**2.1 Study area, sampling strategy and methods**

The Guadiana estuary is a mesotidal system (tidal amplitude: 1.3–3.5 m), with a length of 70 km, a maximum width of ca. 550 m, an average depth of 6.5 m, and an average water residence time of 12 days (Domingues & Galvão, 2007; Vasconcelos et al., 2007). The upstream saltwater intrusion is usually located close to Alcoutim (ca. 38 km from river mouth), whereas tidal influence extends to Mértola (ca. 70 km from river mouth; see Fig. 1). The lower estuary ranges from partially stratified to well-mixed, whereas the upper estuary is generally well-mixed (Cravo et al., 2006; Morais et al., 2009; Oliveira et al., 2006; Rocha et al. 2002). A series of dams has severely restricted its freshwater flow (ca. 75 %), and the recent construction of the large Alqueva dam (ca. 150 km upstream from river mouth) increased flow regulation up to 81% of the total catchment area (55 000 km2) starting in 2003 (Galvão et al., 2008). Since human activity in the Guadiana watershed is mostly agriculture and the main anthropic pressure is associated to dams, the Guadiana is considered one of

Ecological Tools for the Management of Cyanobacteria

some adjustments in the interpretation of chromatograms.

**2.2 Results** 

construction and filling

Blooms in the Guadiana River Watershed, Southwest Iberia 163

0.05% (v/v) TFA (trifluoroacetic acid) in a 25 : 75 ratio. Extract was then evaporated in a rotary evaporator (50-54 ºC). Chromatograms were analyzed between 180 and 900 nm, with main detection at 238 nm for absorption spectrum characteristic of MC-LR. Purified MC-LR (Sigma) was used as standard, and results are expressed in MC-LR equivalents per volume of sample (Meriluoto & Codd, 2005; Ribau-Teixeira & Rosa, 2005; Sobrino et al., 2004). Samples collected during 1999 were analyzed for MC-LR using both ELISA and HPLC techniques in Dr. Wayne Carmichael´s laboratory, Wright State University, Ohio, U.S.A and results confirmed in Prof. Vitor Vasconcelos´ laboratory, Universidade do Porto, Portugal. It is to be noted that the HPLC technique applied for MC-LR determination was carried out in different years by different technical staff and/or students (Master´s, PhD & post-doctoral fellows) in different specialized laboratories. During 1999, MC-LR was analyzed in parallel in two laboratories (Dr. W. Carmichael, Wright State, U.S.A., and Prof. V. Vasconcelos, Universidade do Porto, Portugal). From 2002 onwards, MC-LR analysis was performed in the Environmental Technology Lab., Universidade do Algarve with Prof. M. J. Rosa (2002- 2003) and Dr. M. R. Teixeira (2004-2009). Therefore, although not considered significant in this study, slight variations in the extraction and HPLC methodology existed, as well as

Monthly mean river flow at Pulo do Lobo (ca. 85 km upstream from river mouth) and total monthly rainfall at Alcoutim measured from 1996 to 2009 (Fig. 2) revealed four distinct river

Fig. 2. Monthly mean river flow (m3 s-1) at Pulo do Lobo and total monthly rainfall (mm) at Alcoutim from 1996 to 2009 (data source: http://snirh.pt/). Arrow marks period of dam

the best conserved but also most vulnerable estuaries of the Iberian Peninsula (Vasconcelos et al. 2007).

At different stations (see Fig. 1), vertical profiles of water temperature and salinity were determined in situ using a YSI 556 MPS probe. Vertical profiles of photosynthetically active radiation (PAR) intensity were determined using a LI-COR radiometer. Light extinction coefficient (ke, m-1) was calculated using an exponential function (eq. 1), where Iz represents the light intensity at depth Z (m) and I0 is the light intensity at the surface:

$$\mathbf{I}\_{\mathbf{z}} = \mathbf{I}\_0 \mathbf{e}^{-\mathsf{K}\mathbf{e}\mathbf{Z}} \tag{1}$$

Subsurface water samples (ca. 0.5 m) were collected at different sampling stations (Alcoutim and Mértola) for determination of dissolved inorganic nutrients and phytoplankton variables. For nutrient concentration, samples were immediately filtered through cellulose acetate filters (Whatman, nominal pore diameter 0.2 μm) to acid-cleaned vials. Ammonium (NH4 +), phosphate (PO43-) and silicate (DSi) were determined upon arrival to the laboratory, while samples for nitrate (NO3-) where frozen (-20ºC) until analysis. All nutrient analyses were made in triplicate, according to the spectrophotometric methods described by Grasshoff et al. (1983), using a spectrophotometer Hitachi U-2000 for ammonium, phosphate and silicate, and an autoanalyzer Skalar for nitrate and nitrite.

Chlorophyll *a* concentration was determined spectrophotometrically using glass fiber filters (Whatman GF/F, nominal pore diameter = 0.7 μm). Chlorophyll *a* was extracted overnight at 4ºC with 90% acetone; after centrifugation, absorbance of the supernatant was measured in the spectrophotometer Hitachi U-2000 at 750 and 665 nm, before and after addition of HCl 1 M (Parsons et al., 1984).

Phytoplankton composition (including cyanobacteria), abundance and biomass were determined using epifluorescence (Haas, 1982) and inverted microscopy (Utermöhl, 1958). Samples for enumeration of pico- (<2 µm) and nanophytoplankton (2 - 20 µm) were preserved with glutaraldehyde (final concentration 2%) immediately after collection, stained with proflavine and filtered (1 - 5 mL, depending on the amount of suspended matter) onto black polycarbonate membrane filters (Whatman, nominal pore diameter 0.45 μm). Preparations were made within 24 h of sampling using glass slides and non-fluorescent immersion oil (Cargille type A), and then frozen (-20ºC) in dark conditions, to minimize loss of autofluorescence. Enumeration was made at 787.5x magnification using an epifluorescence microscope (Leica DM LB). Samples for enumeration of microphytoplankton (>20 µm) were preserved with acid Lugol's solution (final concentration ca. 0.003%) immediately after collection, settled in sedimentation chambers (2 - 10 mL, depending on the amount of suspended matter; sedimentation time = 24 hours) and observed at 400x magnification with an inverted microscope (Zeiss Axiovert S100). A minimum of 50 random visual fields, at least 400 cells in total and 50 cells of the most common genus were counted.

For microcystin – LR (MC-LR) determination, 1.5 to 2 L water samples were filtered through Whatman GF/F filters, which were frozen until extraction with 20 mL 75 % (v/v) methanol. High performance liquid chromatography (HPLC) was carried out in a Dionex Summit equipment with photodiode array detector (PDA) and Chromeleon 6.3 software, using a C18 column (Merck Purospher STAR RP18 endcapped, 3 μm particles, LiChro-CART, 55 mm x 4mmm) kept at 40ºC. As a mobile phase, acetonitrile and Milli-Q water were used containing 0.05% (v/v) TFA (trifluoroacetic acid) in a 25 : 75 ratio. Extract was then evaporated in a rotary evaporator (50-54 ºC). Chromatograms were analyzed between 180 and 900 nm, with main detection at 238 nm for absorption spectrum characteristic of MC-LR. Purified MC-LR (Sigma) was used as standard, and results are expressed in MC-LR equivalents per volume of sample (Meriluoto & Codd, 2005; Ribau-Teixeira & Rosa, 2005; Sobrino et al., 2004). Samples collected during 1999 were analyzed for MC-LR using both ELISA and HPLC techniques in Dr. Wayne Carmichael´s laboratory, Wright State University, Ohio, U.S.A and results confirmed in Prof. Vitor Vasconcelos´ laboratory, Universidade do Porto, Portugal.

It is to be noted that the HPLC technique applied for MC-LR determination was carried out in different years by different technical staff and/or students (Master´s, PhD & post-doctoral fellows) in different specialized laboratories. During 1999, MC-LR was analyzed in parallel in two laboratories (Dr. W. Carmichael, Wright State, U.S.A., and Prof. V. Vasconcelos, Universidade do Porto, Portugal). From 2002 onwards, MC-LR analysis was performed in the Environmental Technology Lab., Universidade do Algarve with Prof. M. J. Rosa (2002- 2003) and Dr. M. R. Teixeira (2004-2009). Therefore, although not considered significant in this study, slight variations in the extraction and HPLC methodology existed, as well as some adjustments in the interpretation of chromatograms.
