**4. Materials and methods**

Aiming to resolve the detected intensive eutrophication of Prespa Lake watershed and to produce a management plan that is going to address this issue, the analyses presented in this chapter have actually been conducted in order to resolve the anthropogenic from natural processes of eutrophication. By focusing on the *reference conditions*, we have been able to detect the intensive human impact dated 1,500 years ago through massive deforestation and subsequent washout of nutrients into the Prespa Lake. This influence in combination with the very recent (only some 100 years) intensive pollution impacts in the watershed have triggered the turnover of the lake from the nitrogen towards phosphorus driven ecosystem and corresponding cyanobacterial toxic 'water blooms'.

Environmental Changes in Lakes Catchments

the chosen sampling points on the map.

Fig. 12. Sampling points in Macro Prespa Lake watershed.

reference site of the overall conditions at the border with Greece.

Five lake sampling sites were chosen as the most representative points for establishment of a relevant monitoring system of biological and physic-chemical quality parameters including: site Stenje (L1) village as a reference site for the deepest part of the lake in that area, a site in the vicinity of the mouth of Golema River (L2) for checking the impact of the river on the lake's ecosystem, a site in the vicinity of the mouth of the Kranska River (L3), a site in the vicinity of the mouth of the Brajcinska River (L4) and the Dolno Dupeni site (L5) as a

as a Trigger for Rapid Eutrophication – A Prespa Lake Case Study 79

Development of a representative monitoring network of sampling sites in the Lake Prespa watershed included five lakes, four rivers and seven ground water (wells) sampling points. The most representative lake sampling points were chosen where continuous pressure from various anthropogenic activities was most expected. The river sampling points were the mouth waters of the four main rivers in the Prespa watershed flowing in the Prespa Lake. The ground water sampling points were chosen to represent the quantitative and chemical status of main ground water bodies, with the aim of providing clues as to the possible influence by the continuous pressure of various anthropogenic activities, as well as a certain correlation between the ground water bodies and surface water bodies. Figure 12 represents

Fig. 11. Waste apple dumping in Golema River and persistent foam on the Prespa Lake surface.

Assessment of the ecological status of the water bodies have to be based on the comparison of the level of deviation of current conditions of the biological quality elements in a water body with pristine conditions of the same one which means conditions without any anthropogenic disturbance. The level of deviation in terms of numeric Ecological Quality Ratios (EQR) have to be used for assigning an appropriate ecological class to a water body and describe its ecological status. Due to the usual practice of lack of continuous monitoring data on the biological quality elements which is necessary for description of the trend of the ecological condition of the aquatic ecosystems, the WFD leaves space for two approaches to be used for bridging this gap. A combination of the available historical data with at least a one year period of monthly detailed field monitoring of biological quality parameters gives a firm and reliable starting point in assessment and description of the ecological status of a certain water body. This approach combined with an iterative re-assessment process based on monitoring data of key type-specific biological quality elements to be derived during the river basin management plan implementation will enable a more comprehensive description of the ecological status of the water bodies and more efficient assessment of the effectiveness of the measures and activities implemented aimed at achieving the prescribed environmental objectives.

Bearing in mind the above, the development of the Prespa Lake Watershed Management Plan has been fully accompanied with identification, delineation and categorisation of the main surface and ground water bodies in the basin, and included establishment and implementation of a one year surveillance monitoring system of key biological quality elements with monthly field and laboratory analyses on the most important physical, chemical and biological parameters.

 Fig. 11. Waste apple dumping in Golema River and persistent foam on the Prespa Lake

Assessment of the ecological status of the water bodies have to be based on the comparison of the level of deviation of current conditions of the biological quality elements in a water body with pristine conditions of the same one which means conditions without any anthropogenic disturbance. The level of deviation in terms of numeric Ecological Quality Ratios (EQR) have to be used for assigning an appropriate ecological class to a water body and describe its ecological status. Due to the usual practice of lack of continuous monitoring data on the biological quality elements which is necessary for description of the trend of the ecological condition of the aquatic ecosystems, the WFD leaves space for two approaches to be used for bridging this gap. A combination of the available historical data with at least a one year period of monthly detailed field monitoring of biological quality parameters gives a firm and reliable starting point in assessment and description of the ecological status of a certain water body. This approach combined with an iterative re-assessment process based on monitoring data of key type-specific biological quality elements to be derived during the river basin management plan implementation will enable a more comprehensive description of the ecological status of the water bodies and more efficient assessment of the effectiveness of the measures and activities implemented aimed at achieving the prescribed

Bearing in mind the above, the development of the Prespa Lake Watershed Management Plan has been fully accompanied with identification, delineation and categorisation of the main surface and ground water bodies in the basin, and included establishment and implementation of a one year surveillance monitoring system of key biological quality elements with monthly field and laboratory analyses on the most important physical,

surface.

environmental objectives.

chemical and biological parameters.

Development of a representative monitoring network of sampling sites in the Lake Prespa watershed included five lakes, four rivers and seven ground water (wells) sampling points. The most representative lake sampling points were chosen where continuous pressure from various anthropogenic activities was most expected. The river sampling points were the mouth waters of the four main rivers in the Prespa watershed flowing in the Prespa Lake. The ground water sampling points were chosen to represent the quantitative and chemical status of main ground water bodies, with the aim of providing clues as to the possible influence by the continuous pressure of various anthropogenic activities, as well as a certain correlation between the ground water bodies and surface water bodies. Figure 12 represents the chosen sampling points on the map.

Fig. 12. Sampling points in Macro Prespa Lake watershed.

Five lake sampling sites were chosen as the most representative points for establishment of a relevant monitoring system of biological and physic-chemical quality parameters including: site Stenje (L1) village as a reference site for the deepest part of the lake in that area, a site in the vicinity of the mouth of Golema River (L2) for checking the impact of the river on the lake's ecosystem, a site in the vicinity of the mouth of the Kranska River (L3), a site in the vicinity of the mouth of the Brajcinska River (L4) and the Dolno Dupeni site (L5) as a reference site of the overall conditions at the border with Greece.

Environmental Changes in Lakes Catchments

**Total Microcystins (cell and free)** 

**4.1 Field sampling** 

**4.2 Laboratory analyses** 

analytical range.

Macro Prespa Lake watershed.

COD Annually/12x Annually/12x BOD Annually/12x Annually/12x

as a Trigger for Rapid Eutrophication – A Prespa Lake Case Study 81

Cadmium (Cd) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Lead (Pb) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Mercury (Hg) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Nickel (Ni) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Arsenic (As) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Cooper (Cu) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Chromium (Cr) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Zink (Zn) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x

Pentachlorobenzene Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x Hexachlorobenzene Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x

DDT (6,7) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x DDD (6,7) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x DDE (6,7) Seasonally/4x Seasonally/4x Seasonally/4x Seasonally/4x

Table 5. Parameters and frequencies analysed during the 12 month monitoring period in

Conducting analysis of physic-chemical parameters, priority substances, phytoplankton and phytobenthos quality parameters require a representative water and biological material sample to be collected, preserved and prepared for laboratory analyses. For consistent collection of materials a sampling manual is prepared. After collection, materials were transported to the Laboratory for Ecology of Algae and Hydrobiology at the Institute of

Table 6 describes the analysed parameters, type of the analyses and the methods used

The spectroscopic analyses were conducted on Photometer System Max Direct-LoviBond® (www.tintometar.com) with the use of LoviBond® analytical kits with appropriate

Biology Faculty of Natural Sciences in Skopje for further treatment.

during the 12 month surveillance monitoring in Prespa Lake watershed.

Annually/12x Seasonally/4x Seasonally/4x

**Parameter Rivers Lakes Sediments Biota** 

Iron (Fe) Seasonally/4x Seasonally/4x Seasonally/4x

DEPH Seasonally/4x Seasonally/4x Seasonally/4x Nonylphenols Seasonally/4x Seasonally/4x Seasonally/4x 4-tert-Octylphenol Seasonally/4x Seasonally/4x Seasonally/4x Naphtalene Seasonally/4x Seasonally/4x Seasonally/4x Floranthene Seasonally/4x Seasonally/4x Seasonally/4x

Four major river courses, Istocka, Golema, Kranska and Brajcinska (R1-R4), were permanently monitored during the 12 month sampling period at their mouths in Prespa Lake, in order to determine the overall qualitative and quantitative pressures on the lake's ecosystem. Their reference sites (R1(r)-R4(r)) were checked only once (March 2010) to establish the reference physic-chemical and biological conditions, and delineation of the water bodies.

Table 5 represents the full range of analysed parameters during the 12 month monitoring period and the frequencies used to monitor different environments or media.



Table 5. Parameters and frequencies analysed during the 12 month monitoring period in Macro Prespa Lake watershed.

#### **4.1 Field sampling**

80 Studies on Environmental and Applied Geomorphology

Four major river courses, Istocka, Golema, Kranska and Brajcinska (R1-R4), were permanently monitored during the 12 month sampling period at their mouths in Prespa Lake, in order to determine the overall qualitative and quantitative pressures on the lake's ecosystem. Their reference sites (R1(r)-R4(r)) were checked only once (March 2010) to establish the reference physic-chemical and biological conditions, and delineation of the

Table 5 represents the full range of analysed parameters during the 12 month monitoring

Annually/12x (profiling)

(profiling)

(profiling)

(profiling)

Total Nitrogen Annually/12x Annually/12x Seasonally/4x

Annually/12x Annually/12x

Total phosphorus Annually/12x Annually/12x Seasonally/4x

period and the frequencies used to monitor different environments or media.

Seasonally/4x Seasonally/4x

Phytoplankton Annually/12x

Fish Seasonally/2x Seasonally/2x Phytobenthos Annually/12x Annually/12x Macrophytes Seasonally/2x Seasonally/2x

Depth Annually/12x Annually/12x

(profiling)

Transparency Annually/12x Annually/12x Suspended solids Annually/12x Annually/12x Dissolved oxygen Annually/12x Annually/12x

pH Annually/12x Annually/12x

Conductivity Annually/12x Annually/12x

Alkalinity Annually/12x Annually/12x Ammonium (NH4) Annually/12x Annually/12x Nitrate (NO3) Annually/12x Annually/12x Nitrite (NO2) Annually/12x Annually/12x

Inorganic nitrogen Annually/12x Annually/12x Organic Nitrogen Annually/12x Annually/12x

Sulfate (SO4) Annually/12x Annually/12x Calcium (Ca) Annually/12x Annually/12x Magnesium (Mg) Annually/12x Annually/12x Chloride Annually/12x Annually/12x

**Parameter Rivers Lakes Sediments Biota** 

water bodies.

Benthic

macroinvertebrates

Ortho-phosphate

(PO4)

Flow Annually/12x

Temperature Annually/12x

Conducting analysis of physic-chemical parameters, priority substances, phytoplankton and phytobenthos quality parameters require a representative water and biological material sample to be collected, preserved and prepared for laboratory analyses. For consistent collection of materials a sampling manual is prepared. After collection, materials were transported to the Laboratory for Ecology of Algae and Hydrobiology at the Institute of Biology Faculty of Natural Sciences in Skopje for further treatment.

#### **4.2 Laboratory analyses**

Table 6 describes the analysed parameters, type of the analyses and the methods used during the 12 month surveillance monitoring in Prespa Lake watershed.

The spectroscopic analyses were conducted on Photometer System Max Direct-LoviBond® (www.tintometar.com) with the use of LoviBond® analytical kits with appropriate analytical range.

Environmental Changes in Lakes Catchments

**Total Microcystins (Cell bound** 

Nikon Coolpix 4500 digital camera*.*

10BQ.

**4.3 Hydrology** 

Hydrological Aspects".

 4

on the profile width, such as:

Enzyme-Linked Immuno Sorbent Assay (ELISA)

as a Trigger for Rapid Eutrophication – A Prespa Lake Case Study 83

**Parameter Type of analysis Type of method** 

Floranthene Laboratory Analysis Analytical-GC DDT (6,7) Laboratory Analysis Analytical-GC DDD (6,7) Laboratory Analysis Analytical-GC DDE (6,7) Laboratory Analysis Analytical-GC

monitoring on chosen sampling sites in Prespa Lake watershed.

appropriate procedure and subsequent ELISA analyses.

and flows, using the obtained rating curves Q=f(H).

**and extracellular)** Laboratory Analysis Immunological-ELISA<sup>4</sup>

Table 6. Parameters, analyses and methods used during the 12 month surveillance

The microcystin analyses were conducted with ELISA M956 Microplate Reader Metertech® with the use of Abraxis® Microcystins-DM ELISA 96 Microtiter Plates, product no: 522015 (www.abraxis.com). Analyses of microcystins in sediments used the same method with prior procedure of digestion and pre-treatment of the collected sediments. Analyses of microcystins in biological tissues were conducted by means of their extraction with

Heavy metals' analyses were conducted with Atomic Absorption Spectrophotometer Varian

Determination of chlorophyll pigments was conducted by the trichromatic method (Strickland and Parson, 1968) and measured on Photometer System Max Direct-LoviBond®. Assessment of phytoplankton and phytobenthos species' composition was performed with microscopic analyses of the collected native and preserved algal material. The microscopic analyses of the collected material were performed on a light microscope Nikon E-800 with a

The quantities of the waters in the watershed were followed and measured by permanent water quantity stations and occasional (once per month) measurements of the water levels

On each watercourse where permanent monitoring is not yet established, direct hydrometrical measurements were performed, in parallel with the water quality samplings, in order to determine the overall flux of pollutants into the lake ecosystem. The hydrometrical measurements were performed according to ISO standards: measurements' ranges are based on ISO 748:1979 and ISO2537:1988, using a hydrometrical wing for the flow measurements. Working methodology was undertaken according to the WMO's "Guide to

Water velocities measurements were performed by the usual methodology on several verticals in a perpendicular transect. The number of verticals should be uneven depending

Analyses for priority substances were conducted on Gas Chromatograph Varian.


2 Atomic Absorption Spectrophotometry

3 Gas Chromatography (GC)


Table 6. Parameters, analyses and methods used during the 12 month surveillance monitoring on chosen sampling sites in Prespa Lake watershed.

The microcystin analyses were conducted with ELISA M956 Microplate Reader Metertech® with the use of Abraxis® Microcystins-DM ELISA 96 Microtiter Plates, product no: 522015 (www.abraxis.com). Analyses of microcystins in sediments used the same method with prior procedure of digestion and pre-treatment of the collected sediments. Analyses of microcystins in biological tissues were conducted by means of their extraction with appropriate procedure and subsequent ELISA analyses.

Heavy metals' analyses were conducted with Atomic Absorption Spectrophotometer Varian 10BQ.

Analyses for priority substances were conducted on Gas Chromatograph Varian.

Determination of chlorophyll pigments was conducted by the trichromatic method (Strickland and Parson, 1968) and measured on Photometer System Max Direct-LoviBond®.

Assessment of phytoplankton and phytobenthos species' composition was performed with microscopic analyses of the collected native and preserved algal material. The microscopic analyses of the collected material were performed on a light microscope Nikon E-800 with a Nikon Coolpix 4500 digital camera*.*

#### **4.3 Hydrology**

82 Studies on Environmental and Applied Geomorphology

**Parameter Type of analysis Type of method** 

Flow Field Measurement Hydrometrical wing Depth Field Measurement Van Veen bottom sampler Temperature Field Measurement Tintometar Senso Direct 150

Dissolved oxygen Laboratory Analysis Oxy meter - Tintometar Senso

pH Field Measurement pH meter - Tintometar Senso Direct

Conductivity Field Measurement Conductivity-meter - Tintometar

Ammonium (NH4) Laboratory Analysis Analytical-spectroscopic Nitrate (NO3) Laboratory Analysis Analytical-spectroscopic Nitrite (NO2) Laboratory Analysis Analytical-spectroscopic Total Nitrogen Laboratory Analysis Analytical-spectroscopic Inorganic nitrogen Laboratory Analysis Analytical-spectroscopic Organic Nitrogen Laboratory Analysis Analytical-spectroscopic Ortho-phosphate (PO4) Laboratory Analysis Analytical-spectroscopic Total phosphorus Laboratory Analysis Analytical-spectroscopic Sulfate (SO4) Laboratory Analysis Analytical-spectroscopic Calcium (Ca) Laboratory Analysis Analytical-spectroscopic Chloride Laboratory Analysis Analytical-spectroscopic COD Laboratory Analysis Analytical-spectroscopic BOD Laboratory Analysis Analytical-spectroscopic

Direct 150

Senso Direct 150

150

Transparency Field Measurement Secchi Disk Suspended solids Laboratory Analysis Analytical

Alkalinity Laboratory Analysis Analytical

Magnesium (Mg) Laboratory Analysis Analytical-AAS<sup>2</sup> Cadmium (Cd) Laboratory Analysis Analytical-AAS Lead (Pb) Laboratory Analysis Analytical-AAS Mercury (Hg) Laboratory Analysis Analytical-AAS Nickel (Ni) Laboratory Analysis Analytical-AAS Arsenic (As) Laboratory Analysis Analytical-AAS Cooper (Cu) Laboratory Analysis Analytical-AAS Chromium (Cr) Laboratory Analysis Analytical-AAS Zink (Zn) Laboratory Analysis Analytical-AAS Iron (Fe) Laboratory Analysis Analytical-AAS Pentachlorobenzene Laboratory Analysis Analytical-GC<sup>3</sup> Hexachlorobenzene Laboratory Analysis Analytical-GC DEPH Laboratory Analysis Analytical-GC Nonylphenols Laboratory Analysis Analytical-GC 4-tert-Octylphenol Laboratory Analysis Analytical-GC Naphtalene Laboratory Analysis Analytical-GC

 2

3

Atomic Absorption Spectrophotometry

Gas Chromatography (GC)

The quantities of the waters in the watershed were followed and measured by permanent water quantity stations and occasional (once per month) measurements of the water levels and flows, using the obtained rating curves Q=f(H).

On each watercourse where permanent monitoring is not yet established, direct hydrometrical measurements were performed, in parallel with the water quality samplings, in order to determine the overall flux of pollutants into the lake ecosystem. The hydrometrical measurements were performed according to ISO standards: measurements' ranges are based on ISO 748:1979 and ISO2537:1988, using a hydrometrical wing for the flow measurements. Working methodology was undertaken according to the WMO's "Guide to Hydrological Aspects".

Water velocities measurements were performed by the usual methodology on several verticals in a perpendicular transect. The number of verticals should be uneven depending on the profile width, such as:

<sup>4</sup> Enzyme-Linked Immuno Sorbent Assay (ELISA)

Environmental Changes in Lakes Catchments

additionally processed in the laboratory.

were statistically processed.

**4.7 Benthic invertebrates** 

**watershed** 

and based upon the past experience of the investigator.

as a Trigger for Rapid Eutrophication – A Prespa Lake Case Study 85

The location of each net was selected according to proposed sampling sites in the project

All captured fish were identified, and length and weight was measured. The age and sex composition of the fish population from Lake Prespa were performed with standards ichthyological methods. Analyses of the scales were used for determination of the age structure of the fish populations, as well as length and weight growth. The obtained results

Benthic invertebrates from the Prespa Lake and its main tributaries were collected according

From the Prespa Lake itself, the collection of bottom fauna samples was performed by several different devices: Ekman grab, sediment corer, triangle bottom dredge and hand net. Macroinvertebrate standard methods applicable to lakes were used (ISO 9391:1995 and ISO 7828:1985). Concerning to the main tributaries of Prespa Lake, benthic invertebrate samples were collected with a Surber sampler or hand-net following standard methodology for collection of bottom fauna (ISO 8265:1988 and ISO 7828:1985). For preservation of biological samples 70% ethyl-alcohol or 4% formaldehyde were used, samples properly labelled and

In the laboratory, the animals were flushed with tap water through a standard sieve (280 μm pore size). Material was divided by groups, for further determination, mainly to the lowest taxonomic level (genus/species). Generally, determination to the species level is recommended because the species level logically provides the most detailed and sound information about autecological demands of a certain animal species. Determination was performed using identification manuals. Based on WFD principles, data informing on the communities' taxonomic composition, abundance, diversity and sensitive taxa were taken into consideration, both for Prespa Lake and its tributaries. Biotic indices that are suitable

According to the historical hydrology data and observations, there are four major tributaries in the Macedonian part of Macro Prespa Lake which can be considered as separate rivers of significance and should be subsequently subdivided into the Istočka, Golema, Kranska and Brajčinska Rivers. Although small in it watershed, Kurbinska River was also taken into

All of the other temporal water courses have been proven to be torrent carriers which usually drain forested areas and have little significance in the overall water quality analysis; although they may have a significant role in the water balance of the lake at specific times,

Considering the Prespa Lake itself, there is a littoral plateau of approximately 14-16 metres depth that completely surrounds the lake and two major depressions – one near the village

to the requirements of the EU Water Framework Directive (WFD) 2000/60/EC.

for Prespa Lake and its watershed monitoring purposes were used.

consideration because of it relatively significant quantity of water.

as well as in flood hazard management because of their character.

**5. Results of the performed surveillance monitoring in Prespa Lake** 


The number of measuring points for water velocity on every vertical is dependent on the depth of that vertical, such as:

