Lesley-Ann Martin
email: ann.martin@icr.ac.uk

Giles L S Davies

Marion T Weigel

Nadine Betambeau

Margaret J Hills

Janine Salter

Geraldine Walsh

Roger A ' Hern

Mitch Dowsett

§ Giles

L S Davies

Pre-surgical study of the biological effects of the selective cyclo-oxygenase-2 inhibitor celecoxib in patients with primary breast cancer

Keywords: Estrogen receptor, COX2, celecoxib, pre-surgical study, Ki67

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Introduction

A number of studies over the past two decades have linked the use of non-steroidal antiinflammatory drugs (NSAIDs) and cancer incidence [START_REF] Mehta | Characterization of effective chemo preventive agents in mammary gland in vitro using an initiation-promotion protocol[END_REF]. In particular work relating to NSAIDs in colorectal cancer has reported an inverse relationship between colon cancer incidence and regular use of NSAIDs [START_REF] Thun | Aspirin use and reduced risk of fatal colon cancer[END_REF][START_REF] Rosenberg | A hypothesis: nonsteroidal anti-inflammatory drugs reduce the incidence of large-bowel cancer[END_REF]. Furthermore, randomised placebo-controlled double-blind trials have shown regression in colorectal polyp size after treatment with sulindac [START_REF] Nugent | Randomized controlled trial of the effect of sulindac on duodenal and rectal polyposis and cell proliferation in patients with familial adenomatous polyposis[END_REF][START_REF] Giardiello | Treatment of colonic and rectal adenomas with sulindac in familial adenomatous polyposis[END_REF]. The data in relation to NSAID use and breast cancer incidence are conflicting. For instance a large cohort study of 89,528 registered nurses in the USA showed no association between regular use of asprin and the incidence of breast cancer [START_REF] Egan | Prospective study of regular aspirin use and the risk of breast cancer[END_REF].

By contrast, in a case control study, Harris et al [START_REF] Harris | Nonsteroidal antiinflammatory drugs and breast cancer[END_REF] found a reduced risk of breast cancer associated with the use of any NSAID, three or more times a week for at least a year (RR 0.66). Subsequently a meta-analysis, involving 38 studies supported the inverse association between NSAID use and risk of breast cancer reporting a statistically significant reduction in breast cancer risk associated with use of any NSAID (RR 0.88) and similar associations for aspirin (RR 0.87) and ibuprofen (RR 0.79). No evidence of a dose-response relationship was found and some studies indicated that coxibs were also associated with a lower risk of breast cancer [START_REF] Takkouche | Breast cancer and use of nonsteroidal anti-inflammatory drugs: a meta-analysis[END_REF]. This large-scale meta-analysis is consistent with several smaller meta-analyses [START_REF] Bosetti | Aspirin and cancer risk: an updated quantitative review to 2005[END_REF][START_REF] Gonzalez-Perez | Effects of nonsteroidal anti-inflammatory drugs on cancer sites other than the colon and rectum: a metaanalysis[END_REF][START_REF] Khuder | Breast cancer and NSAID use: a meta-analysis[END_REF][START_REF] Mangiapane | Aspirin use and breast cancer risk: a meta-analysis and meta-regression of observational studies from 2001 to 2005[END_REF].

A chemopreventative effect of NSAIDs for breast cancer would be very important considering their excellent tolerability and inexpensiveness.

The main target of NSAID action is cyclooxgenase (COX), which plays an integral role in the catalysis of arachidonic acid to prostaglandin (PG). Two isoenzymes exist, COX-1, which is constitutively active and COX-2 which is expressed in response to certain stimuli such as tumour promoters, endotoxin, cytokines and hormones (reviewed by Davies et al [START_REF] Davies | Cyclooxygenase (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention[END_REF]). COX-2 expression has been associated with features of aggressive breast cancer [START_REF] Brueggemeier | Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens[END_REF], including large tumour size, positive axillary lymph node metastases and HER-2 postive status [START_REF] Subbaramaiah | Increased expression of cyclooxygenase-2 in HER-2-overexpressing human breast cancer cells[END_REF]. Indirect evidence linking COX-2 to breast cancer has come from studies of tissue PGs, in which high PG levels are associated with poor disease-free and overall survival [START_REF] Bennett | The production of prostanoids in human cancers, and their implications for tumor progression[END_REF][START_REF] Rolland | Prostaglandin in human breast cancer: Evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells[END_REF]. Most notably PGE2, the main catalytic product of COX-2, has been found in high concentrations in tumour cells [START_REF] Schrey | Prostaglandin E2 production and metabolism in human breast cancer cells and breast fibroblasts. Regulation by inflammatory mediators[END_REF] and clinically associated with enhanced metastatic potential and lack of estrogen (ER) and progesterone receptor (PgR) expression [START_REF] Witton | Cyclooxygenase 2(COX2) expression is associated with poor outcome in ER-negative, but not ER-positive breast cancer[END_REF]. Compelling evidence from transgenic models has shown that development of breast tumours is enhanced by expression of COX-2 [START_REF] Liu | Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[END_REF]. A chemopreventive effect of a specific COX-2 inhibitor celecoxib was demonstrated in DMBA-induced mammary tumour models in female rats resulting in a reduction in tumour incidence and tumour volume [START_REF] Harris | Chemoprevention of breast cancer in rats by celecoxib, a cyclooxygenase 2 inhibitor[END_REF]. Whilst more recently the chemopreventive effects of celecoxib have been shown to be limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model [START_REF] Woditschka | The chemopreventive effects of celecoxib are limited to hormonally responsive mammary carcinomas in the neu-induced retroviral rat model[END_REF].

Expression of COX-2 in human solid cancers has also been associated with the neovasculature where significant correlations between COX-2 expression and tumour vascularization and microvessel density have been reported [START_REF] Uefuji | Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer[END_REF]. Of note animal models of angiogenesis have shown that celecoxib reduced both the number and length of sprouting capillaries in a dose dependent manner [START_REF] Masferrer | Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors[END_REF]. In addition to angiogenic effects, COX-2 expression has been shown to inhibit tumour apoptosis an effect negated in human prostate cancer cells by treatment with celecoxib [START_REF] Hsu | The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2[END_REF].

More recently studies have implicated COX-2 with the regulation of aromatase expression [START_REF] Brueggemeier | Aromatase and COX in breast cancer: enzyme inhibitors and beyond[END_REF]. Numerous studies have shown that over 70% of breast tumours are steroid receptor positive and rely on estrogen stimulation for their growth [START_REF] Ali | Endocrine-responsive breast cancer and strategies for combating resistance[END_REF]. In post menopausal women even small differences in serum concentrations of estradiol have been associated risk of breast cancer [START_REF] Thomas | A prospective study of endogenous serum hormone concentrations and breast cancer risk in premenopausal women on the island of Guernsey[END_REF]. Plasma estrogens in postmenopausal women result from aromatase activity in peripheral tissues, particularly adipose. Many breast cancers also contain aromatase [START_REF] Reed | In situ oestrone synthesis in normal breast and breast tumour tissues: effect of treatment with 4-hydroxyandrostenedione[END_REF] suggesting that intratumoural aromatase may be important in breast cancer incidence and progression. There is strong evidence that the promoters that regulate aromatase gene transcription differ between normal and malignant breast tissue [START_REF] Zhou | Aromatase gene expression and its exon I usage in human breast tumors. Detection of aromatase messenger RNA by reverse transcription-polymerase chain reaction[END_REF]. The promoters (exons 1.3 and II) regulating gene transcription in malignant tissue are controlled via cAMP which is known to be stimulated by PGE2 in breast tumour cells, suggesting that PGE2 may induce promoter switching (reviewed by Davies et al [START_REF] Davies | Cyclooxygenase (COX-2), aromatase and breast cancer: a possible role for COX-2 inhibitors in breast cancer chemoprevention[END_REF]). In support of this Bruggemeier's group demonstrated a strong positive correlation between COX-2 and aromatase in 23 human breast tumours using quantitative RT-PCR. Of note however, the highest levels of COX-2 expression were also associated with tumours showing evidence of vascular invasion [START_REF] Brueggemeier | Correlation of aromatase and cyclooxygenase gene expression in human breast cancer specimens[END_REF].

Aromatase inhibitors are currently under investigation in chemoprevention but their systemic side effects such as enhanced bone re-absorption are a significant disadvantage [START_REF] Cuzick | Chemoprevention of breast cancer[END_REF]. Based on the above evidence suppression of COX-2 may provide a means of selectively suppressing estrogen levels in the breast and stimulation of estrogen dependent proliferation in ER+ breast cancer.

Hence there are several putative mechanism by which COX2 inhibitors might act to reduce breast cancer incidence but very limited direct evidence for their involvement in clinical material.

We therefore initiated a randomised controlled pre-surgical study of celecoxib in women with primary breast cancer during the two-week period prior to surgery; The primary purpose was to determine whether COX-2 inhibition led to antiproliferative, proapoptotic and or antiestrogenic effects on molecular markers of biological response.

Materials and Methods

Study

The study design was a randomised phase II trial of celecoxib versus no treatment (2:1).

The drug dose used was 400mg orally twice daily based on studies using celecoxib in Familial Adenomatosis Polyposis [START_REF] Arber | PreSAP Trial Investigators (2006) Celecoxib for the prevention of colorectal adenomatous polyps[END_REF]. The target study size was 40 patients. Using the SD of the log (Ki67) score from a variability study of Ki67, it was estimated that there was an 80% probability of observing a significant Ki67 suppression when the true reduction in Ki67 was ≥ 50%, if complete data on 30 volunteers was derived [START_REF] Harper-Wynne | Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention[END_REF][START_REF] Ellis | Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy[END_REF]. The drug was given in the two-week period between diagnosis and surgical treatment. Patients were eligible for the study if they were postmenopausal, 50-80 years of age, had a clinical diagnosis of stage I or II [American Joint Committee on Cancer (http://www.cancerstaging.org/)], histologically confirmed, primary breast cancer and with a tumour size sufficient to provide a core biopsy, ECOG performance status 0,1 or 2, normal full blood count, normal urea and electrolytes and liver function tests. Patients were excluded if they had recurrent disease, metastases, renal or hepatic impairment, active inflammatory bowel disease or peptic ulceration, current use of NSAIDs including aspirin in the 4 weeks prior to study entry, or use of HRT or oral contraceptives during or 4 weeks prior to study entry.

The Royal Marsden Hospital Research and Ethics Committee approved the study and each patient gave written informed consent.

Biopsy Method

Before the start of the study medication, a core biopsy of the primary breast tumour was obtained using a 14-gauge needle. Ultrasound guidance was used to obtain material from smaller tumours to aid accuracy. At surgery a sample of excised tumour was obtained from the operative specimen. Both specimens were immediately fixed in 10% normal buffered formalin and embedded in paraffin.

Analysis Methods

Measurement of cell proliferation was by immunohistochemical assay using the MIB1 mouse monoclonal antibody to Ki67 [START_REF] Harper-Wynne | Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention[END_REF]. Measurement of apoptosis was by terminal deoxynucleotidyl transferase biotin-dutp nick end labeling technique (TUNEL). The apoptotic index was expressed as a percentage of the number of cells displaying apoptotic bodies [START_REF] Ellis | Reduced apoptosis and proliferation and increased Bcl-2 in residual breast cancer following preoperative chemotherapy[END_REF]. Tumour cells/section were counted for Ki67 (1000 cells) and apoptotic index (3000 cells) based on precision data published previously [START_REF] Detre | Time-related effects of estrogen withdrawal on proliferation-and cell death related events in MCF7 xenografts[END_REF]. ER expression was demonstrated with the Novacastra antibody 6F11 [START_REF] Jotti | Comparison of new immunohistochemical assay for estrogen receptor in paraffin wax embedded breast carcinoma tissue with quantitative enzyme immunoassay[END_REF] and PgR with the Novacastra antibody NCL-PgR clone 1A6 [START_REF] Harper-Wynne | Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention[END_REF]. Samples were also assessed for expression of COX-2 [START_REF] Davies | Correlation between cyclooxygenase-2 expression and angiogenesis in human breast cancer[END_REF] using the antibody PG-27B (Oxford Biomedical Research Inc), and CD31 (DAKO) [START_REF] Davies | Correlation between cyclooxygenase-2 expression and angiogenesis in human breast cancer[END_REF]. ER, PgR and COX2 expression were assessed semi-quantitatively using an H-score as follows: (% of cells intensity 1x1) + (% of cells intensity 2x2) + (% of cells intensity 3x3) providing a score range of 0 to 300. Samples were considered positive for either receptor if the score exceeded 1%. CD31 expression was determined using a Chalkey count [START_REF] Fox | Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis[END_REF].

Two individual observers assessed scoring and a consensus was reached for each biomarker.

Statistical methods

Descriptive statistics were expressed as geometric means. Values at baseline and at 2 weeks were expressed as geometric mean proportions of the baseline and transformed into percentage changes (a negative change denoting a reduction). Comparisons between groups were conducted using the Mann Whitney test with a two-sided 5% significance. The change in each of the parameters and the statistical comparisons are summarised in Table 2.

Results

Recruitment

The change in Ki67 is shown for individual patients according to treatment arm in Figure 1A and B with the ER-negative patients highlighted. The geometric mean change in Ki67 relative to baseline in the celecoxib arm was -16.6% (95%CI -31.4 to +1.4% p=0.056).

Exclusion of the ER-negative patient gave a geometric mean change of -18.1% (95% CI -33.5 to +0.9% p=0.055). The change in the no-treatment group was -8.1% (95%CI -23.2 to +10.1% p=0.24). There was no statistically significant difference in the change between the 2 groups irrespective of the inclusion or not of ER-negative patients.

The change in apoptotic index is shown for individual patients according to treatment arm in Figure 2A and B with the ER negative patients highlighted. Insufficient cells were present in the pre-treatment sample for an estimation of apoptotic index for one patient on the celecoxib arm. There was no significant change in the index with geometric mean changes of +4.0% (95%CI -23.4 to +41.0%, p=1.00) and +8.7% (95%CI -26.1 to +59.7%, p=0.29) in the celecoxib and no-treatment arms respectively.

The effect of treatment on the expression of ER and PgR for those patients showing pretreatment positive status of the respective receptor is shown in Figures 3 and4 respectively. Level of expression and positive/negative status was stable for both receptors during treatment.

CD31 levels increased in 13 of the 14 tumours during celecoxib treatment showing an overall statistically significant increase in expression of 22.2% (95%CI +4.8 to +42.6%, p=0.009; Fig 5). In contrast CD31 expression increased in 4/9 control patients but the difference in change between the 2 arms was not statistically significant. There was no significant effect of treatment on the expression of COX-2 (Fig 6).

Discussion

This study aimed in part to determine whether suppression of COX-2 activity with the selective COX-2 inhibitor celecoxib, would lead to suppression of Ki67 consistent with this providing tissue specific suppression of aromatase activity and an alternative to systemic aromatase inhibition. The target study size was 40 patients but difficulties with competing studies led to its termination after 25 patients. This curtailment of the study inevitably led to loss of statistical power but the data analyzed allowed instructive comparisons with earlier presurgical studies of aromatase inhibitors to be made [START_REF] Eiermann | Preoperative treatment of postmenopausal breast cancer patients with letrozole: A randomized double-blind multicenter study[END_REF][START_REF] Harper-Wynne | Comparison of the systemic and intratumoral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer[END_REF][START_REF] Smith | Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial[END_REF][START_REF] Dowsett | Biomarker changes during neoadjuvant anastrozole, tamoxifen, or the combination: influence of hormonal status and HER-2 in breast cancer--a study from the IMPACT trialists[END_REF].

In these latter studies large reductions in expression of biomarkers in the order of 80% were evident for Ki67 and PgR as early as 2 weeks post treatment. It is clear from our data that the changes with celecoxib were much more modest than this for Ki67 and there was no indication of a reduction in PgR.

Our hypothesis was based on the association between COX-2, PGE2 and aromatase activity. The inhibition of COX-2, should lead to a decrease in PGE2 and cAMP levels. If the hypothesis was correct this would lead to reduced aromatase activity and hence estrogen biosynthesis/signalling, resulting in a decrease in tumour proliferation (Ki67) and other markers of a functional estrogen signalling pathway (eg PgR) in ER+ tumours [START_REF] Smith | Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial[END_REF]. Ki67 and PgR are some of the most studied and robust molecular markers in relation to neoadjuvant endocrine therapy. We have previously shown that the aromatase inhibitors vorozole and anastrozole [START_REF] Harper-Wynne | Comparison of the systemic and intratumoral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer[END_REF][START_REF] Smith | Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial[END_REF] led to decreases in Ki67 of 58% and 75% respectively in the first 2 weeks of treatment accompanied by substantial falls in PgR.

As expected analysis of the no treatment arm showed no significant change in Ki67 or apoptosis during the course of the study in keeping with previous observations [START_REF] Dowsett | Effect of raloxifene on breast cancer cell Ki67 and apoptosis: a double-blind, placebo-controlled, randomized clinical trial in postmenopausal patients[END_REF][START_REF] Bundred | Cyclooxygenase-2 inhibition does not improve the reduction of ductal carcinoma in situ proliferation with aromatase therapy: results of the ERISAC randomized placebo controlled trial[END_REF] Our data revealed that there was a non-significant trend towards a reduced level of Ki67 with the celecoxib arm c. 20%. Ki67 at baseline showed no association with response to celecoxib. However, high Ki67 levels were associated with higher tumour grade in both the no treatment and celecoxib arms. As noted earlier, reduction in Ki67 was less pronounced than expected when compared with previous studies of vorozole or anastrazole [START_REF] Harper-Wynne | Comparison of the systemic and intratumoral effects of tamoxifen and the aromatase inhibitor vorozole in postmenopausal patients with primary breast cancer[END_REF][START_REF] Smith | Neoadjuvant treatment of postmenopausal breast cancer with anastrozole, tamoxifen, or both in combination: the Immediate Preoperative Anastrozole, Tamoxifen, or Combined with Tamoxifen (IMPACT) multicenter double-blind randomized trial[END_REF]. The lack of change in PgR expression suggests that celecoxib had little effect on oestrogen dependent processes in this setting and that any change in Ki67 was probably not dependent on reduced oestrogen signalling. Although the change in Ki67 in response to celecoxib was small, and maybe due to chance it should be noted that we found a change of only 24% with raloxifene, an agent that has subsequently been shown to have chemopreventative activity similar to tamoxifen [START_REF] Dowsett | Effect of raloxifene on breast cancer cell Ki67 and apoptosis: a double-blind, placebo-controlled, randomized clinical trial in postmenopausal patients[END_REF].

The lack of a statistically significant effect on Ki67 in response to 2-weeks treatment with celecoxib may be due to a number of factors. For instance this study was powered to detect a large effect (≥50%) and any effect of COX-2 inhibition appears to be smaller than this and would require larger patient numbers to detect with confidence. The trend towards a reduction in Ki67 after celecoxib merits further investigation given the potenial importance of confirmation; particularly in view of the fact that previous studies addressing the role of celecoxib on Ki67 in ER+ DCIS [START_REF] Bundred | Cyclooxygenase-2 inhibition does not improve the reduction of ductal carcinoma in situ proliferation with aromatase therapy: results of the ERISAC randomized placebo controlled trial[END_REF] and in women with early stage breast cancer [START_REF] Tfayli | Neoadjuvant therapy with celecoxib in women with early stage breast cancer[END_REF] have provided conflicting results. For instance in the recent study by Bundred et al, [START_REF] Bundred | Cyclooxygenase-2 inhibition does not improve the reduction of ductal carcinoma in situ proliferation with aromatase therapy: results of the ERISAC randomized placebo controlled trial[END_REF] celecoxib had no significant effect on Ki67 when given as a single agent whilst in the Tfayli et al [START_REF] Tfayli | Neoadjuvant therapy with celecoxib in women with early stage breast cancer[END_REF] study a significant increase in Ki67 (p<0.009) in response to treatment was noted. However, this study had no placebo arm for comparison.

It is also possible that unlike aromatase inhibitors, the antiproliferative effects of celecoxib may only manifest themselves after a prolonged course of treatment. Duration of treatment was based on that seen to be effective in terms of reduced proliferation caused by aromatase inhibitors in this setting: any effects of celecoxib as a result of aromatase suppression would therefore be expected to be detectable over that time period. However, other putative effects of COX2 inhibition such as angiogenesis may require longer than 2 weeks to become evident.

While the data do not support a major site specific reduction in oestrogen synthesis, a recent study assessing the relationship between current NSAID use and endogenous oestradiol levels in 260 postmenopausal women, showed that age-adjusted and body mass index-adjusted geometic mean serum oestradiol levels among NSAIDs users was significantly lower compared to non-users [START_REF] Hudson | Nonsteroidal anti-inflammatory drug use and serum total estradiol in postmenopausal women[END_REF]. This supports the wider hypothesis that NSAIDs block estrogen signalling and provides a mechanism through which NSAIDs might exert a protective effect against breast cancer.

The available evidence suggests that blockade of COX-2 may provide clinical benefit by increasing apoptosis rather than lowering proliferation, as shown in pre-clinical models [START_REF] Hsu | The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2[END_REF]. However, no significant changes were seen in the level of apoptosis in the celecoxib treated patients. Again we cannot exclude an effect from longer treatment duration.

As discussed previously COX-2 expression has also been strongly associated with neovasculariztion in solid tumours [START_REF] Uefuji | Cyclooxygenase-2 expression is related to prostaglandin biosynthesis and angiogenesis in human gastric cancer[END_REF]. Thus an alternate hypothesis could be that inhibition of COX-2 may result in reductions in tumour vasculature [START_REF] Masferrer | Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors[END_REF] affecting tumour growth. However, our assessment of the effect of celecoxib versus placebo on neovasculatization showed no significant difference between the two arms but there was a statistically significant increase in CD31 expression in the pre-treatment versus the posttreatment samples in the celecoxib arm. The data on CD31 should be viewed with caution as the comparison is made between different types of samples, core biopsy versus the excision biopsy, and vasularity may not be well represented in cores.

Any effect of celecoxib in breast cancer may also be dependent not only on the level of COX-2 expression. The preclinical and clinical evidence suggests that COX-2 over expression is most marked in tumours that are of a poor prognosis [START_REF] Witton | Cyclooxygenase 2(COX2) expression is associated with poor outcome in ER-negative, but not ER-positive breast cancer[END_REF][START_REF] Liu | Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice[END_REF]. The link with COX-2 and angiogenesis may be indicative that patients with metastatic disease, or with large high grade, node positive disease and in particular HER-2 positive patients may benefit from celecoxib. The relatively favourable prognostic group studied here may therefore also account in part, for an absence of a demonstrable COX-2 inhibitory effect.

Similarly the selective effectiveness of celecoxib may be obscured by the procarcinogenic contributions of constitutive COX-1. Preclinical data suggest expression of COX-1 may have implications for the effectiveness of selective COX2 inhibitors in tumours where COX-2 overexpression is not particularly prevalent [START_REF] Tiano | Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis[END_REF].

The strengths of the study reported here include its inclusion of a control no-treatment arm, its randomisation, rigorous pre-recruitment clinical assessment and centralised conduct of the biomarker analyses in a highly experienced laboratory. The lower than intended number of recruits is a substantial weakness.

In summary these data suggest that celecoxib does not provide site-specific oestrogen deprivation in ER+ primary breast cancers when compared to the current third generation aromatase inhibitors. The trend to a reduction in proliferation is however, worthy of further investigation since modest reductions have been associated with substantial chemopreventive effects with raloxifene. The cardiovascular toxicity [START_REF] Mukherjee | Risk of cardiovascular events associated with selective COX-2 inhibitors[END_REF] noted with long-term use of COX-2 inhibitors would however, remain a concern for this widespread chemopreventive application and hence necessitate refocusing COX-directed cancer prevention strategies to avoid this danger

  to the trial was compromised by competing studies and a decision to terminate recruitment after 25 patients was taken blind of any study results. In 2 cases from the treatment arm no malignant tissue was present in the pre-treatment sample leaving 23 paired sets of data, 14 on the treatment arm and 9 on no-treatment arm. Paired treatment and post-treatment data on Ki67 and CD31 was available on all 23 patients. The clinicopathological variables of the 23 patients recruited are shown according to randomisation arm in Table 1. The mean age of the patients in the celecoxib arm was 68 years and in the no treatment arm 71 years. The median (range) tumour sizes were 19.5 mm (10-60) and 20mm (11-29), respectively. Fifty percent and 67% respectively were grade 3. The majority of tumours in both groups were lymph node negative and did not exhibit vascular invasion. At baseline only 3 patients were ER-negative; 1 in the celecoxib arm and 2 in the no treatment. Sixteen patients were PgR-positive (11 and 5, respectively); 2 patients were HER-2 positive (1 in each arm).

Figure 1

 1 Figure 1 Changes in expression of Ki67 were assessed by immunohistochemistry. Tumour

Figure 2

 2 Figure 2 Changes in apoptosis were assessed by TUNEL. Tumour biopsies were screened

Figure 3

 3 Figure 3 Alterations in expression of ER were assessed by immunohistochemistry.

Figure 4

 4 Figure 4 Alterations in expression of PgR were assessed by immunohistochemistry.

Figure 5

 5 Figure5The effect of celecoxib on tumour angiogenesis was assessed by measuring

Figure 6

 6 Figure 6 The effect of celecoxib on COX-2 expression was assessed by

  Figure 1 Figure Click here to download Figure: Cox2 figsii.ppt

Table 1 . Clinicopathological variables of patients in Celecoxib and no treatment arm of 2 week presurgical study

 1

	Baseline characteristics of	No treatment	Celecoxib
	patients
	No Patients	9	14
	Mean age (yr) (range)	71 (59-89)	68 (53-86)
	Mean tumour size (range	20 (11-29)	19.5 (10-60)
	mm)
	Grade II	3 (33%)	7 (50%)
	III	6 (67%)	7 (50%)
	ER-positive	7 (77%)	13 (92%)
	ER negative	2 (22%)	1 (7%)
	PgR positive	5 (56%)	11 (79%)
	HER2 positive	1 (11%)	1 (7%)

Table 2 : Geometric mean percentage change in biomarker expression from baseline at 2 weeks

 2

	Celecoxib	P	No	95%	P	P
		value	Treat.	CI	value	value
	Mean %	95% CI	Mean
	change		%

Acknowledgements

We are grateful to the patients who participated in this trial and thank Breakthrough Breast Cancer and The Mary-Jean Mitchell Green Foundation for generous funding. We also acknowledge NHS funding to the NIHR Biomedical Research Centre.


Ewa Chlebda
email: echlebda@mp.pl

Jan Magdalan

Anna Merwid-Ląd

Malgorzata Trocha

Maria Kopacz

Anna Kuźniar

Dorota Nowak

Adam Szeląg

Anna Merwid-

La Rd

Małgorzata Trocha

Adam Szela Rg

Małgorzata Trocha

Influence of water-soluble flavonoids: Quercetin-5'sulfonic acid sodium salt and morin-5'-sulfonic acid sodium salt on antioxidant parameters in the subacute cadmium intoxication mouse model

Keywords: cadmium intoxication, NaQSA, NaMSA, morin, quercetin, flavonoids, GSH, SOD, antioxidants

come

Introduction

Cadmium (Cd) is a widespread environmental xenobiotic. One of the mechanisms of harmful effects of cadmium exposure is indirect induction of oxidative stress [START_REF] Noor | Superoxide dismutase -applications and relevance to human diseases[END_REF]. Cadmium decreases superoxide dismutase (SOD), catalase, and glutathione peroxidase activity and inhibits antioxidant protein activity via binding with glutathione (GSH) thiol groups [START_REF] Eybl | The effect of curcumin on cadmium-induced oxidative damage and trace elements in the liver of rats and mice[END_REF][START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF].

Despite of availability of chelating agents used in lowering cadmium body burden, there is a great necessity to find new, non-toxic but efficacious substances decreasing cadmium toxicity. In vitro studies have indicated that some compounds belonging to polyhydroxyflavones, e.g. morin and quercetin form complexes with Cd (II) and exhibit strong antioxidant activity. Their sulfonic water-soluble derivatives exert low toxicity therefore could be potential antidotes [START_REF] Kopacz | Complexes of Cadmium(II), Mercury(II) and Lead(II) with Quercetin-5'-sulfonic Acid (QSA)[END_REF]Magdalan et al., 2006a; Magdalan et al., 2006b;[START_REF] Szeląg | Assessment of efficacy of quercetin-5'-sulfonic acid sodium salt in the treatment of acute chromium poisoning: experimental studies[END_REF].

A c c e p t e d m a n u s c r i p t

3

In our previous study we have demonstrated the antagonistic effect of quercetin-5'sulfonic acid sodium salt (NaQSA) and morin-5'-sulfonic acid sodium salt (NaMSA) on Cdinduced hepatic lipid peroxidation in the subacute Cd intoxication in mice [START_REF] Magdalan | Quercetin-5'-sulfonic acid sodium salt and morin-5'-sulfonic acid sodium salt as antidotes in the subacute cadmium intoxication in mice[END_REF].

The aim of the study was to examine the influence of NaQSA and NaMSA on SOD activity and GSH concentration in the mouse liver in the subacute cadmium intoxication model.

Materials and Methods

The study was conducted in 90 Balb/c male mice (19.7±1.6 g), housed at standard laboratory conditions with free access to water and food.

In the experiment were used: cadmium chloride anhydrous (CdCl 2 ) (Fluka Chemie, SOD activity in liver homogenates was assayed spectrophotometrically using RANSOD kit (Randox Laboratories, UK) according to the manufacturer's instructions. SOD activity was expressed as U/mg protein. Total protein concentrations were determined by the Total Protein Kit (Sigma).

Glutathione concentration in liver homogenates was measured using colorimetric assay BIOXYTECH GSH-400 (OxisReseach, USA) according to the manufacturer's instructions. GSH level was expressed as μM/L.

The experiment was performed after approval by the Local Ethics Commission for

Experiments on Animals in Wrocław.

Data were expressed as the mean values ±SD (standard deviation). Statistical analysis was performed using multifactor analysis of variance (ANOVA). Specific comparisons were made with contrast analysis, p value <0.05 was considered to be statistically significant.

Results

In the group receiving only cadmium GSH level and SOD activity in the mouse liver were significantly lower than in groups non-exposed to this metal (group Cd vs. K, Q, M and QM; p≤0.001 in all cases). No significant differences in GSH level and SOD activity between groups non-exposed to cadmium were revealed (groups K, Q, M and QM; p=NS in all cases).

In groups receiving cadmium with one or both examined flavonoids, SOD activity and GSH level were significantly higher than in the group receiving only cadmium (CdQ, CdM and CdQM vs. Cd; p≤0.001 in all cases). Concomitant flavonoids and cadmium administration significantly prevented the cadmium-induced decrease in GSH concentration and SOD activity, however, these changes were not fully reversible (groups CdQ, CdM and CdQM vs.

A c c e p t e d m a n u s c r i p t

5 K, Q, M, and QM; p≤0.001 in all cases). No significant differences in SOD activity and GSH concentration between groups exposed to cadmium receiving one or both of the examined flavonoids were observed (groups CdQ, CdM and CdQM; p=NS in all cases). GSH level and SOD activity were higher in group CdQ than in group CdM, however, all these differences were not statistically significant (Figure 1).

Discussion

In In rats, during chronic cadmium administration, renal superoxide dismutase activity was higher in the group receiving Cd with quercetin than in the group receiving only Cd [START_REF] Morales | Protective effect of quercetin on experimental chronic cadmium nephrotoxicity in rats is based on its antioxidant properties[END_REF]. In our study we also observed similar action. In livers isolated from cadmium-intoxicated mice treated with NaQSA (group CdQ), significantly higher SOD activity was observed in mice receiving only cadmium (group Cd). Till now there was no report on the influence of quercetin or its water-soluble derivatives administered alone on SOD activity. It was observed that a single dose of morin significantly elevated SOD activity in normal mice. Pretreatment with this flavonoids also prevented whole body gamma irradiation-induced drastic decrease in endogenous SOD activity [START_REF] Parihar | Anticlastogenic activity of morin against whole body gamma irradiation in Swiss albino mice[END_REF]. In our work we did not observed significant influence of NaQSA and/or NaMSA on the mouse liver SOD activity (groups M, Q and QM) (Figure 1).

Reduced glutathione, an extremely important cell protectant, is stored mainly in the liver. Glutathione keeps redox-sensitive active sites in enzymes in the reduced state [START_REF] Wu | Glutathione metabolism and its implications for health[END_REF]. It was shown that cadmium produced an increase in intracellular glutathione

A c c e p t e d m a n u s c r i p t

7 concentrations in a dose-and time-dependent manner. At higher Cd doses a decreased GSH level was observed probably as a result of generation of reactive oxygen species exceeding the ability to regenerate reduced glutathione [START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF]. In our work we observed significant glutathione depletion probably as a result of glutathione storage consumption after 28 days of cadmium poisoning (Figure 1).

Quercetin may influence GSH/GSSG (reduced glutathione/glutathione disulfide) ratio and protein thiolation [START_REF] Meyers | Influence of dietary quercetin on glutathione redox status in mice[END_REF]. Quercetin treatment produced an increase in the GSH/GSSG ratio in hepatic tissue but not in the plasma or cardiac tissue. Pretreatment with quercetin may protect against ethanol-induced oxidative stress indirectly by enhancing the production of the endogenous liver GSH [START_REF] Molina | Quercetin, a flavonoid antioxidant, prevents and protects against ethanol-induced oxidative stress in mouse liver[END_REF]. Quercetin administration successfully diminished toxic effects of CCl 4 by influence on GSH concentration in the rat liver [START_REF] Amalia | Quercetin prevents oxidative stress in cirrhosic rats[END_REF]. Quercetin also countered the pro-oxidant effects of galactoseinduced hyperglycemic oxidative stress significantly reversing changes in reduced GSH level [START_REF] Ramana | Effect of quercetin on galactose-induced hyperglycaemic oxidative stress in hepatic and neuronal tissues of Winststar[END_REF].

It was demonstrated that in chronic cadmium administration resulted in higher renal glutathione-reductase activity in the rats receiving Cd with quercetin than those receiving only cadmium [START_REF] Morales | Protective effect of quercetin on experimental chronic cadmium nephrotoxicity in rats is based on its antioxidant properties[END_REF]. In our experiment we observed that NaQSA significantly prevented cadmium-induced GSH depletion in the mouse liver (Figure 1).

It was observed that intraperitoneal administration of morin (100 mg/kg) significantly increased not only SOD activity but also liver GSH level in normal mice. Pretreatment with this flavonoid also prevented whole body gamma irradiation-induced drastic decrease in endogenous GSH concentration [START_REF] Parihar | Anticlastogenic activity of morin against whole body gamma irradiation in Swiss albino mice[END_REF]. Similarly to SOD activity in our work we did not observe significant influence of NaQSA and/or NaMSA on GSH liver amount during 4 weeks of the study (groups Q, M, and QM) (Figure 1). Lack of the influence of these Our stude revealed the significant difference between NaQSA and NaMSA efficacy nor the synergism of both examined flavonoids in the antioxidant action against cadmium toxicity, monitored by GSH levels and SOD activity in mice liver (Figure 1).

Our results may be different from those obtained in other experiments. In previously conducted studies natural, lipophylic quercetin or morin were used, whereas in our experiments water-soluble derivatives of these flavonoids were investigated. Different pharmacokinetic properties may play an important role in gastrointestinal absorption, distribution, metabolism or excretion of these compounds what subsequently could be the reason of various biological effects of hydrophylic and lipophylic compounds. It was shown that sulfonic derivatives of natural morin and quercetin were less potent than the original agents in their cytostatic and cytotoxic activities. However, their solubility in water was greater than that of the original agents and higher culture medium concentrations of NaMSA and NaQSA were obtained [START_REF] Król | Synthesis and tumoricidal activity evaluation of new morin and quercetin sulfonic derivatives[END_REF].

In our study we revealed antioxidant activity of NaMSA or/and NaQSA. Both substances significantly counteracted Cd-induced decrease in SOD activity and GSH depletion in the mouse liver, however, Cd-induced changes were not fully reversible. It could not be excluded that higher doses of NaQSA and/or NaMSA may exert stronger effect. There was no difference in the antioxidant activity between NaQSA and NaMSA against cadmium poisoning in mice probably due to similar (may be even the same) mechanism of action.

These findings seems to be very promising but required further studies.

  Switzerland), NaQSA and NaMSA synthesized in the Department of Inorganic and Analytical Chemistry, University of Technology in Rzeszow, Poland. NaQSA and NaMSA are water soluble substances of which characteristics were described in earlier papers (Kopacz and Kuźniar, 2003; Magdalan et al., 2006a; Magdalan et al., 2006b; Szeląg et al., 2003). Mice were randomized to 8 experimental groups (n=10-12) receiving: K -only 0.9 % saline solution, Q -NaQSA, M -NaMSA, QM -NaQSA and NaMSA, Cd -receiving CdCl 2 , CdQ -CdCl 2 and NaQSA, CdM -CdCl 2 and NaMSA and group CdQM -CdCl 2 and both flavonoids, respectively. Cadmium chloride was administered subcutaneously (sc) at a dose of 0.64 mg/kg b.w. NaQSA and NaMSA were given intraperitoneally (ip) at a dose of 20 mg/kg b.w. in groups: Q, M, CdQ and CdM or 10 mg/kg b.w. in groups QM and CdQM. All substances were dissolved in 0.9% saline solution and administered once a day, for 28 consecutive days. The different routes of administration of CdCl 2 and the studied flavonoids were chosen to avoid complex formation.

A c c e p t e d m a n u s c r i p t 4 Livers

 4 were homogenized on ice, using lysis buffer, centrifuged with 14000 rpm during 25 min. In the obtained supernatants SOD activity and GSH concentration were measured.

A c c e p t e d m a n u s c r i p t 6 inhibition

 6 our previous study we have demonstrated that in the subacute cadmium intoxication model in mice this metal significantly accumulated in the liver and kidneys and exerted Cdinduced prooxidative action. Intraperitoneal administration of NaQSA and NaMSA prevented lipid peroxidation in the liver and decreased Cd level in the organs (Magdalan et al., 2007). Therefore, more detailed studies on the mechanisms of antioxidative action of NaQSA and NaMSA in the same cadmium poisoning mouse model were conducted. Influence of both examined flavonoids on selected nonenzymatic (GSH) and enzymatic (SOD) antioxidative parameters was studied. Animal studies have shown that flavonoids exhibit potent antioxidant activity with mechanisms involving free radical-scavenging, metal chelation, antioxidant enzyme activation, reduction alpha-tocopherol radicals and oxidase inhibition (Fang and Yang, 2002). Mechanism of protective action of NaQSA and NaMSA against Cd-induced prooxidative status could be probably a consequence of direct antioxidative influence and/or Cd chelation by the examined flavonoids. It was demonstrated that both flavonoids were effective antidotes in acute other metal poisoning also e.g. chromium and mercury (Kopacz and Kuźniar, 2003; Magdalan et al., 2006a; Magdalan et al., 2006b; Szeląg et al., 2003).Superoxide dismutase is one of the most important antioxidative enzymes. SOD catalyzes the conversion of single electron reduced species of molecular oxygen to hydrogen peroxide and oxygen[START_REF] Fang | Free radicals, antioxidants, and nutrition[END_REF][START_REF] Noor | Superoxide dismutase -applications and relevance to human diseases[END_REF]. The cadmium-induced of many metalloenzymes is reported to be the result of the displacement of metals from the active site of the enzymes. It was observed that cadmium administration inhibited the activity of SOD in vitro and in vivo in many tissues e.g. erythrocytes, kidneys and liver[START_REF] Stohs | Oxidative mechanisms in the toxicity of metal ions[END_REF]. In our study we have obtained similar results. We demonstrated that subacute cadmium intoxication significantly inhibited superoxide dismutase activity in the mouse liver.Many data indicate protective properties of flavonoids, esp. quercetin against oxidative stress induced by different agents. It was found that quercetin successfully attenuated toxic effects of CCl 4 by influence on SOD activity in the rat liver[START_REF] Amalia | Quercetin prevents oxidative stress in cirrhosic rats[END_REF]. Quercetin also countered the pro-oxidant effects of galactose-induced hyperglycemic oxidative stress in rats, significantly reversing changes in SOD activity[START_REF] Ramana | Effect of quercetin on galactose-induced hyperglycaemic oxidative stress in hepatic and neuronal tissues of Winststar[END_REF].

A c c e p t e d m a n u s c r i p t 8 two

 8 compounds on SOD and GSH levels may be due to lower doses of NaQSA and NaMSA used in our experiment vs. earlier studies.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 11

 11

Figure 1 .A c c e p t e d m a n u s c r i p t 12 Figure 1 .

 1121 Figure 1. Influence of subacute cadmium intoxication and flavonoids (NaMSA and NaQSA)


Tapio Suominen

Harri Tolvanen

Risto Kalliola

Surface layer salinity gradients and flow patterns in the archipelago coast of SW Finland, northern Baltic Sea

Keywords: Archipelago, Baltic Sea, estuaries, GIS, interpolation, monitoring, salinity

The highly fragmented Archipelago Sea in the northern Baltic Sea forms part of a sill area between two large sea basins. In addition to the water exchange between the basins, its waters are influenced by runoff, and thus the sea area has both sill and estuarine characteristics. We studied surface layer salinity gradients and their applicability in defining water exchange patterns through and within the region. A broad scale salinity pattern was detected during two sequential years. The spreading of fresh water in the spring was succeeded by a gradual increase in salinity during the summer. Long term data revealed a non-seasonal salinity fluctuation and diminished salinity stratification in the central and northern parts of the study area.

We concluded that temporally unrepresentative mean values of salinity alone are inadequate for the purposes of coastal management in this region. In addition, both the range of variation and persistence of the conditions define the character of the transitional and coastal waters.

Introduction

The horizontal and vertical gradations of the water properties are characteristic features of the Baltic Sea. The basin has a positive water balance, whose major components are the in-and outflows through the Danish straits (Fig. 1), river runoffs and net precipitation [START_REF] Von Storch | Introduction and summary. The Baltic Sea -Geological history and specifics[END_REF]. The occurrence and intensity of the water exchange through the straits control much of the physical, chemical and eventual biological processes in the Baltic Sea [START_REF] Helcom | Euthrophication in the Baltic Sea -An integrated thematic assessment of the effects of nutrient enrichment and eutrophication in the Baltic Sea region[END_REF]. Further, the sea basin is divided by sills into multiple large sub-basins, which complicates the distribution of the Atlantic waters, along with a weak anti-clockwise surface layer circulation (e.g. [START_REF] Alenius | The physical oceanography of the Gulf of Finland: a review[END_REF][START_REF] Stigebrandt | Physical oceanography of the Baltic Sea[END_REF][START_REF] Maslowski | Circulation of the Baltic Sea and its connection to the Pan-Arctic region -a large scale and high resolution modeling approach[END_REF][START_REF] Myrberg | Modeling of the circulation, water exchange and water age properties of the Gulf of Bothnia[END_REF].

Figure 1.

Surface water salinity in the Baltic Sea decreases from ~9 ‰ in the Arkona basin close to the entrance area to almost freshwater in the northern parts of the Gulf of Bothnia [START_REF] Bock | Monatskarten des salzgehaltes der Ostsee dargestellt für verschiedene tiefenhorizonte[END_REF][START_REF] Rodhe | The Baltic and the North Seas: a process oriented review of the physical oceanography[END_REF]. In general, the less saline waters flow southwards in the surface layer, while the inflowing saline and dense water penetrates into the deeper layers. This results in a permanent stratification with the halocline at a depth of 60-80 metres in the largest basin of the Baltic Sea, the Baltic Proper. The gradient of the surface salinity is rather even in the open sea, with the highest variations occurring in the sill areas between the sub-basins [START_REF] Rodhe | The Baltic and the North Seas: a process oriented review of the physical oceanography[END_REF][START_REF] Stigebrandt | Physical oceanography of the Baltic Sea[END_REF]. The mean surface salinity fluctuations of the Baltic are related to the fresh water input and show an approximate 1 ‰ variation over several decades with no long-term trends [START_REF] Winsor | Baltic Sea ocean climate: an analysis of 100 yr of hydrographic data with focus on the freshwater budget[END_REF][START_REF] Fonselius | One hundred years of hydrographic measurements in the Baltic Sea[END_REF]. In the northern Baltic Sea salinity exhibits a seasonal cycle in near shore areas as in spring the snowmelt runoff diffuses from the mainland. In summer the water is temperature-stratified, while during spring, autumn and in mild winters the water column shows strong vertical circulation above the halocline.

In the coastal and estuarine regions of the northern Baltic Sea, the complex bathymetry associated with different geomorphic forms sets strong prerequisites upon coastal circulations, resulting in highly variable chemical and physical properties of seawater over space and time (e.g. [START_REF] Kirkkala | Variability of nutrient limitation in the Archipelago Sea, SW Finland[END_REF][START_REF] Hänninen | Trends and gradients in nutrient concentrations and loading in the Archipelago Sea, northern Baltic, in 1970-1997[END_REF][START_REF] Weckström | Physical and chemical characteristics of shallow embayments on the southern coast of Finland[END_REF][START_REF] Erkkilä | Patterns and dynamics of coastal waters in multitemporal satellite images: support to water quality monitoring in the Archipelago Sea, Finland[END_REF]. These kinds of transitional changes are ecologically significant with manifold implications for the living environment, fisheries and environmental planning [START_REF] Anon | Common implementation strategy for the Water Framework Directive[END_REF]Schernewski and Wielgat, 2004;Nordic Council of Ministers, 2006). One of the most fragmented coastal areas is the Archipelago Sea between mainland Finland and the island of Åland, forming the eastern part of the sill between the Baltic Proper and the Gulf of Bothnia (Fig. 1). The western side of this sill, formed by the Åland Sea between Åland and Sweden, is relatively deep and wide, whereas the Archipelago Sea is a unique coastal area with a mosaic of islands.

The net water exchange through the Archipelago Sea is estimated to be low compared to the Åland Sea [START_REF] Kullenberg | Physical oceanography[END_REF][START_REF] Omsted | Knowledge of the Baltic Sea physics gained during the BALTEX and related programs[END_REF]. The baroclinic flows combined with river runoffs and net precipitation are of major importance considering the water exchange between the Baltic Proper and the large gulfs as a whole [START_REF] Omsted | Modeling the variations of salinity and temperature in the large Gulfs of the Baltic Sea[END_REF]. Water exchange through and within the Archipelago Sea is further mixed by estuarine circulation with wind-driven surface currents.

Islands and underwater sills form numerous local sea basins at various scales, resulting in a complex transitional system where the fresh water runoff mixes with the brackish sea water of the adjacent main basins.

The intermediate osmotic pressure of the brackish water of the Baltic Sea does not correspond to either a purely marine or limnic environment, with many of the aquatic organisms occurring at the edge of their ecological amplitude. Thus, the horizontal and vertical salinity gradients of the Baltic Sea strongly influence the species composition and abundance of flora and fauna (e.g. [START_REF] Remane | Biology of brackish water[END_REF][START_REF] Bäck | Effects of salinity on growth of Baltic and Atlantic Fucus vesiculosus[END_REF][START_REF] Lappalainen | Coastal-fish communities along the Northern Coast of the Gulf of Finland, Baltic Sea: Responses to salinity and eutrophication[END_REF][START_REF] Hänninen | Atlantic climatic factors control decadal dynamics of a Baltic Sea copepod Temora longicornis[END_REF][START_REF] Gasi Nait | Seasonality of coastal phytoplankton in the Baltic Sea: Influence of salinity and eutrophication[END_REF]. Generally, the lowest biodiversity in the Baltic Sea is reported to occur in conditions where the salinity ranges from 5 to 7 [START_REF] Von Storch | Introduction and summary. The Baltic Sea -Geological history and specifics[END_REF], corresponding to the conditions prevailing in the Archipelago Sea.

The aim of this paper is to provide detailed quantitative information about the surface salinity gradients and their temporal fluctuations in the Archipelago Seaphenomena that create relevant dynamic ecological thresholds in the area. Further, we introduce an interpolation method modified for the archipelagial environment and discuss the applicability of salinity monitoring data in determining long term flow properties through the sill area of the Archipelago Sea.

We identified three main issues concerning the surface layer salinity: gradients, persistence and the magnitude of fluctuations. These issues were studied through three approaches. First, the general surface salinity patterns between the mainland and Åland Island were outlined using salinity data from different sources. The four salinity raster maps show the salinity gradients in July-August in 2007 and 2008.

Second, the succession of the salinity gradients was followed from late spring to early autumn to study the intra-annual persistence of salinity in greater detail in the north eastern Archipelago Sea. Third, to attain an inter-annual perspective, salinity time series data from three intensively monitored stations representing the southern, eastern and northern Archipelago Sea were used.

Material and methods

Physical geography of the study area

Archipelago coasts are typical in the northern Baltic Sea (Frisén et al., 2005). The Archipelago Sea in SW Finland consists of 25 000 islands larger than 500 m 2 and 14 400 km of shoreline in an area of approximately 10 000 km 2 [START_REF] Granö | Environment and land use in the shore zone of the coast of Finland[END_REF] (Fig. 1). The area is structured by fragmented bedrock that has a relative elevation range of about two hundred metres. The bedrock base is partially covered with till, glaciofluvial deposits and marine sediments. The deepest basins in bedrock faults provide channels for water currents through and within the area. The mean depth of the Archipelago Sea is estimated to be only 23 m. The depth is typically ranging from 0 to 50 metres, but some deeps and fault lines exceed 100 m. The Åland Sea in the west is an approximately 40 km wide strait between the Archipelago of Stockholm and the island of Åland. Its maximum depth is 301 m, but there is a sill at a depth of 70 metres at the southern end of the channel. In the Archipelago Sea, the highest surface-layer water temperatures (~20 °C) occur near the mainland in August, while the mean annual period of permanent ice cover extends to 100 days [START_REF] Seinä | Duration of the ice seasons and statistics of fast ice thickness along the Finnish coast 1961-1990[END_REF]. According to the meteorological data from the island of Utö (close to the observation station S in Fig. 1), December is the windiest month with an average wind speed of 8.6 m s -1 , while May, June and July show the lowest average wind speeds [START_REF] Drebs | Climatological statistics of Finland 1971-2000[END_REF].

Description of the salinity data

The Archipelago Sea in July-August in 2007 and 2008

The Southwest Finland Regional Environment Centre (SFREC) collects annual water quality data from 61 stations in the eastern part of the Archipelago Sea (see Fig. 1, Table 1), comprising three sampling visits in the period between July and August. At most stations only the surface layer is sampled, and salinity is rarely measured, since the analyses are focused on indications of eutrophication. However, extended surface-layer (1 m) conductivity sampling was carried out from 27 and 56 stations respectively, in 2007 and 2008. Conductivity was analysed by the laboratory of the Water Protection Association of Southwest Finland, according to the standard SFS-EN 27888. The conductivity values were compensated to 25º C and the laboratory results were given in mS m -1 to an accuracy of three significant figures. The algorithms presented by [START_REF] Fotonoff | Algorithms for computation of fundamental properties of seawater[END_REF] were used to convert conductivity into a practical salinity scale (PSS).

The Environmental Agency of Åland (referred to as ÅL) measures salinity in the western part of the Archipelago Sea, synchronising their sampling regime with SFREC to the same weeks each year. Salinity samples are collected from a depth of one metre at all stations. In this study we used the 38 stations on the eastern side of Åland Island (Fig. 1, Table 1). Conductivity was measured according to the standard SFS-EN 27888 using a WTW inoLab Multi Level 1 instrument, which converts conductivity to salinity. These results were given in per mil to an accuracy of two significant figures.

Table 1.

The 2007 summer season in the NE Archipelago Sea

To obtain spatially and temporally representative salinity data covering the period from late spring to early autumn, we carried out a sampling program in the northeastern part of the Archipelago Sea. The sampling regime consists of 22 stations (Fig. 1, Table 1), a subset of the stations sampled by SFREC. In the sampling network design, we prioritised relatively open sea areas and paid special attention to long and deep straits and their crossings, i.e. flow channels to, from and within the area. At each station, three parallel profiles were measured in a constellation of an equilateral triangle with sides of 300 metres. The measurements were made every third week from mid-May to early October in 2007. One exception to this schedule was made to synchronise our data with the SFREC routine monitoring schedule during weeks 29 and 31 in July. Each station was visited eight times.

The field measurements were made using a multi-parameter sonde (YSI 6600 V2), equipped with sensors for conductivity and temperature (sensor model YSI 6560), and pressure. The readings were recorded at six sampling depths (1, 2, 4, 6, 8 and 10 metres) at each of the three parallel profiles, whose mean values were used. At two stations where the depth was less than 10 metres the measurements were made at one metre intervals until two metres from the sea floor.

To intercalibrate the sonde data with the monitoring program data, water samples were collected from 7 stations during the weeks 29-40. A Limnos water sampler was used to collect 5-10 litres of water to a plastic container, from which the laboratory sample bottles were filled. The sonde was subsequently immersed into the container and readings recorded for 1-2 minutes to get a control reading for the calibration.

The conductivity values measured in the field were compensated to 25º C by an equation provided by the manufacturer; 25º C = /(1+TC(T-25)) where 25º C is the specific conductance compensated to 25º C, is the measured conductance, TC is temperature coefficient 0.0191 and T is the sample temperature at the time of measurement (YSI 2007). The specific conductance values 25º C were calibrated to correspond with the laboratory values with a linear regression model. The field measurements of conductivity showed a good linear correspondence with the laboratory results (r 2 =0.98, n=48). However, although the calibrations with distilled water and the standard solutions differed only marginally from the nominal values, the field readings were typically 20-25 mS m -1 higher than the laboratory results.

According to the manufacturer, the accuracy of the conductivity sensor is ±0.5 % of the reading. The algorithms presented by [START_REF] Fotonoff | Algorithms for computation of fundamental properties of seawater[END_REF] were used to convert the inter-calibrated conductivity to PSS. The reference conductivity was analysed by the laboratory of the Water Protection Association of Southwest Finland according to the standard SFS-EN 27888. An exceptionally high conductivity value at one of the stations in week 26 was assigned as an outlier, and data are missing from the southernmost station in week 37. In these two cases the mean conductivity values from the preceding and subsequent week were used instead.

Long term observations in 1999-2008

The Finnish Environment Institute (SYKE) has 14 intensively sampled monitoring stations in Finnish coastal waters, of which three, i.e. KORP 200, NAU 2361 and BRÄNDÖ 100, are located in the Archipelago Sea. They are referred to here as the southern (S), eastern (E) and northern (N) station, respectively (Fig. 1, Table 1).

These stations are sampled nominally 20 times annually, but due to weather conditions some data are missing, especially during winter periods. The longest timeseries, from 1983 to present, is available from station E (depth 52 metres). The timeseries at stations S (78 m) and N (33 m) start in 1999 and 2000, respectively. In this study, we used data extending from 1999 (2000 at station N) to 2008. We used sample means collected from 1, 5 and 10 metre depths as the surface layer value. This range was found to be homogeneous and by using the mean of the surface layer, we minimised the effects of individual erroneous values obtained as a result of incorrect sampling, analysis or registration. The near-bottom salinities were sampled approximately one metre above the sea floor. Here, salinity was analysed in the laboratory of SYKE with a Guideline Autosal 8400B salinometer and the laboratory results were given as per mil to an accuracy of three significant figures.

Analysis methods

The inverse path distance weighted interpolation

The archipelago conditions require sophisticated interpolation analyses, because the islands and straits restrict free water flow, resulting in an anisotropic distance configuration (see [START_REF] Little | Kriging in estuaries: as the crow flies, or as the fish swims?[END_REF][START_REF] Dunn | Mapping ocean properties in regions of complex topography[END_REF][START_REF] Løland | Spatial covariance modelling in a complex coastal domain by multidimensional scaling[END_REF][START_REF] Krivoruchko | Geostatistical interpolation and simulation in the presence of barriers[END_REF]. We applied a procedure based on the inverse distance weighted (IDW) method (e.g. [START_REF] Longley | Geographic Information Systems and Science[END_REF][START_REF] Chang | Introduction to Geographic Information Systems[END_REF] to interpolate salinity values for the entire study area. The IDW weights the values of sample points linearly according to the inverse distance from the known data point to the raster cell to be estimated. The influence of the distance could be adjusted by raising the distance to a power and by limiting it to a given maximum value. Instead of using Euclidean distances, we calculated path distances along the water surface from each sampling point and named the method as inverse path distance weighted (IPDW). This calculation was made by applying a cost raster surface, in which the water areas have a value of 1, with land areas assigned a high value to prevent the path from crossing land surfaces. The script was written with Python programming language within ArcGIS and utilises GIS functions introduced in this environment.

The execution of the script is described in Table 2, with some of the intermediate and resulting raster surfaces demonstrated in Figure 2.

Table 2. Figure 2.
The cost raster originates from the shoreline vector data (1:20 000) of the National Land Survey of Finland, which was converted to raster format with a cell size of 100 metres. The cells whose centre points lie on land were assigned with the cost value 10 000, and other cells with the cost value 1. Thus, the cost raster allows unrestricted connectivity through narrow straits (<200 m), which does not correspond to the real water exchange potential in such cases. To simulate a more realistic situation where the flow in shallow shores and narrow channels is limited, the cells retaining the cost value 10 000 were buffered by adding an extra row of cells around them.

The error assessment of the model was done with the UTU data (University of Turku, Table 1), which was sampled from 22 stations during the summer of 2007 (see section 2.2.2). The test was performed by omitting one station at time, interpolating a salinity raster surface with this incomplete set of known data points, extracting the interpolated value at the location of the omitted station, and by comparing the deviations of observed and extracted salinity from the observed mean salinity of the corresponding week (Fig. 3). This was done for each of the eight sampling rounds.

However, all stations could not be used in this procedure: the stations at the outer limit of the interpolation area were excluded since they would have been extrapolated instead of interpolated. Thus, only 12 out of 22 stations were used. Since one measurement was regarded as an outlier, the total number of comparisons of modelled and measured salinity values was 95.

We compared the effect of the inverse path distance weighted interpolation (IPDW) against standard IDW by performing the above mentioned test with two cost surfaces. In addition to the cost raster described above, the test was performed with a constant cost raster in which all the cell values were set to value 1. This surface ignores the effect of land and the inverse path distance weighted simulation performs like a standard IDW. In both calculations, the influence of the stations were limited to a maximum distance of 20 km and the effect of the stations were set to decrease linearly according the increasing distance.

Figure 3.

The comparison revealed only minor differences in accuracy between the different interpolation methods. In both cases, the coefficient of determinations (Fig. 3) were r 2 = 0.81, while the mean deviations of the modelled from the observed salinities were also similar, i.e. MD = 0.04. This similarity occurs partly because the stations have initially been selected to represent open sea areas and they are usually connected through straight and wide waterbodies. The main benefits of the IPDW would occur in more complex and sheltered areas. The differences between IDW and IPDW methods were apparent in the straits and their ends (Fig. 4). The IPDW method was used to model continuous raster surfaces on the surface salinity of the Archipelago

Long term time-series analysis

We derived the long term data for the time series analysis from the OIVA database (also known as Hertta-PIVET, see [START_REF] Erkkilä | Spatial and temporal representativeness of water monitoring efforts in the Baltic Sea coast of SW Finland[END_REF][START_REF] Manni | The use of monitoring data -data systems of the Finnish Environmental Administration[END_REF] managed by SYKE. We divided the year into four 13-week quarters (quarter 1 = weeks 1-13, quarter 2 = weeks 14-26, quarter 3 = weeks 27-39, and quarter 4 = weeks 40-52).

The winter observations in particular were occasionally clustered to either end of a given quarter. Since the salinity is considered to have a seasonal cycle, this clustering might lead to erroneous estimations, if the observed values only are used in calculation of quarterly means. Therefore, we calculated the salinity for each week of the year by linear interpolation before calculating the quarterly means.

We studied the long-term salinity patterns by creating two graphs for the period 1999-2008 (2000-2008

Results

The units and accuracy of the conductivity and salinity results differed according to the laboratories and instruments used, as indicated in section 2.2. In the following sections, the salinity values are given without units, an accuracy of two significant figures and are considered to correspond with PSS.

The Archipelago Sea in July-August in 2007 and 2008

The overall surface salinity patterns in the Archipelago Sea indicated similar gradients in mid-July (week 29) and mid-August (week 34) of 2007 (Fig. 5). The highest salinity, 6.1-6.2, occurred in the southern reaches, from where the salinity decreased towards the shallow bays near the mainland. The large islands in the middle of the study area, surrounded by the dense and shallow archipelago, seemed to form a barrier for water exchange. In the more open parts of the archipelago, the isohalines bend northwards, but the salinity values decreased again towards Åland Island in the west.

The 2007 summer season in the NE Archipelago Sea

The surface conductivity data from the summer of 2007 revealed more detailed patterns of salinity variation in the inner parts of the Archipelago Sea. In April and May (week 20 to 23), the isohalines first moved slightly southwards, but during the rest of the study period, the surface layer salinity increased in the inner archipelago (Fig. 7). A major leap in surface salinity occurred in June between weeks 23 and 26, when more saline waters intruded from the south-west and west to the inner archipelago. The large islands of the middle archipelago blocked the inflow of the saline waters, as interpreted also from Figure 5. The greatest salinity range was found in the inner bays near the mainland (~0.8) and in the western edge of the study area (~0.6), with the lowest range (~0.3) occurring in the south (Fig. 8). Station S had the highest overall salinity of the three intensively sampled sites (Fig. 9). The long term average at station S was 6.3 and 7.0 in the surface and bottom layers, respectively. At stations E and N, the surface/bottom layer long-term mean salinities were 5.9/6.1 and 5.9/6.0, respectively. At all stations, salinities were higher in the bottom layer than at the surface. However, at stations N and E the difference was on average only 0.1-0.2 and salinity varied simultaneously in the surface and in the bottom layer. At station S, the salinity difference between the surface and bottom was greater, i.e. 0.8 on average, and the salinities of these layers were not interlinked as at the shallower stations N and E. The salinity range in the surface layer was 0.6 at all stations. At station S, the salinity ranged from 6.0 to 6.6, and from 5.6 to 6.2 at both stations E and N. In the bottom layer, the salinity range was 0.7 at stations E and N (from 5.8 to 6.5, and from 5.6 to 6.3, respectively). The bottom-layer salinity showed more variation at station S, with a range of 2.1 (6.4 to 8.5).

Figure 9.

Although the deviations from the quarterly means revealed a similar pattern of nonseasonal surface layer salinity fluctuation at stations N and S (Fig. 9), the phase of this fluctuation was divergent. The broad scale temporal pattern was similar also at station E, but the short-term fluctuation did not follow the other two stations. In general, there was a period of higher salinity values starting either from the summer of 2003 (stations E and S) or from the beginning of 2004 (station N).

The cross-correlations of salinity between the stations were studied with time-lags of ± 8 quarters (Fig. 10). The cross-correlation between stations N and E showed that the fluctuations occurred simultaneously (r = 0.64 with no lag). The strongest crosscorrelations between stations N and S occurred with lags of -2 or -3 quarters (r = 0.58, r = 0.62, respectively). In other words, station N followed the fluctuation at station S with a lag of 6-9 months. The cross-correlation pattern was unclear between the stations E and S.

Discussion

The presence of a broad-scale surface water salinity gradient in the Archipelago Sea was identified. The general pattern of the gradient was from the lower salinity levels in the shallow inner bays near the mainland to the more saline waters in the open sea areas in the south, towards the Baltic Proper. The gradient was steepest in the semi-enclosed parts of the archipelago, behind a row of large islands and dense island concentrations. The straits that connect the adjacent sub-basins within the Archipelago Sea are usually narrow with shallow sills. This results in a filling-box effect that allows only the surface waters to flow onto the next basin, blocking the more saline waters in the bottom layer. In addition, the runoff of several small rivers leads to lower surface salinity levels within the inner archipelago. Another salinity gradient was detected in the area east of Åland Island, where it was assumed to be caused by southward surface flows of less saline waters from the Gulf of Bothnia.

The terrestrial runoffs from Åland are hardly sufficient to maintain any permanent salinity gradients within this area.

The fingerprint of the anti-clockwise Baltic Sea circulation pattern was apparent. The northward net surface flow in the Archipelago Sea, portrayed as northward lobes of salinity into the middle parts of the study area, where the relatively open sea area allowed the most unrestricted surface flow. This wedge-like plume of more saline water was undisputed in all the spatial interpolations. A similar northward plume has also been recognised by other authors [START_REF] Bock | Monatskarten des salzgehaltes der Ostsee dargestellt für verschiedene tiefenhorizonte[END_REF][START_REF] Helminen | Assessing of long-distance background nutrient load to the Archipelago Sea, northern Baltic, with a hydrodynamic model[END_REF][START_REF] Rodhe | The Baltic and the North Seas: a process oriented review of the physical oceanography[END_REF][START_REF] Erkkilä | Patterns and dynamics of coastal waters in multitemporal satellite images: support to water quality monitoring in the Archipelago Sea, Finland[END_REF] Some parts of the Archipelago Sea showed particularly changeable water salinity levels during a single summer season. The spring runoff was still spreading from the mainland until late May, but from June onwards, the inflow of the more saline surface water from the south compensated the decreased runoff. The dispersion of fresh water from the mainland runoff resulted in varying salinity regimes within the inner archipelago bays. A region of higher salinity variation also occurred on the western edge of the north-eastern study area (Fig. 7), which is occasionally influenced by both the northward flow of water from the Baltic Proper, as well as the westward dispersion of less saline water from the inner archipelago.

The long-term salinity measurements from the three intensively sampled monitoring stations confirmed the inter-annual fluctuations of the surface water salinity, while the deviations from the quarterly means revealed non-seasonal salinity fluctuations. The seemingly regular variation from 2000 to the end of 2003 was followed by a period of elevated salinity levels at all the stations. In August 2002, January 2003 and August 2003, significant inflow events occurred through the entrance area of the Baltic Sea [START_REF] Feistel | Unusual Baltic inflow activity in 2002-2003 and varying deep water properties[END_REF]. These saline pulses are likely seen in our data as a delayed impact of the increased salinity in the Archipelago Sea.

The stations N and E are characterised by rather identical salinity fluctuations. A halocline was not observed at these shallower stations during any season, although salinity was typically 0.1-0.2 higher in the bottom than in the surface layer. At the deeper station S, the average difference between the surface and bottom layer salinity was 0.8. The surface and bottom salinity values did not fluctuate simultaneously, indicating the presence of a halocline and an influence of the bottom layer water of the northern Baltic Proper. These findings further suggest that the underwater sills prevent the intrusion of the denser saline bottom layer waters into the inner archipelago area, allowing only the surface water layer to filter towards the north and the mainland.

Station N followed the fluctuation pattern of station S with a lag of 2-3 quarters, i.e. 6-9 months. Assuming that the surface salinity at these stations is an indication of northward surface water flow, the mean residual flow velocity can be defined: the distance between the stations is approximately 90 km, thus the northward flow component of the surface water flow through the Archipelago Sea is 0.4-0.6 cm s -1 .

There are several studies considering the Baltic Sea circulation patterns based on either observations or modelling. Typical scalar current velocities in the upper layer of the Gulf of Finland are found to be 10 cm s -1 [START_REF] Alenius | The physical oceanography of the Gulf of Finland: a review[END_REF], and near the coast and in the open sea of the Gulf of Bothnia 5-7 cm s -1 and 2-4 cm s -1 , respectively [START_REF] Myrberg | Modeling of the circulation, water exchange and water age properties of the Gulf of Bothnia[END_REF]. Further, in the NE Archipelago Sea, scalar current velocities were typically less than 10 cm s -1 (Virtaustutkimuksen neuvottelukunta, 1979). During the BEVIS project (Kohonen and Mattila, 2007), flow measurements were made on the eastern side of Åland Island with moored instruments for three periods of seven weeks in four locations. They measured surface layer mean scalar velocities of 7-14 cm s -1 [START_REF] Forsius | Water flow measurements in the Archipelago of Åland. Data report[END_REF]. However, particularly in experimental studies periods of measurement are often relatively short. Thus, they do not reflect long term circulation and in moored experiment configurations, the results also depend on the local conditions, such as bathymetry. The circulation pattern of the Baltic Sea is not stable and currents typically oscillate back and forth in different time-scales (e.g. [START_REF] Otsmann | The oscillatory nature of the flows in the system of straits and small semienclosed basins of the Baltic Sea[END_REF].

Therefore, the mean residual flow velocities are significantly lower than the mean scalar velocities. In the early studies of [START_REF] Witting | Zusammenfassende übersicht der hydrographie des Bottnischen und Finnischen Meerbusens und der nördlichen Ostsee[END_REF] and [START_REF] Palmén | Untersuchungen über die Strömungen in den Finnland umgebenden Meeren[END_REF], the annual mean residual vector velocity in the Gulf of Finland and Gulf of Bothnia was reported to be less than 2 cm s -1 , while a similar mean residual flow velocity of 1-2 cm s -1 was also presented by [START_REF] Alenius | The physical oceanography of the Gulf of Finland: a review[END_REF] for the Gulf of Finland. [START_REF] Forsius | Water flow measurements in the Archipelago of Åland. Data report[END_REF] reported the mean northward flow component velocities varying in the range of -4.1 -2.8 cm s -1 in the sea area east of Åland, negative values indicating the southward flow.

Although the residual flow velocity (or mean circulation) is a statistical artefact rather than a physical condition in the Baltic Sea, it is usable when evaluating the distribution of substances [START_REF] Myrberg | Modeling of the circulation, water exchange and water age properties of the Gulf of Bothnia[END_REF][START_REF] Alenius | The physical oceanography of the Gulf of Finland: a review[END_REF]. The northward flow component indirectly derived in this study is low compared to the earlier studies in more pelagic areas. These results indicate that over a long term the Archipelago Sea acts as a relatively strong buffer and that the water retention time is long. This issue calls for further inspection, since it may impact, for instance, on the accumulation of nutrients and thus have implications to the development of the eutrophication process in the Archipelago Sea.

While the salinity gradients discussed above have been intuitively known, they have not been previously studied in such spatial and temporal detail in the Archipelago Sea. On the basis of our results, we argue that the Archipelago Sea can be considered both a transitional, estuary-like environment for its continuous mixing of continental and marine water masses, as well as a sieve-like sill system between two major basins of the Baltic Sea. These parallels may be embedded in the research of the biotic systems in these stress environments, where the local species assemblages constitute a mixture of salinity sensitive fresh water and marine species. The dynamic nature of salinity and other physical and chemical properties of the seawater should be thoroughly considered also in the planning of environmental monitoring in the region [START_REF] Erkkilä | Spatial and temporal representativeness of water monitoring efforts in the Baltic Sea coast of SW Finland[END_REF]. Water protection actions should find the most effective means to reduce nutrient dispersion and eutrophication. In this context, a multidisciplinary understanding of the water exchange dynamics as well as ecological gradients and limits within the Archipelago Sea are required (see Helminen et al., 1998, Kohonen andMattila, 2007). It is also likely that the precipitation in the northern Baltic Sea drainage basin will increase during the century, which would lead to a decrease in the overall salinity of the Baltic Sea [START_REF] Graham | Projections of future anthropogenic climate change[END_REF] and changes in the ecosystem [START_REF] Dippner | Climate related marine ecosystem change[END_REF]. Further understanding of the Archipelago Sea's salinity dynamics in different spatial and temporal scales is needed for the prediction of the sea area's future development.

Conclusions

The combination of estuarine and sill characteristics creates spatially and temporally varying salinity conditions in the Archipelago Sea. Although the range of these fluctuations is narrow, the areas of volatile salinity need to be studied in higher detail, since the ecological implications of the salinity dynamics are manifold in these brackish waters. The highest variations occur in those areas that are influenced by both the occasional marine water intrusions through the area, as well as the runoff from the mainland. We extend the need of further research of spatial and temporal variation also to other key variables of aquatic environment. The predictive environmental modelling in the highly dynamic coastal and estuarine areas of the northern Baltic Sea should be based on the representative sampling and empirical mapping of the aquatic environment with its diverse physical, chemical and biological phenomena. Table 1. Summary of the data sets. -19.7. and 20.-23.8.2007).

Figure 6. Isohalines on weeks 29 and35 in 2008 (14.-17.7. and25.-29.8.2008). Table 2.

Description Equation Resulting raster datasets

Calculate path distances from all sampled stations i along predefined cost surface Path distances P i (i = 1…n)

If the path distance from the station i is less than distance limit (L), preserve the cell value, otherwise set the value to distance limit

  Sea for the late summers of 2007 and 2008 and for the NE corner of the study area in the summer of 2007. The isohalines presented in figures 4-8 are generalised slightly to improve the cartographic presentation.

  Figure 4.

  at station N): the mean salinity of each quarter at each station to show the general pattern of salinity fluctuations, and quarterly deviations from the mean of the corresponding quarter during 1999-2000 to emphasise nonseasonal long-term fluctuations. The temporal relations of the salinity fluctuations were studied by defining the cross-correlations between the stations with different time lags.

Figure 5 .

 5 Figure 5.

Figure 6 .

 6 Figure 6.

  Figure 7.

  Figure 8.

  Figure 10.

Figure 1 .

 1 Figure 1. Location of the study area and the observation stations. For the abbreviations of the data provider, see Table 1. The intensive monitoring stations KORP 200, NAU 2361 and Brändö 100 are indicated with the letters S, E and N, respectively. The major rivers of the study area are indicated with their average discharge.

Table 2 .

 2 A step-wise diagram of the inverse path distance weighted interpolation procedure. The calculations are done locally for each cell separately and letters P, A, B, C, W, D and E refer to raster surfaces. The constant L set by the user defines the maximum distance of an influence. The value S refers to salinity measured in the field.

Figure 2 .

 2 Figure 2. Examples of some of the intermediate raster layers in the inverse path distance weighted interpolation procedure and the resulting estimation of salinity. For explanation, see table 2.

Figure 3 .

 3 Figure 3. Error assessments of the IDW and IPDW methods.

jS

  is the mean of observed salinities on week j, ij S is either the observed salinity at the station i on week j (y-axis), or the modelled salinity at the location of the omitted station i on week j (x-axis).

Figure 4 .

 4 Figure 4. Comparison of the isohalines on week 34 in 2007 produced by the IDW (A) and by the IPDW (B). The dotted lines on land are added for better readability. Field data provided by SFREC.

Figure 5 .

 5 Figure5. Isohalines on weeks29 and 34 in 2007 (16.-19.7. and 20.-23.8.2007).

Figure 7 .

 7 Figure 7. Isohalines in the NE Archipelago Sea on weeks 20-40 (from mid May to the beginning of October) in 2007.

Figure 8 .

 8 Figure 8. The range of surface salinity in the NE Archipelago Sea during the weeks 20-40 in 2007.

Figure 9 .

 9 Figure 9. The quarterly surface salinity (black) and the near bottom salinity (grey) at the intensive monitoring stations N, E and S (left column) and quarterly deviations from the mean of the corresponding quarter (right column).

Figure 10 .

 10 Figure 10. Cross-correlations of surface salinity between the stations N, E and S with time lags of ±8 quarters.

L

  A i (i = 1…n)Calculate the inverse path distance by dividing the distance limit by the limited path distance from the station ii A Inverse path distances B i (i = 1…n)If the inverse path distance is greater than 1, preserve the cell value, otherwise set the value to zero C i (i = 1…n)Calculate the weight of each station in each cell by dividing the stations i inverse path distance with the sum of the stations i = 1…n inverse path distances W i (i = 1…n)Calculate the weighted salinity in each cell by multiplying the weight of a station by measured salinity value of the station (S i )

Acknowledgements

The authors would like to thank the Southwest Finland Regional Environment Centre for co-operation in sampling and laboratory analyses, and Husö biological station of the Åbo Academy University and the Environment Agency of the Government of the Åland Island for providing their field data. Simon Amelinckx, Anne Erkkilä, Jani Helin and Andy Stock are acknowledged for field work assistance, and the Department of Geography and the Archipelago Research Institute of the University of Turku for field work facilities. The study was funded by the Academy of Finland (project 114083), the Turku University Foundation and the Finnish Cultural Foundation.