Kenanga Sari1*, Jafron W Hidayat1,Jerry R. Miller2, Tri Retnaningsih Soeprobowati1,3
1Department Biology, Faculty Sciences and Mathematics Diponegoro Univeristy, Jl. Prof. Soedarto, SH, Kampus Tembalang, Semarang 50275 Indonesia
2Department of Geosciences and Natural Resources, Western Carolina University,Cullowhee, North Carolina 28723, USA 3School of Postgraduate Studies, Diponegoro Univeristy, Semarang 50241, Central Java, Indonesia
*Corresponding author e-mail: [email protected], [email protected]
Diatoms are good indicators of ecological conditions in aquatic ecosystem. The objective of this study was to explain the relationship between diatom and heavy metal in telaga pengilon Dieng and to assess responses of diatoms to changes water quality in tropical lake Indonesia. A sediment was devided into fourteen stratigraphy sequence of 160 cm in length was cored from edge site from the lake close with Warna Lake. the sites were selected to evaluate the heavy metals (Pb, Cr, Cd, Al, Zn,) on sediment and the associated with diatom communities. Redundancy Analysis (RDA) was used to determine environmental gradients along which species vary with heavy metal variables. A total 103 taxa and varieties belonging to 25 genus were identified. Assemblages were dominated by Eunotia 49,7%, Pinnularia 18,1% and Frustulia about 5%. Multivariate analyses indicated that distribution and variation of diatom are strongly associated with heavy metal (0.013, p < 0,05). Furthemore,heavy metal played significant role in structuring diatom community in the Telaga Pengilon and variation in diatom distribution was best explained by) Al (0.031, p< 0,05) and Pb(0.049, p<0.05)
Keywords: Diatom, Biostratigraphy, Warna, Pengilon
Indonesia is an archipelago area and has 130 active volcano, Dieng plateau is a Volcanic complex in central java and series of Quaternary volcanoes, it surronded by 9 volcanoes. Dieng located in Wonosobo and Banjarnegara regency with an altitude about 2000 m above sea level and it is chracterized by a high humidity and low temperature. The effect of vulcanic activity has caused Dieng has unique characteristic with many freshwater ecosystems such as lakes, creater (hydrothermal), valley, hot spring and Vulcan complex. The creater lake has been modification by lava flow from volcanic eruption and devided lake into Telaga Pengilon and Telaga Warna. the lake are separated by Actinoscirpus grossus grass.
Telaga Warna is small shallow creater lake rich of hydrogen sulfide, sulfur dioxide, carbon and chloride filled by biotiteandhesive lava and produced lake with unique charactheristic. The lake has unique colorful appreance like yellow, red, blue, green, caused by mineral, natural phenomenon and combined with the reflection of sunlight and giving colorful effect. Sometime the color of Telaga warna change to red and yellow and it caused by reflection of high sulfur, the influence of limestone deposit such as rocks and quartz change the water into white color, the water ph around 3 sightly acid with depth rate around 16m, The volcanic activity still intensively happend in Telaga Warna it showed by gas bubbles rising on the lake surface (Van Bergen et al., 2000; Soeprobowati et al., 2017; Soeprobowati et al., 2018).
Telaga Pengilon has difference charactheristic the surface area about 7,8 ha and the depth around 18 meter, ph around 6 indicated alkali water. Both Pengilon and Warna lake were located in Dieng, The area around lake is an important habitat and forms a National sanctuary based on Wonosobo Regional Rule No.1/1996, Conservation area based on Regional Rule No.1/2004, Wonosobo Regional Rule No.2/2011. The area is important habitat of protected aves Alchinidae and Ictinaetus malayensis.
The effect of volcanic eruption has caused dieng rich of natural fertilizer and the soil extremly fertile, these potential condition increasing agriculture commodity product such as potato, cabbage, tomato, carrot, onion and contribute 48,96% income. The agriculture product dominated with potato and produced around 16,6 tonnes/hectar. But, the extent of these ecosystems has been reduced by potato cultivation and human impact since the beginning of nineteen century, the effect of agriculture contribute residual fertilizer and pesticide into the lake, high rate sedimentation in these area reduced the catchment area. Besides of that, Telaga Balaikambang, Telaga Swiwi dried out because irrigation redirecting the water from the lake, long term effect causes loss of diversity, decreased water flow, and the catchment area has been graduatelly decreased.
Freshwater environment lake has important value for human, among various types of lake. Lake were located in high mountain are considered as good indicator of the ecological statues of lake and environmental changes (Smol, 2008; Mitchelutti et al., 2015). Aquatic biota is important for lake assesment plant. Moreover, the benthic diatom that preserved in lake sediment are useful to inform an important data of past environental changes because their sensitivy to response environmental change also their abiliy to tolerate under particular condition. Diatoms are microscophic orgnism spread out and dominated in many aquatic environment, bigest contributor oxigen because their function as fundamental primer producer in aquatic. Diatoms has unique cell wall called frustule made of silica, the ornamentation in the cell wall is advantage for microscopic identification and their ability to preserved in sediment usefull for long term monitoring. Diatom is one of the valuable indicator and could be used as indicator of tropical status, indication of acified water because sensitive to pH changes, also diatom could respond the degradation because pollution. Their ability present in sediment at periodic time has become an usefull material to access diversity changes and biomonitoring data of aquatic environment (Bellinger et al., 2010; Wang et al., 2014)
The collaboration between environmental variabel with biological data were analysed can help to understand variability and diversity of diatom community and their relationship with heavy metal variables in order to obtain an integrated information about their function as bioindicator. The objective of this study was to determine the composition and biodiversity of benthic diatom in area between Telaga Warna and Pengilon and to understanding the main heavy metal variables influencing the distribution and diversity of diatom
Dieng plateau is located in Wonosobo, Central Java and related with series of Sumbing and Sindoro volcanoes, The study site was selected as representative of undisturbed area in the border area between Telaga Warna and Pengilon, The area is characterized by shallow water about 1-2 meter undulating terain and the vegetation of the area is covered with Actinoscirpus grossus grass and other macrophyte, The condition of this site is mixture of acid and alkali water surrounded by Telaga warna and Telaga Pengilon, rain season was selected because the water from both of the lake widespread flooding increase water volume and influence for diatom growth rate, average annual temperature about 21,50C, and elevation about 2088 msal, The study area is shown in Figure 1.


Figure 1. Map of Study area
Sampling site is located at coordinat S 07O 12.904″ E 1090 54.894″. Sediment core was collected using manual corer in the deepest part of this wetland area, the 160 cm long core was retrived, the sample were cut with 10 cm interval for diatom analysis. Sedimen samples were llowed to dry for almost 3 days before diatom digestion, Diatom samples were oxidized following the standart of Soeprobowati et al., (2012) modified from Battarbe et al., (2001) using hot acid digestion with 10% HCL and 10% H2O2 . HCl help to removed all material organic and carbonate also to eliminate CaSo4 on the diatom, H2O2 help to remove residu from first digestion and any carbonate also cleaning frustule diatom and make it easier for identification, sample were heating for 2 hours with temperature about 900C-950C and washed with destiled water and repeated at least three times and sample need to leave overnight until settled down separated natan and supernatan and to remove the acid during digestion process. Washing process should repeated several times until the pH reach to neutral. Microscopic slide were prepared using evaporation procesure (Battarbee, 1973). The suspension of diatom were embedded with Naprax at least 400µl, For enumeration process at least 400 valva diatom were counted on each slide, Observation were performed using light Microscop 1000x magnification, digital images were taken and measured with image J, corel draw x7 for editing. Diatom taxonomy for species followed by Krammer and Lange-Bertalot (1986, 1988, 1991a, 1991b), Krammer (2000);Taylor (2007); Gell et al., (1999): Sonneman et al., (2000), Bahls, (2017).

Sediment sample for trace metal analysis were digest with waterbath methods using 3ml of consentrated HNO3 and 1ml HCL. The analysis for trace metal such as Lead (Pb), Cadmium (Cd), Chromium (Cr), Zinc (Zn) and Alumunium (Al) was done with Inductively Coupled Plasma-Optical Emission Spectrometry ICP EOS. The calibration curve were prepared prior analysis trace metal concentration in sample, calibration curve contained set standart sollution with concentration already known and compare it with unknown consentration to help predict concentration in sediment sample.

Environmental and diatom data were analysis using different multivariate methods. Species with relative abundance less than 2% cut off from statistical analysis because species less than 2% relative abundance might be contamined species. Biotic data were transformed as log(x+1) in order to stabilize variance. Thus, tested the normality. Multivariate analysis were used to understand the relationship between trace metal and diatom assemblages. Stratigraphic diatom and trace metal were analyzed using C2 (Juggins, 2003). The next step data were counted with Detrended correspondence analysis or DCA to know the dynamic changes in the diatom composition (Hill and Gauch, 1980) also to determine the length of gradient axis and the result of DCA axes length was bellow than 4SD, indicates that linier methods should be applied in furthur analysis.

Redundancy Correspondance Analysis or RDA was used to give information the main gradient of he variation in diatom assemblages corresponding with trace metal, the significant value of each parameter in environmental variabel was tested by Monte-Carlo permutation test with 999 unrestricted permutations (P ? 0,05) in order to test the significant of the axis. Collinier variables must be analyzed prior RDA analysis, the variance of inflatioon VIF < 10 ( ter Braak & Smilauer,2002) The five variables Pb, Cd, Cr, Zn and Al were used in RDA analysis. In order to know the combinations of trace metal variables which provided the best matches of metal with diatom asseblages were investigated using BioEnv. DCA and RDA were performed using Rstudio software (Oksanen, 2015) Principal Component analyses or PCA should be used because the result was linier ordination based on DCA axis (ter Braak and Prentice, 1988). Hierarchical cluster analysis was examine using Unweighted pair group method with aritmatic mean (UPGMA) that shown the diatom zone based on diatom assemblages with Bray curtis similiarity index were analyzed using PAST softwarae (Clark and Gorley, 2006)
Total the abundance diatom in these site were identified 103 different taxa belonging to 25 genera were recorded in 14 samples, A large group of genus Eunotia dominated around 56% of total diatom assemblages, Pinnularia around 17,2% and Frustulia 4,6%. But, only 11 taxa that reached 2% minimal abundance. In general, Eunotia formica (Ehrenberg) was dominat diatom almost in all stratigraphy site with 25% abundance and Eunotia monodon var. tropica (Lange bertalot) with 20,97% of total diatom assemblages. The primary clustering was subdevided the sediment core into four diatom zone.

Zone 1 (150 cm-110cm) or the lowest zone was dominated with Eunotia monodon var. tropica (Lange bertalot), the abundance of Eunotia monodon var. tropica (Lange bertalot) fluctuated between 2,75% until 17% associated with Eunotia formica (Ehrenberg), Pinnularia gibba (Ehrenberg), Eunotia sulcata (Hustedt) also the abundance of Brachysira brebissonii (Ross) higher 5,9% than any other zone. Eunotia monodon var. tropica (Lange bertalot) is a cosmopolitan species distributed across Indonesia and South Asia. in Indonesia some of research reported this species was found in Lake toba (Sumatra) (Hustedt, 1937), Borneo (Kalimantan) (Schimidt, 1959), Papua (Vyerman, 1995). Also Glushchenko and Kulikovsky (2017) reported Eunotia monodon var. tropica (Lange bertalot) in south eastern Vietnam. The abundance of this species is generally regarded as a warm temperature tolerant. Indonesia is considere the most suitable place for characterising higher average temperaure condition and the abundance Brachysira brebissonii (Ross) indicated that pH fluctuated in this area
Zone II (102cm) no diatoms were found in this zone only breaking frustule of Eunotia monodon var. tropica (Lange bertalot) and Eunotia formica (Ehrenberg), the similiar condition happended in Rawa danau (West Java, Indonesia) around 50-125cm or ca. 181-80 years B.P no diatom are preserved. Vandekars et al., 2001 reported the water level of the lake decreased because initiating pedogenesis process also Puusepp and Kangur (2010) claimed significant change in sediment, interaction with coarse grained meneraloganic and lower carbonate content responsible for breaking frutule. Research from Sajekti (2009) was found evidence of volcanic activity in Dieng from biostratigraphy study of Telaga Cebong and Telaga Balaikambang tephra was found and no polen preserved around 477-478 cm. Both of lake only have less than 4km from telaga Pengilon. In addition, Dieng has had a history of intermittent exploison and there have been 31 Holocene eruptive periods in Dieng with repeat eruptions, at least 8 of eruption caused fatalities.

Zone III (96-60cm) was dominated with Eunotia formica (Ehrenberg), Pinnularia viridiformis (Nitzsch) and Pinnularia viridis (Nitzsch) at the same time the abundance of Achanthes increase about 6%, also Fragillaria and Aulacoseira were increase highly than previous zone. Eunotia formica (Ehrenberg), included in periphytic species tolerate with acid with ph range 4,7 to 7,3 (Taylor et al., 2007; Burge, 2014) and These species are indicators of dystropic until oligotropic condition, with optimal ph arouund 6,7. Eunotia species well known as their ability to tolerate with wide range of ph. The increasing number of periphytic taxa in these area indicated about increase and expansion of aquatic plants close to core site and a lot of shading caused by plant cover (Puusepp and Kangur., 2010; Faria et al., 2013). also Fragillaria is commonly used as indicator as pioneer taxa and represent an successional stage after eruption. The high abundance of Eunotia and peripytic species in the middle of the core indicated a higher water level associated with lower pH because the effect of Ph and catchment soil. Eunotia has ability to respone ph and some eunotia were found in the extreme acid condition.

Zone IV (0 cm) dominated with Eunotia bilunaris var. linearis (Ehrenberg) around 14%, Eunotia bilunaris var. Mucophila (Lange-Bertalot) 11,75%, Eunotia curvata (Kutzing) about 12% and Eunotia incisa (Gregory), all of these species has wide geographic distribution in the world and indicate of acid environment. The abundance of Eunotia bilunaris var. linearis (Ehrenberg) associated with dystrophic waters and increased concentration of sulfate also indicator for less polluted environment (Alles et al., 1991; Lobo et al., 2004a; Bere and Tundisi, 2010), krammer and Lange-bertalot (1999) reported Eunotia bilunaris var. linearis (Ehrenberg) often attached with bryophyta in the acid lake at the mountain, the optimum ph for Eunotia bilunaris var. linearis (Ehrenberg) around 3,36 – 5,3 (Alles, 1991; Novakova and Poulickova, 2004).
Based on biostratigraphy diatom analysis the area was dominated with Eunotia indicated that ph has fluctuated and got influenced by the charactheristic of both lake. Telaga warna consider as acid vulcano lake rich of sulfuric acid, hydrogen sulfide and hydrofluric acid and acidifying influence on the water, in addition the ph extremly low because the lake have no outlet.Telaga pengilon generally have a ph around 6 and have high concentration of mineral, the high precipitation during rainy season for 6 month in tropic has effect for increasing ph. The result of biostratigraphy diatom are shown in Figure 2.

In order to know the relationship between metal and determine the metal stratigraphy, a correlation matrix was calculated for heavy metals in the sediment. The data showed the strong correlation between Cr and Cd (r=0.58, p< 0.01) Cd and Al (r=0.47, p< 0,01) it means that both of those metal tend to accumulate together and were derived from same sources and redistributed in the sediment (Soliman et al., 2015). The result for metal stratigraphy are shown at figure 2, the metal concentration in the sediment varied widely and fluctuated during periode time. The conccentration of metal considerable difference between site were observed for Pb, Zn, Cr, Cd, Al. Increasing trend can be seen from the upper layer, Toward the concentraion of Cr, Cd, Al decreased, while Pb and Zn almost stays at the same level. The consentration of Pb was high at the top layer, The high level of Pb and Zn in the sediment can be come from agriculture and anthropogenic problem, approximately started from 80cm the concentration of Pb and Zn relatively higher. the decreasing trend concentration of Cr and Cd can be seen at site 96 until 0 cm it caused by increasing the number of periphytic diatom taxa and indicated the increasing number of plant cover and succesion area, the present of aquatic plant help to absorb metal from water and sediment.
The volcanic activity in Dieng have had effects increasing trace metal from ash eruption, element preserved in ash material during volcanic eruption. Some elements assosiate with soil create soil rich of mineral (Ruggiery et al., 2011; Canion et al., 2012). But, other element such as Cu, Pb , Cd and Zn leached into water bodies and potentially toxic
for environment. The concentration of Alumunium in Figure 2 relatively higher because Alumunium is major element in the soil. A significant impact on the aluminium concentration in water is exerted by the pH (Yang et al., 2015).

The result of PCA analysis are shown in Figure 3. The PCA included five trace metal variabels in the 14 site. The variance explained 70,30% by component1 about 46,22% respectively of the variance in the environmental data. The first component represented with Cd and Cr correlation coefficients among this group were 0.57 and 0.51 mostly every site were located in the opposite of Cd and Cr. Cadmium is related with site 144cm. Where another site such as 0, 60, 66, 74 are opposite and indicate the lower concentration of Cu. The second component explained 22,08% of the total variance with significant loading on Zn with 0.66, site 0, 60,66, 80 are located in the opposite oh Al indicate that had less concentration of Al.

Figure 3. Principle Component Analysis
The RDA analysis (Figure.4) showed an interesting association between diatom asemblages and trace metal variabel, Axis 1 explained about 21,2% of the variance and axis 2 explained about 34,4% of the variance. The first axis represent with site 122, 138 and 144 located to the left due to their Pb and Zn contain. Species composition such as Eunotia sulcata (Hustedt), Eunotia curvata (Kutzing), Frustulia saxonica (Rabenhorst), Eunotia bilunaris var. linearis (Ehrenberg), Eunotia minor (Kutzing), Pinnularia gibba (Ehrenberg) were associated with the concentration of these metal. Axis 2 is correlated with Al, where species composition such as Pinnularia viridis (Nitzsch), Pinnularia viridiformis (Nitzsch), Nitzschia palea (Kutzing), Eunotia monodon var. tropica (Lange bertalot), Eunotia formica (Ehrenberg). Many studies describe Nitzschia palea has ability to tolerate heavy metal concentration (Kobayashi &Mayama., 1988; Chen et al., 2015). Different diatom composition in the area indicated different reaction of metal, because every species has different range of tolerance. For example the abundance of Nitzschia palea associated with Pinnularia, Gomphonema, Achanthes dominated in the are with heavy metal concentration.

Figure 4. Redundancy analysis
The research about diatom in the area between Telaga Warna and Pengilon found out that based on biostratigraphy diatom the core at 102cm revealed changes in the diatom community and heavy metal. No diatom preserved indicated about disturbance and inhibited diatom to grow. Vulcanic activity proved the high heavy metal anf other evidence from previous research found ash material in Telaga Cebong Dieng. The high abundance of Eunotia indicated that the ph fluctuated and has influence from both of lake. The present of Al (0.031, p<0.05) and Pb (0.049, p <0.05) had influence for diatom.
This study was made possible by Global Innovation Initiative funding and Kelly Ferri, Muhammad Hadi Al amien, Alam Dilazuardi, Geyga Pamrayoga for support during field work and lab work

Canion B., Jacques C., Landsberger S and Taftazani A., 202. Trace analysis of Indonesian volcanic ash using thermal and epithermal neutron activation analysis., Nukleonika Vol 57. No.4 Page 585-589
Chen X, Li C, McGowan, S and Yang X. 2015. Diatom response to heavy metal pollution and nutrient enrichment in an urban lake: evidence from paleolimnology. Annual limnology Vol 50 Page 121-130
Ruggieri F., Fernandez-Turiel,J.L. and Saavedra J. 2011. Environmental geochemistry of recent volcanic ashes from South Andes. Environmental Chemistry Vol 8. Pages 236-247
Soliman, N.F., Nasr S.M., Okbah, M.A. 2015. Potential ecological risk of heavy metals in sediment from the editerranean coast, Egypt. Journal Of Environmental Health Science and Enginering. Vol 13 No. 70
Yang, M., Tan, L., Xu, Y., Zhao, Y., Cheng F., Ye, S., Jiang W. 2015., Effect of low pH and Alumunium Toxicity on the Photosynthetic Charactheristic of Different Fast-Growing Eucalyptud Vegetatively Propogated Clones. Plos One Vol 10 No.6. Pages 1-15
Sajekti, A.S., 2009. A significant impact on the aluminium concentration in water is exerted by the water pH and acidity. Muséum national d’histoire naturelle. Page 1-101