Developing Biological Tools for Monitoring of Lake Ohrid in Accordance to European Water Framework Directive -Final Report

Developing Biological Tools for Monitoring of Lake Ohrid in accordance to European Water Framework Directive

-Final Report-

Ohrid-Durres, 2013

Acknowledgments

Special acknowledgments to the Ministry of Foreign Affairs of Norway as the main and only financer of the project`s activities. Acknowledgment and gratitude to all individuals and institutions participated and contributed for the successful completion of the project.

Table of Contents

Contents Summary ...... 5 Introduction ...... 6 Objectives and Scope ...... 6 PHYSICAL CHEMICAL PARAMETERS ...... 9 Methodology ...... 9 Results and Discussion ...... 9 Conclusion ...... 15 BENTHIC DIATOM ALGAE ...... 16 Methodology ...... 16 Results and Discussions ...... 16 Conclusion ...... 20 MACROPHYTE VEGETATION...... 21 Methodology ...... 21 Results and Discussion ...... 21 Conclusion ...... 24 BENTHIC FAUNA (MACROZOOBENTHOS) ...... 25 Methodology ...... 25 Results and Discussion ...... 25 Conclusion ...... 28 General Recommendations ...... 30

List of Figures

Figure 1: Researched localities in Lake Ohrid ...... 8 -1 Figure 2: Biochemical Oxygen Demand (BOD5), mg l O2 for 2010 ...... 9 -1 Figure 3: Biochemical Oxygen Demand (BOD5), mg l O2 for 2010 ...... 10 -1 Figure 4: Organic matter (consumption of KMnO4) mg l for 2010 ...... 10 -1 Figure 5: Organic matter (consumption of KMnO4) mg l for 2011 ...... 11 3

Figure 6: Concentration of total phosphorus, g l-1 TP for 2010 ...... 11 Figure 7: Concentration of total phosphorus, g l-1 TP for 2011 ...... 12 Figure 8: Concentration of total nitrogen, g l-1 TN for 2010 ...... 13 Figure 9: Concentration of total nitrogen, g l-1 TN for 2011 ...... 13 Figure 10: Trophic state index based on concentration of total phosphorus and transparency for 2010 ...... 14 Figure 11: Trophic state index based on concentration of total phosphorus and transparency for 2011 ...... 14 Figure 12: Number of species in July 2010 ...... 16 Figure 13: Number of species in July 2011 ...... 16 Figure 14: Diversity Index in July 2010 ...... 17 Figure 15: Diversity Index in July 2011 ...... 17 Figure 16: Trophic Index for Diatoms 2010 ...... 18 Figure 17: Trophic Index for Diatoms 2011 ...... 18 Figure 18: Trophic Index for Diatoms ...... 19 Figure 19: Saprobic Index ...... 19 Figure 20: Macrophyte index for entire lake (average)...... 22 Figure 21: Macrophyte index of shallow water 0-2m ...... 23 Figure 22: Macrophyte index for deeper water >10 ...... 23 Figure 23: Endemic and cosmopilitan species ration in Lake Ohrid ...... 26 Figure 24: Biodiversity of benthic fauna in Lake Ohrid-“depth distribution” ...... 26 Figure 25: Density comparison: spring vs. fall ...... 27 Figure 26: Ecological status of the sampling sites in lake Ohrid-spring period ...... 27 Figure 27: Ecological status of the sampling sites in lake Ohrid-fall period ...... 28

List of Tables

Table 1: Number of sites per Trophic Index for Diatoms ...... 19 Table 2: Number of sites per Saprobic Index ...... 19 Table 1: Number of sites per Nutrient Pollution ...... 23

Summary Lake Ohrid is an ancient lake of huge importance for the countries that share it from economic, socio-cultural and other perspectives. Likewise, it is actually a world`s interest as well, since it has been proclaimed as a World Heritage protected site, since 1979. Besides these attributes, the lake is very important and appealing for scientists as well, due to its age, the endemic and relic living world. The need of a project as the Developing Biological Tools for Monitoring of Lake Ohrid according to European Framework Directive, has emerged as logical next step, after considering the continuous anthropogenic impact to the ecosystem`s functionality and the need of harmonization of water legislative between the two countries and the EU. There were considered four mandatory components for the monitoring of Lake Ohrid, at 30 localities, during two-year period (2010-2011). The components include: physic-chemical parameters, benthic diatom algae, macrophyte vegetation and benthic fauna (macrozoobenthos), sampled 4 times, once, once and two times per year, respectively. In the end, there have been collected and analyzed by unified methodology (according to EWFD) 360, 120, 360 and 30 samples from Water Chemistry, Hydrobotany, Macrozoobenthos and Diatoms, in that order. The project resulted in the development of monitoring tools and creation of database, which would be used in the future as the bases for the development of investigative and operational types of monitoring. The valuable results which have been obtained during the project have already been or are in the phase of publishing in numerous, international journals, which will assist in the actualization of the Lake Ohrid environmental issues.

Introduction

Worldwide ancient lakes have been a major focal point of biological and ecological research. Ancient lakes can be found on most continents and climate zones with most actual or putative ancient lakes in Europe being restricted to the Balkan Region. The arguably most outstanding of them is the oligotrophic and karstic Lake Ohrid, a steep-sided graben of rift formation origin situated in the central Balkans. This lake is of an immense importance for the countries of Albania and Macedonia, which share it, from economical, socio-cultural and other aspects. There are approximately 1,200 native species known from the lake, including 586 animals, and at least 212 species are endemic, including 182 animals. In terms of endemic biodiversity, Lake Ohrid is with these 212 known endemic species and a surface area of 358 km2 probably the most diverse lake in the world, taking surface area into account. Moreover, the importance of the lake was further emphasized when it was declared a World Heritage site by UNESCO in 1979. Despite all of its importance and appeal for scientists and ordinary people, Lake Ohrid has not been sufficiently researched with regards to the European Water Framework Directive and there have been no biological and ecological tools developed for its monitoring. Moreover, both Albania and Macedonia are closing to EU membership, so the implementation of the directive is becoming more important, too. All of this has been a great base for beginning of the project Developing Biological Tools according to the European Water Framework Directive, fully financed by the Ministry of Foreign Affairs of Norway and supported by the Ministry of Environment and Physical Planning of Macedonia and the Ministry of Environment of Albania. The Norwegian Institute for Water Research (NIVA), the Agricultural University of Tirana (UBT) and the Hydrobiological Institute Ohrid (HIO) have been the three institutions which were conducting and responsible for the project.

Objectives and Scope The project goals include capacity building in Albania and Macedonia based on the European guidelines (the European Water Framework Directive) and developing common methods for sampling and analysis of samples. All of this has been attained through mutual field work and sometimes mutual laboratory work, too.

The project generated total of 58 protocols and reports, including the Standard Operation Protocols (SOP) for all hydrochemistry, macrophyte, diatom and benthic fauna analyses which will be used as guidelines in Albania and Macedonia, written in Albanian, Macedonian and English. Also, a NIVA technical report has been composed, explaining how to import and display biological data in ENSIS (Environmental Surveillance and Information System). In addition, we have ongoing activities in all three countries, aiming to publish the results both at international and national level. All publications will be composed by representatives from all participating countries, so we established a true collaboration and also friendship among the partners in our project. Likewise, the project`s activities and goals were also supported and attained through the continuous meetings, workshops and conferences organized within the project, including: the workshop in July 2009 in Durres, in December 2009 – kick-off workshop, in January 2011 first ENSIS workshop, in October 2012 – second ENSIS workshop and in February 2013 – the final conference. The respective participants were attending all of these events, but also some representatives from the municipalities, embassies, universities and relevant ministries were in attendance, too. This certainly increased the institutional awareness and interest and the mutual cooperation between the two countries on a political, educational and local-governmental levels. Field work was done using a boat belonging to the Hydrobiological Institute in Ohrid, Macedonia, since this research vessel has proper equipment for sampling. The sampling usually started from Ohrid, Macedonia, and via the border into Albanian territory when sampling the sites on the Albanian side of Lake Ohrid. All necessary formalities were fulfilled, such that there were no experienced problems with the authorities. The field work has been undertaken in 30 sites, 10 of which in Albania and 20 of which in Macedonia (Figure 1.). The number of localities is in accordance to the respective shoreline coverage of each country while the results are:  Water Chemistry: 360 samples were taken and analyzed  Hydrobotany: 120 samples were taken and analyzed  Macrozoobenthos: 360 samples were taken and analyzed  Diatoms: 30 samples were taken and analyzed

Figure 1: Researched localities in Lake Ohrid

As Struga Sateska Kalista

Koselska Daljan Radozda Ohrid Bay Blato

Lin1 Lin2 Racanska Piskupat2 Park

Piskupat1 Metropol

Pestani Velidab Veljapesh Hudenisht Trpejca1 Trpejca2

Memlisht Zaum Cherava Pogradec2 St. Naum Dogana Pogradec1 Tushemisht

Four mandatory components (quality elements) have been chosen according to the WFD Guidelines to monitor in the period 2010-2011: 1. Physico-chemical parameters: 360 samples were taken and analyzed 2. Benthic diatoms: 30 samples were taken and analyzed 3. Macrophyte vegetation: 120 samples were taken and analyzed 4. Macrozoobenthos: 360 samples were taken and analyzed In the following pages the main findings concerning the parameters listed above and in correspondence with the main scope of the Project are going to be ellaborated.

PHYSICAL CHEMICAL PARAMETERS

Methodology The analyzed indicators include: temperature, pH, conductivity, transparency, alkalinity (phenolphthalein, methilorange, and total alkalinity), dissolved oxygen, oxygen saturation, biochemical oxygen demand (BOD5), dissolved biodegradable matter by permanganate consumption, concentration of total phosphorus, nitrogen compounds (nitrate, nitrite, ammonia, nitrogen by Kjeldahl, total nitrogen). The most indicative indicators of all of them are: dissolved oxygen, nitrate, total phosphorus, biochemical oxygen demand, pH and electrical conductivity. All samples and indicators were collected and analyzed in accordance to the methodology described in the protocols developed for the Project`s purposes (for instance, BOD5 according to Winkler method, Total phosphorus according to Persulphuric oxidation method etc.)

Results and Discussion

1. Biochemical Oxygen Demand (BOD5) Generally, according to the obtained results the values of this indicator have been higher in spring and summer periods (for most of the localities the highest values were registered in summer). -1 Figure 2: Biochemical Oxygen Demand (BOD5), mg l O2 for 2010

1 -

L mg demand oxygen Biochemical may'10 july'10

Moreover, the highest registered value for BOD5 (throughout the two-year period) has been in the water from the littoral Grasnica, estimated at 4.105 mg l-1, presumably as a reflection of the influence from the River Vegoska, which delta is nearby. Likewise, relatively high values of this indicator have been registered in Radozda and Kalista.

-1 Figure 3: Biochemical Oxygen Demand (BOD5), mg l O2 for 2010

1 O2 1 -

l mg BOD5

january'11 may'11 july'11 october'11

2. Organic matter (consumption of KMnO4)

The consumption of KMnO4 is an indirect measure for the quantity of organic biodegradable matter in the water. The obtained results for this parameter indicate to small variations of the concentrations of biodegradable matter. The highest values for this indicator have been registered in 2010, in the water from the littorals of Grasnica and River Sateska, which is a result of the influence of River Velgoska and River Sateska, which are inflowing in the respective areas and are the end-recipients of the waste, drainage and households` waters. The graphs below depict the state of the organic matter in the researched localities during the two-year period. -1 Figure 4: Organic matter (consumption of KMnO4) mg l for 2010

KMnO

1 -

l mg matter organic

may'10 july'10

-1 Figure 5: Organic matter (consumption of KMnO4) mg l for 2011

KMnO

1 -

l mg matter organic

january'11 may'11 july'11 october'11

3. Total Phosphorous The nutrient loading of the water has been determined based on the two most important biogenic elements: phosphorus and nitrogen. The graphs bellow show the state in regards to total phosphorous in the two-year period.

Figure 6: Concentration of total phosphorus, g l-1 TP for 2010

g l 

Concentration, TP Concentration,

may'10 july'10 october'10 december'10

As the graphs indicate, the concentrations of total phosphorous from the littoral are generally lower than 11 mg l-1, during both years. The only exceptions were registered in May

2010 and October 2010 in the littoral of Grasnica, with total phosphorous of 49.93 g l-1 and

57.66 g l-1, respectively, as well as in October 2010 and December 2010 in the littoral near

River Sateska, with TP of 28.13 g l-1 and 15.116 g l-1, in that order.

Figure 7: Concentration of total phosphorus, g l-1 TP for 2011

1 1

l g g



Concentration,

january'11 may'11 july'11 october'11

In 2011, the highest values have been registered during winter at Trpejca (11.774 g l-1),

Kalista (11.067 g l-1) and Daljan (8.674 g l-1). By taking into consideration that the highest values were evidenced in the localities which are near to rivers` inflows, it can be concluded that they (the rivers) represent the main factor for this state. The reason for the negative impact of the rivers is to be located in:

 Rivers in the region flow through agricultural areas where agro technical solutions are widely used.  The rivers` watercourses are the end-recipients of communal and households` waste waters (in the areas where the collector system is not functioning).

4. Total Nitrogen

Additionally, the total nitrogen concentrations indicate to similar state as the total phosphorous. The highest concentrations of total nitrogen have been evidenced near the delta of the River Velgoska. The maximum value of the indicator, for this locality has been estimated at 1019.19

g l-1. The total nitrogen concentrations are represented in the following two graphs.

Figure 8: Concentration of total nitrogen, g l-1 TN for 2010

Figure 9: Concentration of total nitrogen, g l-1 TN for 2011

g l 

Concentration,

january'11 may'11 july'11 october'11

5. Trophic State Index

Carlson suggests the method for quantitative determination of the index of trophic state as a function of the separately investigated parameters for the water quality, while the classification of the indexes has been developed by Aizaki. The graphs below represent the values of the index of trophic state based on the transparency and concentration of total phosphorus. The numeric values for the Trophic state index based on transparency indicates to the fact that the water from all investigated localities is characterized by an oligotrophic character.

Figure 10: Trophic state index based on concentration of total phosphorus and transparency for 2010

Figure 11: Trophic state index based on concentration of total phosphorus and transparency for 2011

53-60 eutrophic 45-50

30-40

0-20 ultra-

TSI(TP) TSI(SD)

The values of the trophic state index based on the total phosphorous indicate to a greater variability. Although most are oligotrophic, there are some localities, such as River Grasnica, the littoral near River Cherava, Sateska, Radozda, Dogana, Tushemisht, Hudenisht, Piskupat and Pogradec 2, which have water of mesotrophic character. The most alarming condition has been noticed in the localities of River Grasnica and Pogradec 2, which water is transferring to eutrophic.

Conclusion Generally, the obtained results within the Project`s duration indicate to satisfactory level of water quality (oligotrophic character), based on the researched physic-chemical parameters. However, the mentioned localities (littoral of Grasnica, Daljan, littoral near Sateska River, Cherava) experience increased anthropogenic influence. The most alarming condition (in accordance to all analyzed indicators) has been evidenced in Grasnica, where the water is mainly mesotrophic and sometimes transferring into eutrophic character. As the main cause has been suggested the River Velgoska (which is nearby), representing the end-recipient of waste, drainage and households` waters. During summer time (touristic season) the indicators are increased, too, especially total phosphorous and nitrogen and the organic matter. This condition occurs as a result of the increased number of people in that period and raised number of touristic facilities, both of which contribute to the pollution of the ecosystem.

BENTHIC DIATOM ALGAE

Methodology The bio-monitoring activities have been focused on attached diatoms living on stones from the lake. In fact, cobbles, pebbles and boulders without filamentous algal growth and sedimentary organic or inorganic matter were the preferred substratum for sampling. The removal of diatom community from the substratum has been conducted by vigorous brushing of the upper surface and the resulting suspensions were collected and preserved in formalin (3-4 %). Then, diatoms frustules were cleaned of organic and inorganic materials based on acid method by Krammer & Lange – Bertalot (1986 – 2001) - boiling the material, first with HClcc (for about 20 min) and then boiling with H2SO4cc,(for about 20 min) with addition of a few crystals of KNO3. Benthic diatoms, cleaned of cell contents and mounted in Naphrax are identified and counted with optic microscope. The determination of species was based on four volumes of KRAMMER & LANGE-BERTALOT (1986–1991) keys and other available literature, as described in the protocol.

The Trophic index for diatoms (TI DIA) and Saprobic index (SI) were calculated with the application of the formula of Zelinka & Marvin (1961), considering the relative trophic and saprobic classes after the classification made by Root et al. (1997; 1999; 2003). In addition, the diversity index of Shannon & Weaver (H’) has been calculated, too.

Results and Discussions Throughout the research, there have been evidenced total of 110 species in Lake Ohrid. The number of species that have been registered follows a stable path in both years and is depicted in the figures below. Figure 12: Number of species in July 2010 Figure 13: Number of species in July 2011

The most frequent centric Diatom was Cyclotella ocellata. Moreover, from the order Pennales, genus Navicula, have the largest number of species, followed by genus Cymbella and Gomphonema. The number of species fluctuate from 23 (Udenisht, Velidab) to 42 (Auto camp As, Sateska river) with an average value of 35 species per station. Furthermore, the species composition varies among sampling localities, while most of the registered species in Lake Ohrid are indicating an oligotrophic character of the ecosystem. For instance, some of the species that have been registered are: Diploneis ovalis, Achnanthes minutissima, Navicula cryptotenella etc. These species have been sampled at Cape of Lini, Dogana-Tushemisht, Hotel Park, St. Zaum and Radozda. Besides the so-called oligotrophic diatoms, there have been evidenced some mesotrophic to eutrophic species, including: Amphora pediculus, Cocconeis pediculus, Cymbella minuta etc. registered at the localities Piskupat, Udenisht, Piskupat 2, Memlisht, Ohrid bay, Pestani and Kalista. In the end, there have been evidenced some poly-hypertrophic species, but they are not widely spread. These species include Cymatopleura solea, Fragilaria ulna, Gomphonema parvulum, Nitzchia palea, and have been evidenced in Lini village, Grasnica and Pogradec 2. The H’ - Diversity Index varied from 0.98 to 1.40. Low values of the Diversity index (H') resulted from a small number of genera and high abundance of a few common species, which were observed in Ohrid bay, Udenisht, St. Zaum, Blato etc. (under 1.00). On the other hand, higher diversity values were observed in River Sateska, Pogradec 2, Grasnica, River Racanska, and Autocamp As with about 1.30 and River Koselska (about 1.40). The Diversity Index in July 2010 and 2011 is shown below. Figure 14: Diversity Index in July 2010 Figure 15: Diversity Index in July 2011

The trophic index for diatoms varied between 1.5 (oligo-mesotrophic) and 2.7 (eu- polytrophic). The lowest values for trophic index (ranged from oligo-mesotrophic to mesotrophic) were registered for Dogana, St. Zaum (1.5), Udenisht (1.7), Velidab, Piskupat and Tushemisht (1.8). More of the stations ranged from mesotrophic to meso-eutrophic, while River Racanska (2.6); St. Naum (2.5), Ohrid bay (2.4), Pogradec town (2.3) ranged from mesotrophic to eutrophic. In Lini-village and Grasnica TI DIA was the highest (2.7) and indicates to eu- polytrophic class. The following figures show the TI DIA for 2010 and 2011. Figure 16: Trophic Index for Diatoms 2010

Figure 17: Trophic Index for Diatoms 2011

Therefore, the sampling sites, according to the Trophic Index for Diatoms were described as follows: Figure 18: Trophic Index for Diatoms Table 1: Number of sites per Trophic Index for Diatoms Trophic Index for # of Diatoms localities

Oligo-mesotrophic 2

Mesotrophic 4

Meso-eutrophic 13

Eutrophic 9

Polytrophic 2

According to the Saprobic Index (SI) which varied from 1.4 to 2.1, the water from the littoral zone of Lake Ohrid is classified into three saprobic classes, i.e. oligosaprobic, oligo – β mesosaprobic and β mesosaprobic. The lowest value of the Saprobic Index was recorded in St. Zaum (1.4) and Park (1.5) - oligosaprobic class. Most of the stations ranged from oligosaprobic to oligo - β mesosaprobic, including Pogradec 2, Udenisht, Lini-village, Grasnica, Ohrid Bay, and River Cerava in 2010 and Pogradec 1, Piskupat, Tushemisht, Memelisht, Kalista, Pestani in 2011 were classified from oligo-β-mesosaprobic to -mesosaprobic, which shows a slight pollution.Therefore, the sampling sites, according to the Saprobic Index were described as follows: Figure 19: Saprobic Index Table 2: Number of sites per Saprobic Index

Saprobic Index # of localities

Oligosaprobic 1

Oligo-β-mesosaprobic 10

β-mesosaprobic 19

Conclusion Littoral habitats in Lake Ohrid are oligotrophic or mesotrophic and comprise species such as: Cyclotella ocellata, Achnanthes minutissima etc. In addition to those, there are some eutrophic species registered in the lake, including: Amphora pediculus, Cocconeis pediculus, Cymbella minuta etc., but there are not widely spread. Trophic diatom index ranged from 1.5 (mesotrophic) in Dogana, Udenisht and St. Zaum to 2.7 (eu-polytrophic) in Lini-village and Grasnica. Saprobic index ranged from 1.4 (oligosaprobic) in St. Zaum and 1.6 (oligo- β- mesosaprobic), in Dogana, Cape of Lini; to 2.0 (β-mesosaprobic), in Lini-village. The water of Lake Ohrid near Pogradec town and Grasnica is dominated by polytrophic species but there were not spread around the lake. Generally, the composition of diatoms shows a slight pollution with organic matter and moderate polluted with inorganic matter. In Lini-village and Pogradec the urban wastewater is directly transported without any treatment in the lake.

MACROPHYTE VEGETATION

Methodology Submerged macrophytes, including monocotyledonous and dicotyledonous plants, charophytes and macroscopic filamentous algae Cladophora glomerata (L.) Kützing, have been surveyed in belt transects of approximately 10 meters width - perpendicularly to the shoreline - from the upper littoral to the lower vegetation limit. Each transect was divided into depth zones: 0-2 m, 2-4 m, 4-10 m, and >10 m depth. In deeper waters, plants have been collected by a Van-veen grab, while in the shallow waters by snorkeling. Species occurrence has been registered in each transect and each depth zone, and the abundance of each species was estimated according to a five degree scale (1 = very rare, 2 = infrequent, 3 = common, 4 = frequent, 5 = abundant, predominant). For the purpose of determination of vascular macrophytes there have been used different floristic books (floras) in accordance to the protocols, such as: Hayek, ed. (1924-1933); Јordanov, ed. (1963-1970) etc. The macrophyte index was calculated according to the formula described by Melzer (1999), but with updated indicator values and class boundaries as described by Melzer and Schneider (2001).

Results and Discussion There have been recorded total of 29 macrophyte species in all researched localities of the littoral region of Lake Ohrid. Out of those species, 17 were vascular macrophytes, 11 charophytes and only one (Cladophora sp.) is classified under the group of non-charophyte macroscopic algae. The dominant species of vascular macrophytes in Lake Ohrid is Potamogeton perfoliatus, which has been recorded in total of 27 sampling sites. At six (6) of the localities this species` abundance has been estimated as “abundant”, at five (5) it has been “common”, at five (5) it has been “frequent”, at nine (9) – “rare” and at only 2 of the sites it has been “very rare”. The most abundant charophyte has been Chara tomentosa, which has been present in total of 25 sampling sites, at 21 of which it has been “abundant”, at two (2) of which has been “common” in one (1) of which it has been “frequent” and in one (1) of them has been “very rare”. The presence of Chara tomentosa indicates to the fact that the water in the littoral is still to be considered as water with higher quality. 21

Elodea canadensis, an invasive species in Lake Ohrid, has been registered in total of 16 sampling sites, at five (5) of which it has been marked as “common”, at one (1) of the sites has been “frequent”, at eight (8) of them it has been “rare” and at two (2) of the sites it has been marked as “very rare”. The macrophyte index for the entire lake is shown in the figure below. Figure 20: Macrophyte index for entire lake (average)

As it can be seen, the macrophyte index in average indicates to “low to medium” nutrient pollution in the littoral of Lake Ohrid, but there are considerable differences among sites. .). 8 of the sampling sites have “very low” nutrient pollution, in 7 of them there is a “low” nutrient pollution, in 8 sites there is “moderate” nutrient pollution, in 5 sites there has been recorded “moderate-immense” nutrient pollution, and in only two of them there is an “immense” nutrient pollution. The highest values of nutrient pollution have been registered at Kalista, rivers Sateska and Koselska, at Trpejca site, near St Naum site in Macedonia, and Dogana and Pogradec in Albania.

Table 3: Number of sites per Nutrient Pollution Macrophyte index for Nutrient pollution Shallow Waters (0-2 m) Deeper Waters (>10) shallow (0-2 m) and Very low 6 8 deeper (>10 m) Low 1 Moderate 4 waters have been Moderate-Immense 5 calculated in order to Immense 4 Heavy 7 compare and grasp a Massive 3 better image regarding the state of such pollution. For depths larger than 10 m, all sites had low or very low nutrient pollution (the sites not represented on the map did not have macrophyte vegetation below 10 m depth). The following figures depict the macrophyte index for shallow and deeper waters.

Figure 21: Macrophyte index of shallow Figure 22: Macrophyte index for deeper water 0-2m water >10

Furthermore, submerged macrophytes are of particular importance in aquatic ecosystems, since with their well-defined ecological optima and ranges; they are widely used for bio- indication of lake and rivers trophic status (Kohler & Schneider 2003). For instance, Zannichellia palustris, a species that indicates increased eutrophication, has been found at 15 out of the 30 sampling sites. Moreover, the presence of the invasive species in Lake Ohrid, Elodea canadensis, also indicates to an increased trophic state of the water. This species has been registered at 16 out of total 30 sampling sites.

The macrophyte index is important for determination of trophic state because unlike water chemistry, it may indicate eutrophication processes in the littoral ecosystem, combining water and sediment. The average values of the macrophyte index for all investigated sites indicate generally good trophic status of the lake, due to the fact that in 15 sites the water is described as very low or low nutrient pollution. However, it is evident that pollution originates from the shore, because macrophytes indices in shallow water are consistently higher than in deeper waters. Schneider et al. (submitted) have shown that the macrophyte index in shallow water does not correspond with water total phosphorus concentrations. Thus, presumably, part of the nutrients is incorporated in the plants biomass.

Conclusion In all researched localities, there have been evidenced total of 29 macrophyte species, classified into nine (9) families. Out of those, 17 are categorized under the macrophyte indicator groups (Meter and Schneider, 2001) while the rest (12) do not belong to this group. Furthermore, the maximum depth vegetation limit in all localities is 14 m (Chara tomentosa) while the minimum is 6 m (Potamogeton perfoliatus). Potamogeton perfoliatus was present in 27, and it is most abundant in the depth zone 2-4 m while Chara tomentosa was present in 25, and it is most abundant in the depth zone 4-10 m. Likewise, Chara ohridana, a Balkan endemic species, has been evidenced in 16 of the researched sites, as well as Vallisneria spiralis and Elodea canadensis. The values of the macrophyte index for the entire lake indicate that nutrient pollution is very low or low for the majority of the researched sites. This indicator was lower in deeper waters, if compared to shallow waters, i.e. deeper waters have lower nutrient pollution. Therefore, the nutrient pollution is mainly driven by the shallow waters. In the end, according to the macrophyte index the trophic state of the littoral region of Lake Ohrid is 50% oligotrophic and oligo-mesotrophic.

BENTHIC FAUNA (MACROZOOBENTHOS) Methodology The sampling methods used have been in accordance to the standard limnlogical methods and the protocol according to Lind (1985), Wetzel (1975), Wetzel&Likens (1979) etc. Depending of the bottom, two sampling methods have been used during the 2 years period of sampling, including: kick and sweep method and grab sampling method (van Veen grab). The first one includes kicking up the substrate and then sweeping above the disturbed area to capture dislodged or escaping invertebrates, but leaving behind much of the debris. On the other hand, the second method of sampling is especially effective in fine-grained, soft substrate. Grab samples are taken from a boat on all transects starting from 2, 5, 10, 15 and 20 m. The samples have been transported from the field to the laboratory within a few hours of collection. To determine the structure of the macrozoobenthos community the following structural indices were used: index of species richness - d, Shannon-Wiener Diversity Index - H '(diversity), index of evenness J(e) (evenly representation of macrozoobenthos groups of individuals) in different habitats and localities. Next, the metrics of the zoobenthos from Lake Ohrid have been calculated according the above mentioned indices is further processed using software Saprosoft.

Results and Discussion The total number of species identified during the researched period was 111 species. They belong to 8 systematic groups: Turbellaria, Oligocheata, Hirudinea, Gastropoda, Bivalvia, Amphipoda, Isopoda and Insecta. Even 62 percent of the total number of taxa belong to the group of endemic ones. The rest of the species are cosmopolitan belonging to the following groups based to their indicator valences (Fig.23): - 24 species indicate mesotrophic conditions - 7 species indicate oligotrophic conditions and, - 11 species indicate eutrophic conditions of the water. The results in the researches have confirmed the influence of the habitat and the season on the benthic communities’ structures in Lake Ohrid. More details about the diversity and distribution of the benthic communities are shown on the figures 23-25. In spring, the total number of identified species is 103 while in the fall, the diversity is slightly lower-101 species have been recorded.

Figure 23: Endemic and cosmopilitan species ration in Lake Ohrid

Concerning the density, it was found that the density is significantly higher in fall compared with the spring one In both seasons, the highest density has been recorded on depth of 1 m, which corresponds with the bottom type covered by sandy facies with presence of detrital material. Figure 24: Biodiversity of benthic fauna in Lake Ohrid-“depth distribution”

Total number of species 83 Endemc species Cosmopolitan species 70 Cosmopolitan eutrophic species

Cosmopolitan oligotrophic species 63 Cosmopolitan mesotrophic species 42 45 41

38 22 25 12 25 7 15 6 4 15 8 4 1 5 15 Based on the figure, it is noticeable that more or less, the diversity of the endemic species is not affected by the depth (at least not in the littoral zone), and the endemic species are uniformly distributed on all depth points in the Lake. Unlike, the diversity of the cosmopolitan species is highest on the shallowest point and it decreases with increasing of the depth. The general density of the benthic fauna has been also influenced by the seasons and it has been found that in fall, it is higher than in spring. Precisely (Fig.25) the density of the macrozoobenths on Lake Ohrid is higher for almost one third in fall than in spring, a fact that could be related to the food availability as well as the life cycle of the benthic organisms.

Figure 25: Density comparison: spring vs. fall

Figure 26: Ecological status of the sampling sites in lake Ohrid-spring period

One of the main goals in the researches of the benthic fauna was to assess the ecological status of the sampling sites using the metric characteristics. Using the structural indexes (index of species richness - d, Shannon-Wiener Diversity Index - H ', index of evenness J(e) ) and by extrapolating their values in the colorful scale for ecological status it was found that none of the researched sampling sites characterizes bad ecological status. So, in spring, 4 of the sites were with poor, 6 with moderate while 20 with good or high ecological status. Three of the sites with poor status are on Macedonian while 1 on Albanian side of the Lake. All of them have already been considered as “black” spot of the Lake concerning their values for the physical chemical parameters. In fall, the number of the sites with poor status is lower (3) and they are all on Macedonian side of the Lake. Two sites (Radozda and River Grashnica) persist in the same status as it was case in the spring, while Trpejca 2 “appears” as a new one with poor ecological status. However the general picture about the ecological status of the sampling sites in fall is better in fall due to the less sites with poor ecological status and more sites with good-high status.

Figure 27: Ecological status of the sampling sites in lake Ohrid-fall period

Conclusion The benthic fauna of Lake Ohrid is characterized by both high level of biodiversity and high level of endemism. During the research, there have been identified total of 111 different species, 69 of which (62%) were endemic, seven (7) of which were oligotrophic species, 24 of which were mesotrophic and 11 – eutrophic species. Moreover, 7 different classes of benthic organisams are identified in our samples, including species of TURBELLARIA, OLIGOCHAETA, HIRUDINEA, BIVALVIA, GASTROPODA, CRUSTACEA (AMPHIPODA AND ISOPODA), INSECTA. The macrozoobenthos diversity is higher in spring season (103 species) and highest diversity is recorded on 1 m depth point. Density of macrozoobenthos is lower in Fall, (5 m depth highest) with the most abundant species Dreissena prespensis (814 ind∙m²). The species diversity is influenced by the habitat, i.e. stony-sandy bottom in 1 meter depth and sandy-muddy bottom with high vegetation in 5 meters depth are the most preferred habitats. Finally, the research shows that there are no sites with bad ecological status. In fact, there were 13 sites with “high” in both seasons; four (4) sites in spring and three (3) in fall were characterized by ”poor” status while two (2) sites persist in “poor” status throughout the year.

General Recommendations Based on the research and the gained experience and best practices of the project, the general recommendations include: 1. 2/3 of the lake`s area belong to the deep zone (sub-littoral and profundal), fact which encourages further researches that would be directed into investigating of these regions. 2. The results obtained in the project indicated that the Lake Ohrid is a specific aquatic ecosystem; consequently it is more than needed, a development of a specific system for assessment of the ecological status of the ancient lake. Needless to say, this system should be based on specific indicator indices for the endemic flora and fauna. 3. One of the basic premises of the European Water Framework Directive is the development of integrated basin management. In that regard, the entire watershed (not only of Lake Ohrid, but including the watershed of River Drim) should be taken into consideration as a subject of monitoring. 4. There is a need of introduction of activities which would result in the development and implementation of self-sustainable and long-term monitoring processes of the region. 5. Finally, the political ellites in both countries (and for the watersheds in question of the five countries) should pay more attention to this issue, thus facilitating the cooperation between scientists for monitoring and prevention, since the water and its benefits are borderless and essential.