Plankton community structure diversity indices and similarity relationship with reference to industrial wastewater pollution by using correspondence analysis

ADVANCED EDUCATION PROGRAM TRAN THI MINH HA PLANKTON COMMUNITY STRUCTURE, DIVERSITY INDICES, AND SIMILARITY RELATIONSHIP WITH REFERENCE TO INDUSTRIAL WASTEWATER POLLUTION BY USING CORR

ADVANCED EDUCATION PROGRAM

TRAN THI MINH HA

PLANKTON COMMUNITY STRUCTURE, DIVERSITY INDICES, AND SIMILARITY RELATIONSHIP WITH REFERENCE TO INDUSTRIAL WASTEWATER POLLUTION BY USING CORRESPONDENCE ANALYSIS

JANUARY – 2015

ADVANCED EDUCATION PROGRAM

PLANKTON COMMUNITY STRUCTURE, DIVERSITY INDICES, AND SIMILARITY RELATIONSHIP WITH REFERENCE TO INDUSTRIAL WASTEWATER POLLUTION BY USING CORRESPONDENCE ANALYSIS

TABLE OF CONTENTS

I Introduction 1

1.1 Rationale of the study 1

1.2 Aims of the study 2

1.3 Research questions 2

II Literature review 3

2.1 Introduction to water 3

2.1.1 The importance of water 3

2.1.2 Water quality 3

2.2 Plankton community 4

2.3 Impact of wastewater in plankton community 7

III Methodology 12

3.1 Collection and sample 14

3.1.1 Place and time 14

3.1.2 Materials and Equipments 14

3.1.3 Plankton samplings 15

3.1.4 Water samplings 15

3.2 Tools and Identification process 15

3.2.1 Tools 15

3.2.2 Identification plankton process 16

3.2.3 Water analysis 16

3.3 Data analysis 19

IV Results and Discussion 20

4.1 Results of water analysis in 6 stations 20

4.2 Biodiversity Indices Measurement 25

4.3 Similarity relationship with reference to industrial wastewater pollution by using Correspondence Analysis 32

4.4 Diversity indices 35

V Conclusions and recommendations 40

5.1 Conclusions 40

5.2 Further research 40

ABBREVIATIONS

BOD : Biochemical Oxygen Demand

COD : Chemical Oxygen Demand

DO : Dissolved Oxygen

TSS : Total Suspended Solids

SNI : Indonesia national standard

PDAM : Drinking Water Company Installation

LIST OF FIGURES

Figure 1: Musi River 12

Graph 1: The distribution of planktons on 6 stations in the first observation 34 Graph 2: The distribution of planktons on 6 stations in the second observation 35

LIST OF TABLES

Table 1: Water analysis in soy sauce industry 20

Table 2: Water analysis in Crumb rubber 21

Table 3: Water analysis in Ship dock 22

Table 4: Water analysis in Drinking Water Company Installation 23

Table 5: Water analysis in Cement Company 24

Table 6: Water analysis in Stock pile 25

Table 7: Planktons in Soy Sauce 26

Table 8: Planktons in Crumb Rubber 26

Table 9 : Planktons in Ship Dock 27

Table10: Planktons in PDAM 28

Table 11: Planktons in Cement Company 29

Table 12: Planktons in Stockpile 29

Table 13: Observation 1 30

Table 14: Observation 2 31

Table 15: Diversity Indices 37

Table 13: The first observation 1

Table 14: the second observation 2

Thai Nguyen University of Agriculture and Forestry Degree Program : Bachelor of Environmental Science and Management

Student name: Tran Thi Minh Ha Student ID: DTN 1053110064

Thesis Title: PLANKTON COMMUNITY STRUCTURE, DIVERSITY INDICES, AND SIMILARITY RELATIONSHIP WITH REFERENCE TO

INDUSTRIAL WASTEWATER POLLUTION BY USING

Planktons are the main the primary producers which can be easily found in all kinds

of water bodies Plankton community is firstly influenced and involved in water pollution As planktons are very sensitive to the chemicals in water, the functions of planktons as the food source for many aquatic animals restrict

The present study focused on phytoplankton species composition in Musi River, Palembang, Indonesia, where alongside the river many industries were hosted Two water and plankton samplings were carried out to collect planktons from six selected sites or stations, i.e soy sauce industry, crumb rubber industry, ship dock, regional drinking water company, cement industry and coal stockpile from

November 7, 2014 to November 16, 2014.The physicochemical parameters were Dissolved Oxygen (DO), Chemical Oxygen Demand (COD), pH and Total Suspended Solids (TSS) The study also included Biochemical Oxygen Demand (BOD5)

The result showed that at every station the population community of planktons varied Several planktons were found at one station, and were not found at the other stations For the first observation the number of plankton species found was 22, but for the second observation was 39 species The most abundant planktons found

were Ankistrodesmus acicularis (Monoraphidium aciculare) and Ankistrodesmus

angustus with 150 individuals and 83 individuals, respectively, for first observation,

and Ankistrodesmus acicularis, Striatella interrupta, Koliella Longiseta with 300,

217 and 166 individuals, respectively, for second observation

The study concluded that discharged wastewater from industries contributed significant effect on the plankton community

Keywords: Planktons; Water Quality, Wastewater, Physicochemical parameters,

Biological parameter

Number of pages: 50 pages

Date of submission: January 15, 2015

SIMILARITY RELATIONSHIP WITH REFERENCE TO INDUSTRIAL WASTEWATER POLLUTION BY USING CORRESPONDENCE ANALYSIS

I Introduction

1.1 Rationale of the study

Planktons are composed of phytoplankton and zooplankton which are typically found near the surface in aquatic environments Planktons form the most sensitive components of the ecosystems Phytoplankton plays a vital role in primary production They also play an important role as food for herbivorous animals

(Reddy, et al., 2013) Zooplankton plays an essential role in water ecosystems

including river The planktonic animals take part in the transformation and

circulation of organic matter (Ejsmont-Karabin et al., 2004), regulate the biomass of phytoplankton (Lair, 2005; Kentzer et al., 2010) and provide food for fish, especially for their larval stages and for fish fry (Pourriot et al., 1997)

Furthermore, the phytoplankton serves as a producer in the food chain Their productivity depends upon the quality of water Many species of zooplankton are primary consumers and feed on phytoplankton, thus playing an important role in energy and transfer In a water ecosystem, the diversity of phytoplankton can

influence the diversity of zooplankton, or vice versa and both can be affected by the environment factors (Chou et al., 2011) Companies which produce goods and

directly discharge wastewater to the river are responsible for disturbance and diversity of plankton In fact, water quality is a strong determinate of phytoplankton and zooplankton dynamics, as well as diversity in aquatic system (Nasrollahzadeh

et al., 2008; Ramdani et al., 2009)

1.2 Aims of the study

1 To identify and measurement the diversity of planktons in Musi River, Palembang, Indonesia

2 To determine the relationship among planktons by using Correspondence Analysis

1 The activities of industries can influence the diversity species of planktons

2 Pollutants discharged by industries can influence the value of diversity indices of planktons

3 The types of industries which discharge wastewater will have different closeness relationship with plankton species

II Literature review

2.1 Introduction to water

2.1.1 The importance of water

Water has always been a vital material of Man’s existence It is used for drinking , cooking, agriculture, transport, industry, and recreation show immediately the extent

to which it is an integral part of our life (Hunt, et al.,1974) However, the abundance

and quality of the world’s freshwater resources are declining rapidly Changes in land use degrade natural freshwaters and reduce biodiversity by eliminating valuable

habitats and adding excess nutrients (Patrick, et al., 2012)

A lot of wastewater sources are caused of water pollution One of the important pollution sources in the pollution of the water environment is industrial wastewater There are many types of industrial wastewater based on different industries and contaminants, each sector produces its own particular combination of pollutants (Hanchang, 2009) With the rapid development of various industries, a huge amount of fresh water is used as a raw material So in fact the wastewater is an

“essential by-product” of modern industry, and it plays a majors role as a pollution sources in the pollution of water environment (Hanchang, 2009) As a consequence, the plankton populations of the river has been affected in terms of abundance and

diversity The water quality criteria with reference to freshwater bodies was

polluted by various industries ( Mathivanan, et al 2007)

Water pollution is the biggest menace of urbanization, industrialization and agricultural practices It leads to alteration in physical, chemical and biological properties of water bodies as well as that of the environment Chemical toxicants directly and indirectly affects the life processes of flora and fauna of the water body

(Kumari et al., 2006; Krishnan et al., 2007)

2.2 Plankton community

The study of plankton as an index of water quality with respect to industrial,

municipal and domestic pollution has been reported earlier (Acharjee et al., 1995, Jha et al., 1997) Planktons are microscopic organisms that float freely with oceanic

currents and in other bodies of water Plankton is made up of tiny plants (called phytoplankton) and tiny animals (called zooplankton) The word plankton comes from the Greek word "planktos" which means "drifting." Phytoplanktons are the autotrophic components of the plankton community and a key factor of oceans, seas and freshwater basins ecosystems The name comes from the Greek words φυτόν (phyton), meaning "plant", and πλαγκτός (planktos), meaning "wanderer" or

"drifter" Most phytoplankton are too small to be individually seen with the unaided eye However, when present in high enough numbers, they may appear as a green discoloration of the water due to the presence of chlorophyll within their cells (although the actual color may vary with the species of phytoplankton present due

to varying levels of chlorophyll or the presence of accessory pigments such as phycobiliproteins, xanthophylls, etc (Thurman, 2007)

“Phytoplankton are primary producers (also called autotrophs) As the base of the oceanic food web, phytoplankton use chlorophyll to convert energy (from sunlight), inorganic chemicals (like nitrogen), and dissolved carbon dioxide gas into

carbohydrates” Phytoplankters need a supply of inorganic nutrients from the water These they absorb from the water layer, a few micrometres thick, immediately in contact with the cell wall or membrane Molecules forces tend to preserve this layer intact and it soon becomes depleted of nutrients which are not rapidly replaces by diffusion alone Continuous movement of the cell through the water, as it sinks and

is retrieved by upwardly directed eddy currents, sloughs away the depleted nutrient shell and maintains a continual supply of undepleted water at the cell surface (Moss, 1979)

Plankton also contains zooplankton, “zooplankton are microscopic animals that eat other plankton Some zooplankton are larval or very immature stages of larger animals, including mollusks (like snails and squid), crustaceans (like crabs and lobsters), fish, jellyfish, sea cucumbers, and seastars (these are called meroplankton) Some zooplankton are single-celled animals, like foraminifera and radiolarians Other

zooplankton are tiny crustaceans, like Daphnia

In food web, “plankton is the first link in the marine food chain; it is eaten by many organisms, including mussels, fish, birds, and mammals (for example, baleen whales) The plankton encompasses an incredibly diverse group of organisms, ranging in size from viruses to large jellyfish, united only by the fact they are all weak swimmers and so are largely transported by the movement of the water, drifting about in the sea (John, 2012)

On details, fresh water phytoplankton includes single cells, colonies of cells and filaments (linear strings of cells) that are usually converts solar radiant energy

into biological energy through photosynthesis as primary production (Reddy, et al,

2013) The plankton community is a very dynamic one Not only are the relative

positions of all of its particles, live or dead, changing from second to second, but also dozens of chemical changes are going on simultaneously Phytoplankton is one

of the most rapid detectors of environmental changes due to their quick response to toxins and other chemical Pollution stress reduces the number of algal species but increases the number of individuals A marked change in the algal community severely affects the species diversity (Biligrami, 1988)

Phytoplanktons are the foundation of the aquatic food web, the primary producers, feeding everything from microscopic, animal-like zooplankton to multi-ton whales Small fish and invertebrates also graze on the plant-like organisms, and then those smaller animals are eaten by bigger ones Phytoplankton can also be the harbingers of death or disease Certain species of phytoplankton produce powerful biotoxins, making them responsible for so-called “red tides,” or harmful algal blooms These toxic blooms can kill marine life and people who eat contaminated seafood(Lindsey and Scott, 2010)

Through photosynthesis, phytoplankton consume carbon dioxide on a scale equivalent to forests and other land plants Some of this carbon is carried to the deep ocean when phytoplankton die, and some is transferred to different layers of the ocean as phytoplankton are eaten by other creatures, which themselves reproduce, generate waste, and die (Lindsey and Scott, 2010)

With zooplankton, the protozoa, rotifers and crustaceans are the major groups

of freshwater zooplankton (Moss, 1979) Zooplankton diversity is one of the most important ecological parameters in water quality assessment The distribution of zooplanktons depends on a complex factors such as, change of climatic condition,

physical and chemical parameters and vegeration cover (Salve et al., 2013) The

importance of zooplankton as food resource of fish is not limited to pelagic planktivorous species The survival of the young herbivorous fishes such as tilapia may depend on the availability of abundant littoral zooplankton and benthos aided

by omnivorous and high ecological efficiencies Planktonic fish larvae prey on zooplankton and occasionally phytoplankton Several families of fish consume zooplankton wholly or partly in various stage of their life histories (Lazzaro, 1987) Zooplankton are thus crucial to achieving high fish yields in the tropics even if their role seems to be mainly through the young stages of fish (Fernando, 1994) Zooplankton is one of the fascinating groups of microorganism found in the aquatic environment They include a varied assemblage of taxonomically unrelated organism, their common ecological characteristic being their habit, are found freely

drifting in epilimnion (Salve et al., 2013) Zooplanktons are also sensitive to various

substances that enrich or pollute water They have been used as indicators to monitor and assess the condition and change of the fresh water environment Partic Zooplankton have been frequently used as ecotoxicological test organisms to assess the acute chronic effect of various toxic substances that are found in the freshwater environment

2.3 Impact of wastewater in plankton community

The impacts of wastewater in plankton are physico-chemical (such as pH, temperature, salinity, and various inorganic nutrients) and biological factors which are often identified as the major factors influencing the dynamics of plankton

(Ramdani et al 2009) Plankton are very sensitive to the environment they live in

and any alteration in the environment leads to the change in the plankton communities in terms of tolerance, abundance, diversity and dominance in the habitat Therefore, plankton population observation may be used as reliable tool for biomonitoring studies to assess the pollutionstatus, of aquatic bodies (Mathivanan and Jayakumar, 1995)

However, impact of wastewater in plankton is very clear The River and marine receive pollution from different sources in all countries along the River, but especially from industrialized areas Industrial wastewater contributes most pollutants to the River However, oil pollution, municipal wastewater and agriculture pollution also contribute heavy metals, pesticides, herbicides, and microbes to the water (El-Sheekh, 2009) Poisons are frequently present in freshwater at concentrations too low to cause rapid death directly but they may impair the functioning of organisms These sublethal effects may be observed at the biochemical, physiological, behavioral or life cycle level Many small changes in these parameters have been related to pollution but it is essential to show that they have ecological meaning, that they reduce the fitness of an organism in its environment and are not merely within its range of adaptation The biochemical effects of pollution are basic, and these can then be related to the efficiency of tissues and organs, which can in turn be examined in relation to the performance of the organism and whether this has any adverse effect on the natural population (Sprague, 1971)

At the ecosystem level, natural water bodies may be described by the properties (Gross Primary Production, Autotrophic Respiration, Net Primary Production, Heterotrophic Respiration, Net Ecosystem Production, Ecosystem Respiration, Production Efficiency, Effective Production, Maintenance Efficiency,

Respiration Allocation, Ecosystem Productivity) These properties are associated with rates of energy utilization, nutrient cycling, predator-prey relationships, and size of the energy reservoir within the system A pollutant that affects anyone of them can affect the others in relation to the resiliency of the system

Many studies on algal species and specific pollutants have been published Most have described effects upon population growth or photosynthesis and indicate that, generally, algae are as sensitive to pollutants as animals Growth and photosynthesis are closely related, each being a function of the utilization of light and nutrients (Dugdale, 1975) described the growth of an algal population as being proportional to the effect of light on photosynthesis (Yentsch, 1974), the concentration of nutrients, and the maximum specific growth rate

Population pressure, urbanization, industrialization and increased agricultural practices have significantly contributed to the pollution and toxicity of aquatic ecosystems Pollutants bring about a change not only in physic and chemical quality of water but also modify the biotic components, resulting in the

elimination of some, probably valuable, and species (Desai, et al., 2008)

Pollutants may also affect species composition of the plankton community Adult zooplanktons are affected by pollutants and accumulate relatively large amounts Uptake occur either through ingestion of contaminated food or directly from water Varying sensitivities to pollutants among zooplankton species could cause changes in community structure by affecting variables such as rate of increase, rate of predation, mortality, and population density (Walsh, 1978)

The quantitative and qualitative abundance of the phytoplankton and zooplankton were significantly correlated by existing water quality conditions

Phytoplankton and zooplankton are good indicators for changes in nutrient pollution over time because they respond quickly to changes in nutrient input to aquatic ecosystem The biological analysis of coastal waters, especially the phytoplankton analysis will describe clearly about the pollutant materials impact on the aquatic life and a decrease in biological diversity Furthermore, the phytoplankton will reflect the condition of the waters, not only at the time of sampling, but also the condition

at a previous time point Moreover, planktons are sensitive to many environmental conditions such as salinity, rainfall, temperature, dissolved oxygen levels, turbidity, and other factors (Harris and Vinobaba , 2012) On the other hand, zooplankton is the most important components of the aquatic ecosystem, playing a major role in energy transfer between the phytoplankton and the economically important fish populations The abundance of zooplankton depends on a great variety of abiotic and biotic factors, which collectively affect individual species of the zooplankton community Generally zooplankton populations were poor compared to phytoplankton Many environmental factors can affect zooplankton assemblages, including water temperature, nutrient concentrations, and salinity (Harris and Vinobaba, 2012)

With water analysis, samples needed to be collected Plankton samples were collected by standard methods (APHA, 1989) from predetermined sampling sites from the point of effluent outfall along with the downstream water stretch The collected samples were fixed in 3-4% Lugol’s solution and brought to the laboratory for plankton analysis Counting and identification were done as per APHA (1989) Species diversity index was obtained by following Shannon Weaver methodology (Nath, 1997)

Planktons play an important role in water ecosystem which are very sensitive with changing environment However, the environmental conditions are different among areas, rivers and wastewater sources So, the adaption level of plankton communities is also different The study will emphasize the effluence of industrial wastewater into plankton communities

III Methodology

Musi River (figure 1) locates in South Sumatra, Indonesia Its long is 750 km and it is biggest River on South Sumatra Island This River divides Palembang city into two parts The River has an average depth of about 6.5 meters, suitable for large ships can go into inland and far Palembang, where there is a major port with many functions, including the export sector oil, rubber and coal

Figure 1: Musi River

River brings an enormous benefit about economic for Palembang and Indonesia However it also affected to all organisms that live in this River, in a long period, aquatic ecosystem will become damage and people who live on Musi River also can affect

Six stations in Musi River were chosen to take samples These stations were big companies which discharged chemical and wastewater to the river Those stations were:

Station 1: Soy Sauce Industry

With soy sauce industry, the main substances of soy sauce wastewater was salt (about 10.000-50.000mg/L) which keeps a high level of salinity Furthermore, wastewater from Soy Sauce Industry had high protein concentration because of fermentation process which contributed to strong smell on wastewater

Station 2: Crumb Rubber

In the second station, because all production need to add acid on processes , crumb rubber wastewater had high acid concentration characteristic

Station 3: Ship Dock

Waste generated from the ships in dock bilge water (water that collects in the lowest part of the ship’s hull and may contain oil, grease, and other contaminants), sewage, gray water (wastewater from showers, sinks, laundries and kitchens), ballast water (water taken on board or discharged from a vessel to maintain its stability), and solid waste (food waste and garbage) The potential environmental impacts of these waste streams generates should be concerned

Station 4: Drinking Water Company Installation (PDAM)

PDAM Tirta Musi provides water supply to 1.2 million people in the city of Palembang, Indonesia Drawing water from the Musi River, the PDAM channels the raw water through six waterworks and distributes treated water through several stations

Station 5: Cement Company

Cement wastewater has a high pH level that is normally range within 11 and

12 (the standard of pH level of fresh water is 6-7)

Station 6: Stockpile

This stockpile is used to store coal which wastewater discharged several heavy metals such as manganese, phosphorous, and other metal with high Total Suspended solid (TSS) The amount of wastewater from washing coal in stockpile lead to acidic wastewater (high pH) which can influence aquatic organisms(Sari 2013)

3.1 Collection and sample

3.1.1 Place and time

For the present study, water samples were collected from 6 stations on Musi River From the first observation, water samples were taken from 8.00 am to 11.00 am, on November 7th 2014 from station 1 (Soy sauce) to station 6 (Stock pile) With the second observation, water samples were taken from 8 am to 11 am, on November 16, 2014 Place: Musi River at 6 stations: Soy Sauce, Crumb Rubber, Ship Dock, PDAM, Cement and Stock Pile

3.1.2 Materials and Equipments

1 6 plastic containers (1 liter)

2 6 glass bottles (250 ml) which were dark and thick glass to keep water and planktons couldn't be affected by sunshine

3 Film bottles for plankton

4 Plankton net

5 Label paper

6 Chemical: Lugol solution

7 Pipette tube

8 Ice box which used to keep all samples in a cold environment

9 Ice which was necessary to keep environment inside ice box around 4 degree to protect sample before analysis in the laboratory

3.1.3 Plankton samplings

The first step, planktons net was tied with film bottles for plankton

The second step, water was taken by bucket, which was filtered through a plankton net in order to obtain one sample of plankton, 20 liters of water were filtered All samples were preserved with 3 drops of Lugol solution

The third step, label papers sticked on all samples

3.1.4 Water samplings

The first step, plastic containers used to take sample for BOD5, which needed

to keep under River water and take full bottle without bubble Covering a slight plastic before closing lids

The second step, glass bottles were taken water sample to analysis for TSS, COD chemical oxygen demand, DO and pH All samples were taken from the depth

of 0.5 to 1m using standard methods

The third step, sticking label paper on all samples

The last, all sample kept in ice boxes

3.2 Tools and Identification process

3.2.1 Tools

All tools were used to identify planktons which contained:

1 Microscope

2 Glass

3 Pipette tube

4 Tissue

3.2.2 Identification plankton process

For this process, all samples were sharked before identifying

Pipette tube was used to take water inside the sample before dropping two drops ( 6ml) onto slides , after that wastewater put under Microscope at objective of 10x which used to identify planktons

When identification planktons were implemented under Microscopes, all shapes of planktons which compared to picture on 2 books “An illustrated guide to River Phytoplankton” (Belcher and Swale, 1979), and “The marine and fresh-water plankton” by Davis, 1955 After that, name of the plankton was indentified

Interpret data: Number of planktons counted in samples is number of plankton found in 6ml, which needed to be converted to the population of planktons into 100ml 6ml-number of plankton

2 COD: chemical oxygen demand is amount of oxygen needed to oxidize the chemical compounds in water, including inorganic and organic

Method which uses for COD is oxidation titration with KMnO4 Chemical oxygen demand is amount of oxygen which has KMnO4 used to oxidant organic matter in water It indicates the mass of oxygen which consumed per liter in the solution

3 TSS (Total Suspended Solids): The total amount of organic matter and inorganic suspended (silt, organic detritus, algae) suspended in water Concentration

of Total Suspended Solids or solids deposition is likely indicator of the degree of water pollution

Method of determining: TSS was determined by the method of weight

Conduct quantitative:

Dry the filter paper at a temperature of 105oC for 2 hours

Weighing just finished drying the filter paper (m1 /mg)

Filtered water sample volume through the filter paper which was determined volume

m1 = initial weight of filter paper (mg)

m2 = Weight of filter paper and filter material composition (mg)

V = volume of sample straining water (ml)

1000 = coefficient changed to 1 liter

4 DO is the dissolved oxygen in the water needed for respiration of aquatic organisms To identify DO, the method needs to use is electrode dissolved oxygen with oxy measuring machine

DO electrode meter operates under the principle that appear in the electrode is proportional to the amount of oxygen dissolved in the water diffuses through the membrane electrode, during which oxygen diffuses through the membrane is proportional to the concentration of dissolved oxygen Measure amperage allows appears determined DO

5 pH is measured by a machine, sensor head need to put into pure water which concentration of pH is 7 before measuring pH in wastewater sample

IV Results and Discussion

4.1 Results of water analysis in 6 stations

From water analysis, the study has been conducted in 6 stations, which show differently results among tables 1, table 2, table 3, table 4, table 5, and table 6

Table 1 showed water analysis of TSS, COD, BOD5 , DO and pH Indeed, TSS

in 2 observations was satisfied SNI standard with 50 mg/l pH and DO also reached standard from 6-9 with pH and approximately 6 mg/l with DO However, COD fluctuated widely from 2 mg/l to 19 mg/l in 2 observations with only 10 mg/l of SNI standard Furthermore, BOD5 increased 3 times with 5.5 mg/l in the first observation

Table 1: Water analysis in soy sauce industry

The maximum level of the River water class I

Result

Of the first time 7/11

Result

of the second time 16/11

Examination methods