**3.4 Methods**

These reservoirs have been monitored for standard physical, chemical and microbiological water quality, including, since 2003, determination of phytoplankton biomass and composition. Monthly surface and bottom samples were taken, from 2003 to 2009, at Choça Queimada tower for Odeleite reservoir, and at the extraction tower of the Beliche reservoir. During 2009 and 2010, a new sampling site in the middle of the lake, 500m upstream from the dam wall, was added according to new guidelines from the European Med GIG (INAG, 2009). At each of these sampling sites, vertical profiles were determined *in situ* using a YSI

Ecological Tools for the Management of Cyanobacteria

http://www.drapalg.min-agricultura.pt/

**Month/year Bottom discharge** 

source: http://snirh.pt/)

**(dam3)** 

Blooms in the Guadiana River Watershed, Southwest Iberia 177

Fig. 9. Evolution of stored water volume (hm3) and mean monthly rainfall from July 2003 to November 2010 in Odeleite and Beliche reservoirs. Data source: http://snirh.pt/ and

> **Surface discharge (dam3)**

Jul/2003 0 0 13 0 Aug/2003 0 0 26 0 Sep/2003 0 0 13 0 Oct/2003 0 0 1 0 Nov/2003 1234 8722 1818 0 Dec/2003 3894 9821 382 0 Feb/2004 6648 0 1562 0 Mar/2006 5970 0 1409 0 Nov/2006 15196 19868 3536 1776 Dec/2006 8685 0 543 0 Feb/2007 2364 0 0 0 Aug/2007 74 0 0 0 Mar/2008 0 261 0 0 Apr/2008 6095 18388 0 1012 Feb/2009 8495 0 0 3502 Dec/2009 0 22450 0 5105 Dec/2010 0 29821 40 6714 Jan/2011 22528 0 0 2186 Mar/2011 50139 0 0 12577 Table 2. Surface and bottom water discharged from Odeleite and Beliche since 2003 (Data

**Bottom discharge (dam3)** 

**Surface discharge (dam3)** 

**Reservoir Odeleite Beliche** 

650 MDS probe, for water temperature, dissolved oxygen, pH and conductivity. Nutrient concentrations analysis were performed at the accredited (EN 17025) water analysis laboratory from Administração da Região Hidrográfica do Algarve (ARH Algarve), who was also responsible for all the sampling campaigns. Phytopigments including chlorophyll *a* were analyzed at Huelva University (Forján et al., 2008) through HPLC, according to Young et al. (1997). Microcystin detection and quantification was performed according to Carmichael & An (1999) using the micro-ELISA kit Microcystin Plate Kit from Adgen – Agrifood Diagnostic. Phytoplankton composition was determined by the same methods referred in 2.1. Phytoplankton biovolumes following European guidelines were calculated on the basis of predefined 3-dimensional shapes and their respective stereometric formulas as recommended by Edler (1979a, 1979b) and Hillebrand et al. (1999), according to the CEN/TC230/WG2/TG3 N108 Water Quality and Olenina et al. (2006).

Berger Parker dominance index was determined by calculating the proportion of the most abundant species over the total phytoplankton cell density (Magurran, 1988). Carlson Trophic State Index (TSI) was calculated for chlorophyll *a* values and both for total phosphorus (TP) and soluble reactive phosphorus (SRP) concentrations, according to Carlson (1977). Contribution of cyanobacteria to total phytoplankton is given by the percentage of total biovolume attributed to cyanobacteria. Catalán Index for Algal Groups (InGA) was determined by using biovolume proportions of colonial and non-colonial algal groups (Catalán et al., 2003). The MedPTI index was calculated according to Marchetto (2009).

## **3.5 Results and discussion**

## **3.5.1 Hydrometric features**

Monitoring data for the last 7 years included a severe 18 months drought from 2004 to 2006, and an exceptional rainy year in 2010. As seen in Fig. 9, water level at Odeleite reservoir had to be lowered in November 2006 for maintenance works, originating intense bottom and surface discharges. Surface overflow was released through the stream bed into the Guadiana estuary, but bottom discharged water flowed through the underground channel into Beliche reservoir, causing more surface and bottom discharges at this reservoir. Water volumes discharges at surface and at bottom of both reservoirs are enlisted in Table 2, revealing how water level regulation in both reservoirs is interconnected. Apart from water extraction for municipal consumption, no water outflow occurred from February 2004 to March 2006, during the drought (see Table 2). In fact, there is no ecological flow stipulated for these streams, such that, downstream from the dam walls, only estuarine water flows in during high tide.

Bottom discharges from Odeleite to Beliche, through the underground channel, induced mixing of water column, with ressuspension of sediment and nutrients. Indeed, both reservoirs did not behave as warm monomyctic lakes, but rather as artificially polimyctic, since water column mixed in the winter and also partially, whenever channel sluices were opened. Water level regulation and Beliche water withdrawal for drinking water were seemingly the main impacts on the water quality of these reservoirs. Nevertheless, sharp shifts in water level also affected margin vegetation, contributing to increased nutrient leaching from soils.

650 MDS probe, for water temperature, dissolved oxygen, pH and conductivity. Nutrient concentrations analysis were performed at the accredited (EN 17025) water analysis laboratory from Administração da Região Hidrográfica do Algarve (ARH Algarve), who was also responsible for all the sampling campaigns. Phytopigments including chlorophyll *a* were analyzed at Huelva University (Forján et al., 2008) through HPLC, according to Young et al. (1997). Microcystin detection and quantification was performed according to Carmichael & An (1999) using the micro-ELISA kit Microcystin Plate Kit from Adgen – Agrifood Diagnostic. Phytoplankton composition was determined by the same methods referred in 2.1. Phytoplankton biovolumes following European guidelines were calculated on the basis of predefined 3-dimensional shapes and their respective stereometric formulas as recommended by Edler (1979a, 1979b) and Hillebrand et al. (1999), according to the

Berger Parker dominance index was determined by calculating the proportion of the most abundant species over the total phytoplankton cell density (Magurran, 1988). Carlson Trophic State Index (TSI) was calculated for chlorophyll *a* values and both for total phosphorus (TP) and soluble reactive phosphorus (SRP) concentrations, according to Carlson (1977). Contribution of cyanobacteria to total phytoplankton is given by the percentage of total biovolume attributed to cyanobacteria. Catalán Index for Algal Groups (InGA) was determined by using biovolume proportions of colonial and non-colonial algal groups (Catalán et al., 2003). The MedPTI index was calculated according to Marchetto

Monitoring data for the last 7 years included a severe 18 months drought from 2004 to 2006, and an exceptional rainy year in 2010. As seen in Fig. 9, water level at Odeleite reservoir had to be lowered in November 2006 for maintenance works, originating intense bottom and surface discharges. Surface overflow was released through the stream bed into the Guadiana estuary, but bottom discharged water flowed through the underground channel into Beliche reservoir, causing more surface and bottom discharges at this reservoir. Water volumes discharges at surface and at bottom of both reservoirs are enlisted in Table 2, revealing how water level regulation in both reservoirs is interconnected. Apart from water extraction for municipal consumption, no water outflow occurred from February 2004 to March 2006, during the drought (see Table 2). In fact, there is no ecological flow stipulated for these streams, such that, downstream from the dam walls, only estuarine water flows in during

Bottom discharges from Odeleite to Beliche, through the underground channel, induced mixing of water column, with ressuspension of sediment and nutrients. Indeed, both reservoirs did not behave as warm monomyctic lakes, but rather as artificially polimyctic, since water column mixed in the winter and also partially, whenever channel sluices were opened. Water level regulation and Beliche water withdrawal for drinking water were seemingly the main impacts on the water quality of these reservoirs. Nevertheless, sharp shifts in water level also affected margin vegetation, contributing to increased nutrient

CEN/TC230/WG2/TG3 N108 Water Quality and Olenina et al. (2006).

(2009).

high tide.

leaching from soils.

**3.5 Results and discussion 3.5.1 Hydrometric features** 



Table 2. Surface and bottom water discharged from Odeleite and Beliche since 2003 (Data source: http://snirh.pt/)

Ecological Tools for the Management of Cyanobacteria

biovolume proportion (Fig. 12) in at least 50% of the samples.

Blooms in the Guadiana River Watershed, Southwest Iberia 179

gathered during 2009 and 2010 showed diatom (Bacillariophycea) dominance in terms of

Fig. 11. Cyanobacteria dynamics in Odeleite and Beliche reservoirs. Total phytoplankton

In terms of cell abundance, *Microcystis* spp. dominated both reservoirs until spring 2008, but Oscillatoriales dominated in terms of biovolume. Cyanobacteria cell densities above WHO alert level 1 of 2000 cells mL-1, occurred in 62 to 63% of all samples, with episodes of *Microcystis* blooms in June 2004 for Odeleite and July 2004 for Beliche. In summer 2006, a *Microcystis* spp. bloom was toxic with microystin concentrations at the bottom of Beliche reservoir reaching 3.5 µg L-1. Despite high cyanobacteria abundances, no significant levels of

Biovolume proportions for main algal groups (Fig. 12) confirmed cyanobacterial dominance from August to October 2009 in Beliche and from October to December 2009 in Odeleite.

abundance (Log cells mL-1) compared with total Cyanobacteria, *Microcystis* and

*Oscillatoria*/*Planktothryx* spp. abundances

microcystins were detected under other bloom situations.

#### **3.5.2 Nutrient dynamics**

Despite these hydrographical fluctuations, no nutrient accumulation or eutrophication trend was detected. Yearly turn-over of Dissolved Inorganic Nitrogen (DIN) and Soluble Reactive Phosphorus (SRP) was clear in Fig. 10, where water temperature at the surface can be used as reference for seasonal changes in nutrient dynamics in Beliche reservoir.

Fig. 10. Dissolved Inorganic Nitrogen (DIN; mg\*L-1), Soluble Reactive Phosphorus (SRP; mg\*L-1) and water temperature (ºC) during 2003 -2010 in Beliche reservoir. Drought months are highlighted in light grey. Months were unusual Odeleite to Beliche discharges occurred are highlighted in darker grey

The increase in DIN levels during the drought years was fictitious (see light grey box Fig. 10), since water level was so low that surface and bottom samples were almost undistinguishable. Shallower depth allowed for oxygen diffusion to the bottom inhibiting deep summer denitrification. Unusual surface and bottom DIN levels occurred in November 2006 due to exceptional water transfer from Odeleite to Beliche. As stated in Galvão et al, 2008, management of the underground channel between the two reservoirs has been associated with conditions favoring blooms through bottom sediment and nutrient resuspension. Consequent water column mixing was revealed by similar bottom and surface temperatures. Comparing Fig. 9 with Fig. 10 also indicated that the increase in DIN concentration in March 2010 was linked to high precipitation levels. Overall low nutrient concentrations in both reservoirs were associated with oligotrophic conditions. In spite of phosphorus limitations and low median and mode values for DIN:SRP ratios, high average N:P ratios were observed, due to outlier values observed in 2005, 2006 and March 2010.

#### **3.5.3 Phytoplankton dynamics**

In terms of cell abundance, more than 80% of monthly water samples during last eight years from Odeleite and Beliche reservoirs, were dominated by cyanobacteria (Fig. 11), but data

Despite these hydrographical fluctuations, no nutrient accumulation or eutrophication trend was detected. Yearly turn-over of Dissolved Inorganic Nitrogen (DIN) and Soluble Reactive Phosphorus (SRP) was clear in Fig. 10, where water temperature at the surface can be used

Fig. 10. Dissolved Inorganic Nitrogen (DIN; mg\*L-1), Soluble Reactive Phosphorus (SRP; mg\*L-1) and water temperature (ºC) during 2003 -2010 in Beliche reservoir. Drought months are highlighted in light grey. Months were unusual Odeleite to Beliche discharges occurred

The increase in DIN levels during the drought years was fictitious (see light grey box Fig. 10), since water level was so low that surface and bottom samples were almost undistinguishable. Shallower depth allowed for oxygen diffusion to the bottom inhibiting deep summer denitrification. Unusual surface and bottom DIN levels occurred in November 2006 due to exceptional water transfer from Odeleite to Beliche. As stated in Galvão et al, 2008, management of the underground channel between the two reservoirs has been associated with conditions favoring blooms through bottom sediment and nutrient resuspension. Consequent water column mixing was revealed by similar bottom and surface temperatures. Comparing Fig. 9 with Fig. 10 also indicated that the increase in DIN concentration in March 2010 was linked to high precipitation levels. Overall low nutrient concentrations in both reservoirs were associated with oligotrophic conditions. In spite of phosphorus limitations and low median and mode values for DIN:SRP ratios, high average N:P ratios were observed, due to outlier values observed in 2005, 2006 and March 2010.

In terms of cell abundance, more than 80% of monthly water samples during last eight years from Odeleite and Beliche reservoirs, were dominated by cyanobacteria (Fig. 11), but data

as reference for seasonal changes in nutrient dynamics in Beliche reservoir.

**3.5.2 Nutrient dynamics** 

are highlighted in darker grey

**3.5.3 Phytoplankton dynamics** 

gathered during 2009 and 2010 showed diatom (Bacillariophycea) dominance in terms of biovolume proportion (Fig. 12) in at least 50% of the samples.

Fig. 11. Cyanobacteria dynamics in Odeleite and Beliche reservoirs. Total phytoplankton abundance (Log cells mL-1) compared with total Cyanobacteria, *Microcystis* and *Oscillatoria*/*Planktothryx* spp. abundances

In terms of cell abundance, *Microcystis* spp. dominated both reservoirs until spring 2008, but Oscillatoriales dominated in terms of biovolume. Cyanobacteria cell densities above WHO alert level 1 of 2000 cells mL-1, occurred in 62 to 63% of all samples, with episodes of *Microcystis* blooms in June 2004 for Odeleite and July 2004 for Beliche. In summer 2006, a *Microcystis* spp. bloom was toxic with microystin concentrations at the bottom of Beliche reservoir reaching 3.5 µg L-1. Despite high cyanobacteria abundances, no significant levels of microcystins were detected under other bloom situations.

Biovolume proportions for main algal groups (Fig. 12) confirmed cyanobacterial dominance from August to October 2009 in Beliche and from October to December 2009 in Odeleite.

Ecological Tools for the Management of Cyanobacteria

index applied to Beliche and Odeleite.

**B)** 

**MedPTI classes and upper limits** 

interpretation

**MedPTI** 

Blooms in the Guadiana River Watershed, Southwest Iberia 181

Monthly values for the MedPTI index are illustrated in Fig. 13, with open circles and boxes representing non-valid values based on lower than 70% species contribution to total phytoplankton biovolume. This figure constituted a test to the robustness of the MedPTI

**Beliche** 30 47 3,05

**Odeleite** 19 47 2,90

good – moderate (<2.45)

moderate – poor (<2.13)

poor – bad (1.81)

**TSI classes** <30 30-40 40-50 50-60 60-70

(<2.77)

Fig. 13. Monthly values for MedPTI. Dashed lines indicate lower boundaries of "excellent" and "good" classifications. Open circles and boxes correspond to non-valid values for

Odeleite and Beliche, respectively. See text for explanation

Table 3. A) Determined values for Carlson Trophic State Index (TSI) based on total P concentrations and on chlorophyll a content, and for phytoplankton composition MedPTI index. B) Classification boundary values for TSI and MedPTI with a color code to facilitate

excelent high – good

**A) Reservoir/Index TSI (Tota P) TSI (Chl-a) MedPTI** 

Summer bloom absence in 2010 could be linked to high water discharges in consequence of an exceptional rainy winter and spring. (see Fig. 9 and Table 2).Thus, during the study period both Beliche and Odeleite reservoirs were susceptible to CHABs.

Fig. 12. Relative contribution (%) of main algal groups to total phytoplankton biovolume

### **3.5.4 Ecological indices**

Table 3, compiles Carlson Trophic State Indices (TSI) calculated for Beliche and Odeleite based on Chlorophyll *a* (Chl-a) and on total phosphorus (TP) as well as for phytoplankton ecological index (MedPTI) proposed by Marchetto et al. (2009) for Italian deep lakes in the Mediterranean region. This index is based on the proportion of biovolumes of species, listed in Italian lakes, and should not be applied in situations where the biovolume of listed existing species does not exceed 70% of total phytoplankton biovolume. Since the contribution of Marchetto species to total phytoplankton biovolume reached 77% in Beliche during 2010 and 71% in Odeleite, the MedPTI index was also calculated for comparison. TSI based on transparency measured by Secchi depth was not calculated, since it has long been established that torrential hydrographic regimes promote high values for this index without any correlation with eutrophication. Total Phosphorus concentrations were also misleading, since bottom sediment resuspension promoted by artificially induced polimyctic behavior, released organic phosphorus unavailable for phytoplankton into the water column. Low chlorophyll *a* values in spite of high cellular cyanobacteria abundance, as referred previously, was due to low chlorophyll content of cyanobacteria.

Summer bloom absence in 2010 could be linked to high water discharges in consequence of an exceptional rainy winter and spring. (see Fig. 9 and Table 2).Thus, during the study

> Bacillariophyceae Chlorophyceae Chrysopyceae Conjugatophyceae Cryptophyceae Cyanobacteria Dinophyceae Euglenophyceae Klebsormidiophyceae

Trebouxiophyceae Unidentified phytoplankton

previously, was due to low chlorophyll content of cyanobacteria.

Fig. 12. Relative contribution (%) of main algal groups to total phytoplankton biovolume

Table 3, compiles Carlson Trophic State Indices (TSI) calculated for Beliche and Odeleite based on Chlorophyll *a* (Chl-a) and on total phosphorus (TP) as well as for phytoplankton ecological index (MedPTI) proposed by Marchetto et al. (2009) for Italian deep lakes in the Mediterranean region. This index is based on the proportion of biovolumes of species, listed in Italian lakes, and should not be applied in situations where the biovolume of listed existing species does not exceed 70% of total phytoplankton biovolume. Since the contribution of Marchetto species to total phytoplankton biovolume reached 77% in Beliche during 2010 and 71% in Odeleite, the MedPTI index was also calculated for comparison. TSI based on transparency measured by Secchi depth was not calculated, since it has long been established that torrential hydrographic regimes promote high values for this index without any correlation with eutrophication. Total Phosphorus concentrations were also misleading, since bottom sediment resuspension promoted by artificially induced polimyctic behavior, released organic phosphorus unavailable for phytoplankton into the water column. Low chlorophyll *a* values in spite of high cellular cyanobacteria abundance, as referred

period both Beliche and Odeleite reservoirs were susceptible to CHABs.

**0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%**

**% Biovolume of algal groups**

**0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%**

**3.5.4 Ecological indices** 

**Odeleite**

**Beliche**

Monthly values for the MedPTI index are illustrated in Fig. 13, with open circles and boxes representing non-valid values based on lower than 70% species contribution to total phytoplankton biovolume. This figure constituted a test to the robustness of the MedPTI index applied to Beliche and Odeleite.



Table 3. A) Determined values for Carlson Trophic State Index (TSI) based on total P concentrations and on chlorophyll a content, and for phytoplankton composition MedPTI index. B) Classification boundary values for TSI and MedPTI with a color code to facilitate interpretation

Fig. 13. Monthly values for MedPTI. Dashed lines indicate lower boundaries of "excellent" and "good" classifications. Open circles and boxes correspond to non-valid values for Odeleite and Beliche, respectively. See text for explanation

Ecological Tools for the Management of Cyanobacteria

allowance of natural variability and (v) reliability.

should be reevaluated, at least for warm semi-arid regions.

contribution of cyanobacteria blooms to phytoplankton biomass.

(Eq. 2)

phytoplankton biovolume.

Blooms in the Guadiana River Watershed, Southwest Iberia 183

As discussed by Jackson et al. (2000), an index that does not allow for temporal and spatial variability, might lose reliability. The decreased InGA value calculated for summer 2009 (Fig. 14), reflected the weight of 4 given to cyanobacterial biovolumes in the index formula

> 1 0.1 \* Cr Cc 2 \* (Dc Chc) 3 \* Vc 4 \* Cia + ++ + <sup>=</sup> + ++ + + +

In fact, whenever a lake or reservoir has a cyanobacterial bloom, InGA value will indicate it, but so will simply calculating the relative contribution of cyanobacteria to total

Another problem emerging from the application of multi-metric indices is the loss of interpretability. With InGA, high contributions to total biovolumes of Dinophyceae and non- colonial phytoplankton groups are assumed to improve the ecological status, whereas colonial forms and cyanobacteria worsen it. Thus, high biovolume proportions of non-toxic Chroococcales, are given the same negative weight as toxic filamentous cyanobacteria.

It is well known and accepted that different metrics applied to the same ecological condition can attribute different classifications. This is obviously linked to the information provided by the variables selected in each metric analysis. Criteria for the selection of these metrics should consider the prerequisites previously mentioned (see section 1.), such as: (i) obtainability, (ii) relevance in term of specific objectives, (iii) discriminant capacity, (iv)

Results obtained for dominance (Fig. 11) and cyanobacterial contribution (> 9%) to total phytoplankton biovolume, (Fig. 12), indicate eutrophication under WFD guidelines (JRC EC, 2009). Considering the oligotrophic state of both reservoirs, the reliability of such indicators

Solimini et al. (2006) considered the contribution of cyanobacteria to total phytoplankton biomass, as a reliable and simple indicator of trophic state, based on the following assumptions: (i) most cyanobacteria species show a strong preference for eutrophic conditions, (ii) due to toxicity of some taxa, blooms can pose serious water quality, animal and human health risks, as well as environmental problems, and finally, (iii) the large

This study contradicts the first assumption, since the genera and species typically linked to eutrophication were found associated to oligotrophy. The second assumption was partially verified, but toxin production seemed to be limited in oligotrophic conditions, despite high cell abundances. Potentially toxic cyanobacteria do not always produce cyanotoxins, so toxicity needs to be confirmed. Finally, the last assumption which links eutrophication to

(2)

1 2 \* (D Cnc) Chnc Dnc InGa

D Dinophyceae Cc Chrysophyceae colonial Cnc Chrysophyceae non colonial Dc Bacillariophyceae colonial Chnc Chlorococcales non colonial Chc Chlorococcales colonial DnC Bacillariophyceae non colonial Vc Volvocales colonial Cr Cryptophyceae Cia Cyanobacteria

Since MedPTI was specifically developed for deep natural lakes in Italy, this index should probably not be applied to reservoirs without adjustments to existing species lists. Nevertheless, MedPT1 classifications obtained in this study seemed more consistent than those obtained using General Algal Group Index (InGA) proposed by Catalàn et al (2003).

 Table 4 compiles values for InGA Catalàn et al. 2003). These authors recommended that the use of this multi-metric index of phytoplankton composition for ecological status classification should be calculated for late summer and fall samples. Trophic state metrics, as recommended by several water authorities in Portugal and in Europe, apply a color code for easy comparison of different classifications, which was used in Tables 3 and 4. In the case of Beliche and Odeleite reservoirs, fall values represented a worst case scenario and artificially attributed a good or moderate classification to waters that would otherwise be recognized as very good. Fig. 14 illustrates monthly variability of Catalàn InGA values.


Table 4. Catalàn InGA values determined for Beliche and Odeleite reservoirs for several groups of samples

Fig. 14. Monthly values of the General Algal Groups Index (InGA) determined for Beliche and Odeleite Reservoir 2009-2010 data. Dashed lines indicate the lower limits for Very Good (0,1) and Good (0,01) classification

Since MedPTI was specifically developed for deep natural lakes in Italy, this index should probably not be applied to reservoirs without adjustments to existing species lists. Nevertheless, MedPT1 classifications obtained in this study seemed more consistent than those obtained using General Algal Group Index (InGA) proposed by Catalàn et al (2003). Table 4 compiles values for InGA Catalàn et al. 2003). These authors recommended that the use of this multi-metric index of phytoplankton composition for ecological status classification should be calculated for late summer and fall samples. Trophic state metrics, as recommended by several water authorities in Portugal and in Europe, apply a color code for easy comparison of different classifications, which was used in Tables 3 and 4. In the case of Beliche and Odeleite reservoirs, fall values represented a worst case scenario and artificially attributed a good or moderate classification to waters that would otherwise be recognized as very good. Fig. 14 illustrates monthly variability of Catalàn InGA values.

**Period Beliche Odeleite InGA limits InGA classes 2009-2010** 0.167 0.237 >0.1 very good **2009** 0.098 0.067 0.01-0.1 good **2010** 0.196 0.403 0.005-0.01 moderate

**October 2009** 0.022 0.008 0.003-0.005 poor **October 2010** 0.536 1.864 <0.003 bad

Table 4. Catalàn InGA values determined for Beliche and Odeleite reservoirs for several

Fig. 14. Monthly values of the General Algal Groups Index (InGA) determined for Beliche and Odeleite Reservoir 2009-2010 data. Dashed lines indicate the lower limits for Very Good

groups of samples

**InGA** 

(0,1) and Good (0,01) classification

As discussed by Jackson et al. (2000), an index that does not allow for temporal and spatial variability, might lose reliability. The decreased InGA value calculated for summer 2009 (Fig. 14), reflected the weight of 4 given to cyanobacterial biovolumes in the index formula (Eq. 2)

$$\text{InGa} = \frac{1 + 2 \,\text{\*} \,(\text{D} + \text{Cnc}) + \text{Chnc} + \text{Dnc}}{1 + 0.1 \,\text{\*} \,\text{Cr} + \text{Cc} + 2 \,\text{\*} \,(\text{Dc} + \text{Chc}) + 3 \,\text{\*} \,\text{Vc} + 4 \,\text{\*} \,\text{Ca}} \tag{2}$$


In fact, whenever a lake or reservoir has a cyanobacterial bloom, InGA value will indicate it, but so will simply calculating the relative contribution of cyanobacteria to total phytoplankton biovolume.

Another problem emerging from the application of multi-metric indices is the loss of interpretability. With InGA, high contributions to total biovolumes of Dinophyceae and non- colonial phytoplankton groups are assumed to improve the ecological status, whereas colonial forms and cyanobacteria worsen it. Thus, high biovolume proportions of non-toxic Chroococcales, are given the same negative weight as toxic filamentous cyanobacteria.

It is well known and accepted that different metrics applied to the same ecological condition can attribute different classifications. This is obviously linked to the information provided by the variables selected in each metric analysis. Criteria for the selection of these metrics should consider the prerequisites previously mentioned (see section 1.), such as: (i) obtainability, (ii) relevance in term of specific objectives, (iii) discriminant capacity, (iv) allowance of natural variability and (v) reliability.

Results obtained for dominance (Fig. 11) and cyanobacterial contribution (> 9%) to total phytoplankton biovolume, (Fig. 12), indicate eutrophication under WFD guidelines (JRC EC, 2009). Considering the oligotrophic state of both reservoirs, the reliability of such indicators should be reevaluated, at least for warm semi-arid regions.

Solimini et al. (2006) considered the contribution of cyanobacteria to total phytoplankton biomass, as a reliable and simple indicator of trophic state, based on the following assumptions: (i) most cyanobacteria species show a strong preference for eutrophic conditions, (ii) due to toxicity of some taxa, blooms can pose serious water quality, animal and human health risks, as well as environmental problems, and finally, (iii) the large contribution of cyanobacteria blooms to phytoplankton biomass.

This study contradicts the first assumption, since the genera and species typically linked to eutrophication were found associated to oligotrophy. The second assumption was partially verified, but toxin production seemed to be limited in oligotrophic conditions, despite high cell abundances. Potentially toxic cyanobacteria do not always produce cyanotoxins, so toxicity needs to be confirmed. Finally, the last assumption which links eutrophication to

Ecological Tools for the Management of Cyanobacteria

Vasconcelos was greatly appreciated.

**6. References** 

Blooms in the Guadiana River Watershed, Southwest Iberia 185

45 ("Risk evaluation of toxic blooms in lower Guadiana") from LEADER+ program, project DYNCYANO (PTDC/AMB/64747/2006) funded by the Portuguese Science and Technology Foundation (FCT). We also thank past post-doctoral fellows Dr. Alexandre Matthiensen, Dr. Carlos Rocha, and Dr. Cristina Sobrino for contributing to different parts of this study at different times, and the following Master´s and PhD students Rute Miguel, Pedro Mendes, Vânia Sousa and Teresa Cecílio, as well as research assistants Cátia Luis, Tânia Anselmo, Erika Almeida and Cátia Guerra. Collaboration from Dr. Wayne Carmichael and Prof. Vitor

R. Domingues acknowledges PhD (SFRH/BD/27536/2006) and Post-Doctoral (SFRH/BPD/ 68688/2010) fellowships from FCT, and S. Mesquita acknowledges PhD grant (SFHR/BD/ 18921/2004) and the research grant financed in the scope of the Southwest European

Algarve reservoirs were studied in the scope of 4 research projects, namely CIANOALERTA I, II and III (2003-2008) funded through INTERREG IIIA, contracts nº SP5/P35/01, SP5/P19/02 and SP5/P138/03 and CIANOTOOLS financed in the scope of the Southwest European Research Net (RISE) through the POCTEP UE Program in collaboration with

Admnistração da Região Hidrográfica do Alentejo (ARH Alentejo), (2011). Planos de gestão

http://www.arhalentejo.pt/downloads/part\_publi\_pgrh/fase\_final/RH7/t09122\_

Alves, M. H. & Bernardo, J. M. (1998). Novas perspectivas para a determination do caudal

Alves, M. H. & Gonçalves, H. (1994). O caudal ecológico como medida de minimização dos

Barbosa, A.B., Domingues, R.B. & Galvão, H.M. (2010). Environmental forcing of

Berg, K., Carmichael, W.W., Skulber, O.M., Benestad, C. & Underdal, B. (1987). Investigation

Blaha, L., Babica, P. & Maršálek, B. (2009). Toxins produced in cyanobacterial water blooms – toxicity and risks. *Intersisciplinary Toxicology*, Vol. 2, No. 2, (May 2009), pp.36-41

das bacias hidrográficas integradas nas regiões hidrográficas 5, 6 e 7-região hidrográfica 7. In: Relatório técnico para efeitos de participação pública, t09122/01,

ecológico em regiões semi-áridas. Proceedings of *Seminário sobre barragens e ambiente*. Comissão nacional Portuguesa da Grandes barragens, ISBN 92-894-5122-

impactes nos ecossitemas lóticos. Métodos para a sua determinação e aplicações. Proceedings of: "*Actas do 6ºSILUSB/1ºSILUSBA, Simpósio de Hidráulica e Recursos dos* 

phytoplankton in a semi-arid estuary (Guadiana estuary, south-western Iberia): a decadal study of climatic and anthropogenic influences. *Estuaries and Coasts*, Vol.

of a toxic-water bloom of *Microcystis aeruginosa* (Cyanophyceae) in Lake Akersvatn,

Research Net (RISE) through the I2TEP UE Program- subprogram CIANOTOOLS.

Algarve Regional Hydrographycal Administration and University of Huelva.

(Junho 2011), Acessed in August 1st 2011, Available from

*Países de Língua oficial Portuguesa*", (Abril de 1994), Lisboa

33, No. 2, (March 2010), pp. 324-341, ISSN 1559-2723

Norway. *Hydrobiologia*, Vol. 144, pp. 97–103

01\_PGBH\_RH7\_Relatorio\_Tecnico.pdf

X, Porto, Maio de 1998

large contribution of cyanobacteria to phytoplankton biomass, should be re-assessed in Mediterranean regions, where, even under oligotrophic conditions, cyanobacteria are favoured in naturally warm waters.
