Stepan Boitsov

Svein A Mjøs

Sonnich Meier

Identification of Estrogen-Like Alkylphenols in Produced Water from Offshore Oil Installations

Keywords: Produced water, alkylphenols, endocrine disruptors, isomers, GC-MS, retention indices, APRI

Produced water released into the sea from oil installations contains a vast number of organic compounds. This work focuses on the analysis and identification of phenols in produced water, in particular long-chain para-substituted alkylphenols, which act as endocrine disruptors for marine biota. Some alkylphenol standards, unavailable commercially, have been synthesised and some compounds of interest identified. However, a complete identification is not possible since conventional GC techniques cannot achieve the desired degree of separation. An overview of the levels of the 52 known alkylphenols in produced water from 9 oil installations in the North and Norwegian Seas has been made. The previously unidentified alkylphenols in produced water have been characterised by means of alkylphenol retention indices (APRI) and mass spectrometry, and their total amounts estimated for the same 9 locations. Our results confirm the presence of naphthols and other as yet unidentified compounds in produced water, while thiophenols were not detected by the used technique.

Introduction

In oil exploration activities, produced water is the water that comes up with oil from sea bed reservoirs, and is then separated from the oil and released into the marine environment. The quantities are large (147×10 6 m 3 from Norwegian offshore oil activities in 2005) and ever increasing due to a larger water content in maturating production wells. The cited 2005 releases are 4×10 6 m 3 higher than those in 2004 [START_REF] Npd | Norwegian Petroleum Directorate[END_REF]. Total releases from all national sectors throughout the North Sea are larger still, having reached 560×10 6 m 3 already in 2002 (Thomas et al., 2004a). The water released in the Norwegian sector each year contains tens of thousands of tonnes of organic compounds, including hundreds of tonnes of alkylphenols [START_REF] Olf | Recommended guidelines for the sampling and analysis of produced water[END_REF]. Alkylphenols are potential endocrine disruptors, and are thus recognized as a * To whom the correspondence is to be addressed. Fax: +47-55238555, email: stepan.boitsov@imr.no matter of concern for marine biota [START_REF] Nimrod | Environmental estrogenic effects of alkylphenol ethoxylates[END_REF][START_REF] Meier | Effects of alkylphenols on the reproductive system of Atlantic cod (Gadus morhua L.)[END_REF]. Produced water has been shown to contain estrogen receptor agonists, and alkylphenols have been identified as major contributors to this effect (Thomas et al., 2004a,b).

A sensitive and selective method for determination of alkylphenols in produced water has recently been described [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF]. The method is based on solid-phase extraction with derivatisation by pentafluorobenzoyl chloride and gas chromatography-mass spectrometry (GC-MS) analysis of the derivates using negative ion chemical ionisation, NCI.

The method has detection limits at low ng/l level and now uses 52 alkylphenols of different chain length, from phenol (C 0 ) to nonylphenol (C 9 ), as standards. However, a large number of alkylphenols are found in an average produced water sample. Theoretically, hundreds of isomers may be present. Most of these are not commercially available and have not been identified.

The primary aim of this work has been to help determine the endocrine-disrupting potential of produced water, since only some alkylphenol isomers have been shown to exert this effect. Namely, long-chain, branched (especially tertiary) para-substituted alkylphenols have the strongest xenoestrogen effect, 4-tert-octylphenol being the most potent xenoestrogen of all alkylphenols [START_REF] Routledge | Structural features of alkylphenolic chemicals associated with estrogenic activity[END_REF]. Ortho-substituted and short-chain alkylphenols have little or no xenoestrogen effect [START_REF] Routledge | Structural features of alkylphenolic chemicals associated with estrogenic activity[END_REF].

Here we describe the qualitative analysis of the total alkylphenol matrix in produced water, together with the quantitative analysis of known alkylphenols in samples from various Norwegian offshore oil installations. For the qualitative analysis, large sample volumes and improved sample purification were used to achieve a high degree of analytical sensitivity and selectivity for detecting as many alkylphenol isomers as possible. Several approaches were then used to carry out identifications.

The first way to identify the unknown chromatographic peaks is to synthesise some of the alkylphenol isomers that are not currently available commercially. Having synthesised a few tertiary para-substituted long-chained alkylphenols, it may be possible to determine a significant part of xenoestrogens in produced water. However, this leaves the rest of the alkylphenol matrix unidentified.

Identification of alkylphenols in produced water may be facilitated by mapping all the unidentified isomers to provide a reliable basis for the analysis of hundreds of compounds for which standards do not exist. An efficient way of achieving such systematisation is by using retention indices. The retention index is the retention time of the analysed compound expressed relative to the retention times of a series of reference compounds. A method of alkylphenol retention indices (APRI) has been developed and applied to alkylphenols in produced water [START_REF] Mjøs | Alkylphenol retention indices[END_REF]. It has been shown to be more robust for this purpose than the traditionally used Kovats indices, and allows accurate characterisation of alkylphenol isomers despite changes in chromatographic properties such as retention times. Thus, a systematisation of separate alkylphenol isomers becomes possible, resulting in unequivocal determination of any given alkylphenol isomer in various samples analysed under various chromatographic conditions, without the use of standards.

In addition to alkylphenols, thiophenols and naphthols have also been included in the analysis. These compounds have not previously been studied in produced water; however, their presence in oil has been shown [START_REF] Beens | The characterization and quantitation of sulfurcontaining compounds in (heavy) middle distillates by LC-GC-FID-SCD[END_REF][START_REF] Thomson | Determination of sulfides and thiols in petroleum distillates using solid-phase extraction and derivatization with pentafluorobenzoyl chloride[END_REF] and their toxicity towards bacteria or higher marine organisms investigated [START_REF] Londry | Toxicity effects of organosulfur compounds on anaerobic microbial metabolism[END_REF][START_REF] Falk-Petersen | Toxic effects of hydroxilated aromatic hydrocarbons on marine embryos[END_REF][START_REF] Cajaraville | Stability of 1-naphthol in seawater solutions and its uptake by the marine prosobranch gastropod Littorina-Littorea (L)[END_REF]. These two classes of compounds can undergo pentafluorobenzoyl derivatisation [START_REF] Thomson | Determination of sulfides and thiols in petroleum distillates using solid-phase extraction and derivatization with pentafluorobenzoyl chloride[END_REF] and may therefore be analysed by the same procedure as the one used for alkylphenols.

Materials and Methods

Sampling strategy

Large volumes (100 l) of produced water samples have been used to provide sufficient concentrations for analysing even the minor compounds. This was necessary, since many alkylphenols, particularly long-chain ones, are found only in small quantities in produced water. This study was not quantitative due to the novelty of the task, the absence of standards for establishing response factors, and because of the complicated multi-step procedure. However, smaller volumes of samples originating from 9 oil installations in the North and Norwegian Seas have been analysed for levels of the known alkylphenols by a previously described quantitative procedure [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF], and the amounts of the unknown isomers have also been estimated. All together, the following 13 samples of produced water from 10 oil installations (see Fig. A blank sample of 100 ml distilled water has been analysed together with regular volume samples for quantitative analysis purposes. All samples except the ones mentioned in point 3 above were added 1% vol. of HCl:water 1:1 mixture for conservation immediately upon sample collection. After that, the samples were sent without airing to the laboratory and stored for 1 to 3 weeks at +2°C in the dark until analysis.

(Figure 1) 2.2. Chemicals and syntheses

Chemicals

Pure standards (95-99.9%) of 49 alkylphenols and 5 deuterated alkylphenols, 3 thiophenols and 2-naphthol were from Sigma-Aldrich (Oslo, Norway) and Chiron (Trondheim, Norway).

Standard solutions were prepared in methanol. The derivatisation agent, pentafluorobenzoyl chloride (PFBC) was from Sigma-Aldrich. Solvents (methanol, hexane, tert-butyl-methyl ether, formic acid, toluene, isopropanol) were analytical grade from Merck (Oslo, Norway) and Arcus (Vestby, Norway). Sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3 ) and hydrochloric acid (HCl) were from Merck. Alcohols and alkenes used for syntheses were from Sigma-Aldrich (Oslo, Norway) and Alfa Aesar (Karlsruhe, Germany). The catalysts used for syntheses, AlCl 3 and Amberlite IR-112 resin, were from Sigma-Aldrich (Oslo, Norway). The distilled water used was purified with NANOPURE Ultrapure Water Systems (USA). Solid-phase extraction Oasis® cartridges with anion-exchange MAX sorbent were from Waters (Milford, MA, USA).

Syntheses

The general procedure for the synthesis of para-substituted alkylphenols is well known, involving Friedel-Crafts reaction and certain catalytic and/or physical conditions to achieve the desired selectivity. Syntheses of long-chain tertiary alkylphenols have been carried out at the University of Bergen according to the two following procedures. An acid-catalysed reaction between phenol and substituted alcohols leads to pure single isomer products [START_REF] Huston | Condensation of aliphatic alcohols with aromatic compounds in the presence of aluminium chloride[END_REF]; another one with phenol and an isoalkene as substrates and an ionexchange resin as catalyst yields a mixture of the corresponding ortho-or para-alkylphenol isomers [START_REF] Loev | Cation exchange resins as catalysts in the alkylation of phenols[END_REF]. The first procedure was used for specific syntheses of 3 para-tert-alkylphenols and the second one for non-specific syntheses of mixtures of various tertiary alkylphenol isomers. Products were recrystallised consecutively from pure dichloromethane and a hexane-dichloromethane 3:1 mixture and dried in oil vacuum (in case of solid product), or the solvent was removed by rotor vapour (in case of liquid product).

Products were then analysed by GC-MS (full scan) and by 400 MHz 1 H nuclear magnetic resonance (NMR) with deuterochloroform (CDCl 3 ) used as NMR solvent and reference. Only pure products were analysed by NMR since NMR spectra of the mixtures could not be resolved. The products of the syntheses are described in Table 1.

(Table 1)

Extraction and purification

The 100 ml samples were purified by filtration and solid-phase extraction, with subsequent derivatisation by pentafluorobenzoyl chloride, according to the procedure described previously [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF]. A method blank, made in distilled water, was processed together with the produced water samples. Quantitative data obtained for the produced water samples has then been corrected for blank values. The following detailed description of the experimental procedure concerns only the 100 l samples.

In this work we improved our regular purification procedure by introducing two extra steps, gel-permeation chromatography (GPC) and normal-phase liquid chromatography (NP-LC). GPC was used to remove small molecules like elementary sulphur and large polymer compounds that could interfere with the analysis. The following NP-LC step prevents an overlap of the signals from alkylphenols with those from other compounds [START_REF] Snyder | Identification and quantitation method for nonylphenol and lower oligomer nonylphenol ethoxylates in fish tissues[END_REF]. All alkylphenols were found in the same NP-LC fraction, well separated from PAH and other compounds. Samples were then sufficiently clean to provide MS spectra of good quality.

Each of the 100 l samples was first extracted with 2x50 ml dichloromethane (DCM) for every 2 l water, the extract volume was then reduced to approximately 0.5 ml under gentle nitrogen gas flow in a water bath at 40° C. The extracts were mixed together and the volume was again reduced to approximately 5 ml. The two resulting extracts were then filtered, the filters washed with DCM and the filtrates extracted with acetonitrile-hexane, and the polar fraction was further fractioned by GPC, with the solvent exchanged to DCM. Both DCM and acetonitrile-hexane extraction efficiency was checked by analysing standard solutions of alkylphenols extracted in a similar way, achieving recoveries usual for this method [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF].

GPC

Gel permeation chromatography (GPC) was used for the 100 l samples to remove sulphur and other interfering compounds. The GPC cleanup was performed using systems from Gilson fluorescence detector, Pharmacia LKB, Sweden). Two GPC columns from Waters (Envirogel GPC cleanup 19 mm x 300 mm) were used, coupled together using Gilson 232 autoinjector as a switch vent. The setup and cleanup procedure are described in more detail elsewhere [START_REF] Meier | Gas chromatography-mass spectrometry analysis of alkylphenols in cod (Gadus morhua) tissues as pentafluorobenzoate derivatives[END_REF]. The elution was performed with DCM at a flow rate of 5 ml/min. Samples were subjected to GPC twice and the resulting purity of the alkylphenol fraction was confirmed by GC-FID.

NP-LC

After GPC cleanup, the solvent was exchanged to hexane and samples were subjected to NP-LC cleanup and fractionation. For this, the same Gilson fractioning system and Pharmacia fluorescence detector as for GPC was used, but the NP-LC pump was from Shimadzu (LC-9A with FCV-9AL low-pressure solvent mixer). The NP-LC column (150x4.6mm, LiChrosorb 60-5 Si) was from Varian (Middelburg, the Netherlands). Injection volume was 500 µl at a time. A gradient of 0 to 6% isopropanol in hexane was used as mobile phase. Fractions of 4 ml were collected from 0 to 30 min. 15 fractions were obtained, with alkylphenols being concentrated in fraction 8 (from 14 to 16 min), up to 97% according to GC-FID measurements. Other polar compounds eluted in fractions 4-15, while PAH and other non-polar hydrocarbons eluted in fractions 1-3. All the NP-LC fractions were then derivatised by pentafluorobenzoyl chloride as described previously [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF]. 2 compounds in fraction 8 have been identified as belonging to the naphthol class, one of them being 2-naphthol.

GC-MS analysis

All analyses were performed on an Agilent 6890 GC system connected to an Agilent 5973 mass selective detector with either electron impact (EI) ionization, or negative chemical ionisation (NCI), in full scan mode, scanning from m/z 50 to 500, or in selected ion monitoring (SIM) mode. Samples of 0.1 l were analysed in NCI SIM mode according to the quantitative procedure described earlier [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF], routinely used for this purpose at our laboratory. The following deuterated internal standards were used for quantification: phenol- A Varian Factor Four VF-5ms (Varian, Lake Forest, CA, USA), L = 50 m, I.D. = 0.25 mm, df = 0.25 µm, was used as analytical column. Helium (99.9%) was used as carrier gas in 22 cm/s constant flow mode. The samples (1 µl hexane solution) were injected in splitless mode and the split valve was opened after 2 min. The GC program was optimised for alkylphenol analyses as described by [START_REF] Mjøs | Alkylphenol retention indices[END_REF]. The oven temperature was 60°C at injection. After 2 min the temperature was increased to 100°C by a temperature programming rate of 30°C/min, which was followed by a second rate of 2°C/min to a final temperature of 260°C. Dwell time per ion for all ions was 100 ms. In SIM mode, the total chromatogram was divided into 5 windows (after solvent delay, 10 min), each of these scanned for 4 to 5 selected ions to achieve better sensitivity. The corresponding parameters are shown in Table 2.

(Table 2) For naphthol analysis, the derivatised NP-LC fractions have been scanned in NCI mode for ion mass 338, corresponding to pentafluorobenzoyl derivates of C 1 -naphthols, and analysed in EI mode. 2-naphthol was used as a standard for determining compounds of naphthol type in produced water.

For thiophenol analysis, the derivatised NP-LC fractions have been scanned in NCI mode for ion masses 318, 332, 346, 360, 374, 388, 402 and 416, corresponding to pentafluorobenzoyl derivates of C 1 -C 8 thiophenols, and analysed in EI mode. 4-Thiocresol, 4ethylthiophenol and 3,4-dimethylthiophenol were used as standards.

Results and discussion

3.1. Qualitative analysis: large-volume samples.

Alkylphenols.

A large number of alkylphenol isomers is present in produced water, only a portion of which are identifiable from available standards (see Fig. 2). The number of non-identified alkylphenols grows rapidly with alkyl chain length, as shown in Table 3.

(Figure 2) (Table 3)

Here we have employed a variety of methods in an attempt to characterize the unidentified alkylphenols. Large produced water samples (100 l) from Statfjord A and Statfjord B platforms were used for the detailed, non-quantitative investigations described below. More than 350 compounds have been definitely or tentatively identified as alkylphenols with up to 8 carbon atoms in the alkyl substituents, by means of visual investigation of mass spectra. Of these, 305 peaks belonged to unidentified alkylphenols, which is apparent from m/z 195 (EI) belonging to the pentafluorobenzoate fragment, and the corresponding molecular peak for each alkylphenol; the rest were identified by means of the available standard.

The alkylphenol retention indices, APRI, developed for this work [START_REF] Mjøs | Alkylphenol retention indices[END_REF], have been used to systematise all the detected alkylphenols. APRI can be calculated by the following equation (1):

APRI x = n t R(x) -t R(z) t R(z+n) -t R(z) + z APRI x = n t R(x) -t R(z) t R(z+n) -t R(z) + z (1)
where t R is retention times of the compound of interest, x, and two para-substituted nalkylphenols eluting on each side of the compound. z represents the number of carbon atoms in the alkyl chains of the para-alkylphenols eluting before x, and n is the difference in the number of carbon atoms between the two references. z is zero if the first reference compound is phenol.

This systematisation provides a standard list of alkylphenols found in produced water, each compound on this list being assigned a unique number in accordance to its APRI value. Even though the precise isomeric structure of each particular compound remains unknown, this allows a determination of the same compound in any new sample on a similar stationary phase (for details, see [START_REF] Mjøs | Alkylphenol retention indices[END_REF]). A selection of unidentified alkylphenols systematised in this way is shown in Section 3.2.

The biggest problem when attempting to identify the previously unidentified longchain alkylphenol isomers in produced water was the poor resolution of the peaks, despite the optimised GC conditions. Obviously, conventional GC-MS techniques do not provide sufficient data quality for this type of analysis, and supplementary or different techniques have to be used to achieve complete peak separation. In recent years, there have been substantial achievements in the development of methods for the mathematical resolution of overlapping chromatographic peaks [START_REF] De Juan | Chemometrics applied to unravel multicomponent processes and mixtures -Revisiting latest trends in multivariate resolution[END_REF]. Most of these methods are multivariate procedures that reconstruct both chromatograms and spectra of the overlapping chromatographic peaks [START_REF] Liang | White, gray and black multicomponent systems -a classification of mixture problems and methods for their quantitative analysis[END_REF]. It has been shown that with the proper selection of instrument parameters and pretreatment of data, it is possible to resolve overlapping peaks with very similar spectra [START_REF] Mjøs | Spectral transformations for deconvolution methods applied on gas chromatography-mass spectrometry data[END_REF]. However, this is a rather time-consuming procedure when complex matrices such as produced water are studied, in which hundreds of peaks need to be resolved. An alternative solution to the problem is the use of a two-dimensional separation technique. Among these, GCxGC seems to be the most efficient technique that is suitable for analysis of complex samples [START_REF] Dallüge | Comprehensive two-dimensional gas chromatography: a powerful and versatile analytical tool[END_REF]. Thus, 40 isomers of 4nonylphenol present in technical nonylphenol mixture have been successfully separated by GCxGC coupled to time-of-flight mass spectrometer, although precise structural elucidation remained impossible since the samples were not derivatised [START_REF] Moeder | Separation of technical 4-nonylphenols and their biodegradation products by comprehensive twodimensional gas chromatography coupled to time-of-oflight mass spectrometry[END_REF].

The mixtures of (ortho-, para-)hexyl-and heptylphenols were synthesised to determine xenoestrogenic para-isomers of long-chained alkylphenols in produced water and compare their relative amount to that of their less potent ortho-and meta-isomers. It has been observed for technical nonylphenol, representing a mixture of known nonylphenol isomers [START_REF] Ruβ | Synthesis of branched paranonylphenol isomers: occurrence and quantification in two commercial mixtures[END_REF], when derivatised with PFBC and analysed together with other standards, that the majority of para-isomers have APRI values in the range of 7.1 to 7.7, eluting in a narrow chromatographic "window" as a cluster of peaks. These are strongly branched alkylphenols, while para-n-nonylphenol has APRI=9. Therefore, we expected the paraisomers in the synthesised mixtures to elute closely together as well. However, their APRI values were found to span a wide range, unlike those of technical nonylphenol, and the samples therefore did not provide a basis for identification of clusters of para-substituted hexyl-or heptylphenols.

Thiophenols.

No compounds of thiophenol class have been found in the studied samples. The absence of thiophenols could be partly due to the pre-analysis sample preparation procedure that was not optimised for this type of compounds. However, the large volume of the analysed samples, 1000 times larger than the regular volume used for routine analysis of produced water at our laboratory, allows to suggest that the amounts of thiophenols in produced water are insignificant.

Naphthols.

Two compounds in the studied samples have been identified as belonging to the naphthol class (C1-naphthols, derivate molecular mass 338), one of them identified as 2-naphthol by means of the used standard. Qualitative results suggest that the concentrations of naphthol are rather limited, possibly 10 to 50 times smaller than the concentrations of alkylphenols of similar mass (C 3 -alkylphenols, derivate molecular mass 330), i.e. at µg/l or below.

Other compounds.

A large number of unidentified compounds with molecular masses 328, 342, 356, 370 and 384 have been determined in the produced water samples. The same type of compounds have previously been observed in produced water from the Oseberg C oil installation (Boitsov et. al., unpublished results). The masses differ by 2 from C 3 -C 7 alkylphenol pentafluorobenzoates; however, the relative amounts of the compounds are comparable to or even larger than those of the corresponding alkylphenols (Fig. 3). Similar compounds with masses differing by 4 from masses of alkylphenol derivates have also been observed.

(Figure 3)

A similarity between the mass spectra and chromatographic parameters of these compounds and those of alkylphenol derivates has given rise to a suggestion that the unknown compounds may be alkylphenols with unsaturated alkyl chains, i.e. alkenylphenols and alkynylphenols.

Alkenylphenols are known to exist in nature, for instance in certain tropical plants (e.g., gibbilimbols, compounds with cytotoxic and antibacterial properties [START_REF] Orjala | Gibbilimbols A-D, cytotoxic and antibacterial alkenylphenols from Piper gibbilimbum[END_REF]) and are also known to be formed as by-products during the photodegradation of alkylphenols or alkylation of phenol [START_REF] Kohtani | Adsorptive and photocatalytic properties of Ag-loaded BiVO4 on the degradation of 4-nalkylphenols under visible light irradiation[END_REF][START_REF] Brodskii | Determination of isomer and homolog composition of alkylphenols by molecular mass spectromery[END_REF]. Confirming the identity of the unknown compounds still remains to be done. 3.2. Quantitative analysis: regular volume samples. Forty-nine alkylphenols have been identified and quantified in 100 ml produced water samples from 9 oil platforms by means of commercially available alkylphenol standards or by those synthesised in our laboratory. It is worth pointing out that the previously described analytical method used in this study [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF] allows for more precise measurements than some other commonly used methods not involving derivatisation of analytes, such as the one recommended to oil companies by the Norwegian Oil Industry Association (OLF) [START_REF] Olf | Recommended guidelines for the sampling and analysis of produced water[END_REF]. The results presented below are thus different from those reported by Norwegian oil companies or by studies that use the OLF-endorsed method, which may result in a considerable underestimation of short-chain alkylphenol levels and an overestimation of long-chain alkylphenol levels (e.g., [START_REF] Olf | Recommended guidelines for the sampling and analysis of produced water[END_REF].

The results from our study are given in Table 4, with APRI values given for each alkylphenol. The amounts of alkylphenols in the samples vary significantly from platform to platform, up to 3 orders of magnitude in some cases, thus reflecting a strong dependence of produced water composition on the origin of the oil. The difference in concentrations of longchained alkylphenols is, however, much smaller than that of phenol and short-chained alkylphenols, indicating that the endocrine-disrupting ability of produced water does not change as much from platform to platform as the overall toxicity.

(Table 4)

The main results of this study include a description of the levels of tertiary para- 4), which remains true even if 4-tert-butylphenol is not taken into account. A recent bioassay study of produced water from the same 10 platforms has also found samples from these 3 platforms to have the highest xenoestrogen potency, up to an equivalent of 4.0 ng/l 17-β-estradiol [START_REF] Tollefsen | Estrogen receptor (ER) agonists and androgen receptor (AR) antagonists in effluents from Norwegian North Sea oil production platforms[END_REF]. This points to the crucial role that alkylphenols play in xenoestrogen potency of produced water.

Earlier in vitro screening of produced water samples from the UK sector of the North Sea has revealed even higher estrogen equivalents, up to 91 ng/l 17-β-estradiol for some samples (Thomas et al., 2004a). This significant xenoestrogen potency of produced water has a clear endocrine-disrupting effect on marine biota like codfish [START_REF] Meier | Effects of alkylphenols on the reproductive system of Atlantic cod (Gadus morhua L.)[END_REF]; however, upon release into the sea and consequent rapid dilution, the effect disappears. A risk assessment modelling study has predicted no effect on codfish except in close proximity of the discharge points [START_REF] Myhre | Risk assessment of reproductive effects of alkylphenols in produced water on fish stocks in the North Sea[END_REF]; this has now been confirmed both by in vivo experiments [START_REF] Meier | Effects of alkylphenols on the reproductive system of Atlantic cod (Gadus morhua L.)[END_REF] and field experiments studying codfish caged near the discharge sites [START_REF] Børseth | Water column monitoring 2003 -summary report[END_REF][START_REF] Scott | Assessment of oestrogen exposure in cod (Gadus morhua) and saithe (Pollachius virens) in relation to their proximity to an oilfield[END_REF]. The field experiments have shown a weak induction of estrogen biomarker, vitellogenin, in the fish from cages put closest to the oil platform, within 500 m from the point of discharge [START_REF] Scott | Assessment of oestrogen exposure in cod (Gadus morhua) and saithe (Pollachius virens) in relation to their proximity to an oilfield[END_REF]. Current work reported no alkylphenols found in liver of wild North Sea codfish [START_REF] Grøsvik | Condition monitoring in the water column 2005: Oil hydrocarbons in fish from Norwegian waters[END_REF].

Samples from two of the oil platforms, Statfjord C and Oseberg C, were tested for stability when the conservation agent, HCl, was not added. Two separate samples, one with and one without HCl, were obtained from each platform. No significant differences in the concentrations were observed between the two samples in either case, except some for orthosubstituted alkylphenols, due to the lowered efficiency of the method for these compounds [START_REF] Boitsov | Gas chromatography-mass spectrometry analysis of alkylphenols in produced water from offshore oil installations as pentafluorobenzoate derivatives[END_REF]. This confirms the reliability of analysis, which is best suited for metaand para-substituted alkylphenols. Thus, alkylphenols in produced water seem to be stable towards bacterial or other degradation when kept cool and in darkness in tightly sealed glass bottles for up to 3 weeks.

The 4-nonylphenol that was found in some, but not all, samples in quantifiable amounts (see Table 4) is thought to originate from sources other than produced water, due to the omnipresence of nonylphenols in the laboratory environment [START_REF] Meier | Gas chromatography-mass spectrometry analysis of alkylphenols in cod (Gadus morhua) tissues as pentafluorobenzoate derivatives[END_REF].

An estimate of the amounts of unidentified alkylphenols has also been made for the nine 100-ml samples. The established APRI system provided a confirmation of the presence of the same compounds in different samples for all the unknown alkylphenols. However, since these compounds are not available as standards, it was not possible to establish response factors for them, so a response factor of 1 was used. This cannot be regarded as an absolute quantification; however, it may be helpful to give a rough estimate of the amount of the compound present, and especially to compare relative amounts between produced water samples from different locations. The total amounts for each molecular mass are shown in Table 4, while concentration estimates for the selected separate unknown alkylphenols (given as an average for the 9 locations) are shown in Table 5. All the unidentified C3-alkylphenols are shown. For other alkylphenols, due to the large number of peaks (see Table 3), only two of each are shown: those which, on average, appear in the largest amounts in the samples.

(Table 5)

Conclusion

In this work, an attempt has been made to investigate the previously unidentified alkylphenols in produced water released into the sea from offshore oil installations. Two 100 l samples from 2 locations in the North Sea have been used for a detailed qualitative analysis. Three alkylphenols of high xenoestrogenic power were synthesised and identified in produced water. A system of alkylphenol retention indices, APRI, developed for this work, has been used to systematise all the observed alkylphenols, including the numerous unidentified ones.

This may facilitate further work to identify the unknown isomers. However, poor peak separation precludes such analysis from being comprehensive, and other separation techniques, such as GCxGC, are likely to be necessary. Other compounds, such as naphthols, were also found in the samples, while thiophenols were not observed.

We also determined the concentrations of 52 alkylphenols found in produced water from 9 offshore platforms in the North and Norwegian Seas, including the three newly synthesised ones. While the levels of low-weight alkylphenols vary greatly from location to location, only moderate differences have been observed in the levels of long-chained alkylphenols. The results seem to support recent biological in vivo, in vitro, and field study data in their conclusion that estrogenically induced endocrine-disruption in the marine biota, due to produced water discharges, is only probable in the immediate vicinity of the point of discharge.

  1) in the Norwegian sector of the North Sea and the Norwegian Sea have been analysed in this work: 1. 2 large-volume (100 l) samples from Statfjord A and Statfjord B oil installations (sampled on January 28 and 29, 2005), to be used for qualitative analysis of even minute concentrations of unknown alkylphenols; 2. 9 regular volume (100 ml) samples from all oil installations except Statfjord B (sampled on various dates in August 2005), to be used for quantitative analysis of known alkylphenols; 3. 2 regular volume (100 ml) samples from Statfjord C and Oseberg C oil installations, without HCl added (sampled in August 2005, simultaneously with the samples to which HCl was added), to be used for checking the efficiency of the conservation procedure.

(

  Gilson 232 autoinjector, injector Gilson 401 dilutor, Gilson 202 fraction collector, Gilson, France) and Pharmacia (LKB 2150 HPLC pump, LKB 2252 LC controller, LKB 2144

  d 5 , as reference for phenol; cresol-d 8 , as reference for cresols; 4-ethylphenol-d 8 , as reference for C 2 -alkylphenols; 4-propylphenol-d 12 , as reference for C 3 -C 6 alkylphenols; 4-nonylphenold 4 , as reference for C 7 -C 9 alkylphenols.Samples of 100 l were analysed non-quantitatively by both EI and NCI, both in SIM and full scan mode, as described below. The analyses of the EI mass spectra yield structure information while the NCI spectra lead to improved sensitivity and selectivity.

  alkylphenols, the most potent xenoestrogens found in produced water. Significantly, the 3 tertiary para-alkylphenols, synthesised in the course of this work, 4-(1,1dimehtylbutyl)phenol, 4-(1,2,2-trimethylpropyl)phenol and 4-(1,1-dimethylpentyl)phenol have all been identified in produced water. The quantity of these compounds seemed to be relatively low, when compared to other alkylphenols of similar alkyl chain length. The total amount of the eight quantified para-tert-alkylphenols does not exceed 1.75 µg/l in any sample and is, as expected, heavily dominated by the least potent, but the most water-soluble compound, 4-tert-butylphenol. The largest amounts of the 8 xenoestrogens were found in discharges from Statfjord A, Statfjod C and Oseberg C platforms (see Table

Figure captions Figure 1 .

 captions1 Figure captions Figure 1. The map of oil installations used for produced water sampling.

Figure 2 .

 2 Figure 2. Negative chemical ionisation (NCI), selected ion monitoring (SIM) ion chromatograms of a standard solution of known alkylphenols (a) as compared to produced water samples (b) for ion masses 330 (C3-alkylphenols), 344 (C4-alkylphenols) and 358 (C5alkylphenols).

Figure 3 .

 3 Figure 3. Negative chemical ionisation (NCI), selected ion monitoring (SIM) ion chromatograms of derivatised produced water sample from Statfjord B oil platform, for two ion masses. Ion 358: C7-alkylphenol derivates. Ion 356: the unknown compounds.

Table 1 .

 1 A description of the products of the syntheses. 578

	Type of product	Main product	Description		1 H NMR chemical shifts, ppm
	Pure C 6 -alkylphenol 4-(1,1-dimehtylbutyl)phenol.	Black-green	oily	0.80-0.87 (t), 1.04-1.15 (m),
			liquid.			1.27 (s), 1.52-1.58 (m), 3.98
						(s), 6.76-6.81 (d), 7.18-7.24
						(d).
	Pure C 6 -alkylphenol 4-(1-methyl-2,2-	Yellow crystals.		0.73-0.78 (d), 1.22 (s), 1.81-
		dimethylpropyl)phenol.				1.89 (m), 3.99 (s), 6.73-6.80
						(d), 7.16-7.23 (d).
	Pure C 7 -alkylphenol 4-(1,1-dimethylpentyl)phenol.	Black oily liquid.	0.80-0.87 (t), 0.99-1.08 (m),
						1.17-1.25 (m, unres.) 1.27 (s),
						1.52-1.60 (m), 3.98 (s), 6.74-
						6.80 (d), 7.17-7.23 (d).
	Mixture of (ortho-,	4-(1-methyl-2,2-	Clear	colourless	-
	para-)hexylphenols	dimethylpropyl)phenol.	liquid.
	Mixture of (ortho-,	4-(1,1-dimethylbutyl)phenol.	Clear red liquid.		-
	para-)hexylphenols
	Mixture of (ortho-,	4-(1-methyl-3,3-	Clear pink liquid.	-
	para-)heptylphenols	dimethylbutyl)phenol.

Table 2 .

 2 Selected ion monitoring (SIM) mode parameters used for alkylphenol analysis. 580

	No. Ion masses

Table 3 .

 3 Number of alkylphenol peaks in GC-MS chromatograms of produced water samples. 584

	Alkylphenol

chain length Alkylphenol derivate mass Identified alkylphenol peaks Non-identified alkylphenol peaks

					Total	Of them	Theoretically
						clusters	possible
	C0	288	1	0	1	0
	C1	302	3	0	3	0
	C2	316	9	0	9	1
	C3	330	11	9	20	3
	C4	344	9	35	44	6
	C5	358	6	45	51	14
	C6	372	5	93	98	45
	C7	386	3	88	91	18
	C8	400	2	35	37	21
	Total		49	305	354	108
	585

Table 4 .

 4 Concentrations of alkylphenols (with their APRI values) in produced water from 9 oil platforms in the North and Norwegian Seas, µg/l

		Para-substituted	et al. (2004).		Statfjord Oseberg	C* C*	2468.4 8227.2		1267.4 3984.1	1136.9 3735.9	1064.9 2534.4	3469.2 10254.4		27.70 49.76	76.51 147.54	100.54 160.62	115.48 191.19	79.97 159.79	66.30 164.73	39.59 72.89	35.79 56.01	50.20 96.17	592.10 1098.70		11.62 10.05	2.31 3.99	12.63 21.89	15.28 12.64	42.74 40.31	30.59 43.91	4.36 5.83	10.40 13.97	14.95 19.00
		(nq -not quantifiable, i.e. peaks below the limits of quantification; nd -not detected, i.e. peaks below the limits of detection).	tertiary alkylphenols are shown in italics. For limits of detection and quantification, and the method uncertainties, see Boitsov	Oil installations	Compound APRI Statfjord Troll B A Statfjord C Åsgard B Grane Oseberg C Heidrun Snorre A Gullfaks B	Phenol 0.0000 18.9 2033.2 2426.1 20228.8 325.7 8748.8 2060.7 2126.8 272.9	C1-alkylphenols	o-Cresol 0.6847 7.9 861.2 658.8 5402.0 164.6 2954.8 297.4 1026.3 285.5	m-Cresol 0.8990 11.5 1034.9 1011.5 4073.3 142.8 3667.1 317.6 818.5 232.8	p-Cresol 1.0000 10.2 1187.3 1102.2 4264.8 55.0 2553.3 861.4 466.1 330.4	Sum total C1-alkylphenols 29.6 3083.3 2772.5 13740.2 362.4 9175.2 1476.4 2310.9 848.7	C2-alkylphenols	2-Ethylphenol 1.3203 0.58 22.37 16.92 62.69 2.39 45.13 14.14 10.18 11.31	2,6-Dimethylphenol 1.4847 6.08 48.62 27.26 68.84 11.64 86.60 9.62 43.38 14.24	2,5-Dimethylphenol 1.6379 18.06 99.81 70.32 79.96 10.87 154.76 26.49 40.74 22.03	2,4-Dimethylphenol 1.7354 21.59 143.14 102.69 133.08 6.33 198.76 40.26 29.66 64.01	3-Ethylphenol 1.7772 20.52 61.49 61.42 167.09 5.35 156.52 39.88 21.80 49.57	3,5-Dimethylphenol 1.8774 37.52 79.86 63.89 149.48 7.30 167.62 24.45 35.12 24.68	4-Ethylphenol 2.0000 9.03 42.08 30.67 63.99 6.13 61.74 35.36 17.40 23.21	2,3-Dimethylphenol 2.0214 6.82 28.80 29.17 59.71 4.85 49.46 28.44 14.67 17.14	3,4-Dimethylphenol 2.3089 5.91 63.23 55.86 50.37 3.80 96.62 30.43 19.30 24.26	Sum total C2-alkylphenols 126.11 589.41 458.20 835.21 58.66 1017.23 249.06 232.26 250.45	C3-alkylphenols	2-Isopropylphenol 1.6379 2.39 13.56 11.54 2.34 0.48 9.75 1.74 3.45 2.46	2-n-Propylphenol 2.0550 0.25 2.48 2.13 6.50 0.11 3.88 0.75 1.12 0.51	3-Isopropylphenol 2.2813 7.85 15.18 11.96 15.85 0.84 21.42 3.40 2.45 7.75	2,4,6-Trimethylphenol 2.5474 8.45 16.60 10.87 3.70 0.16 12.05 2.71 2.78 4.42	4-Isopropylphenol 2.6361 0.05 45.47 40.31 10.41 0.12 39.08 6.36 3.87 7.67	3-n-Propylphenol + 3-ethyl-5-2.7187 methylphenol 11.09 34.17 29.29 55.08 0.92 43.16 10.34 5.98 19.17	3-Ethyl-4-methylphenol 2.8716 nq 4.52 3.24 2.04 0.36 5.64 1.26 2.61 1.34	2,3,6-Trimethylphenol 2.8720 2.34 10.76 7.70 4.83 0.85 13.52 3.00 6.24 3.17	2,3,5-Trimethylphenol 2.9908 0.02 18.09 13.70 17.90 1.39 19.13 5.75 3.11 9.78
	587	588	589

Table 5 .

 5 A selection of unidentified alkylphenols, found in produced water.

	Unidentified alkylphenols	APRI	Concentration, µg/l
	(arbitrarily assigned number)		(Average for 9 locations)
	C3
	1	2.1560	0.490
	2	2.3425	3.510
	3	2.4771	0.574
	4	2.5168	5.969
	5	3.0471	5.678
	6	3.0748	1.219
	7	3.1939	0.693
	8	3.4598	0.939
	9	3.6094	1.464
	C4
	10	3.2465	6.587
	12	3.4571	1.336
	C5
	17	3.8615	0.657
	19	4.0000	1.087
	C6
	22cl	4.6175	0.118
	29cl	4.8584	0.077
	C7
	1cl	4.4940	0.021
	8cl	4.7590	0.029
	C8
	29	7.7682	0.027
	35	8.6786	0.028
	cl -cluster.

Fig.1

Akcnowledgements

We are grateful to Research Council of Norway for financial support, to Professor Jon Songstad for valuable discussion and for laboratory facilities, to Dr. Nils Åge Frøystein for the help with interpretation of NMR spectra, to Dr. Craig Morton for the help with the English and to Julie Andersen for the help with creating the map.


Jia-Jang Hung
email: hungjj@mail.nsysu.edu.tw

Chia-Sui Hung

Huei-Meei Su

Biogeochemical responses to the removal of maricultural structures from an eutrophic lagoon (Tapong Bay) in Taiwan

Keywords: Carbon, Nutrient, Biogeochemical processes, Budgets, Environmental change, Tapong Bay

The biogeochemistry of Tapong Bay, one of the major lagoons in southern Taiwan, was studied from 1999 to 2004, encompassing a period in 2003 in which aquaculture activities were terminated and the associated structures removed.

Removal of the maricultural structures resulted in the reduction of the mean time for water exchange time in Tapong Bay from about 10±2 days to 6±2 days. The annual mean concentration of measured nutrients (DIN, DIP & DSi) also decreased significantly, likely due to improved water exchange, ceased feeding and increased biological utilization. An overall high primary production was maintained and likely to be constrained by temperature, light availability and turbidity rather than by abundant nutrients throughout the study period. The change in stoichiometric ratios among inorganic and organic nutrients (C/N, Si/N, N/P) may be attributed to the shift of plankton community between the two periods. The annual mean of POC/ PON was 8.1, a little bit larger than that (7.3) before removal, also possibly resulting from the shift of planktonic community (from diatom-dominated to flagellate-dominated and increase of total phytoplankton & zooplankton) and removal of periphyton and oysters. The Tapong Bay shows a 37% increase (from 5.6 to 7.7 mol C m-2 yr-1) in net ecosystem production (NEP) after structure removal, although the increase was not statistically significant. The change in environmental conditions has therefore ACCEPTED MANUSCRIPT influenced profoundly the carbon and nutrient biogeochemical processes and budgets in the semi-enclosed ecosystem.

Introduction

Tapong Bay is a tropical lagoon located on the southwestern coast of Taiwan.

The lagoon is relatively shallow and has been extensively exploited as maricultural farms for decades, during which the lagoon was occupied largely by oyster culture racks and caged fishery structures. The cultured oysters and fishes tend to release nutrients into the lagoon via recycling from excretion (Souchu et al., 2001) and remineralization of feces and pseudofeces (Reusch & Williams, 1998). The lagoon ecosystem has undergone eutrophication due to poor circulation and continuous inputs of nutrients and organic matter from internal and external sources, as the lagoon was also surrounded by watersheds extensively used for urban and aquacultural purposes (Hung & Hung 2003;Hung et al., 2003). In recent years, the government has initiated a grand plan to develop Tapong Bay as a central part of the National Scenic and Recreational Park. The lagoon-associated environments were cleaned up with the complete removal of the maricultural structures from the lagoon around January 2003.

Tapong Bay was previously reported to be an autotrophic ecosystem with a sink for carbon dioxide (Hung & Hung, 2003). A net nitrogen fixation was also found before the removal of maricultural structures (Hung & Hung, 2003). There was no evidence of any change in biogeochemical processes and ecosystem functioning after the removal of surface structures. Although the water quality of the lagoon was expected to improve, primary production could change significantly and shift the trophic states, as oysters were totally removed and rack-associated periphytons would largely vanish after the removal of surface structures.

Accordingly, Tapong Bay was a near ideal site for documenting the biogeochemical responses to controlled ecosystem changes. Thus, this study aims to illustrate and compare the biogeochemical dynamics and budgets of carbon and nutrients before and after the removal of maricultural structures from the lagoon.

Materials and methods

Study site

Tapong Bay is a small and semi-enclosed coastal lagoon in southwestern Taiwan (22°27'N, 120°26'E) (Fig. 1). The total area of the lagoon is around 5.32 km 2 , and it has a volume of approximately 11.6 x 10 6 m 3 . The depth ranges from 1 m near the tidal inlet to 6 m in the inner bay with a mean depth of 2.2 m. Water exchange between Tapong Bay and Taiwan Strait is driven primarily by a semi-diurnal dominated tide, which is somewhat restricted by exchanging water through a narrow tidal inlet. In addition to direct input of precipitation, the terrestrial water input via the Lipan Dike is derived mainly from urban and aquacultural wastewater with a moderate salinity (<20). The lagoon was farmed extensively with hanged oysters and caged fishes, but all maricultural structures were completely removed around January

2003.

Temperature and rainfall patterns differed between dry (October -April) and wet (May -September) seasons. The temperature of lagoon water, which might respond quickly to the change of air temperature, and ranged from about 22 °C in winter (dry season) to 32 °C in summer (wet season) (Hung & Hung, 2003). Spring and fall seasons are transition periods and last only 1-2 months each. Northeastern and southwestern monsoons prevail respectively during the dry and wet seasons, which also affect the mixing of lagoon water. Table 1 shows that total inputs of precipitation and wastewater are much greater in the wet season than in the dry season.

Because of small lagoon volume, lagoon salinity was subject to seasonal variability, ranging from 25.4 in August 2000 to 35.1 in May 2003, regardless of removal of the structures during the study period. Samples were taken bi-monthly before structure removal but were taken either bi-monthly or quarterly after structure removal because of restricted access. Water samples were collected from upper, middle and bottom layers using a peristaltic pump and a pre-cleaned silicone tube. Dissolved oxygen (DO) was measured in situ with a portable DO meter (YSI 52) calibrated by the method of direct spectrophotometry of total iodine [START_REF] Pai | Determination of dissolved oxygen in seawater by direct spectrophotometry of total iodine[END_REF]. The reproducibility was better than 3%. Water column pH was measured in situ with a portable pH meter (Mettler MP-120) with reproducibility better than ±0.02. Salinity was determined with an Autosal salinometer (Guildine 8400B) in the laboratory to gain precise salinity values (±0.002) for deriving salt and water budgets. Chlorophyll a samples were collected in situ by filtering 100 ml water through GF/F filters that were frozen in the dark until analyses in the laboratory. Another four liters of each sample were stored in a polyethylene bottle and brought back to the laboratory immediately for further quantitative filtration and analyses.

Sampling and analytical methods

A part of each water sample was filtered through pre-combusted GF/F filters (at 450 °C, 4 hr). The filtered water was used to measure dissolved nutrients, dissolved organic carbon (DOC), nitrogen (DON) and phosphorus (DOP). Three replicate measurements were made for each chemical analysis. Dissolved inorganic nitrogen (NO 3 +NO 2 +NH 4 , hereafter DIN), phosphate (PO 4 3-, hereafter DIP) and silicate (H 4 SiO 4 , hereafter Dsi) were determined colorimetrically [START_REF] Grasshoff | Methods of Seawater Analysis[END_REF] with an UV-VIS spectrophotometer equipped with a flow injection analysis.

Precision was better than 5%. DOC was measured by the high temperature catalytic oxidation method (Shimadzu TOC 5000) [START_REF] Hung | Dissolved and particulate organic carbon in the southern East China Sea[END_REF] with precision better than 4%. DON was determined from the difference between total dissolved nitrogen (TDN) and dissolved inorganic nitrogen. TDN was measured with the high temperature oxidation and chemiluminescent detection (Antek N/S analyzer) with reproducibility within ±8% for seawater (n = 8). DOP was determined from the difference between dissolved inorganic phosphate and total dissolved phosphorus (TDP) that was measured by the UV-persulfate oxidation and colorimetric method [START_REF] Ridal | A re-examination of the measurement of dissolved organic phosphorus in seawater[END_REF]. Reproducibility of TDP measurement in seawater (n = 8) was ±7%.

Chlorophyll a was extracted with 90% acetone and determined with a Turner Design 10-AU fluorometer [START_REF] Welschmeyer | Fluormetric analysis of chlorophyll a on the presence of chlorophyll b and pheopigments[END_REF]. Phytoplankton production and dark community respiration were determined with oxygen evolution and consumption, respectively, by conducting incubation experiments conducted outdoors near the Tapong Bay. Seawater samples were generally collected at low tide in the early morning, separated into 300-ml BOD bottles, and incubated under light and dark conditions by running surface seawater to maintain temperature at ambient levels.

The light bottles were incubated at 100%, 50% and 20% of the surface irradiance by shading with different black screens. Both light and dark incubations were run for at least 6 hrs. Net production (NP) and respiration rates (mmol O 2 m -2 h -1 ) were derived from changes in DO concentrations monitored at initial and final incubations.

Concentrations of DO were measured by a spectrophotometric method adapted from the Winkler titration [START_REF] Pai | Determination of dissolved oxygen in seawater by direct spectrophotometry of total iodine[END_REF]. Net production and respiration rates were expressed as carbon equivalent rates by using 1.2 as photosynthetic and respiration quotients. Gross production (GP) was estimated as the sum of NP and respiration rate.

Particulate matter samples collected on GF/F filters were used for analyses of particulate organic carbon (POC) and nitrogen (PON). The filtered samples were placed in silver boats and then determined for POC and PON with a C/N/S analyzer (Fisons NCS 1500) after removing the inorganic carbon with 2 M hydrochloric acid [START_REF] Hung | Lateral transport of lithogenic particles from the continental margin of the southern East China Sea[END_REF]. The blank value attributed to pre-combusted GF/F filter and silver boat were deduced from the raw value of a sample concentration; the precision was ±0.3 µM C and ±0.2 µM N.

ACCEPTED MANUSCRIPT

Biogeochemical modeling approach

The biogeochemical fluxes and metabolism of nutrients and carbon in the lagoon were evaluated by using the LOICZ biogeochemical budget model [START_REF] Smith | Tomales Bay metabolism: C-N-P stoichiometry and ecosystem heterotrophy at the land-sea interface[END_REF][START_REF] Gordon | LOICZ Biogeochemical Modelling Guidelines[END_REF]Smith & Hallibaugh, 1997). The fundamental principles of modeling are that non-conservative nutrient and carbon budgets can be constructed from non-conservative distributions of nutrients and water budgets, which in turn are constrained from the salt balance. The non-conservative flux of a material is estimated from the flux deviation between inputs and outputs based on salt and water balances. The non-conservative flux of dissolved inorganic phosphorus is assumed to be an approximation of net metabolism, as phosphorus is not involved in gas-phase reactions.

Nitrogen and carbon both have other major pathways, such as denitrification, nitrogen fixation, and gas exchange across the air-sea interface, and calcification. The biogeochemical pathways of carbon can be approximated from non-conservative phosphorus flux and C-P stoichiometric ratio of reactive particles in the lagoon. Details of modeling and carbon-budget derivation can be found from the LOICZ web site (http://www.loicz.org/) and Smith & Hallibaugh (1997) or from Hung & Hung (2003) for those budgets in the before removal period. Because the distinct variability in wastewater and material inputs with time, the non-steady state method (Smith & Hallibaugh ,1997) was applied to derive water, nutrient and carbon budgets for each sampling period. Those two-month and/or quarter block-averaged budgets were integrated to give an annual budget for both before and after structure removal..

Results and discussion

ACCEPTED MANUSCRIPT

Hydrological variability throughout the study period

As Tapong Bay is a small and choked lagoon with only one tidal inlet for water exchange and a dike (Lipan) allowing brackish water to flow in, the spatial and temporal variability of hydrological conditions were likely to be determined by the seasonal rainfall pattern and tidal dynamics. The study area encompasses a wet season with southwest monsoon prevailing from May to September, and a dry season with northeast monsoon prevailing from October to April (Water Resources Planning Commission, 1997). Thus, temporal variability of lagoon-averaged temperature was parallel to the seasonal change in air temperature through the whole study period (Table 1). Although the structure removal may increase surface temperature by removing the shedding effect, it may also pose a cooling effect by improving flushing.

Consequently, the temperature difference between before and after periods was not statistically significant (Table 4). Salinity distributions were closely related to precipitation and seawater exchange. Dominant semi-diurnal and diurnal tides controlled primary water exchange and subsequently drove the lagoon circulation.

Two sub-anti-clockwise circulation patterns were separated generally by the middle area of lagoon in a northeast-southwest direction [START_REF] Yu | Dynamic interaction and numeric simulation among Tapong Bay[END_REF]. This circulation pattern showed no significant difference between before and after removal of the maricultural structures (Yu, C.-S., personal communication). Therefore, the spatial pattern of salinity was similar, with higher salinity near the tidal inlet and lower salinity near the Lipan Dike in surface and bottom waters for both periods. However, the spatial gradient appears to be more pronounced in surface water than in bottom water. The salinity in summer (August) was slightly lower in 2003 (after removal) than in 1999 (before removal) (Fig. 2), primarily due to the higher net precipitation in 2003.

Spatial distribution of salinity in the dry season also shows a decreasing pattern from the outer to the inner lagoon (data omitted for brevity).

The hydrochemistry and water budgets of Tapong Bay before removal of the maricultural structures have been described by Hung and Hung (2003). Briefly, the water exchange time ranged from 7 days (summer) to 13 days (winter) with a mean of 10±2 days. It was also longer in the inner lagoon (7-24 days) than in the outer lagoon (4-12 days). The water exchange and circulation were apparently restricted by the densely occupied maricultural structures. After removal of the maricultural structures, the water exchange time decreased to the range of 4-9 days with a mean of 6±2 days. The differences were only significant at the 10% probability level (Table 4). As the major circulation pattern was quite similar to the previous condition before the structure removal, the water exchange time in the inner and outer lagoons after removal of the structures was reduced proportionately to the previous conditions to 5-13 days and 3-7 days, respectively. Apparently, the flushing efficiency of lagoon water was significantly improved after the surface structures were removed completely from the lagoon.

Distributions of lagoon-averaged pH and DO were always higher in surface than in bottom waters, resulting more likely from higher primary production and air exchange in surface than in bottom waters (Fig. 2). In addition, lower concentrations of pH and DO may also be caused by intensive respiration and organic matter decay occurring in bottom water and/or surface sediments, as more than 50% of lagoon surface sediments contain rather high TOC (4-9%) contents [START_REF] Hsieh | The detrital food web on the Kaoping coast (II): Food web of tapong lagoon[END_REF]. This inference was supported by the data showing higher pH and DO in surface water in summer than in winter. Summer stratification may be also responsible for lower DO concentrations in bottom water. The surface-bottom differences of DO distributions were significant at the 10% probability level for the before period and 5% probability level for the after period. However, the surface-bottom differences of pH were not statistically significant, likely due to small ranges of distributions. There was no particular trend throughout the time span of this study. Nevertheless, concentrations of DO showed a significant increase most likely due to a better condition of water flushing after the removal of surface structures (Table 1). The differences were significant at the 5% probability level for both surface and bottom waters (Table 4).

Taking summer as an example, spatial variations of DO display a strong gradient decreasing from the tidal inlet to the inner zone for both surface and bottom waters during the two study periods (Fig. 3). The differences between the highest (near tidal inlet) and lowest concentrations (inner lagoon) were significant (<5% level) in both periods. The spatial variation of pH was less significant. In general, the pH value was slightly lower in the inner lagoon than in the outer lagoon, in bottom water than in surface water, and in summer than in winter (data omitted for brevity).

However, the pH differences between before and after periods were insignificant (Table 4). Seasonal variations of flushing rates, water temperature, terrestrial inputs, biological production and respiration appeared to control the temporal and spatial distributions of DO and pH in the lagoon throughout the study period.

Hydrochemical changes may have subsequently influenced biogeochemical processes and budgets of nutrients and carbon after the surface structures were removed from the lagoon.

Effects of structural removals on nutrient dynamics

Spatial patterns of DIN and DIP (DIP omitted for brevity) distributions showed a similarly pronounced increase from the tidal inlet to the inner lagoon during the summer season (Fig. 4), reflecting the strong influence of external inputs from Lipan

Dike and tidal flushing. The concentrations were significantly higher (<10% probability level) in bottom water than in surface water, attributable to intensive biological uptake in surface water and remineralization in bottom water and sediments.

Spatial distributions of DIN and DIP in other seasons, although not shown, exhibited the same patterns as those in summer. After structure removal, the spatial patterns of nutrient distributions showed no obvious change in the lagoon. The differences between the highest (inner lagoon) and lowest (near tidal inlet) concentrations of nutrients were all significant at the 5% probability level (data not shown) in both before and after periods.

Concentrations of DIN and DIP decreased significantly over the entire lagoon in summer (Figs. 4) and in other seasons (Table 2) after removal of the surface structures.

The differences were significant at the 1% probability level for DIP and 5% probability level for DSi, but were at the 10% probability level for DIN. 10.0 µM) for DSi after removal of the structures. Despite significant decreases in concentrations, the concentration levels are still higher than those reported for non-eutrophic lagoons (Smith et al., 1997;Smith & Crossland, 1999;Dupra et al., 2000ab;[START_REF] Mcglathery | Magnitude and variability of benthic and pelagic metabolism in a temperate coastal lagoon[END_REF], and comparable to those in the state of eutrophication [START_REF] Tada | Standing stock and production rate of phytoplankton and a red tide outbreak in a heavily eutrophic embayment, Dokai Bay, Japan[END_REF][START_REF] Hung | Temporal variability of carbon and nutrient budgets from a tropical lagoon in Chiku, southwestern Taiwan[END_REF][START_REF] Newton | Evaluation of eutrophication in the Rio Formosa coastal lagoon, Portugal[END_REF]. The decrease in concentrations was attributed mainly to improved water flushing, which resulted in a period of 4 days shorter compared with the previous condition in water exchange time after removal of the surface structures. The increased NEP, which will be discussed in the final section, might also enhance biological uptake of nutrients and lead to lower nutrient concentrations after removal of the surface structures. This inference can be supported by the occurrence of a larger difference in concentrations between the two periods during summer with higher NEP, relative to the conditions of other seasons. In addition, the nutrient decrease could also be due to the termination of fish feeding after structure removal, although the area of caged fish farming was relatively small compared to that of oyster culture.

Compared with DIN and DIP distributions, DON distributions appear to be higher than those of DIN in both before and after removal periods. Such difference was commonly reported in most surface waters, as DON is generally derived from biological production [START_REF] Hansell | Biogeochemistry of total organic carbon and nitrogen in the Sargasso Sea: control by convective overturn[END_REF]Hung et al., 2003). Similar to other eutrophic systems [START_REF] Tada | Standing stock and production rate of phytoplankton and a red tide outbreak in a heavily eutrophic embayment, Dokai Bay, Japan[END_REF][START_REF] Anderson | Nutrient pulses, plankton bloom, and seasonal hypoxia in western Long Island Sound[END_REF], the concentration of ammonium species was frequently equal to or greater than that of nitrate, while DON distributions generally dominated the total dissolved nitrogen in the lagoon throughout the study period. In the Tapong Lagoon, DON distributions were likely to be derived from both external sources (Lipan Dike) and in-situ production (Hung & Hung, 2003). This was also true for DOC distributions. Consequently, the spatial distributions of DOC and DON followed closely that of DIN, with higher concentrations (5% probability level) in the inner lagoon than in the outer lagoon.

Despite a less distinct gradient, the concentration of DOP was also higher in the inner lagoon than in the outer lagoon for both periods.

The significant increase of DOC and DON concentrations after structure removal was largely attributed to the enhancement of gross production and nitrogen fixation (Table 3), as external inputs were not much difference between the two periods.

Both gross production and nitrogen fixation not only fix carbon and nitrogen as the particulate organic form but also release them into water as the dissolved organic form.

Such inference also supports the fact that DOP distributions, on the contrary, were lower than those of DIP, as well as lower in the after period than in the before period, as nitrogen fixation didn't involve DOP release. Thus, the decrease of DOP distribution may be largely determined by a better flushing condition (higher dilution effect). The differences between the two periods were only significant at the 10% probability level for DOC, DON and DOP.

Variability in elemental ratios of nutrients

Elemental ratios of DIN, DIP and Si are displayed in Fig. 5. The DIN/DIP ratio ranges from 1.5 to 9.2 throughout the study period, which is much lower than the Redfield ratio (16) [START_REF] Redfield | The influence of organisms on the composition of sea water[END_REF] (Fig. 5c). The low DIN/DIP ratios were likely to have been influenced by inputs of P-contaminated wastewater through the Lipan Dike with very low DIN/DIP ratios (< 2.5). Such ratios imply that the system was either biologically limited by DIN or under the condition of DIP excess. As both DIN and DIP concentrations were much higher than the critical levels (DIN < 1 µM; DIP < 0.1 µM; DSi < 2 µM) of nutrient limitation (Harrison et al., 1977;[START_REF] Perry | Phosphate uptake by phytoplankton in the central North Pacific Ocean[END_REF][START_REF] Nelson | Kinetics of silicic acid uptake by natural diatom assemblages in two Gulf Stream worm-core rings[END_REF][START_REF] Justic | Stoichiometric nutrient balance and origin of coastal eutrophication[END_REF] regardless whether the structures were removed or not, the lagoon appears to be under the condition of DIP excess. Low DIN/DIP ratio is one of necessary conditions for nitrogen fixation, and this could lead to net nitrogen fixation throughout the study period. There was no particular trend in temporal variability even after the structure removal, although the DIN/DIP ratio was slightly higher in summer than in other seasons possibly due to intensive uptake of nutrients in summer.

It was much clearer that the Si/DIN ratio was generally higher in summer

(1.6-2.5) than in other seasons (0.6-1.5), except for the period of July 2002 when a lower precipitation rate perhaps induced a lower silicon input. Higher Si input and intensive DIN uptake during most summer seasons could result in Si/DIN greater than 1.0, corresponding to the ratio of diatoms [START_REF] Brzezinski | The Si:C:N ratio of marine diatoms: interspecific variability and effect of some environmental variables[END_REF]. The shift of plankton community structures may determine the seasonal variations of Si/DIN ratios, as Su et al. ( 2004) reported a significant difference in plankton community between summer and winter (p = 0.05). In summer, the uptake of Si may decrease as the occurrence frequency of diatoms (Skeletonema costatum) decreases, and cyanobacterium Oscillatoria sp. and Chaetoceros sp. become the most common species (Su et al., 2004).

Distributions of Si/DIP ratios (1.2-18.5) follow closely the seasonal patterns of Si/DIN. The consistently higher ratios in summer may also be due to higher input of Si and intensively biological DIP uptake in the same season. With the exception of first sampling, the ratios are lower than the typical ratio (16) in diatoms (Brzezeniski, 1985) throughout the study period, which is consistent with the above-mentioned DIP excess in the lagoon. No significant difference was found before and after removal of the surface structures.

Although we did not make systematic measurements of particulate organic phosphorus, the lagoon-averaged particulate organic C/N ratio was slightly lower in the period before (C/N= 7.3) than after (C/N= 8.1) removal of the structures, although the differences were not statistically significant. Such ratios imply that particulate organic matter in the lagoon may be mainly of biological origin, as the C/N ratios of external detritus were much greater (> 9.2) than the Redfield ratio. [START_REF] Hsieh | The detrital food web on the Kaoping coast (II): Food web of tapong lagoon[END_REF] reported the values of 13 C in suspended POC ranging from -19.5 to -20.7 ‰ in both periods, supporting strongly the biological origins of POC. Both C and N may not be highly enriched through biological utilization. The change in grazing impacts of zooplanktons (Lo, 2004) and periphyton distribution [START_REF] Lin | Relative importance of phytoplankton and periphyton on oyster-culture pens in a eutrophic tropical lagoon[END_REF] may determine the slight change of particulate C/N ratio after the removal of the structures.

Comparison of gross production and biomass between the two periods

Spatial distributions of total planktonic cell number, biomass (Chl-a) and integrated gross production (IGP) before and after removal of the structures were compared (Fig. 6). Significant correlations were found between these three parameters (p < 0.05). The number of cell, Chl-a and IGP increased consistently from the tidal inlet zone to the inner lagoon, and the increase was more pronounced after than before the period of structure removal. Thus, distributions of gross production, number of cell and Chl-a for individual samples (volumetric rates) generally correlate inversely with salinity (p < 0.01) but not significantly with nutrients (p > 0.05). The spatial distribution patterns for these three parameters are likely to be from the result of spatial gradients of salinity, water depth, water stability and/or nutrients.

All three parameters are elevated after structure removal, attributed largely to improved water quality. The increase in gross production may be mainly due to increases in DO concentrations and solar radiation availability by improving flushing efficiency and removing shading effect from the surface. Although temporal distributions of lagoon-wide integrated Chl-a do not co-vary with integrated DIN and DIP, the integrated Chl-a correlates very well with temperature, solar radiation and DO before structure removal (Fig. 7). Such relationships are largely maintained after the structure removal (data omitted for brevity). Although many experiments conducted in mesocosms and coastal systems have established the positive relationship between nutrient inputs and phytoplankton biomass or production [START_REF] Twilley | Nutrient enrichment of estuarine submersed vascular plant communities: 1. Algal growth and effects on production of plants and associated communities[END_REF][START_REF] Monbet | Control of phytoplankton biomass in estuaries: A comparative analysis of microtidal and macrotidal estuaries[END_REF], these systems do not generally suffer from over enrichment of nutrients. Because of high abundance of nutrients and contrasting distributions between IGP (or Chl-a) and nutrients in bottom waters of most regions of Tapong Lagoon, the lagoon-wide integrated DIN and DIP become non-critical in determining gross production. Distributions of Chl-a significantly correlate inversely with total suspended matter (r<-0.56, p<0.0001), but correlate poorly with nutrients. Thus, gross production and biomass (Chl-a) may be primarily controlled by light availability and temperature in this lagoon with high turbidity and abundant nutrients. Therefore, summer distributions of cell number, Chl-a and IGP are higher than those in other seasons, and the structure removal enhances gross production and abundance of phytoplankton biomass.

It is worth noting, however, that the periphyton production attributable up to 6% of gross production before the structure removal [START_REF] Lin | Relative importance of phytoplankton and periphyton on oyster-culture pens in a eutrophic tropical lagoon[END_REF] may eventually vanish after removal of the structures. The removal of major suspension feeder (oysters) and change of habitat environments may subsequently alter the food web structure and lagoon carbon budgets as well.

Effects of structure removals on nutrient and carbon budgets

Nutrient and carbon budgets were determined principally by water budget, nutrient and carbon distributions and internal biogeochemical processes. For a nutrient budget, the difference between total inputs and total outputs is an indication of source (inputs < outputs; positive ∆Y) or sink (inputs > outputs; negative ∆Y) in the ecosystem. Individual budget of each component for a sampling time was estimated by using the non-steady state method (Smith & Hallibaugh, 1997) and the time interval-averaged budgets were listed in Table 3. Data show that the lagoon was a sink for DIP (∆DIP < 0) for most sampling periods, and consequently a sink for an annual basis in the period before and after structure removal. There was no particular trend in seasonal patterns as ∆DIP was determined by many physical (tidal fluctuation, external inputs of water and nutrients) and biogeochemical factors (internal sources, production/respiration, biological community structures). The magnitude of DIP sink is larger in the period after than before removal of the structures (Table 3). This is particularly interesting because the ∆DIP can be scaled up to the carbon budget according to LOICZ modeling.

Distributions of ∆DOP are variable for different sampling events and exhibit a positive value (0.01 mol m -2 yr -1 ) and a negative value (-0.07 mol m -2 yr -1 ) before and after the structure removal, respectively. With ∆DIP and ∆DOP, the lagoon system provides a sink for total phosphorus (∆P < 0) for both periods. However, the lagoon system appears to be a source for total dissolved nitrogen (∆N > 0) in both periods before and after the structure removal, and the magnitude is greater for the latter period than for the former period. This is largely due to significant sources of DON but contrasting conditions for DIN in both separated periods. This may imply that various denitrification may occur in the lagoon throughout the year, but nitrogen fixation contributes to DON pool.

The net ecosystem metabolism (NEP) derived from ∆DIP (p-r = -106∆DIP) may provide a short cut to understanding carbon budgets through nutrient budgets, as direct observations of NEP involve complicated experiments. The basic assumption is that the internal reaction flux of DIP should be proportional to production and consumption of particulate organic matter (POM) [START_REF] Gordon | LOICZ Biogeochemical Modelling Guidelines[END_REF], although the adsorption/desorption of DIP onto/from particulates is possible under certain environmental conditions. Assuming that DIP adsorption was almost balanced by DIP desorption during any two sampling events, the system ∆DIP may then approximate the NEP of the lagoon. The system with a negative ∆DIP is generally regarded as an autotrophic and net CO 2 consuming system via a net production of organic matter (production > respiration). In contrast, the system is heterotrophic showing a net CO 2 production if ∆DIP is positive. The temporal variation of lagoon NEP is rather pronounced throughout the experiment (Fig. 8). The NEP is all positive except for one being negative and two approaching the balance. Thus, Tapong

Lagoon exhibits clearly an autotrophic system while individual NEP(s) are averaged from two separated periods (Table 3). Before structure removals, the overall NEP

(5.6±6.1 mol C m -2 yr -1 ) is equivalent to 6.0±6.5% of the annual gross production (mol C m -2 yr -1 ). After removal of the surface structures, the overall NEP (7.7±20.4 mol C m -2 yr -1 ) is about 5.8±15 % of annual gross production

Although the mean value of NEP increased obviously (37%) after removal of the maricultural structures, the increase (difference) was not statistically significant, primarily due to large temporal variations (standard deviations) in both before and after removal periods. It may be conservatively said that the small increase of NEP was likely due to alteration in biological community structures, leading to just a slight excess for gross production over community respiration. As producer (periphytons)

and important phytoplankton consumer (oysters) were completely removed from the lagoon, the food web of lagoon should change significantly. Actually, the jellyfish, which was an important feeder of zooplanktons, almost disappeared after oyster racks were removed from the lagoon (Lo, 2004). Consequently, distributions of phytoplankton biomass and zooplankton population increased significantly after maricultural structures were removed from the lagoon. The lagoon ecosystem tends to be unstable once the structures were removed; thus, a longer period of experiment may be necessary to clarify the change of carbon budget. At current situation, the lagoon appears to be a carbon sink with a small increased value compared with its previous condition.

Conclusion

Tapong Bay is a semi-enclosed lagoon and an ideal site for studying the effects of environmental changes on ecosystem carbon and nutrient biogeochemical processes and budgets, as a critical land-use change occurred during the time course of this study. Before the structure removal, the lagoon was autotrophic with a net organic carbon production (5.6±6.1 mol C m -2 yr -1 ). However, the lagoon system was a source of total dissolved nitrogen. After the structure removal, the lagoon remained autotrophic and the net organic carbon production increased up to 7.7±20. Before removal Aug-1999 -0.58 3.40 -0.06 0.26 2.82 0.20 -0.38 -6.36

Oct-1999 -1.00 0.62 -0.13 0.64 -0.46 0.51 -8.62 -13.8

Dec-1999 -1.15 3.28 -0.36 -0.32 2.25 -0.68 13.1 -38.2

Feb-2000 -0.28 3.21 0.02 -0.38 3.04 -0.36 8.80 2.12 Apr-2000 0.92 1.51 -0.02 0.06 2.52 0.04 1.88 -2.12 Jun-2000 -0.08 1.89 -0.08 0.26 1.60 0.18 -1.28 -8.48

Aug-2000 2.28 -1.53 -0.42 -0.25 0.66 -0.67 1999-2001) and after (2003-2004)

  Water samples were collected from Tapong Bay (Sta. 1 ~ Sta. 10, Fig. 1), Lipan Dike, and the adjacent coastal sea, before (from August 1999 to July 2002) and after (from February 2003 to September 2004) removal of the maricultural structures.

  Concentrations of DIN, DIP and DSi ranged from 9.1 to 30 µM (ave.16.4 µM), from 1.6 to 7.6 µM (ave. 4.0 µM) and from 11.0 to 39.6 µM (ave. 20.3 µM), respectively, before removal of the surface structures. The concentrations decreased to 8.0-18.2 µM (ave. 11.4 µM) for DIN, 0.2-3.0 µM (ave. 1.5 µM) for DIP, and 4.2-19.7 µM (ave.

  4 mol C m -2 yr -1 , despite a slight increase in the state of nitrogen source. Significant changes in tidal flushing, light availability and water quality following the structure removal were likely responsible for the enhancement of gross production. The shift of biological community structures due to alteration of habitat environments may eventually change the regimes of dark community respiration. The improvement of water quality and natural ecosystem production may provide sustainable values to compensate the economic loss caused by the exclusion of oyster and caged fishery farming previously practiced in the lagoon.

Fig. 1 .

 1 Figure captions.

Fig. 2 .

 2 Fig. 2. Temporal variations of lagoon-averaged salinity, temperature, pH and DO in

Fig. 3 .

 3 Fig. 3. Spatial distributions of DO in surface and bottom during summer for

Fig. 4 .

 4 Fig. 4. Spatial distributions of DIN in surface and bottom waters during summer for

Fig. 5 .

 5 Fig. 5. Temporal variations of nutrient ratios for Si/DIP (a), Si/DIN (b) and DIN/DIP

Fig. 6 .

 6 Fig. 6. Comparisons of distributions for averaged total number of cell, Chl-a and

Fig. 7 .

 7 Fig. 7. Relation plots among temperature, solar irradiance, integrated DO and Chl-a

Fig. 8 .

 8 Fig. 8. Temporal variability of NEP (p-r) in Tapong lagoon. The vertical dashed line

  Fig.1

  Fig. 7

Table 1

 1 Temporal variability of freshwater inputs and hydrochemical conditions in lagoon water.

			Freshwater input			Lagoon water
		Sampling	(10 3 m 3 d -1 )
		time	Precipi-tation	Evapo-ration	Salinity	pH	DO (mg l -1 ) surface bottom	(day) #
	Before	Aug-1999	90	22	31.3	8.24	6.38	5.36	7
	structure	Oct-1999	28	16	31.8	8.22	4.44	3.20	12
	removal	Dec-1999	4	12	32.9	8.1	6.33	6.36	12
		Feb-2000	4	12	33.3	7.95	3.99	3.14	13
		Apr-2000	10	21	33.9	8.06	3.87	3.63	11
		Jun-2000	30	26	31.5	8.36	10.7	3.98	10
		Aug-2000	93	18	25.4	8.04	5.27	4.96	9
		Dec-2001	1	12	33.4	8.19	5.05	4.79	9
		July-2002	68	25	32.7	8.44	5.32	3.14	6
		Mean±sd*	36±37	18±6	31.8±2.5 8.22±0.15 5.71±2.07 4.28±1.14 10±2
	After	Feb-2003	2	17	34.4	8.20	8.04	7.79	7
	structure	May-2003	8	28	34.6	8.19	6.12	4.20	5
	removal Aug-2003	128	21	30.4	8.19	10.3	5.71	6
		Nov-2003	10	15	32.7	7.89	6.55	4.31	9
		Jun-2004	6	22	32.1	8.18	7.17	5.00	4
		Sep-2004	143	22	31.3	8.18	8.35	5.65	5
		Mean±sd	49±66	21±5	33±1.6	8.14±0.12 5.44±1.50 6.10±1.31 6±2
	*sd: standard deviation # : water exchange time

  Comparisons of hydrochemical variables, nutrients, and ecosystem properties measured before and after removal of maricultural rafts. P values are results from One-Way ANOVA tests comparing variables (means of all stations for each sampling date) before (

	Table 4
									11.4 -44.5
		Dec-2001 1.21	-	-0.04	-	1.21 -0.04 1.85 -4.66
		Jul-2002	3.74	-	-0.22	-	3.74 -0.22 7.26 -23.0
		Mean	0.56	1.77	-0.15	0.04	1.93	-0.12	3.78	-15.4
		±sd*	±1.63	±1.79	±0.15	±0.37	±1.31	±0.40		±16.4
		Annual	0.21	0.65	-0.05	0.01	0.70	-0.04	1.38	-5.6
		mean#	±0.61	±0.67	±0.05	±0.14	±0.49	±0.15	±2.60	±6.1
	After	Feb-2003 -4.90 -5.70 -1.20 0.02 -10.6 -1.18 8.35	-127
	removal	May-2003 -1.88 12.6 -0.02 -0.51 10.74 -0.53 19.2 -2.01
		Aug-2003 -2.05 6.27	0.25 -1.16 4.22 -0.91 18.7	26.7
		Nov-2003 -1.23 10.0 -0.23 0.01 8.79 -0.23 12.4 -24.7
		Jul-2004	-3.47 4.15 -0.05 0.48 0.68 0.44 -6.30	-5.30
		Sep-2004	-1.69	3.75	0.044	-0.06	2.06	-0.012	2.25	4.16
		Mean	-2.54	5.18	-0.20	-0.20	2.65	-0.40	9.1	-21.2
		±sd	±1.38	±6.33	±0.51	±0.56	±7.55	±0.59	±9.9	±54.3
		Annual	-0.93	1.89	-0.07	-0.07	0.97	-0.15	3.32	-7.7
		mean#	±0.51	±2.37	±0.19	±0.21	±2.83	±0.22	±3.71	±20.4
	#Annual mean (mol m -2 yr -1 ) *sd: standard deviation

Acknowledgements

The authors greatly appreciate Drs. R. Spies (editor) and S. V. Smith (reviewer) for constructive comments in manuscript revision and Dr. J. Purcell for data analysis and discussion. We also thank Mr. S.-M. Wang, C.-M. Ho and Y.-C. Wang for their assistance in sampling and the analyses. This study was supported by the National Science Council, Taiwan, through Contracts: NSC 92-2621-Z110-003-and NSC 93-2621-Z110-001-. A partial support was given by the ''Aim for the Top University Plan''of the National Sun Yet-sen University and Ministry of Education, Taiwan,