**2.2 Results**

162 Studies on Water Management Issues

the best conserved but also most vulnerable estuaries of the Iberian Peninsula (Vasconcelos

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

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

were made in triplicate, according to the spectrophotometric methods described by Grasshoff et al. (1983), using a spectrophotometer Hitachi U-2000 for ammonium, phosphate

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

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

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

least 400 cells in total and 50 cells of the most common genus were counted.

KeZ

3-) and silicate (DSi) were determined upon arrival to the laboratory,

) where frozen (-20ºC) until analysis. All nutrient analyses

z 0 I Ie<sup>−</sup> <sup>=</sup> (1)

the light intensity at depth Z (m) and I0 is the light intensity at the surface:


and silicate, and an autoanalyzer Skalar for nitrate and nitrite.

et al. 2007).

(NH4+), phosphate (PO4

while samples for nitrate (NO3

HCl 1 M (Parsons et al., 1984).

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 construction and filling

Ecological Tools for the Management of Cyanobacteria

Monthly River flow (m s )

 3 -1

300

250

NO , DSi (μM)

3

200

150

100

50

0

Light extinction coefficient Ke (m ) -1

year 1997) were omitted for clarity

NO3 DSi

Blooms in the Guadiana River Watershed, Southwest Iberia 165

**A**

**B**

**C**

**1997 1999 2000 2002 2004** 

**1998 2001 2003 2010** 

Fig. 3. Box and whisker plots showing the distribution of monthly Guadiana river flow (A), subsurface nitrate (NO3-) and silicate (DSi) concentration (B), and light extinction coefficient, Ke (C) in the Guadiana upper estuary, binned into different periods. Median value is represented by the line within the box, 25th to 75th percentiles are denoted by box edges, 5th to 90th percentiles are depicted by the error bars, outliers are indicated by circles, and extreme values by diamonds. Extreme values of monthly river flow (maximum 1258 m3s-1,

flow regimes: period before Alqueva dam construction (1996-1998), period during dam construction (1999 – 2000) and filling (2001 - 2003) and severely regulated river flow afterwards. Before Alqueva, river flow fluctuated widely from torrential winters to dry summers typical of Mediterranean flood - drought rainfall regime. However, starting with construction and filling of Alqueva dam, river flow became abruptly restricted particularly during winter months despite heavy rainfall. Mean river flow during summer reached 20 – 25 m3 s -1 during 1997 – 1998 previous to Alqueva, and decreased below 10 m3 s -1 from 1999 to 2003 during Alqueva construction and filling. Afterwards, summer river flow increased to 10 – 15 m3 s -1 during 2004 – 2005 reaching 20 – 25 m3 s -1 during 2007 – 2008, but decreased back below 10 m3 s -1 during 2008 – 2009.

Box plots in Fig. 3A also revealed these oscillations with respect to median values and overall distribution during these periods. Light extinction coefficient (Ke, see Fig. 3C), which is tightly correlated to sediment load, was generally low during 1997- 1998 previous to Alqueva; however, during dam construction in 1999 and part of 2000, light extinction reached maximum values in extremely turbid waters with very high sediment load (Suspended Particulate Material, SPM, peak values of 140 mg L-1; data not shown). After this period of dam construction and extensive soil movement, waters tended to clear with Ke values decreasing from 2000 to 2010, but with wide intra-annual fluctuations due to winter summer oscillations in rainfall and river flow. It is to be noted that the composition of SPM varied markedly between river mouth (Vila Real Sto António) and upper estuary (Alcoutim and Mértola). In the lower estuary, SPM was mainly composed by quartz, which contributed minimally to light attenuation in the water column. On the contrary, in the middle and upper estuarine regions, SPM was mostly dominated by clays (Machado et al., 2007), which usually play an important role in light absorption.

Nitrate concentration, the predominant form of total dissolved inorganic nitrogen (ca. 65% - 89% of total inorganic N), showed a decreasing trend after construction of Alqueva dam (see Fig. 3B). Nitrate annual means during 1996-2001 in Alcoutim ranged between 65.0-73.6 µM, whereas mean NO3- was 56.2 µM in 2002, further decreasing to 30.4 ± 17.3 µM in 2005 (Barbosa et al., 2010), and remaining relatively low (32.23 ± 20.7 µM) during 2007-2009 (Domingues, 2010).

As referred in previous studies (Barbosa et al., 2010), silicate concentration was usually correlated with rainfall and river flow, and negatively correlated to chlorophyll *a*. Both DSi and nitrate exhibited seasonality with higher values during winter and lower values between midspring and summer. In contrast with nitrate, DSi exhibited an obvious increase during the period of the Alqueva dam filling (2002–2003) that led to a significant increase in the Si:N and Si:P molar ratios (data not shown), and a subsequent decline after its completion from 2004 to 2010 (see Fig. 3B).

Chlorophyll *a* (Fig. 4A) and total cyanobacteria abundance (Fig. 4B) in the upper estuary revealed a sharp collapse in 1999 during Alqueva construction, increasing during dam filling (2000 – 2001). Afterwards, chlorophyll *a* decreased again markedly from 2002 to 2010. Cyanobacterial abundance since Alqueva dam completion did not recuperate to high values observed previously (1997-1998). Furthermore, potentially toxic species, such as *Microcystis* spp., which were previously abundant exceeding WHO alert level 2 (≥100 000 cells mL-1) during 1997 and 1998, have remained at very low densities if not practically absent from water samples collected in the upper estuary after Alqueva dam completion.

flow regimes: period before Alqueva dam construction (1996-1998), period during dam construction (1999 – 2000) and filling (2001 - 2003) and severely regulated river flow afterwards. Before Alqueva, river flow fluctuated widely from torrential winters to dry summers typical of Mediterranean flood - drought rainfall regime. However, starting with construction and filling of Alqueva dam, river flow became abruptly restricted particularly during winter months despite heavy rainfall. Mean river flow during summer reached 20 – 25 m3 s -1 during 1997 – 1998 previous to Alqueva, and decreased below 10 m3 s -1 from 1999 to 2003 during Alqueva construction and filling. Afterwards, summer river flow increased to 10 – 15 m3 s -1 during 2004 – 2005 reaching 20 – 25 m3 s -1 during 2007 – 2008, but

Box plots in Fig. 3A also revealed these oscillations with respect to median values and overall distribution during these periods. Light extinction coefficient (Ke, see Fig. 3C), which is tightly correlated to sediment load, was generally low during 1997- 1998 previous to Alqueva; however, during dam construction in 1999 and part of 2000, light extinction reached maximum values in extremely turbid waters with very high sediment load (Suspended Particulate Material, SPM, peak values of 140 mg L-1; data not shown). After this period of dam construction and extensive soil movement, waters tended to clear with Ke values decreasing from 2000 to 2010, but with wide intra-annual fluctuations due to winter summer oscillations in rainfall and river flow. It is to be noted that the composition of SPM varied markedly between river mouth (Vila Real Sto António) and upper estuary (Alcoutim and Mértola). In the lower estuary, SPM was mainly composed by quartz, which contributed minimally to light attenuation in the water column. On the contrary, in the middle and upper estuarine regions, SPM was mostly dominated by clays (Machado et al.,

Nitrate concentration, the predominant form of total dissolved inorganic nitrogen (ca. 65% - 89% of total inorganic N), showed a decreasing trend after construction of Alqueva dam (see Fig. 3B). Nitrate annual means during 1996-2001 in Alcoutim ranged between 65.0-73.6 µM,

(Barbosa et al., 2010), and remaining relatively low (32.23 ± 20.7 µM) during 2007-2009

As referred in previous studies (Barbosa et al., 2010), silicate concentration was usually correlated with rainfall and river flow, and negatively correlated to chlorophyll *a*. Both DSi and nitrate exhibited seasonality with higher values during winter and lower values between midspring and summer. In contrast with nitrate, DSi exhibited an obvious increase during the period of the Alqueva dam filling (2002–2003) that led to a significant increase in the Si:N and Si:P molar ratios (data not shown), and a subsequent decline after its

Chlorophyll *a* (Fig. 4A) and total cyanobacteria abundance (Fig. 4B) in the upper estuary revealed a sharp collapse in 1999 during Alqueva construction, increasing during dam filling (2000 – 2001). Afterwards, chlorophyll *a* decreased again markedly from 2002 to 2010. Cyanobacterial abundance since Alqueva dam completion did not recuperate to high values observed previously (1997-1998). Furthermore, potentially toxic species, such as *Microcystis* spp., which were previously abundant exceeding WHO alert level 2 (≥100 000 cells mL-1) during 1997 and 1998, have remained at very low densities if not practically absent from

water samples collected in the upper estuary after Alqueva dam completion.


decreased back below 10 m3 s -1 during 2008 – 2009.

2007), which usually play an important role in light absorption.

whereas mean NO3

(Domingues, 2010).

completion from 2004 to 2010 (see Fig. 3B).

Fig. 3. Box and whisker plots showing the distribution of monthly Guadiana river flow (A), subsurface nitrate (NO3-) and silicate (DSi) concentration (B), and light extinction coefficient, Ke (C) in the Guadiana upper estuary, binned into different periods. Median value is represented by the line within the box, 25th to 75th percentiles are denoted by box edges, 5th to 90th percentiles are depicted by the error bars, outliers are indicated by circles, and extreme values by diamonds. Extreme values of monthly river flow (maximum 1258 m3s-1, year 1997) were omitted for clarity

Ecological Tools for the Management of Cyanobacteria

never surpassed 1 µg L-1.

**2.3 Discussion** 

abundance or chlorophyll *a* (see Fig. 7).

chlorophyll maxima usually occurred.

growth was nitrogen limited (Domingues et al., 2011).

Blooms in the Guadiana River Watershed, Southwest Iberia 167

Furthermore, the number of taxa observed not only in cyanobacteria, but also in phytoplankton populations has declined significantly. During summer of 1997 and 1998 the following genera were observed in numbers >1000 cells mL-1: *Microcystis, Anabaena, Oscillatoria, Merismopedia, Lyngbya, Gomphosphaeria, Coelosphaerium, Syenchococcus*, and several unidentified species of *Chroococcales.* In contrast, during 2007 – 2009, besides

Total cyanobacterial abundance included abundant small chroococcoid species and more

As for microcystin – LR (MC-LR) concentrations in suspended particulate material (Fig. 4C), distribution in different periods showed highest values during 1999, often surpassing the 1 µg L-1 limit for drinking water (WHO 1998 guidelines). Yet, in 1999 both cyanobacteria abundance and chlorophyll *a* reached overall minimum values observed in the study period from 1997 to 2010. MC-LR decreased from 2002 onwards with concentrations frequently below detection limit. In fact, after 2004, MC-LR concentrations in the particulate fraction

The variation of MC- LR concentration over time (see Fig. 6) revealed the same decreasing trend as box plots in Fig. 4C. The frequency of samples where microcystins were nondetectable increased over time particularly during 2004 and 2005. Gap years (2000, 2001,

Microcystin concentration during the study period was not correlated to total cyanobacteria

In past published reports dealing with the microbial ecology of the Guadiana estuary (Domingues et al., 2005; Domingues & Galvão, 2007; Rocha et al., 2002), the impact of Alqueva dam construction was predicted to increase eutrophication conditions and possibly promote cyanobacterial blooms and associated cyanotoxins. In fact, this has not been observed during the seven-year period after dam completion. Not only cyanobacteria, but overall phytoplankton abundance, biomass and chlorophyll *a* concentrations have decreased markedly and have remained at low levels even in the upper estuary, where peak

Typical estuarine phytoplankton succession observed in the Guadiana estuary from diatoms in early spring, to chlorophytes and finally cyanobacteria in late summer and fall was driven by nutrient regime with high winter loads of nitrogen and phosphorus discharged downriver, and silica depletion after the spring diatom bloom (Rocha et al., 2002). These authors also referred that cyanobacteria dominated the chlorophyll maximum zone in the upper estuary in late summer- early fall, due to warm waters, reduced sinking and grazing, as well as N limitation with low N:P ratio. Nitrogen limitation during summer increased in the period after Alqueva in the upper estuary (Barbosa et al., 2010). Additionally, nutrient enrichment experiments performed during 2008 clearly demonstrated that phytoplankton

Contrary to more stringent nitrogen limitation, the improved light regime with lower extinction coefficients should have promoted overall phytoplankton growth from 2003

2006 and 2007) are due to lack of funding for regular monitoring in estuarine waters.

unidentified Chroococcales, only *Planktothr*ix could be occasionally identified.

rare large filamentous forms, and was not correlated with chlorophyll a (see Fig. 5).

Fig. 4. Box and whisker plots showing the distribution of chlorophyll *a* concentration (A), total cyanobacteria abundance (B), and microcystin-LR concentration (C) in the Guadiana upper estuary, binned into different periods. Median value is represented by the line within the box, 25th to 75th percentiles are denoted by box edges, 5th to 90th percentiles are depicted by the error bars, outliers are indicated by circles, and extreme values by diamonds. An extreme chlorophyll *a* value (216.0 µgL-1, year 2001) was omitted for clarity

Furthermore, the number of taxa observed not only in cyanobacteria, but also in phytoplankton populations has declined significantly. During summer of 1997 and 1998 the following genera were observed in numbers >1000 cells mL-1: *Microcystis, Anabaena, Oscillatoria, Merismopedia, Lyngbya, Gomphosphaeria, Coelosphaerium, Syenchococcus*, and several unidentified species of *Chroococcales.* In contrast, during 2007 – 2009, besides unidentified Chroococcales, only *Planktothr*ix could be occasionally identified.

Total cyanobacterial abundance included abundant small chroococcoid species and more rare large filamentous forms, and was not correlated with chlorophyll a (see Fig. 5).

As for microcystin – LR (MC-LR) concentrations in suspended particulate material (Fig. 4C), distribution in different periods showed highest values during 1999, often surpassing the 1 µg L-1 limit for drinking water (WHO 1998 guidelines). Yet, in 1999 both cyanobacteria abundance and chlorophyll *a* reached overall minimum values observed in the study period from 1997 to 2010. MC-LR decreased from 2002 onwards with concentrations frequently below detection limit. In fact, after 2004, MC-LR concentrations in the particulate fraction never surpassed 1 µg L-1.

The variation of MC- LR concentration over time (see Fig. 6) revealed the same decreasing trend as box plots in Fig. 4C. The frequency of samples where microcystins were nondetectable increased over time particularly during 2004 and 2005. Gap years (2000, 2001, 2006 and 2007) are due to lack of funding for regular monitoring in estuarine waters.

Microcystin concentration during the study period was not correlated to total cyanobacteria abundance or chlorophyll *a* (see Fig. 7).
