Aquaculture Research, 2004, 35, 1339^1348

doi:10.1111/j.1365-2109.2004.01157.x

Fractionation of phosphate in marine aquaculture sediments: optimization of the ethylenediaminetetraacetic acid method and comparison with other procedures
Jawad Kassila & Jerome Hussenot
' Centre de Recherche sur les Ecosystemes Marins et Aquacoles (CNRS-Ifremer), LHoumeau, France
Correspondence and present address: J Kassila, 376 Avenue Mohamed V 23000 Beni Mellal, Morocco. E-mail: kassilajawad@ , yahoo.com

Abstract
The ethylenediaminetetraacetic acid (EDTA) method was compared with two other P-fractionation methods, Sedex and Hieltjes & Lijklema (H. & L.), in order to choose a suitable method that extracts better the inorganic (inorg-P) and organic (org-P) phosphate from marine aquaculture sediments. The EDTA method gave reliable results and did not change the P-composition of the sediments during the analysis. The Sedex method can be improved if the quantity of pre-extracted org-P is separately determined after digestion, and the org-P can thereby corrected. The Sedex method underestimates the org-P present in the sediments ( 59%), whereas the calcium-bound phosphate (CaCO3 % P) is overestimated (117%) in comparison with the EDTA method. The NaOH and HCl used in H. & L. method are not specic to extract inorg-P from the sediments. To provide optimal extractions of inorg-P in muddy sediments containing 1% org-C and 15% CaCO3, the EDTA method was optimized according to extraction times, dithionite concentration and solute/solid ratio. Five extractions of 2-h duration each with Ca-EDTA are required to extract more than 95% of the iron-bound phosphate (Fe(OOH) % P) while the extraction of CaCO3 % P with Na-EDTA takes more than 96 h. The concentration of dithionite up to 1% did not inuence the amount of P and Fe extracted (P 5 0.098 and 0.174 respectively), whereas a solute/solid ratio of 40:1 was best suitable for the optimal extraction of Fe(OOH) % P. These conditions can be applied to analyse P composition of other marine pond sediments having similar texture and chemical composition.

Keywords: P fractionation, EDTA, Sedex, phosphate, ponds, marine

Introduction Phosphate is often the limiting nutrient for phytoplankton productivity in aquaculture ponds (Boyd 1990). Their sediments strongly adsorb the phosphate produced by the sh culture and large quantities of P fertilizer must therefore be added to support micro-algae production (Boyd 1995). Masuda and Boyd (1994) found about 67% of phosphate applied to freshwater ponds in feed accumulates in bottom soils. By a decrease in pH during the mineralization of organic matter, the o-P can be released from sediments particularly from calcium-bound phosphate (CaCO3 % P) (Staudinger, Peier, Avnimeleck & Berman 1990; Kassila, Hasnaoui, Droussi, Loudiki & Yahyaoui 2001). In order to optimize the addition of fertilizers in aquaculture ponds, it is necessary to understand the P dynamics at the water^sediment interface. As the P fractions in sediment have dierent chemical and biological properties, for a better knowledge of the P cycle in aquaculture systems, it is essential to measure them separately. Fe(OOH) plays an important role in aquatic systems through their interaction with P (Torrent, Schwertmann & Barron 1994) and organic matter (Day, Hart, Mc Kelvie & Beckett1994). The presence of CaCO3 % P in sediment is essentially due to the precipitation of P with Ca21 as apatite. When sediment becomes anoxic, a decrease of pH will dissolve the apatite. Part of the

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released phosphate will be re-adsorbed onto Fe(OOH), which is also present in sediments (Golterman 2001). Organic phosphate (org-P) has often been neglected and underestimated in sediment chemistry. Bstrom, Jansson and Forsberg (1982) presented an idealized distribution of Psed fractions in which orgP was estimated at approximately16%. Dierent studies have shown after the presence of large quantities of org-P, accounting for more than 50% of total phosphate (Tot-P) (De Groot & Fabre 1993; Moutin, Picot, Ximenes & Bontoux 1993). In a recent study, Kassila (2003) estimated the pool of org-P at only 6% of Tot-P in freshwater aquaculture sediments. To understand the P dynamics in aquaculture sediments, it is critical to choose a sediment P-fractionation procedure suitable for separating dierent P fractions. At present, there is no standard method for the extraction of the dierent P fractions in sediments. A discussion has been started on the standardization of the fractionation of sediment-bound inorganic phosphates (inorg-P) which consist of iron-bound P (Fe(OOH) % P) and CaCO3 % P respectively. If the choice of extractants can be standardized, the extraction time and extractant/sediment ratio depend on the type of sediment and amount of P (Golterman 1996). The main objective of this work was to compare the eciency of the three P-fractionation methods, ethylenediaminetetraacetic acid (EDTA) (Golterman 1996), Hieltjes and Lijklema (1980) and Sedex (Ruttenberg 1992), to extract inorg-P and org-P from sh pond sediments. The suitable method was optimized according to dierent factors such as the extraction time, dithionite (reducing agent) concentration, solute/solid ratio and type of sediment.

Materials and methods The ponds of CREAA (Regional Centre for Aquaculture Experiments) are located on the French Atlantic coast (Oleron Island) and are used to evaluate new marine land-based integrated aquaculture systems and then they are transferred for commercial application (European Innovation Project GENESIS). The system is proposed as an alternative to open sea cage systems, for which environmental concerns are likely to become an increasing constraint. The system consists of three components: Sea bass (Dicentrarchus Labrax L.) is the nuclear culture species. The euent from this rst compartment is treated by micro-algae ponds after a rapid settling. The grown algae are

transferred to feed shellsh, which convert the lowvalue algal by product into a high-value commodity (Hussenot & Shpigel 2003). Sediments (0^0.5 cm) were collected in May 2003 from the sh pond of CREAA with a core sampler. They contained about 16% ne sand, 67% silt, 17% clay, 15% CaCO3 and 1% org-C. Sediments were wet sieved through a mesh of 0.2-mm pore size to remove larger particles and used for P fractionation in ve replicates. Within 24 h after collection, about 0.7^1.0 g was mixed with 20 mL of the extractants and used in EDTA, Sedex and Hieltjes & Lijklema (H. & L.) methods. Sequential extractions were carried out with the pellets that remained in the centrifuge tube. In each step, the extractions were repeated several times until the quantity of a particular form of P extracted was less than 5% of the amount extracted. In the EDTA procedure (Golterman 1996) Fe(OOH) % P is extracted with a mixed solution of 0.5-M Ca-EDTA and dithionite, buered with TRIS. As dithionite decomposes auto-catalytically in H2O by the acidity produced, it must be dissolved in a buffer solution (Golterman & Booman1988). This extraction was performed during 2 h at the same pH as found in the sediment (7.2). The supernatants were split into two portions: one was analysed for dissolved Fe and the other for P. Calcium-bound P (CaCO3 % P) is extracted with 0.1-M Na-EDTA at pH 4.5 in order to reduce the extraction time. The EDTA method extracts org-P in two steps, after the inorg-P extractions had been removed. The rst removes an acid soluble organic fraction (org-P! ac) with 0.5-M HCl and the second removes a fraction which is soluble in 2-M NaOH: org-P! alk. The residual organic P (ROP) was obtained after the digestion of the nal pellet with H2SO4/K2S2O8 at 120 1C. The major steps of the two fractionation methods to be compared with the EDTA method are: (1) The Sedex method (Ruttenberg 1992) uses MgCl2 and citrate-dithionite-bicarbonate (CDB) to extract soluble P and Fe(OOH) % P, respectively, at pH 7.2 as found in the sediments (Table 1). The pH is adjusted at sediment pH to reduce the dissolution of carbonates before the next step. The acetate buer and HCl are used to extract CaCO3 % P in two steps. Organic phosphate is measured after heating the nal pellet at 550 1C followed by an extraction with 1-M HCl. (2) The Hieltjes and Lijklema (1980) method consists of using 1-M NH4Cl, 0.1-M NaOH and 0.5-M HCl solutions to extract soluble P, Fe(OOH) % P and CaCO3 % P respectively. The residual P is

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P fractionation of marine aquaculture sediments J Kassila & J Hussenot

Table 1 P and Fe extractions with dierent methods of fractionation

EDTA Sedex MgCl2 Extraction no.
Soluble P 1st 2nd 3rd Sub-total

H. & L. NH4Cl SD Mean SD

Mean

13.1 11.1 11.8 36.0

1.1 1.0 0.8

8.1 9.1 7.6 24.8

1.4 0.7 1.0

Extractant Ca-EDTA P Extraction no.

Fe(OOH) % P 1st 2nd 3rd 4th Sub-total

CDB Fe SD Mean SD P Mean SD Fe Mean SD

0.1M NaOH P Mean SD Fe Mean SD

Mean

154 41 11.9 15.1 222

7.4 1.3 7.6 2.0

5.13 1.80 0.85 0.59 8.37

0.10 0.05 0.03 0.02

124 56.5 23.1 5.1 209

6.2 2.7 3.6 2.0

3.95 3.00 2.89 0.52 10.36

0.06 0.05 0.09 0.11

103 3.8 17.5 0.8 9.5 2.7 4.3 0.8 134 (126.8 org-P)

0.36 0.07 0.06 0.08 0.57

0.02 0.01 0.01 0.02

Extractant Na-EDTA Extraction no.

CaCO3 % P 1st 2nd 3rd 4th Sub-total Total CaCO3 % P Inorg-P

Na-acetate SD Mean SD

1.0 M HCl Mean SD

0.5 M HCl Mean SD

Mean

143 118 45.7 49.6 356 356 578

7.7 8.0 15.3 9.9

27.8 28.5 80.1 52.4 188 419 628

11.5 15.6 33.1 17.7

219 10.1 7.9 0.7 3.1 1.3 0.6 1.4 231 (120.0 org-P)

266 166 31.5 22.9 487 487 621

12.3 5.4 5.6 1.2

Extractant 0.25 M H2SO4 Extraction no.

Org-P 1st 2nd 3rd Sub-total Org-P

Ashing1Ext.1M HCl SD Mean SD

2 M NaOH Mean SD

Mean

43.5 22.4 7.9 73.8 115.1

7.3 1.5 0.1

47.4

3.4

29.9 9.1 2.3 41.3

3.5 1.8 0.7

47.4

Extractant H2SO41K2S2O8 Mean

Residual P Total P 27.9 693 1.4 675 39.0 660 2.0

1M NaOH SD Mean SD

Mean values of ve replicates and SD, P in mg g 1 d.w. and Fe in mg g 1 d.w. EDTA, ethylenediaminetetraacetic acid; H. & L., Hieltjes & Lijklema; Fe(OOH) % P, iron-bound phosphate; CDB, citrate-dithionite-bicarbonate; CaCO3 % P, calcium-bound phosphate; org-P, organic phosphate; inorg-P, inorganic phosphate; SD, standard deviation.

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extracted from the nal pellet by using a strong alkali solution at 90 1C. P concentrations of the supernatants were determined colorimetrically using the blue-molybdate method (Murphy & Riley 1962). Fe was determined after centrifugation and neutralization with o-phenantroline using ascorbic acid as reductant (Golterman, Clymo & Ohnstad 1978). Ethylenediaminetetraacetic acid solutions have the disadvantage of interfering with the P and Fe determinations. For the P determination, not more than 2 mL of 0.1-M EDTA can be used without interference (Golterman & Booman 1988). The Fe-phenantroline colour develops slowly in the presence of EDTA, but not if 1mL of 2-M Na-acetate solution is added to the 50-mL nal volume (Golterman 1996).

Optimization of EDTA method To optimize the EDTA method for the sediments of CREAA, dierent factors were tested as follows:  Dierent extraction times were evaluated to extract Fe(OOH) % P (1, 2 and 4 h), CaCO3 % P (4, 8, 16 and 24 h), org-P! ac (15 and 30 min) and orgP! alk (15 and 30 min).  Under the suitable extraction time, dierent concentrations of dithionite (0.5%, 1.0% and 2.0%) were applied to optimize the extraction of Fe(OOH) % P and Fe(OOH), bound to sediments. The dithionite was applied in dissolved and solid forms.  To extract Fe(OOH) % P and Fe(OOH), dierent solute/solid ratios were also tested in this study.

Fe(OOH) % P extraction (P 5 0.370, ANOVA); the dierence was larger for Fe(OOH) (Po0.010). Even after three subsequent extractions with Ca-EDTA, more than 7% of Fe(OOH) % P and Fe(OOH) remained in the pellet. The mineralization of the supernatants demonstrated that the extraction with 0.1-M NaOH extracts about 27 mg org-P g 1 d.w. after hydrolysis (Table 1). The amount of CaCO3 % P extracted by the dierent methods ranged from 360 to 490 mg g 1 (Table 1). For the Sedex procedure, this extraction is usually made in two steps: the rst extracts autogenic carbonate ouroapatite plus biogenic apatite with acid Naacetate, and the second extracts detrital apatite with HCl. The results show that the second fraction represents 55% of the total CaCO3 % P extracted. More than four subsequent extractions with Na-EDTA are needed to extract eciently the CaCO3 % P present in the sediments, whereas only three extractions with 0.5-M HCl yielded over 95% of this fraction. No signicant dierence was found between the amount of the inorg-P extracted using the Sedex and H. & L. procedures. The org-P was better extracted using the EDTA procedure in two steps. The org-P! ac represents 64% of the total org-P extracted. The Sedex method extracted only 47.4 mg g 1 after the mineralization. The org-P represents 17% of Tot-P when extracted with the EDTA procedure, but it is only 7% with the Sedex procedure. The P remaining in the nal pellet as residual phosphate was under 40 mg g 1 in all procedures. The Tot-P extracted using dierent procedures was between 660 and 693 mg g 1 d.w. Optimization of the EDTA method

Statistical analysis The data from the three P-fractionation methods were compared using ANOVA. Po0.05 was judged to be indicative of a signicant dierence between the values.

Results Comparison of dierent methods The loosely sorbed P extracted in three subsequent extractions represents only 5.5% and 3.8% of Tot-P in the Sedex and H. & L. procedures respectively (Table 1). The Ca-EDTA and CDB extracted more Fe(OOH) % P and Fe(OOH), than NaOH. No signicant dierence was found between Ca-EDTA and CDB for

During the rst extraction, about 56% of Fe(OOH) % P was removed during 1-, 2- and 4-h extraction (Fig. 1a). The amount of P extracted after six subsequent extractions was not signicantly dierent for the three extraction times (P40.070). The rst extractions remove between 50% and 60% of Fe(OOH) (Fig. 1b). No signicant dierence between 2- and 4-h extractions was found (P 5 0.650). More than four subsequent extractions were required to remove up to 95% of Fe(OOH) % P and Fe(OOH). The concentration of CaCO3 % P extracted after four subsequent extractions still increased with time (Fig. 1c). The extraction yield was highest during the rst extraction. The amount of P extracted during 24 h tended to stabilize after the third extraction. The amounts of org-P extracted after 15 or 30 min of

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P fractionation of marine aquaculture sediments J Kassila & J Hussenot

(a)

(b)
600 1h 500 400 2h 4h mg Fe/g d.w. 10 8 6 4 2 0 1 2 3 4 5 Number of total extractions 6 7 1 2 3 4 5 Number of total extractions 6 7 14 12 1h 2h 4h

g P/g d.w.

300 200 100 0

(c)
600 500 400 g P/g d.w. 300 200 100 0 0 1 2 3 4 5 4H 8H 16 H

(d)
600 500 400 g P/g d.w. 15 min 300 200 100 0 0 1 2 3 4 Number of total extractions 30 min

24 H

Number of total extractions

(e)
600 500 400 g P/g d.w. 300 200 100 0 0 1 2 3 4 Number of total extractions 15 min 30 min

Figure 1 Extraction of Fe(OOH) % P (a), Fe(OOH) (b), CaCO3 % P (c), org-P! ac (d) and org-P! extraction times. Mean values of ve replicates.

alk

(e) during dierent

shaking was not signicantly dierent (P 5 0.066 for org-P! ac and P 5 0.144 for org-P! alk) (Fig. 1d). The treatment with 0.5% of dithionite was less ecient to extract both Fe(OOH) % P and Fe(OOH) (Fig. 2a and b). Only 20% of Fe(OOH) was removed in comparison with the amount extracted with 2% of dithionite. Furthermore, no signicant dierence

was found between the treatments of 1% and 2% (P 5 0.314 for Fe(OOH) % P and P 5 0.061 for Fe(OOH)). The concentration of dithionite (41.0%) did not inuence the concentration of soluble P and Fe. Both Fe(OOH) % P and Fe(OOH) were better reduced with the dithionite in solid form (Fig. 3a

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Figure 2 Extraction of Fe(OOH) % P (a) and Fe(OOH) (b) with dierent concentrations of dithionite (0.5%,1.0% and 2.0%). Mean values of ve replicates.

Figure 3 Results of the extraction of Fe(OOH) % P (a) and Fe(OOH) (b) with two forms of dithionite (dissolved, D.D.; solid, S.D.). Mean values of ve replicates.

and b). ANOVA showed a statistically signicant dierence between the two treatments (P 5 0.0025 for Fe(OOH) % P and P 5 0.0001 for Fe(OOH)). Using 20:0.75 and 20:1.0 as solute/solid ratios, less P and Fe were solubilized during the extraction with Ca-EDTA (Fig.4a and b). More than of 28% and13% of P and Fe, respectively, were additionally extracted when 20:0.5 as ratio was used. The solute/solid ratio was found to have an inuence on the solubilized P and Fe. We have checked the eciency of EDTA method to extract Pand Fe from the other sediments collected in the sedimentation and micro-algae ponds. More than 600 mg P g 1 d.w. of Fe(OOH) % P was extracted from the algae pond sediments (Fig. 5a). Five extractions are sucient to give the complete extraction of Fe(OOH) % P and Fe(OOH) in the sh- and sedimentation-pond sediments. However, an additional application must be applied to give a suitable extraction of Fe(OOH) % P from the micro-algae sediments. Five extractions were required to extract eciently CaCO3 % P. At the last extraction, less than 6% of CaCO3 % P was removed from the dierent sediments
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(Fig. 5c). The org-P! ac was largely solubilized at the rst extraction in all sediments. Negligible quantities of P were found in the supernatants after three extractions. Discussion Comparison of dierent methods The loosely sorbed P, which was extracted with MgCl2 and NH4Cl at constant rate during three subsequent extractions came probably from the interstitial P. The neutral pH of the extractions prevents the dissolution of CaCO3 % P. The Ca-EDTA/CDB solutions have therefore an advantage over NaOH to remove both Fe(OOH) and Fe(OOH)P with optimal eciency. Extractions with 0.1-M NaOH yielded much lower quantities of Fe(OOH) % P. This observation is in agreement with the results reported by De Groot and Golterman (1990), Salvia-Castelvi, Scholer and Homann (2002). The mineralization of the supernatant indicates that NaOH extract some org-P present in sediment as well (Table 1). The results of Golterman and Booman (1988) have also shown that NaOH is not an accurate extractant as both the concentration of NaOH and the duration of the

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P fractionation of marine aquaculture sediments J Kassila & J Hussenot

(a)

800 700 20:0.5 600 500 20:0.75 20:1.0

400 300 200 100 0 1 2 3 4 5 6 7 Number of total extractions

(b)

10 9 8 20:0.5 7 20:0.75 20:1.0

mg Fe/g d.w.

6 5 4 3 2 1 0 1 2 3 4 5 6

be accepted as the sum of CaCO3 % P and org-P! ac extracted using the EDTA method was not dierent from the total CaCO3 % P extracted using the Sedex method. Moreover, mineralization of supernatant demonstrated that about 20 mg g 1 came from the org-P (Table 1). overestimation of CaCO3 % P in H. & L. comes from neglecting org-P extraction in fractionation steps. Na-EDTA solution gave an optimal extraction of CaCO3 % P and needed a longer extraction time in comparison with solutions used in Sedex and H. & L. methods. Ethylenediaminetetraacetic acid method extracts a large quantity of org-P as org-P! ac. Polyphosphates, if present, may represent the major part of the org-P! ac while phytate P and humic-bound P dominates the composition of org-P! alk (Golterman & De Groot 1994). Sedex method underestimated orgP; a large part may be extracted during inorg-P extractions. Extraction of detrital apatite with HCl causes a loss of 17% of org-P before the mineralization step. Residual P, less than 40 mg g 1 in all methods, did not give any indication about extraction methods, as all experimental errors accumulate in this fraction. Optimization of the EDTA method One-hour extractions were less ecient to extract both Fe(OOH) and Fe(OOH) % P even after six extractions. The highest extraction eciency was obtained using ve sequential extractions of 2-h duration each. Except for 24 h, all extraction times applied to sediments were not sucient to give a suitable extraction of CaCO3 % P. One single extraction can never give a suitable recovery. Four extractions of 24-h duration each are recommended to assess most of CaCO3 % P (495%) present in the sediments. For the organic fraction, three subsequent extractions were required to extract most of org-P! ac (495%), whereas only two extractions were enough to remove org-P! alk. Treatment with 0.5% of dithionite was less ecient to extract both Fe(OOH) % P and Fe(OOH) and was hence omitted. Inuence of dithionite was larger on Fe extractability than on P extractability. Free Fe(OOH) seems to be easily reduced in comparison with Fe(OOH) % P complex. This observation was in agreement with Golterman (1984) who argued that the high stability of Fe(OOH) % P complex protects it against reduction. Five extractions of 2-h duration each and 1% of dithionite will be applied to the sediments of CREAA to give an optimal extraction of Fe(OOH) % P and Fe(OOH).

g P/g d.w.

Number of total extractions

Figure 4 Extraction of Fe(OOH) % P (a) and Fe(OOH) (b) with dierent extractant/sediment ratios. Mean values of ve replicates.

extraction have an inuence on the quantity of phosphate extracted, due to the hydrolysis of org-P. The extraction with NaOH does not only extract Fe(OOH) % P but phytate phosphate as well (Jackman & Black 1951; Golterman, Paing, Serrano & Gomez 1998). It is considered an advantage of the H. & L. that only one extraction with NaOH is needed to extract all Fe(OOH) % P. Repeating the treatment, however, we found that following extractions extracted 30% of the rst. The amount of CaCO3 % P extracted using dierent methods is low in comparison with the results reported by Kassila (2003) in freshwater pond sediments. The increase in pH during the extraction of Fe(OOH) % P with NaOH favoured a back precipitation of P extracted from this fraction onto CaCO3 % P. This re-adsorption is rarely taken into account and explains the large quantity of CaCO3 % P extracted using the H. & L. procedure. The Sedex extraction yielded results similar to that of the EDTA method for Fe(OOH) % P, but extracted more CaCO3 % P. Probably that this overestimation is caused by the hydrolysis of org-P! ac with 1-M HCl. This assumption can

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(a)

800 700 600

(b)

14 12 10

mg Fe/g d.w.

g P/g d.w.

500 M. pond 400 300 200 100 0 1 2 3 4 5 6 Number of total extractions S. pond F. pond

8 6 4 2 0 1 2 3 4 5 6 Number of total extractions M. pond S. pond F. pond

(c)

800 700 600 M. pond S. pond

(d)

200 180 160 140 M. pond S. pond 120 100 80 60 40 F. pond

g P/g d.w.

F. pond

400 300 200 100 0 1 2 3 4 Number of total extractions 5 6

g P/g d.w.

500

20 0 1 2 3 4 Number of total extractions

ac

Figure 5 Extraction of Fe(OOH) % P (a), Fe(OOH) (b), CaCO3 % P (c) and org-P! and micro-algae (M.) pond sediments. Mean values of ve replicates.

(d) from the sh (F.), sedimentation (S.)

Use of dithionite in solid form to extract Fe(OOH) % P and Fe(OOH) is more ecient than in dissolved form, as dithionite decomposes rapidly in H2O by the acidity produced (Golterman & Booman 1988). Even in the dry state, e.g., in pots that are often opened, dithionite deteriorates slowly. Golterman (1996) suggested to check their activity regularly using an Fe(OOH) suspension. Only the 20:0.5 ratio can give a suitable extraction of Fe(OOH) % P and Fe(OOH). Golterman and Booman (1988) have found that the concentration of dithionite inuenced the quantity of P solubilized if 2000 mg of sediment was used, but not if 500 mg was used. Consequently, more dithionite (41%) is needed when more than 500 mg of sediment is used as in 20:0.75 and 20:1 ratios. The eect of dierent concentrations of dithionite must be tested to see whether an extraction will yield some extra P and Fe. The P fertilizer added (H3PO4, 5 mM) in the micro-algae pond increased the concentration of Fe(OOH) % P in sediments. Six extractions are

sucient to give the complete extraction of Fe(OOH) % P and Fe(OOH). The Fe(OOH) % P removed from the micro-algae sediments seems to be not proportional to Fe(OOH). This result led to consider that P was not bounded to Fe(OOH) but simply adsorbed on sediment particles and dead cell algae. To avoid this confusion in future studies, it is necessary to begin the P fractionation with an extraction of loosely sorbed P as in Sedex or H. & L. methods. Less precipitation of P with apatite occurred in the sedimentation pond while the relatively higher values of pH ( $ 8.4) enhanced probably this precipitation in the micro-algae ponds. The phosphates that may constitute a fraction of org-P! ac in sediment (Golterman et al. 1998) were largely solubilized at the rst extraction in all sediments. The amount of org-P! ac removed in the sh pond sediments was in agreement with the results reported by Kassila (2003) in an earthen sh pond in Morocco. In conclusion, the EDTA method is more suitable than the Sedex and H. & L. methods for P

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P fractionation of marine aquaculture sediments J Kassila & J Hussenot

Sediment (20:0.5 as solute/solid ratio) 3 extractions of 2 h with 1 M MgCl2 Pellet 1 5 extractions of 2 h with 0.5 M Ca-EDTA + 1 % of dithionite in solid form Pellet 2 5 extractions of 24 h with 0.1 M Na-EDTA CaCO3 P Fe(OOH) P Loosely-sorbed P

Pellet 3

3 extractions of 15 min with 0.25 M HCl Pellet 4

org-Pac

2 extractions of 15 min with 2 M NaOH Final pellet

org-Palk

Mineralization with H2SO4 + K2S2O8

ROP

Figure 6 Fractionation scheme proposed to extract dierent fractions of P from the sediments.

fractionation of the aquaculture sediments. To apply this method, it is necessary to extract before the loosely sorbed P to avoid confusion with Fe(OOH) % P. The EDTA solutions have a disadvantage i.e. the longer extraction time in comparison with the solutions used in Sedex and H. & L. methods. A P fractionation scheme may be recommended as in Fig. 6 for marine pond sediments having similar characteristics like the sediments of CREAA. Conclusion The overall results show that the EDTA method developed by Golterman (1996) for brackish marshes is also suitable for marine aquaculture ponds. This method allows the identication of changes in the P fractions due to seasonal variations in freshwater systems (Moutin et al.1993). Using the Sedex method, only the rst step with CDB can be applied to extract both Fe(OOH) % P and Fe(OOH). The extractants used to remove CaCO3 % P disturb the org-P. The Sedex

method can be improved if the quantity of pre-extracted org-P is separately determined after digestion, and the org-P can thereby be corrected. The EDTA method provides optimal extractions and results in the most meaningful interpretation if it is optimized for a particular sediment. The optimal conditions for P fractionation obtained in this study are generally similar with those recommended by Golterman (1996) for calcareous sediments in brackish water systems. These conditions can be applied to analyse the P composition in other marine pond sediments having both similar texture and chemical composition.

Acknowledgments The authors acknowledge the nancial help of Con seil regional de Poitou-Charentes, Ifremer and the European Community (Genesis Innovation Project). We would like to thank Dr M. Laima for critical remarks and English correction.

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