Seydisehir? :
Treatment Schedule Blank Cd(II) 2.5;10\ M Alumina plant 0.5% soil alumina plant (SS1) 0.5% soil near reactor (SS2) 0.5% red sludge (pellet) (SS3) 2% red sludge (supernatant) (SS3) Red sludge holding pond 1% water column (SS4) 2% water column (SS4) 0.5% lake sediment pore water (SS5) 2% lake sediment pore water (SS5) 0.5% beach sediment pellet (SS6) 2% beach sediment pore water (SS6) ?Quadruplicate experiment. FR 92.4$0.7 2.0$1.2 86.0$0.9 89.0$0.7 88.0$0.6 38.0$3.5 P1 6.17$1.7 ND 3.0$1.5 5.3$1.7 5.0$3.0 24.8$11.6 P2 3.4$0.7 ND 4.3$2.7 5.3$1.3 3.8$2.6 53.5$17.8

97.0$0.4 98.0$1.3 65.0$2.9 95.0$0.8 99.0$0.4 98.0$1.2

7.5$2.2 9.8$4.5 7.8$2.8 16.8$5.0 4.5$1.2 4.5$1.2

2.8$0.6 3.0$2.3 2.0$1.7 2.5$0.9 2.8$1.1 4.0$1.7

BAUXITE BY-PRODUCTS IN SEA URCHINS

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components that may remain immobilized in the solid structure and, thus, may not contribute to solid residueassociated environmental e!ects. By comparing the data reported in Tables 1 and 2, respectively, some inorganic components, e.g., Cr and Pb, may result in noticeable levels if analyzed following strong-acid extraction, yet their levels were below detection limits when extraction was carried out by soaking samples in seawater for 24 h. Thus, the present as well as previous experience (Pagano et al., in press) point to the need to reconsider the strong-acid extraction procedure which may itself provide some misleading information relative to the toxicity of complex mixtures. On the other hand, a &&mild'' extraction procedure, e.g., seawater extraction, may provide some more realistic information on the levels of environmentally available contaminants released from the complex mixtures being examined. Based on the results of the present study and on previous analytical datasets from bauxite sludge and solid residues (Trie! et al., 1995; Pagano et al., in press), the roles of the mixture components may appear to di!er somewhat among the di!erent facilities (Seydisehir vs Gardanne vs Por' tovesme). Namely, the chemical composition and environmentally availability of the inorganics involved may change from one plant to another plant, in that (a) the main nominal components, i.e., Al and Fe, may (or may not) be released from solid residues to a di!erent extent, possibly as a function of mineralogic di!erences in bauxite ores used at the di!erent facilities; and (b) the role(s) for the &&minor'' components may be relevant to the resulting toxicity, due to components that may vary in their absolute levels and, again, in their release from the mixture. As a clear example of this observation, Zn was present at very low levels in both Seydisehir samples and in bauxite sludge from Gardanne, ' whereas high Zn levels and extensive release in seawater were directly associated with sample toxicity at the Portovesme facilities (Pagano et al., in press). The subject of metal speciation has not been considered in the present study, yet its relevance in evaluating environmental availability and toxicity deserves further investigations. Sea Urchin Toxicity Testing The most severe developmental toxicity was exerted by two wastewater samples, namely, the water column from the red sludge lake (SS4) and the supernatant from the red sludge (SS3), the former resulting in early embryolethality (100% D2). This e!ect could possibly be attributed to a very high pH (:12), at which aluminum has high solubility in the Al(OH)\ form, resulting in a pH shift of approx imately one unit in bioassay medium at the concentration tested. At the same time, sample SS4 was found to be contaminated by a relatively high Al(III) level (1700 g L\, or :5;10\ M); thus, both increased pH and increased

Al(III) contamination may have contributed to SS4-associated embryolethality (Pagano et al., 1985, 1996). The respective contributions of high Al(III) levels and high pH toxicity await disentanglement in a further study. Among solid samples, only the soil sample collected near the reactor (SS2) resulted in a signi"cant increase in developmental defects (P1#P2), ranging from 30 to 70% of larvae (Tables 3 and 4); it is worth noting that the SS2 sample showed a concentration-dependent release of Fe(III) and Mn(II) (Table 2). Conversely, the SS6 sample (beach soil at the red sludge holding pond) resulted in no toxicity and failed to show any seawater release of Fe(III) or Mn(II), consistent with weathering processes having occurred in SS6. The overall lack of solid residue-associated toxicity in Seydisehir samples (excepted for SS2) di!ered from the data ' previously obtained on bauxite solid residues in Gardanne and Portovesme, while it was consistent with an analogous lack of severe toxicity observed in soil samples collected outside the facilities at Aghios Nikolaos. Comparative testing of solid residue toxicity provided con"rmation of the previously observed di!erences in sample toxicity, which were una!ected by sample aging, since the bioassay (data in Table 4) was conducted in October 1999 on samples having aged from 1 years (Seydisehir and Portovesme samples) to  ' 3 years (Gardanne samples). Thus, it could be observed  that the relative toxicity (or lack of toxicity) was maintained in samples tested more than 3 years later. Among the di!erent sites, it could be seen that the most toxic sites corresponded to samples IT-011 (Portovesme), causing 100% of developmental arrest (P2), and F-20 (Gardanne) resulting in approximately 90% malformations (P1) plus developmental arrest (P2). Thus, both Seydisehir ' and Aghios Nikolaos samples were con"rmed to result in lesser developmental toxicity, when compared with the most toxic samples from Portovesme and Gardanne. Consistent with the above-discussed variation in sample composition, the di!erences in toxicity outcomes could be related to possible di!erences in the environmental availability of the contaminants present in the solid residues at di!erent levels due to seawater release, as was the case for the high zinc levels in Portovesme (Pagano et al., in press). Consistent with the outcomes of an overall lack of developmental toxicity for solid residues in Seydisehir samples, only red sludge supernatant (SS3) exerted a' spermiotoxic e!ect, which was followed by the observation of increased malformations and prelarval arrest in the o!spring of treated sperm (Table 5). A general statement should be made regarding the choice of using a marine organism in toxicity testing for a terrestrial environment. First, it should be stressed that bauxite sludge is a marine contaminant as in the Gardanne, Aghios Nikolaos, and Portovesme facilities, whose sludge is disposed of in marine coastal areas. The

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PAGANO ET AL. His, E., Beiras, R., Seaman, M. N., Pagano, G., and Trie!, N. M. (1996). Sublethal and lethal toxicity of aluminum industry e%uents to early developmental stages of the Crassostrea gigas oyster. Arch. Environ. Contam. oxicol. 30, 335}339. Hudson, L. K. (1987). Alumina production. In Production of Aluminium and Alumina (A. R. Burkin, Ed.), pp. 11}46. Wiley, Chichester, UK. Pagano, G., Esposito, A., and Giordano, G. G. (1982). Fertilization and larval development in sea urchins following exposure of gametes and embryos to cadmium. Arch. Environ. Contam. oxicol. 11, 47}55. Pagano, G., Esposito, A., Bove, P., deAngelis, M., Rota, A., and Giordano, G. G. (1983). The e!ects of hexavalent and trivalent chromium on fertilization and development in sea urchins. Environ. Res. 30, 442}452. Pagano, G., Cipollaro, M., Corsale, G., Esposito, A., Ragucci, E., and Giordano, G. G. (1985). pH-induced changes in mitotic and developmental patterns in sea urchin embryogenesis. I. Exposure of embryos. Teratogen. Carcinogen. Mutagen. 5, 101}122. Pagano, G., Cipollaro, M., Corsale, G., Esposito, A., Ragucci, E., Giordano, G. G., and Trie!, N. M. (1986). The sea urchin: Bioassay for the assessment of damage from environmental contaminants. In Community Toxicity Testing (J. Cairns, Jr., Ed.), pp. 67}92. ASTM, Philadelphia. Pagano, G., Corsale, G., Dinnel, P. A., Esposito, A., and Romana, L.-A.  (1989). Use of sea urchin sperm and embryo bioassay in testing the sublethal toxicity of realistic pollutant levels. In Carcinogenic, Mutagenic, and Teratogenic Marine Pollutants: Impact on Human Health and Environment (E. Grandjean, Ed.), pp. 153}164. WHO EURO/UNEP, Adv. Appl. Biotechnol. Series, Vol. 5. Gulf, Houston. Pagano, G., and Trie!, N. M. (1992). Use of echinoids in in vitro embryotoxicity and teratogenicity studies. oxicol. in <itro 6, 177}178. Pagano, G., Anselmi, B., Dinnel, P. A., Esposito, A., Guida, M., Iaccarino, M., Melluso, G., Pascale, M., and Trie!, N. M. (1993). E!ects on sea urchin fertilization and embryogenesis of water and sediment from two rivers in Campania, Italy. Arch. Environ. Contam. oxicol. 25, 20}26. Pagano, G., His, E., Beiras, R., De Biase, A., Korkina, L. G., Iaccarino, M., Oral, R., Quiniou, F., Warnau, M., and Trie!, N. M. (1996). Cytogenetic, developmental and biochemical e!ects of aluminum, iron and their mixture in sea urchins and mussels. Arch. Environ. Contam. oxicol. 31, 466}474. Pagano, G., Korkina, L. G., Iaccarino, M., De Biase, A., Deeva, I. B., Doronin, Y. K., Guida, M., Melluso, G., Merii, S., Oral, R., Trie!, N. M., and Warnau, M. (2000). Developmental, cytogenetic and biochemical e!ects of spiked or environmentally polluted sediments in sea urchin bioassays. In Biomarkers in Marine Ecosystems: A Practical Approach (P. Garrigues, C. H. Walker, and H. Barth, Eds.), Elsevier, Amsterdam, in press. Pagano, G., De Biase, A., Doronin, Y. K., Iaccarino, M., Merii, S., Petruzzelli, D., Tunay, O., Warnau, M., and Trie!, N. M. Bauxite manuK facturing residues from Gardanne (France) and Portovesme (Italy) exert di!erent patterns of pollution and toxicity to sea urchin embryos. Environ. oxicol. Chem., in press. Tessier, A., Campbell, P. G. C., and Bisson, M. (1979). Sequential extraction procedure for the speciation of particulate trace metals. Anal. Chem. 51, 844}851. Trie!, N. M., Romana, L. A., Esposito, A., Oral, R., Quiniou, F., Iaccarino, M.,  Alcock, N., Ramanujam, V. M. S., and Pagano, G. (1995). E%uent from bauxite factory induces developmental and reproductive damage in sea urchins. Arch. Environ. Contam. Toxicol. 28, 173}177. Whorton, E. B. (1985). Some experimental design and analysis considerations for cytogenetics studies. Environ. Mutagen. 7, 9}15. Zar, J. H. (1996). Biostatistical Analysis. Prentice-Hall. Englewood Cli!s, NJ.

Seydisehir facilities are an exception due to their distance ' ('200 km) from the coast; hence, the two holding ponds are used as dumping sites. A second, more general argument relates to the utilization of the sea urchin test system in a variety of subjects and substrates, e.g., in testing pharmaceutical drugs, industrial chemicals, and complex mixtures not con"ned to the marine environment, e.g., river sediment and industrial sludge (Pagano and Trie!, 1992; Pagano et al., 1993, 2000; Graillet et al., 1993; Trie! et al., 1995). Thus, the utilization of sea urchin bioassays in testing Seydisehir samples of sludge, water column, and solid resi' dues may be envisaged as one additional case for evaluating complex mixture toxicity, independently of whether bauxite by-products are disposed of in the marine environment.
CONCLUSIONS

Bauxite manufacturing by-products can be viewed as a matter of environmental concern that remains to be elucidated further. This holds true for both bauxite sludge and solid residues. A striking variability in bauxite by-products can be recognized, both among samples from the same facilities and among samples from di!erent factories. Major sources of variability, with respect to both analytical and the toxicity outcomes, may be variable ore composition and variable release of toxic contaminants from bauxite byproducts, including their main components (Al and/or Fe) and some &&minor'' components, such as Mn, Zn and Pb. The use of mild extraction procedures prior to analytical determinations is strongly suggested by the present studies, since strong-acid extraction may lead to unrealistic information in terms of environmental availability of complex mixture components. Thus, two related overall lessons from the present and previous studies point to (i) the need for appropriate and realistic extraction procedures, and (ii) the variability in the environmental e!ects of bauxite by-products as related to the recognized variability in ore composition.
ACKNOWLEDGMENTS
The authors thank the ETIBANK, Ankara, for kindly providing access to their facilities in Seydisehir. This study was supported by the European ' Commission, Projects EV5V-CT94-0550 and ENV4-CT96-0300 and, in part, by the Italian Labor Ministry. Thanks are due Dr. Norman M. Trie! for critical revision of the manuscript. The Zoological Station, Naples, provided support by their "shery service (Pasquale Sansone and coworkers).

REFERENCES
APHAAWWAWEF (1992). Standard Methods for the Examination of =ater and =astewater, 18th ed. Washington, DC. Graillet, C., Pagano, G., and Girard, J. P. (1993). Stage-speci"c e!ects of teratogens on sea urchin embryogenesis. Teratogen. Carcinogen. Mutagen. 13, 1}14.