Fraction at Ion of Phosphate in Marine Aquaculture Sediments

8/6/2019 Fraction at Ion of Phosphate in Marine Aquaculture Sediments

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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 Ecosyste' mes Marins et Aquacoles (CN RS-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 meth-

ods, Sedex and Hieltjes & Lijklema (H. & L.), in order to

choose a suitable method that extracts better the in-

organic (inorg-P) and organic (org-P) phosphate f rom

marine aquaculture sediments. The EDTA method

gave reliable results and did not change the P-composi-

tion of the sediments during the analysis. The Sedex

method can be improved if the quantity of pre-ex-

tracted org-P is separately determined after digestion,

andthe org-P cantherebycorrected.The Sedex meth-od 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.methodare not specic toextract inorg-Pfrom

the sediments. To provide optimal extractions of in-

org-P in muddy sediments containing 1% org-C and

15% CaCO3, the EDTA method was optimizedaccord-

ing 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 to1% did not inuence the amount of P and Fe ex-

tracted (P50.098 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, phos-phate, ponds, marine

Introduction

Phosphate is often the limiting nutrient for phyto-

plankton productivity in aquaculture ponds (Boyd

1990). Their sediments strongly adsorb the phos-

phate produced by the sh culture and large quant-

ities of P fertilizer must therefore be added to support

micro-algae production (Boyd 1995). Masuda andBoyd (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 sedi-

ments particularly from calcium-bound phosphate

(CaCO3%P) (Staudinger, Peier, Avnimeleck & Ber-

man 1990; Kassila, Hasnaoui, Droussi, Loudiki &

Yahyaoui 2001). In order to optimize the addition of

fertilizers in aquaculture ponds, it is necessary to un-

derstand the P dynamics at the water^sediment in-

terface. 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 Kel-

vie & Beckett1994). The presence of CaCO3%P insedi-

ment 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

Aquaculture Research, 2004, 35, 1339^1348 doi: 10.1111/j.1365-2109.2004.01157.x

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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 neg-

lected and underestimated in sediment chemistry.

Bstrom, Jansson and Forsberg (1982) presented an

idealized distribution of Psed fractions in which org-P was estimated at approximately16%. Dierent stud-

ies have shown after the presence of large quantities

of org-P, accounting for more than 50% of total phos-

phate (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 understandthe P dynamics in aquaculture sedi-

ments, it is critical to choose a sediment P-fractiona-

tion procedure suitable for separating dierent P

fractions. At present, there is no standard method

for the extraction of the dierent P fractions in sedi-

ments. A discussion has been started on the stand-

ardization of the fractionation of sediment-bound

inorganic phosphates (inorg-P) which consist of

iron-bound P (Fe(OOH)%P) and CaCO3%P respect-

ively. 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).

Themain objectiveof this work was to compare the

eciency of the three P-fractionation methods,

ethylenediaminetetraacetic acid (EDTA) (Golterman

1996), Hieltjes and Lijklema (1980) and Sedex (Rut-

tenberg 1992), to extract inorg-P and org-P from shpond sediments. The suitable method was optimized

according to dierent factors such as the extraction

time, dithionite (reducing agent) concentration, sol-

ute/solid ratio and type of sediment.

Materials and methods

The ponds of CREAA (Regional Centre for Aquacul-

ture 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 appli-

cation (European Innovation Project GENESIS). The

system is proposed as an alternative to open sea cage

systems, for whichenvironmentalconcerns are likely

to become an increasing constraint. The system con-

sists 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 low-

value algal by product into a high-value commodity

(Hussenot & Shpigel 2003).

Sediments (0^0.5 cm) were collec ted 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 particlesand used for P fractionation in ve rep-

licates. 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 buf-

fer solution (Golterman & Booman1988). This extrac-

tion was performed during 2 h at the same pH as

found in the sediment (7.2). The supernatants were

split into twoportions: onewas 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 ex-

tracts org-P in two steps, after the inorg-P extractions

had been removed. The rst removes an acid solubleorganic 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 at120 1C.

The major steps of the two fractionation methods

to be compared with the EDTA method are:

(1) The Sedex method (Ruttenberg1992) uses MgCl2

and citrate-dithionite-bicarbonate (CDB) to ex-

tract 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 byan extraction with1-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

P fractionation of marine aquaculture sediments J Kassila & J Hussenot Aquaculture Research, 2004, 35, 1339^1348

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Table 1 P and Fe extractions with dierent methods of fractionation

Extraction no.

EDTA Sedex H. & L.

MgCl2 NH4Cl

Mean SD Mean SD

Soluble P

1st 13.1 1.1 8.1 1.4

2nd 11.1 1.0 9.1 0.7

3rd 11.8 0.8 7.6 1.0

Sub-total 36.0 24.8

Extraction no.

Extractant

Ca-EDTA CDB 0.1 M NaOH

P Fe P Fe P Fe

Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD

Fe(OOH) % P

1st 154 7.4 5.13 0.10 124 6.2 3.95 0.06 103 3.8 0.36 0.02

2nd 41 1.3 1.80 0.05 56.5 2.7 3.00 0.05 17.5 0.8 0.07 0.013rd 11.9 7.6 0.85 0.03 23.1 3.6 2.89 0.09 9.5 2.7 0.06 0.01

4th 15.1 2.0 0.59 0.02 5.1 2.0 0.52 0.11 4.3 0.8 0.08 0.02

Sub-total 222 8.37 209 10.36 134 0.57

(126.8 org-P)

Extraction no.

Extractant

Na-EDTA Na-acetate 1.0 M HCl 0.5 M HCl

Mean SD Mean SD Mean SD Mean SD

CaCO3 % P

1st 143 7.7 27.8 11.5 219 10.1 266 12.3

2nd 118 8.0 28.5 15.6 7.9 0.7 166 5.4

3rd 45.7 15.3 80.1 33.1 3.1 1.3 31.5 5.6

4th 49.6 9.9 52.4 17.7 0.6 1.4 22.9 1.2

Sub-total 356 188 231 487

(120.0 org-P)Total CaCO3 % P 356 419 487

Inorg-P 578 628 621

Extraction no.

Extractant

0.25 M H2SO4 Ashing1Ext.1M HCl 2 M NaOH

Mean SD Mean SD Mean SD

Org-P

1st 43.5 7.3 47.4 3.4 29.9 3.5

2nd 22.4 1.5 9.1 1.8

3rd 7.9 0.1 2.3 0.7

Sub-total 73.8 41.3

Org-P 115.1 47.4

Extractant

H2SO41K2S2O8 1M NaOH

Mean SD Mean SD

Residual P

27.9 1.4 39.0 2.0

Total P 693 675 660

Mean values of ve replicates and SD, P in mg g1d.w. and Fe in mg g1d.w.

EDTA, ethylenediaminetetraacetic acid; H. & L., Hieltjes & Lijklema; Fe(OOH) % P, iron-bound phosphate; CDB, citrate-dithionite-bicar-

bonate; CaCO3 % P, calcium-bound phosphate; org-P, organic phos phate; inorg-P, inorganic phos phate; SD, standard deviation.

Aquaculture Research, 2004, 35, 1339^1348 P fractionation of marine aquaculture sediments J Kassila & J Hussenot

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extracted from the nal pellet by using a strong

alkali solution at 90 1C.

P concentrations of the supernatants were deter-

mined colorimetrically using the blue-molybdate

method (Murphy & Riley 1962). Fe was determined

after centrifugation and neutralization with o-phen-antroline using ascorbicacid as reductant (Golterman,

Clymo & Ohnstad 1978). Ethylenediaminetetraacetic

acid solutions have the disadvantage of interfering

with the P and Fe determinations. For the P determi-

nation, 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 ex-

tract Fe(OOH)%P (1, 2 and 4 h), CaCO3%P (4, 8,

16 and 24 h), org-P! ac (15 and 30 min) and org-

P! 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.

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

Fe(OOH)%P extraction (P50.370, ANOVA); the dier-

ence 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 de-

monstrated that the extraction with 0.1-M NaOHextracts about 27 mg org-P g1d.w. after hydrolysis

(Table 1).

The amount of CaCO3%P extracted by the dier-

ent methods ranged from 360 to 490 mg g1 (Table

1). For the Sedex proce dure, this extraction is usually

made intwo steps:the rstextractsautogenic carbon-

ate ouroapatite plus biogenic apatite with acid Na-

acetate, and the second extracts detrital apatite with

HCl. The results show that the second fraction rep-

resents 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 signi-

cant 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 g1 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 wasunder 40mg g1

inall pro-cedures. The Tot-P extracted using dierent proced-

ures was between 660 and 693 mg g1d.w.

Optimization of the EDTA method

During the rst extraction, about 56% of

Fe(OOH)%P was removed during 1-, 2- and 4-h ex-

traction (Fig. 1a). The amount of P extracted after six

subsequent extractions was not signicantly dier-

ent 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 (P50.650). 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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shaking was not signicantly dierent (P50.066 for

org-P! ac and P50.144 for org-P! alk) (Fig.1d).

The treatment with 0.5% of dithionite was less e-

cient 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% (P50.314 for Fe(OOH)%P and P50.061 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 re-

duced with the dithionite in solid form (Fig. 3a

0

100

200

300

400

500

600

1 2 3 4 5 6 7

Number of total extractions

gP/gd.w

.

1 h

2 h

4 h

0

2

4

6

8

10

12

14

1 2 3 4 5 6 7


mgFe/gd

.w.

1 h

2 h

4 h

0

100

200

300

400

500

600

0 1 2 3 4 5


gP/gd.w.

4 H

8 H

16 H

24 H

0

100

200

300

400

500

600

0 1 2 3 4


gP/gd.w. 15 min

30 min

(e)

(c) (d)

(a) (b)

0

100

200

300

400

500

600

0 1 2 3 4


gP/gd.w.

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! alk (e) during dierent

extraction times. Mean values of ve replicates.




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and b). ANOVA showed a statistically signicant dier-

ence between the two treatments (P50.0025 forFe(OOH)%Pand P50.0001 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 thanof 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 g1d.w. of Fe(OOH)%P was extracted

from the algae pond sediments (Fig. 5a). Five extrac-

tions are sucient to give the complete extraction of

Fe(OOH)%P and Fe(OOH) in the sh- and sedimenta-

tion-pond sediments. However, an additional applica-

tion 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

(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 sub-

sequent extractions came probably from the intersti-

tial P. The neutral pH of the extractions prevents the

dissolution of CaCO3%P. The Ca-EDTA/CDB solu-

tions 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 observa-

tion 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

Figure 2 Extraction of Fe(OOH)%P (a) and Fe(OOH) (b)

with dierentconcentrations of dithionite (0.5%,1.0% and

2.0%). Mean values of ve replicates.

Figure 3 Resultsof theextractionof Fe(OOH)%P (a) and

Fe(OOH) (b) with two forms of dithionite (dissolved, D.D.;

solid, S.D.). Mean values of ve replicates.




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extraction have an inuence on the quantity of phos-

phate extracted, due to the hydrolysis of org-P. The ex-traction with NaOH does not only extract Fe(OOH)

%P but phytate phosphate as well (Jackman & Black

1951; Golterman, Paing, Se rrano & 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 re-

ported by Kassila (2003) in freshwater pond sedi-

ments. The increase in pH during the extraction of

Fe(OOH)%P with NaOH favoured a back precipita-

tion 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 extrac ted more CaCO3%P. Prob-

ably that this overestimation is caused by the hydro-

lysis of org-P! ac with 1-M HCl. This assumption can

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 dem-

onstrated that about 20 mg g1 came from the org-P

(Table 1). overestimation of CaCO3%P in H. & L.comes from neglecting org-P extraction in fractiona-

tion steps. Na-EDTA solution gave an optimal extrac-

tion 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. Polyphos-

phates, 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 org-

P; 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 mineraliza-

tion step. Residual P, less than 40mg g1 in all meth-

ods, 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 extrac-

tions. The highest extraction eciency was obtained

using ve sequential extractions of 2-hduration each.Except for 24 h,all extractiontimes appliedto sedi-

ments 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.

Treatmentwith 0.5% of dithionitewas 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 sedi-

ments of CREAA to give an optimal extraction of

Fe(OOH)%P and Fe(OOH).

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6 7


gP/gd.w.

20:0.5

20:0.75

20:1.0

0

1

2

3

4

5

6

7

8

9

10

1 2 3 4 5 6 7


mgFe/g

d.w.

20:0.5

20:0.75

20:1.0

(a)

(b)

Figure 4 Extraction of Fe(OOH)%P (a) and Fe(OOH) (b)

with dierent extractant/sediment ratios. Mean values of

ve replicates.




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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 bythe 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 Boo-

man (1988) have found that the concentration of

dithionite inuenced the quantity of P solubilized if

2000mg 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 pro-

portional to Fe(OOH). This result led to consider that

P was not bounded to Fe(OOH) but s imply adsorbed

on sediment particles and dead cell algae. To avoid

this confusion in future studies, it is necessary to be-

gin the P fractionation with an extraction of loosely

sorbed Pas in Sedex or H. & L. methods.

Less precipitation of P with apatite occurred in the

sedimentation pond while the relatively higher val-

ues of pH ($ 8.4) enhanced probably this precipita-

tion in the micro-algae ponds. The phosphates that

may constitute a fraction of org-P! ac in sediment

(Golterman etal. 1998) were largely solubiliz ed 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 suit-

able than the Sedex and H. & L. methods for P

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6


gP/gd.w.

M. pondS. pond

F. pond

0

2

4

6

8

10

12

14

1 2 3 4 5 6


mgFe/gd.w.

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6


gP/gd.w.

0

20

40

60

80

100

120140

160

180

200

1 2 3 4 5 6Number of total extractions

gP/gd.w.

(a) (b)

(d)(c)

M. pond

S. pond

F. pond

M. pond

S. pond

F. pond

M. pond

S. pond

F. pond

Figure 5 Extraction of Fe(OOH)%P (a), Fe(OOH) (b), CaCO3%P (c) and org-P! ac (d) from the sh (F.), sedimentation (S.)

and micro-algae (M.) pond sediments. Mean values of ve replicates.




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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 solu-

tions used in Sedex and H. & L. methods. A P fractio-

nation scheme may be recommended as in Fig. 6 for

marine pond sediments having similar characteris-

tics like the sediments of CREAA.

Conclusion

The overall results show that the EDTA method de-

veloped 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 etal. 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-ex-

tracted 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 general-

ly similar with those recommended by Golterman

(1996) for calcareous sediments in brackish water sys-

tems.These conditions canbe appliedto analyse theP

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.

Sediment (20:0.5 as solute/solid ratio)

3 extractions of 2 h with 1 M Loosely-sorbed P

MgCl2

Pellet 1

5 extractions of 2 h with 0.5 M Fe(OOH) PCa-EDTA + 1 % of dithionite

in solid form

Pellet 2

5 extractions of 24 h with 0.1 M CaCO3 P

Na-EDTA

Pellet 3

3 extractions of 15 min with org-Pac

0.25 M HCl

Pellet 4

2 extractions of 15 min with org-Palk

2 M NaOH

Final pellet

Mineralization with H2SO

4+ K

2S

2O

8ROP

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




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Fraction at Ion of Phosphate in Marine Aquaculture Sediments

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