PRELIMINARY RESULTS ON THE CYANOTOXICITY IN THE “NEW” LAKE KARLA (THESSALY-GREECE) Th.Papadimitriou1, Z.Stampouli2 and If. Kagalou 1,2 1. Management Body of Lake Karla, Stefanovikeio Magnisias, Greece. 2. Dept. of Ichthyology & Aquatic Environ., Univ. of Thessaly, Volos, Greece e-mail: [email protected] EXTENDED ABSTRACT

The appearance of massive water blooms has become a worldwide problem coinciding with the eutrophication of aquatic ecosystems. In Mediterranean eutrophic lakes, cyanobacteria can form dense blooms which are able to persist from spring to late autumn. This bloom forming process can be caused by increased levels of nutrients like nitrogen and phosphorous resulting from anthropogenic pressures. Cyanotoxins, representing mainly by microcystins are of great environmental as well as health concern since they can released in high concentrations during cell lysis causing serious adverse effects in aquatic organisms and human. It is known that former Lake Karla (Thessaly) was completely drained in 1962 while at 1990s the first restoration plans were proposed addressed to the re-establishment of a functional wetland. At the present time, Lake Karla is almost refilled while restoration project of the wetland is still ongoing. Because of its conservation value Lake Karla is listed in the network of the Greek protected areas. In the present study preliminary results concerning the occurrence of microcystins in the “re-established” Lake Karla along with the key -eutrophication variables are presented. Water quality analyses were performed, from April 2010 to November 2010, focusing on the following parameters: Temperature, pH, dissolved oxygen, conductivity, soluble reactive phosphorus (SRP), ammonium + nitrite+ nitrate nitrogen (Dissolved Inorganic Nitrogen) and chlorophyll a (chl a) concentrations. Extra-cellular and intra-cellular microcystin concentrations were analyzed using an enzyme-linked immunosorbent assay (ELISA). Eutrophication signals were found indicating possible anthropogenic inputs. Concerning microcystin content, based on the W.H.O guidelines, the presence of cyanotoxicity it is suggested exhibiting higher risk during the warm period. Lake Karla’s restoration project includes future uses and services as irrigation, drinking water supply along with the ecological function of the wetland and recreation activities. Since the presence of microcystins in drinking water supplies, irrigation and recreational activities poses a potential hazard to human health and agricultural products for animal and human consumption, attention should be addressed to the water quality monitoring at catchment scale, as well as to the limnological features in the new established ecosystem. Worldwide, in most countries, measures to protect public health as well as agricultural products and livestock from cyanotoxins have been implemented following the W.H.O suggestions. In Greece, to date, there is no legislation about it. We strongly suggest that cyanobacteria and cyanotoxins should be incorporated in the water quality regulations as parameters that must to be monitored for water quality control. Key words: Cyanotoxins, Lake Karla, eutrophication, microcystins, risk.

1.INTRODUCTION Eutrophication is considered as the main water quality problem in lakes and reservoirs. Lake eutrophication along with the land use changes may result in deterioration of water clarity, and loss of ecological value and often causes algal community shifts towards bloom-forming cyanobacteria species that can be highly toxic (Codd, 2000). Cyanobacterial mass occurrences and detrimental effects on human and animal life have been associated at least since the early Middle Ages (Chorus & Bartram, 1999). Since this time, the awareness of toxic cyanobacteria has increased continuously. At first, by a rising number of scientific documentations of fish kills and livestock mortalities of animals living in and drinking from lakes and ponds containing toxic cyanobacteria, and since the nineteen-eighties, by the chemical and toxicological characterization of numerous toxic metabolites which were isolated from cyanobacterial blooms (Dow & Swoboda, 2000). Almost all cyanobacterial toxins are intracellular toxins and therefore, they are primarily released into the water by natural bloom senescence. The most widespread cyanobacterial toxins are the MCYST (peptides) and neurotoxins (alkaloids) and the related nodularins (Chorus, 2001). Microcystins (MCYST) are produced by freshwater cyanobacteria species such as Microcystis, Anabaena, Oscillatoria, and Nostoc (Carmichael, 2001). Epidemiological evidence results from studies of human populations that have shown symptoms of poisoning or injury were attributed to the presence of microcystins in drinking water or other sources of water. In Mediterranean countries cyanobacterial water blooms and subsequently production of toxins may be expected to be of extended or even continuous duration throughout the year, particularly in eutrophic freshwaters experiencing high temperatures, stratification of water column, high retention time of water and low zooplankton grazing pressure (Cook et al., 2004). Concerning Greek lakes there is evidence about the presence of microcystins in both water and fish species (Gkelis et al., 2006; Kagalou et al., 2008; Papadimitriou et al., 2010). Lake Karla is considered as a very important aquatic ecosystem both in terms of its biodiversity (Natura site, GR1420004) but also because it is a new re-established lake in the place of the former Lake Karla which was drained at 1960s. Reviewing the literature, there are available data about the hydrology of the new Karla basin (Gerakis, 1992; Loukas et al., 2007) as well as information on habitat types and fauna (Gerakis & Koutrakis, 1996). The present paper is the first evidence about water quality issues since 1956 (Ananiadis, 1956), thus serving also as a baseline for further study. The aim of the present study is to summarize, the limnological history of the former Lake Karla and to present the preliminary results concerning the occurrence of microcystins in the new “re-established” Lake Karla along with the key -eutrophication variables. 2. MATERIALS AND METHODS 2.1 Lake description and limnological history Lake Karla (the ancient lake Voiviis) occupied the lower depression plain of Thessaly region and was one of the most important wetlands in Greece until 1962 (Zalidis et al., 2005).The watershed of the former- Lake Karla covered an area of 1500 km2 of which more than 600km2 made up a southern flat plain while the north-eastern part is surrounded by low mountains and hills. The lake was situated at 47 m a.s.l, with a surface area varying between 40-108 km2 due to high water level fluctuations and the land-slope. The recorded depth, on 1956, was about 2.5 m (Ananiadis, 1956) while at 1930’s the depth was between 4-6 m. As regards the geological origin of the lake, it is

suggested that is of tectonic origin formed during the Tertiary of Cenozoic period (Ananiadis, 1956). Lake Karla used to be a closed basin, without any surface outlet, while the water level of the former Lake Karla has a seasonal fluctuation varying between 2- 6m. There is also an evidence about completely dryness on 1908. Pinios river was the most important inflow source while run-off from the catchment area along with springs (Kanalia, Asmaki) and temporary streams (east-south part) were also feed the lake. Concerning the limnological features of the former Lake Karla, Ananiadis (1956) classified the lake as eutrophic, with low transparency, poor species diversity and frequent appearance of algal blooms. Lake Karla was completely drained in 1962 and has experienced a number of anthropogenic impacts including wetland loss, intrusion of salt waters into its aquifer, subsequent soil salinization, loss of ecological and aesthetic value. Restoration efforts have been started at 1980’s, with the approach of re-establish a reservoir for irrigation, but finally taking into consideration the multi- functional role of the ecosystem, a more holistic approach was applied in order to re-establish an restore an aquatic ecosystem. The suggested plan proposed the creation of a reservoir of about 38 km2, fed by Pinios river and from the surface water from the watershed. The restoration of destroyed habitats was also included along with the scenarios about the sustainable development of the catchment area. An extended restoration plan has been reported by Zalidis et al. (2005). It is worth note, that the restoration of Lake Karla is listed among the most important environmental projects in Europe. 2.2 Sampling and toxin analysis For MCYST determination, integrated water samples (500 mL of volume) were collected, by filling 1L polyethylene bottles 10-20 cm below the water surface monthly at three littoral stations in Lake Karla (Figure 1). The collection was performed from April 2010 to October 2010. MCYST were analyzed in two forms: dissolved in water (extra-cellular microcystins) and cell-bound in suspended matter (intra-cellular microcystins). For intracellular MCYST, the water sample was filtered by a Whatmann GF/C 0.45-µm filter, which was immediately frozen at -20°C. Intra-cellular MCYST extracted from the filter papers by placing in 100% methanol and stirring overnight at room temperature (20-22οC) followed by centrifugation at 1300 x g for 15 min. Extraction procedure was repeated three times and the supernatants of the extractions were pooled. The organic solvent was removed by placing the extract under nitrogen-stream. The concentrated sample extract remaining after removing of the organic solvent was subjected to an enzyme-linked immunosorbent assay (ELISA). Results are expressed as micrograms of cellular MCYST equivalents per Liter. For analysis of extra-cellular microcystins, the filtered water was applied directly to ELISA. Results are expressed as micrograms of extracellular MCYST equivalents per Liter. A commercial ABRAXIS-Microcystin ELISA kit was used (520011, USA) following the instructions of the manufacturer. The ELISA is an indirect-competitive method used for the quantitative analysis of all the microcystin analogues and nodularins. This ELISA test, using the β-amino acid 6E-ADDA as the epitope for antibody recognition, has a limit of quantification of 0.02-0.07 ng/mL, lower than the WHO-proposed guideline (1ng/mL) for drinking water. Water samples for the determination of nutrients (nitrite-N, nitrate-N, ammonium-N, Soluble Reactive Phosphorus (SRP)) and chlorophyll- α (chl-α) were also collected. APHA (1998) methodology was applied to perform the chemical analysis. Water temperature, pH, Dissolved Oxygen and conductivity were measured in situ using portable YSI instruments.

Figure 1. Map of Lake Karla

3. CALCULATIONS PERFORMED Carlsons’ trophic state index was calculated for Lake Karla. The calculation was based on chl- α concentrations for the period April 2010 to November 2010. It was used the above equation:

TSI(Chl-α)=10*[6-(2,04-0,68LnChl-α)/Ln2](Carlson,1977) The GLM (general linear model) was used in order to examine if there are significant differences between stations in relation to the MCYST concentrations and environmental parameters. The association of extra- and intra-cellular MCYST was tested with Spearman correlation. Data were explored using the software SPSS 17.0 (Chicago, Illinois, U.S.A.) for Windows. 4.RESULTS 4.1 Water quality of Lake Karla Water temperature fluctuated from 13°C in November to 35.5°C in July. Dissolved Oxygen ranged between 3.3 mg/L (in October) and 7.2 (in November) (Figure 2). The p H values were (8.2-9.2) without any apparent seasonal variation. Water conductivity was relatively high, ranging between 2.9 mS/cm (in May) and 4.9 mS/cm (in August) (Figure 2). The values of the in situ parameters showed no differences among stations. With regard to nutrient concentrations, nitrite was the least important form in terms of percentage contribution of dissolved inorganic nitrogen (DIN) in the lake and ranged from 0.003 mg/L to 0.06 mg/L, while Ammonia-N was the most important form in the DIN pool varying between 0.38 mg/L to 1.99 mg/L, exhibiting higher values during the summer months. Nitrate-N fluctuated between 0.26 mg/L and 0.8 mg/L and peaking in November. Soluble Reactive Phosphorus fluctuated between 0.07 mg/L and 3.17 mg/L and peaking in July (Figure 2). Chlorophyll- α ranged between 77.97 mg/m3 and 525.24 mg/m3, with higher values during the warmer months (Figure 2). As regards the stoichiometry of the nutrients, the DIN/SRP ratio ranged between 0.35 and 23.56, with an average value of 5.28, suggesting a nitrogen limitation.

Average TSI based on chlorophyll a concentration during the investigated year in Lake Karla was 85.8, which indicates conditions of hypereutrophy.

Figure 2. Monthly variation of conductivity, pH, temperature, dissolved oxygen, conductivity, nitrite-N, nitrate-N, ammonia-N, Soluble Reactive Phosphorus and Chl-α for the year 2010.

4.2 Microcystin dynamics Extra-cellular MCYST concentrations in lake water were detected in all monthly samples (Figure 3). Concentrations ranged between 0.75 µg/L and 3.90 µg/L. Extra-cellular MCYST increased over the warm months, peaking in October. Intra-cellular MCYST concentrations in ranged between 1.01 µg/L and 9.83 µg/L. Generally intra-cellular MCYST values followed the seasonal pattern observed in dissolved fraction, showing peaking values during late summer when the lake surface is characterized by the presence of cyanobacterial blooms (Figure 3). There were no statistical differences between the sampling stations, concerning extra-cellular MCYST (P=0.654, F=0.435) and intra-cellular MCYST (P=0.191, F= 0.191). Also, a significant correlation was found between extra-cellular and intra-cellular MCYST (P<0.01, r=0.916).

Figure 3. Monthly variation of exta-cellular and intra-cellular MCYST Concentrations for the year 2010

5. CONCLUSIONS During the sampling period, the maximum depth of the lake was 2m, so Lake Karla is consider as a shallow lake appearing the typical temperature pattern of the Mediterranean region, with high temperatures in warm months. This is accompanied by the decrease of dissolved oxygen concentration during the warm, dry period. pH was found to be generally, high coupling with photosynthesis in shallow lake ecosystems (Moss,1980). High conductivity values during warm months are associated with high concentrations of nutrients found in Lake Karla, during the same period. Lake Karla is now supplied with large quantities of water, from Pinios river which is characterized by eutrophicated conditions (Bellos & Savvidis, 2005). Additionally, the river occasionally overflowed, and floodwaters rich in nutrients drained into Karla (Zalidis et al., 2005). High concentrations of S.R.P. in warm months maybe the result of both decrease of the water volume and of phosphorus release from the rich lake sediment which, mostly, takes part at high temperatures (Sondergaard et al., 2003). High DIN concentrations in summer are associated with high concentrations of ammonia, found in the lake during summer, indicating high decomposition rates. Phytoplankton biomass and Carlsons’ trophic state index as estimated by Chl-a values, reflect the hypertrophic status of the lake. Oikonomou et al., (2010), found that Lake Karlas’ colonist consisted a diverse microbial community indicating an hypertrophic status. The same authors confirmed the occurrence and dominance of toxic cyanobacterial species of Anabaenopsis and Planktothrix (Oikonomou et al., 2010). Our results also suggest that nutrient-rich conditions found in Lake Karla could support the bloom of toxic cyanobacteria in lake water. Furthermore, we found that the mean value of in-lake nitrogen/ phosphorous concentrations ratio is less than the threshold of 10:1 which is considered as indicator for strong nitrogen-limiting conditions, thus favouring the growth of N2-fixing cyanobacteria (Havens et al., 2003). In the present study, massive surface blooms of cyanobacteria were recorded from early summer to late autumn. According to the present results, extra-cellular and intra-cellular MCYST were present in Lake Karla during all months examined. However, warm months were characterized by higher MCYST concentrations. This temporal variation in MCYST concentrations is probably due to the population dynamics of the existed cyanobacterial species or/and strains (Chorus, 2001). This study is the first evidence concerning the monthly fluctuation of MCYST in Lake Karla and thus there are no other comparable data. Concentrations of intra-cellular MCYST found in lake, were higher than intra-cellular MCYST due to the fact that microcystins are considered endotoxins because the majority of the toxin is found within cells (Kotak et al., 1996). Additionaly, the relationships founded between extra-

cellular and intra-cellular MCYST support the opinion that the function of MCYST in cyanobacterial cells is still unclear depending, mainly, on biochemical processes (Lam et al., 1995).The occurrence of MCYST has previously been reported in Greek lakes (Cook et al., 2004; Gkelis et al., 2005; Kagalou et al., 2008; Papadimitriou et al., 2010). MCYST concentrations found in Lake Karla are similar to those found in the eutrophic lakes Pamvotis and Kastoria and also similar to those reported for other Mediterranean lakes (Chorus, 2001). The presence of MCYST in drinking water supplies and irrigation poses a potential hazard to human health and agricultural products for animal and human consumption (Crush et al., 2008). Farmers are potentially in danger from the inhalation of droplets containing toxins during spray irrigation or through dermatological contact with toxin containing water (Chorus & Bartram, 1999). World Health Organization (W.H.O., 1998) has proposed a safety guideline value of approximately 1 µg/L drinking water. MCYST concentrations in water of Lake Karla are above the WHO Guide level for drinking water, during warm period. A series of guidelines associated with probability of adverse effects from cyanobacteria in recreational waters has been defined on three levels (W.H.O., 2003). Relatively low probabilities, of adverse health effects deal mainly with the irritant or allergenic effects of cyanobacterial compounds, and intra-cellular MCYSTs may range from 2-10 µg/L. ‘Moderate probability” and “High risk” of adverse health effects concern MCYST concentrations in recreational waters in the range of 10-40 µg/L and >40 µg/L, respectively (W.H.O., 2003). Intra-cellular MCYST concentrations in Lake Karla pose a low risk of adverse health effects (Figure 4). Hypertrophicated conditions along with high microcystin concentrations found in Lake Karla indicate that water quality and microcystin monitoring is essential in order to protect the reconstructed ecosystem and to avoid possible human health risks.

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