Blood and Micronuclei Analysis of Tawes Fish (Barbonymus gonionotus) Correlation with Water Quality in the Boon Pring Ecotourism Area, Turen, Malang Regency

Tawes (Barbonymus gonionotus) is a type of endemic fish that lives in the Boon Pring Reservoir, Turen, and Malang Regency. This study aims to analyze the water quality and blood profile of Tawes Fish (Barbonymus gonionotus) in Boon Pring Ecotourism and to determine the relationship between water quality and fish health through their blood profile using the Canonical Correspondence Analysis (CCA) method. The results obtained after measuring the water quality during two samplings at both locations were that the temperature ranged from 21.7–27C, TDS 111–145 mg/l, TSS 2-4 mg/l, pH 6-6.7, DO 8–10.1 mg/l, BOD 0.44-0.98 mg/l, and ammonia 0.026–0.075 mg/l. Both locations are classified as having good or normal conditions. Observations on the blood profile of Tawes fish (Barbonymus gonionotus) are healthy and normal. The average number of erythrocytes was 2,586,667 cells/mm3, leukocytes were 60,500 cells/mm3, lymphocytes were 76.8%, monocytes were 1.4%, neutrophils were 3.3%, hemoglobin was 9.8 g/mL, hematocrit was 34.6%, and micronuclei were 12.3 cells/1000. The results of data analysis using CCA showed that all blood and micronuclei profiles of fish were affected by water quality parameters, both significantly and moderately to severely.


Introduction
Boon Pring is an ecotourism area located in Sanankerto Village, Turen District, Malang Regency, East Java which has an area of around 36.8 hectares [1].Boon Pring is an ecotourism region that focuses on education, empowerment, and conservation, with a strong commitment to environmental sustainability.Before becoming a quite popular tourist attraction, this location was a bamboo forest that had more than 60 types of bamboo [2].This tourist attraction is separated into two sections, namely the ecotourism area and the conservation area.The ecotourism area used by tourists to play water bikes and and renting motorboats for the purpose of exploring the lake.Furthermore, Boon Pring boasts picnic spaces and swimming pools that are extensively employed for family vacations.Meanwhile, in the conservation areas, specific regions are designated to safeguard and sustain the population of native fish species, ensuring their continued survival.There is a daily rise in human activity in Boon Pring.The influx of tourists to this location is steadily growing on a daily basis.In 2017, the number of visitors to the Boon Pring ecotourism attraction was documented to be at least 76 thousand.In 2018, the approximate number of tourists increased to around 90 thousand [3].This leads to the enhancement of Boon Pring's service facilities and infrastructure by the management.The expansion of these services and infrastructure may compromise the ecological integrity of Boon Pring as a protected area for bamboo forests and endemic fish especially on water quality.Alterations in water quality directly affect the survival of the aquatic organisms inhabiting it [4].Therefore, this needs to be studied to determine its impact on the aquatic environment.One way to determine the degradation of the aquatic environment is to use bioindicators [5].
Fish are a type of aquatic species that can serve as bioindicators for detecting alterations in water quality [6].Fish are easily found organisms that predominantly inhabit freshwater river habitats [7].The Tawes fish (Barbonymus gonionotus) is an endemic fish species exclusively located in the Boon Pring ecotourism region.Tawes fish often inhabit rivers characterized by strong currents, because this fish has biological traits that necessitate a high level of oxygen and dwells in freshwater with tropical temperatures [8].The fish possesses a body that is somewhat flattened and elongated, allowing it to adapt to the swift currents of its natural habitat [9].The blood profile of tawes fish (Barbonymus gonionotus) serves the purpose of facilitating adaptability and ensuring the fish's survival, without exerting any influence on its genetics.The blood profile serves as a reliable signal for detecting alterations in water quality [10].Furthermore, an examination of the physical, chemical, and biological factors is conducted to get comprehensive data regarding the state of the water.
Therefore, it is important to evaluate the health of tawes fish (Barbonymus gonionotus) based on blood profiles in the Boon Pring ecotourism area, Malang Regency, East Java in connection with changes in water quality including temperature, pH, DO, BOD, ammonia, TDS and TSS.The aims of this research are 1) to analyze the water quality of the Boon Pring ecotourism area, Malang Regency, 2) to analyze the blood profile of tawes fish (Barbonymus gonionotus) in the Boon Pring ecotourism area, Malang Regency, 3) to investigate the correlation between water quality and the blood profile of tawes fish (Barbonymus gonionotus) in the Boon Pring ecotourism area, Malang Regency.

Study area and sampling sites
The study was conducted from March to May 2023 in the Boon Pring ecotourism area.The sampling was conducted in two specific places, the ecotourism area and the conservation area, which accurately reflect the water conditions.Two times of fish and water sampling were conducted within a one-month period, with a duration of two weeks between each time.The method used in this research is descriptive and uses purposive sampling techniques.The research sampling station are presented in the Figure 1.

Water quality measurements
The water quality measurements in this research include the parameters of temperature, pH, dissolved oxygen (DO), Biological Oxygen Demand (BOD), Total Suspended Solids (TSS), Total Dissolved Solids (TDS), and ammonia (NH3).The techniques and tools employed for assessing water quality are outlined in Table 1 Ammonia (ppm) Spectrophotometer

Data analysis
The Canonical Correspondence Analysis (CCA) approach is employed to examine the link between water quality and fish blood samples.Canonical Correspondence Analysis (CCA) is a widely employed multivariate analysis technique that offers insights into the correlation between biological species and their surroundings [11].The CCA analysis was conducted using the PAST 4.03 program.Canonical Correspondence Analysis (CCA) is a statistical technique used to examine and evaluate the association between a group of independent factors and a group of dependent variables.It aims to identify and measure the influence of these variables on each other [12].The research focuses on water quality, specifically examining variables such as temperature, pH, dissolved oxygen (DO), biochemical oxygen demand (BOD), total suspended solids (TSS), total dissolved solids (TDS), and ammonia.The fish blood profile variables being studied in this research include erythrocytes, leukocytes, leukocyte differentiation, hemoglobin, hematocrit, and micronuclei.

Erythrocytes
The procedure for observing red blood cells (erythrocytes) in fish is done by taking a fish blood sample using an erythrocyte pipette to a limit of 0.5, then adding hayem solution as a dye solution to the fish blood sample until the pipette limit reaches 101 and stirring the blood until it is evenly mixed by means of swing the pipette by 10 to form a figure eight.Next, throw away the first three drops, then put the remaining solution into the hemocytometer and cover with a cover glass.The final step is to count erythrocytes under a microscope by counting erythrocyte cells in 5 fields of view with 400x magnification [13].= dilution factor 0,004 = konstanta

Leukocytes
The procedure for observing white blood cells (leukocytes) in fish is carried out by taking a sample of fish blood from an Eppendorf tube using a leukocyte thoma pipette to a scale of 0.5, then adding Turk's solution to a scale of 101 and stirring the blood until it is evenly mixed by swinging the pipette in a figure eight shape.Next, remove the first two drops of blood solution from the leukocyte thoma pipette in order to remove air bubbles.After that, add the blood solution to the hemocytometer by dripping it and then cover it with a cover glass.The final step is to observe white blood cells with a microscope with 400x magnification.Leukocyte counting is done by counting cells in 4 fields of view [14].

Leukocyte Differentiation
The procedure for observing white blood cells (leukocytes) in fish is carried out by preparing an object glass and a blood sample of the fish to be observed.Hold the first glass object using your thumb and forefinger, then drop a little blood on the right and place the second glass object at an angle of 300 on the left.Pull the glass object until the blood spreads along the edge of the second glass object to make a blood preparation.Dry the blood on the glass object with air fixation.Fix the glass object with methanol for 5 minutes, then soak the blood preparation in Giemsa solution for 15 minutes.Rinse the blood preparation using distilled water, then observe under a microscope with 400x magnification.Perform calculations of leukocyte differentiation [14].

Hematocrit
Observation of hematocrit values can be done using the microhematocrit method with a procedure, namely the first step is to prepare a fish blood sample and a microcapillary tube, then fill the microcapillary tube with ¾ of the fish blood sample and close the end of the tube with a covering material (wax).Next, put the tube in a hematocrit centrifuge machine for 3 minutes at a speed of 5000 rpm.Then, turn off the machine and remove the tube from the hematocrit centrifuge machine.The final step is to measure the hematocrit value using the microhematocrit scale and the results are expressed in percent (%) [15].

Haemoglobin
The data analysis method used to analyze the correlation between water quality and fish blood profiles is preparing fish blood samples and sahli pipettes.Prepare the Hb-meter tube, then fill the tube with 0.1 N HCl up to a scale of 10.Put the fish blood sample into the Hb-meter tube using a sahli pipette with a scale of 0.2 ml or up to a scale of 20 mm 3 .Stir the fish blood sample for 3-5 minutes with a stir stick.Add distilled water to the Hb-meter tube until the blood color changes to the color of the standard solution in the Hb-meter tube.Observe the hemoglobin scale on the gr% line (yellow) [16].

Micronuclei
Micronuclei observations were carried out by taking fish blood samples and making blood smear preparations on a glass object, then drying them, then fixing them with 95% methanol solution and then drying them.After drying, stain 50% Giemsa for 2 minutes and rinse using distilled water then dry again.Observe under a microscope with 1000x magnification [17].

Result and Discussion
Water quality parameters serve as supplementary data in the estimation of contamination.The blood profile of tawes fish is influenced by water quality characteristics.Consequently, unfavorable water conditions will have an adverse impact on the health of tawes fish in the Boon Pring Ecotourism region.

Water Quality
The results of water quality measurements carried out at 2 research locations are presented in Figure 2.
The temperatures recorded at location 1, namely the Ecotourism region, were 27 °C and 22.4 °C.The temperature readings recorded at position 2, namely the Conservation area, were 27 °C and 21.7 °C.The ideal temperature range for the survival of Tawes fish is 24 -27 °C [16].These findings indicate that the water temperature at both observation sites remains conducive to the survival of Tawes fish.Typically, every fish species has a certain temperature range that is most suitable for their optimal growth and activity.While fish are capable of surviving in a broad range of temperatures, too low or high temperatures can result in the demise of the fish.Elevated temperatures can induce stress in fish due to a reduction in dissolved oxygen levels in the water.Elevated temperatures can induce alterations in hematocrit and hemoglobin concentrations in the bloodstream.The reason for this is the inefficiency of oxygen transport, which leads to a significant rise in fish metabolism [19].
The pH parameter values at position 1 varied from 6 to 6.6, while at location 2 they ranged from 6.7 to 6.The pH value of 6.7 was recorded at position 2, indicating the maximum alkalinity.There was a decline in pH at both locations, but this change was not particularly substantial.The pH levels obtained are considered within the normal range, despite experiencing oscillations, and are conducive to the survival of Tawes fish.The optimal water acidity level for fish growth is greater than 5 mg/L [20].
The DO values at position 1 ranged from 8 mg/L to 9.8 mg/L, whereas the DO levels at location 2 were approximately from 9.6 mg/L to 10.1 mg/L.As to Government Regulation Number 22 of 2021, the water quality requirements classify the optimal amount of DO parameters as class 2, with a minimum requirement of 4 mg/L.These findings indicate that the DO levels at both monitoring stations remain below the established quality criteria, posing a potential risk to aquatic life.The activity of microorganisms in converting organic molecules into inorganic ones is the primary factor that can affect DO levels in water.In addition, DO levels can be affected by several parameters including temperature, salinity, organism respiration, water turbidity, water turbulence, easily oxidizable chemicals, and air pressure [21].
The BOD value measured at site 1 ranged from 0.83 to 0.98 mg/L.The BOD value measured at site 2 ranged from 0.44 to 0.62 mg/L.According to Government Regulation Number 22 of 2021, which deals with the execution of environmental protection and management, the highest permissible BOD value for class II quality requirements is 3 mg/L.These findings indicate that the BOD values at both observation sites remain below the established quality requirements, ensuring the safety of aquatic species.An elevated BOD level can suggest a deficiency of dissolved oxygen in a water body.Elevated amounts of organic matter in water can contribute to the increase in BOD levels in water [22].
The TSS parameter values at position 1 varied between 3 and 4 mg/l, while at location 2 they ranged from 4 to 2 mg/l.According to Government Regulation Number 22 of 2021, the highest permissible level of TSS for class II spring water is 50 mg/L.This indicates that the TSS findings obtained are within the expected range and appropriate for the survival and well-being of Tawes fish.The TDS values measured at the two sampling locations during two sampling events at location 1 were 145 mg/L and 123 mg/L, respectively.At location 2, the TDS levels were 111 mg/L and 112 mg/L.As per Government Regulation Number 22 of 2021, the ideal level of the TDS criterion in water quality standards falls within class 2, namely at 1000 mg/L.These findings indicate that the TDS levels at both monitoring sites remain below the established quality limits, ensuring the safety of aquatic life.The level of TDS in water is directly correlated with the clarity of the water.Elevated levels of TDS in water lead to decreased penetration of sunlight, hence reducing photosynthesis and ultimately leading in a decline in aquatic production.Elevated TDS can also impact the hue of the water's surface, as it contains several particles that absorb light [23].The ammonia readings at position 1 yielded a concentration of 0.049 ppm, while at location 2, the concentration was 0.026 ppm.For the second measurement, the recorded ammonia levels were 0.075 ppm at site 1 and 0.032 ppm at location 2. According to Regulation PP no.22 of 2021, the acceptable concentration of ammonia in class II water quality is 0.2 ppm.Therefore, it may be inferred that the ammonia levels from both locations are still within the expected range.Ammonia is derived via industrial waste, animal excretion processes, decomposition of microbes, and similar sources.Fish and other aquatic organisms typically generate ammonia through protein metabolism or gill discharge.Fish possess a physiological system for the elimination of ammonia to prevent its accumulation within their bodies, hence averting toxicity.Elevated amounts of ammonia can lead to a decrease in hematological parameters, including hematocrit and hemoglobin [24].

Profile of Blood Tawes Fish (Barbonymus gononiotus)
The results of blood profile examinations carried out on Tawes fish are presented in Figure 3.This examination encompasses the assessment of total erythrocytes, total leukocytes, leukocyte differentiation, hemoglobin (Hb), hematocrit, and micronuclei.The total number of erythrocytes in Tawes Fish at position 1 varied between 2,200,000 and 2,970,000 cells per cubic millimeter, while at location 2, it ranged from 2,370,000 to 2,860,000 cells per cubic millimeter.The mean number of erythrocytes of Tawes Fish in sampling 1 and sampling 2 at both locations was 2,615,000 cells/mm3 and 2,558,333 cells/mm3, respectively.The usual range of erythrocytes in teleost fish is between 1.05 and 3.0×106 cells/mm3 [25].The erythrocyte count in Tawes Fish in both locations was within the usual range for teleost fish.The abundance of erythrocytes in fish is highly influenced by environmental factors such as temperature, dissolved oxygen concentrations, pH, and salt.In addition, the quantity of erythrocytes in fish is influenced by internal factors like species variation, age, gender, and reproductive condition.Alterations in environmental conditions [26] can lead to a reduction or elevation in erythrocyte levels.
The leukocyte count or number of white blood cells in Tawes fish at position 1 varied between 39,000 and 57,500 cells per cubic millimeter.At site 2, the total leukocyte count of Tawes Fish ranged from 65,000 to 80,000 cells per cubic millimeter.The mean leukocyte count in sampling 1 was 58,500 cells per cubic millimeter at both locations, while in sampling 2 the mean count was 62,500 cells per cubic millimeter.The leukocyte count in fish varies from 20,000 to 150,000 cells per cubic millimeter [27].These findings indicate that the leukocyte or white blood cell counts of Tawes fish from both observation sites are within the expected range.Elevated leukocyte count might result from the fish's physiological reaction to unfavorable environmental conditions, disease pathogens, and stressors.An exceptionally significant rise in the overall leukocyte count, or white blood cells, may suggest the presence of an anomaly in the fish, namely an autoimmune occurrence within the body.Conversely, a reduction in the overall leukocyte count might result from disruptions in the fish's kidney and lymphatic system, which impede the production of leukocytes due to infectious infections [28].
The leukocyte differentiation investigation of Tawes fish revealed the presence of lymphocytes, monocytes, and neutrophils.The percentages of lymphocytes, monocytes, and neutrophils in Tawes fish at position 1 varied from 76-78%, 1-2%, and 3-4%, respectively.At location 2, the percentages ranged from 74-79%, 1-2%, and 3-4% for lymphocytes, monocytes, and neutrophils, respectively.The average percentages of lymphocytes, monocytes, and neutrophils at Location 1 were 76.7%, 1.5%, and 3.3%, respectively.The average percentages of lymphocytes, monocytes, and neutrophils observed at site 2 were 77%, 1.3%, and 3.3%, respectively.In teleost fish, the typical percentage of monocytes is below 5%, whereas the proportion of lymphocytes ranges from 60% to 80%.Additionally, the proportion of neutrophils is approximately 6% to 8% of the total leukocyte count [15].These findings indicate that the lymphocyte, monocyte, and neutrophil counts seen in this investigation fell within the normal range.Lymphocytes serve as immunological effectors, acting as a protective mechanism against the invasion of foreign entities into the body [29].The drop in monocyte count is attributed to the fish's overall health, which eliminates the requirement for monocyte cells to engage in phagocytosis.This is because there is no infection or foreign items stimulating the production of monocytes [30].The drop in the proportion of monocytes is attributed to an increase in the number of other types of leukocytes, specifically lymphocytes, resulting in a shift in the blood balance.It is worth noting that the monocyte nucleus exhibits variations in structure, with some resembling a kidney and having two big lobes [15].The cytoplasmic volume typically contains less than 50% of the monocyte nucleus.A reduction in neutrophil count suggests that the fish is in a healthy state.This suggests the absence of any microbial contamination [30].The hematocrit levels of Tawes Fish (Barbonymus gonionotus) at position 1 varied between 26% and 37%, while at location 2 they ranged from 26% to 45%.The mean hematocrit levels of Tawes Fish (Barbonymus gonionotus) at the two observed sites during sampling 1 and 2 were 31% and 39%, respectively.The hematocrit value in teleost fish varies between 22% and 60% [31].The hematocrits collected from observations on Tawes Fish (Barbonymus gonionotus) from both locations were deemed satisfactory.Hematocrit refers to the proportion of red blood cells within the total volume of blood in a fish.A hematocrit score below 22% signifies anemia in the fish.Fish exhibit low hematocrit as a physiological reaction to alterations in environmental circumstances or contamination [32].The variation in hematocrit levels in the fish's body is determined by the size and quantity of erythrocytes present.Pathogenic germs can also contribute to a drop in the hematocrit number [33].
The hemoglobin levels of Tawes Fish at position 1 varied between 8 and 13.8 G%, while at location 2, they ranged from 8 to 12.2 G%.The mean hemoglobin concentration of Tawes Fish in sampling 1 and sampling 2 at both locations was 11.3 G% and 8.5 G%, respectively.The typical range for hemoglobin levels in fish is 2.0-14.0g/dL [34].The hemoglobin levels in a fish's body can be affected by various factors, including species, age, gender, physical conditions, season, air pressure, and lifestyle behaviors [35].The concentration of hemoglobin in red blood cells is directly correlated with the number of erythrocytes, as hemoglobin serves as the pigment within these cells.A high hemoglobin level within the usual range suggests a healthy fish with abundant oxygen, protein molecules, and iron [36].
The count of micronuclei in Tawes fish at site 1 varied between 8 and 22 cells per 1000.At site 2, the number of micronuclei in Tawes fish ranged from 8 to 16 cells per 1000.The mean number of Tawes fish micronuclei in sampling 1 was 14 cells per 1000 at both locations, while in sampling 2, the average value was 10 cells per 1000.The normal fish often have a range of 8 to 23 cells per 100 micronuclei [25].These findings indicate that the quantity of micronuclei in Tawes fish from both observation locations remains within the expected range.Micronuclei are chromosomal fragments that do not successfully integrate into the cell nucleus during the process of cell division [37].Micronuclei are an indirect indicator of chromosomal damage.Micronuclei arise from mitotic abnormalities.The incidence of micronuclei can fluctuate based on the specific pollutant and the kind of fish.Elevated micronuclei count indicates chromosomal structural disturbance in the organism, which can be attributed to several sources.An influential issue is the escalating level of environmental contamination.Polluted waters have a higher likelihood of causing mutagenic disturbances compared to pure waters [38].

Relationship between levels of water quality and blood profile
The research was conducted by employing the Canonical Correlation Analysis (CCA) method to analyze the data, as depicted in Figure 4.A Canonical Correspondence Analysis (CCA) was conducted to examine the impact of water quality, as independent environmental parameters, on the conditions of hematological parameters, which serve as dependent variables.This relationship is visually depicted on the two axes of the diagram [39].The length of the line representing the environmental parameter reflects the strength of the correlation, while the direction of the line indicates its association with the dependent variable.There is a positive correlation between environmental parameters situated in the same direction as the dependent variable, and conversely [40].The fish erythrocytes, is significantly affected by optimal circumstances of many water quality indicators.These factors include temperature, TSS, TDS, pH level, DO, BOD, and ammonia concentration.The leukocyte dependent variable is typically affected by high DO levels, as well as moderate to low temperatures, TDS, BOD, ammonia levels, pH, and TSS.The abundance of lymphocytes and neutrophils is affected by water parameters with moderate to low dissolved oxygen levels, high total suspended solids, elevated ammonia concentrations, high pH values, increased temperature, high biological oxygen demand, and elevated total dissolved solids.The abundance of fish monocytes is affected by moderate to low levels of ammonia and high levels of TSS, TDS, BOD, temperature, DO, and pH.Elevated levels of TDS, BOD, temperature, pH, TSS, ammonia, and reduced DO have an impact on hemoglobin levels and the occurrence of micronuclei in fish.The hematocrit value is affected by TSS, total dissolved solids TDS, ammonia concentration, pH level, temperature, moderate to low BOD, and high DO levels.Hematological characteristics serve as a toxicity index for monitoring the aquatic environment [41].The utilization of blood parameters to unify physiological and pathological changes in fish is both effective and highly sensitive.Blood serves as a vital diagnostic tool for assessing health, as it exhibits varying levels of fish blood response and is influenced by diverse stress conditions [42].

Conclusion
The water quality measurements conducted during two sampling sessions at the two locations were categorized as either good or normal conditions, in accordance with the quality criteria outlined in PP Number 22 of 2021, specifically for class II quality standards.The blood profile of Tawes Fish (Barbonymus gonionotus) shows healthy and normal values.The mean erythrocyte count was 2,586,667 cells/mm3, leukocyte count 60,500 cells/mm3, lymphocyte percentage 76.8%,monocyte percentage 1.4%, neutrophil percentage 3.3%, hemoglobin level 9.8 gram%, hematocrit level 34.6%, and micronucleus count 12.3 cells/1000.The data analysis utilizing CCA indicates that erythrocytes are influenced by favorable water quality circumstances as a whole.Leukocytes are affected by elevated dissolved oxygen levels, temperature, total dissolved solids, total suspended solids, biochemical oxygen demand, pH, and moderate to low levels of ammonia.The abundance of lymphocytes and neutrophils is affected by elevated levels of TSS, ammonia, pH, temperature, BOD, and TDS.Additionally, their abundance is regulated by conditions of moderate to low DO.The abundance of fish monocytes is typically affected by TSS, TDS, BOD, DO, temperature, elevated pH levels, and ammonia concentrations ranging from moderate to low.High levels of TDS, BOD, temperature, pH, TSS, ammonia, and moderate to low levels of DO have a tendency to affect hemoglobin and micronuclei.Hematocrit levels are affected by high DO levels, as well as moderate to low levels of TSS, TDS, ammonia, temperature, pH, and BOD.

Figure 4 .
Figure 4. Result of CCA (Canonical Correspondence Analysis) between water quality and Tawes fish hematological profile

Table 1 .
. Water Quality Measurement Methods and Instruments