Journal of Coastal Development ISSN: 1410-5217 Volume 6, Number 1, October 2002 : 47-54 Accredited: 69/Di ti/Kep/2000 Original paper PRELIMINARY STUDY ON POTENCY OF COPROSTANOL AND COLIFORM BACTERIA IN SEMARANG COASTAL AREA By: Tonny Bachtiar *) (1, 2, 3) (1) Oceanography Study Program, Department of Marine Science, (2) Center for Tropical Coastal and Marine Studies, Research Institute, (3) raduate School of Environmental Science, Diponegoro University, Semarang 50239, Indonesia Received: December 23, 2002 ; Accepted: January 26, 2003 ABSTRACT Feca co iform bacteria have been wide y used as a bio ogica indicator of sewage (domestic waste) po ution. (owever, as a bio ogica indicator in urban coasta waters, it has disadvantage, in particu ar because of increased vo ume of industria wastes that are to)ic and heated, increase of sa inity, and ow disso ved o)ygen. These conditions may affect the growth rate of most bacteria, inc uding feca co iform bacteria that becomes under representative in sewage po ution in urban coasta waters. It is necessary to find a ternative indicator that can be used to better understand the sewage po ution in urban coasta waters. Many researchers have proposed coprostano as a chemica indicator of sewage po ution. To understand the e)istence of coprostano and co iform bacteria, a pre iminary study has been done on water and sediment samp es from the river, river mouth, and seawater of Ban,ir -ana Timur .emarang coasta waters. The resu ts showed that coprostano cou d be detected in sediment from a sites, on the other hand co iform bacteria decreased with the increase of sa inity, and were not detected in the seawater. Key words: Coprostanol, co iform, coastal, pollution, sewage *) Correspondence: Phone/,a-: ./021.241 7474092, 2453035 Email: tonny_bachtiar5hotmail.com the environment 7uality. Pollution of INTRODUCTION organic substance, such as domestic Identification of sewage .domestic waste1 wastes, affects the dissolved o-ygen into pollution in the environment is important insufficient condition for the organism, as regarding health, esthetical, and other well as contains a lot of other materials ecological reasons. Increased intensity and that are hazardous wastes, such as variety of human activities in coastal pathogen organism. regions re7uire specific environment Co iform bacteria have been conditions. 8ased on particular conditions, widely used as a biological indicator of better understanding of the condition of sewage pollution. To determine each sewage pollution in urban coastal waters pathogen organism directly is not easy and become very important. The main problem sometimes is e-pensive, so that in the of pollution is not only based on the purpose of water 7uality management, it is to-icity of the pollutants .Alloway and necessary to have an indicator. Co iform Ayers 19941, but also the indirect impact to bacteria have been used as an indicator 47 Journal of Coastal Development ISSN: 1410-5217 Volume 6, Number 1, October 2002 : 47-54 Accredited: 69/Di ti/Kep/2000 because they are found abundant in the e-istence and persistence of coprostanol. It fecal of human and animal, and in general is because as organic material, coprostanol are relatively easy to determine. The will be degraded in the environment, occurrences of co iform bacteria indicate especially in tropical region. 8ased on that the contamination of domestic wastes, fact, the e-istence of coprostanol and therefore the e-istence of pathogen co iform bacteria in 8anCir Danal Timur organism should be concerned .Dutka et Semarang coastal waters was e-amined. a . 1974, Chapra 19971. However, using co iform bacteria as an indicator of domestic waste pollution in the environment with high environmental MAT RIAL AND M THODS stress, such as urban coastal waters, has created other problems that may be Sampling affected by several factors: these are a1 the increase of salinity from freshwater to Sampling of water and sediments were seawater, b1 increase of industrial wastes carried out at 5 stations in three that are to-ic and heated, and c1 low environmental conditions: namely a1 dissolved o-ygen .Walker et a . 1922, rivers, b1 riverEs mouth, and c1 seawater, of 8artlett 19271. These conditions may affect 8anCir Danal Timur Semarang coastal the growth rate of bacteria, so that the waters. There were three stations in the e-isting fecal co iform bacteria are under river environment: 8anCir Danal Timur representative in sewage polluted urban .8DT1, Dali Tambak Lorok .DTL1, and coastal waters. To solve these problems, Dali Tenggang .DT1G one station was in several researchers proposed coprostanol riverEs mouth environment, and one station as a chemical indicator of sewage pollution was in seawater environment. Si- liters of .Hatcher et a . 1977, Hatcher and water samples were collected for each Mc illivary 1979, 8rown and Wade 1924, station by using Niskin 8ottle water D?reth et a . 1920, Holm and Windsor sampler at 10 cm below the surface. One 1990, Coakley and Poulton 1991, Coakley liter was prepared for co iform analysis. et a . 1992, 8achtiar 1993, 8achtiar et a . Surface bottom sediment samples were 1990, Jeng and Hang 1994, Jeng et a . collected using van Ieen rab sampler, 1990, Chan et a . 19921. and sampled from the top to 2 cm of Coprostanol .5bAcholestanA3bAol1, surface sediment. The samples were a derivative of cholesterol by intestinal immediately put in dark bottle and stored microorganism, is dominant fecal sterol in in a cooler .5èC1 during the fieldwork. human faces .40A00 B of total sterol1, and ,urther all samples were immediately put was also detected in mammal and chicken, into cold storage at 5èC until analysis. but it was not produced by marine organism .Walker et a . 19221. The Coprostanol Analysis e-istence of coprostanol in coastal water environment indicates that domestic wastes Coprostanol analyses were done for both have reached that area. Coprostanol had sediment and water samples. ,ive liters of been used to indicate and to trace the water samples for each station were domestic wastes, and the results showed pumped through the glass fiber filter paper that coprostanol has a high performance as .2.0 mm, Ahlstrom1 using a hand vacuum an indicator and a natural tracer of pump to get the suspended material for domestic wastes However, all of the coprostanol analysis. The sediment and studies had been done in high latitude suspended materials were prepared for as regions. To use coprostanol in Indonesia, it Chromatography . C1 analysis as follows is necessary to well understand the .8achtiar 20021: 42 Journal of Coastal Development ISSN: 1410-5217 Volume 6, Number 1, October 2002 : 47-54 Accredited: 69/Di ti/Kep/2000 a. So-hlet e-traction chloroform, and fractions that contain Sediment and suspended sediment samples sterol were isolated with 10 B methanol in were dried using freezeAdried method. A chloroform. The fractions were collected in minimum of 5 gram dried sample was 9.5 dram vials, and stored in a fridge until needed for e-traction. The samples were preparation for as Chromatography . C1 e-tracted in benzene:methanol .1:1, v/v1 in analysis. so-hlet apparatus for 24 hours. f. Sample Preparation for C Heptadecanol was added as internal Sterol fractions were evaporated Cust to standard to the e-tract. E-tracted dryness, and later the samples were sediments were dried in the oven .00èC1 transferred to HP septumJcapped vial for several days. The weights were using 2 - 0.5 ml Heptane. 8ST,A continuously recorded until their values .8is.trimethylsilyl1Atrifluoroacetamide1 have no longer changed. 100 ml was added, and then the samples b. Evaporation were heated at 130 èC for 15 minutes to The e-tracts were evaporated until near make them more responsive on the C dryness by using rotary evaporator. capillary column. ,inally, the samples c. Saponification were allowed to cool. Once cooled, the The e-tracts were dissolved in samples were ready for C analysis. benzene:methanol .1:1, 2 ml, 3A4 times1 in g. as Chromatography a 50 ml centrifuge tube and were then Analyses of coprostanol were carried on saponified with 5 ml methanolic DOH .0.5 Hitachi 203A50 as Chromatography with N DOH in 95B methanol, 5 B H2O1. The an SEA30 column capillary, and standard tube was placed in boiling water bath for flameAionization detector .,ID1. Nitrogen 20 minutes and allowed to cool. was used as carrier gas .50 ml/minute1. d. E-traction The as Chromatography was The saponification was followed by programmed that the inCector and detector e-traction by using nAhe-ane .5 ml1 4 was 300èC, and the oven was 150èC J times. The tubes were centrifuged for few 220èC with increasing 5èC/minute. minutes. The top organic phase was Coprostanol concentration was calculated transferred into a pear shape flask .50 ml1 based on relative response factor .RR,1 using a long pipette. Methanol phase was from a reference solution containing discarded. coprostanol standard and reference e. ,ractionation standard. RR, was determined by using E-tract lipids were fractionated using silica the following formula .Telford et a . gel .deactivated with 5 B water1 in 19931: chromatography column. Less polar lipids were fractionated using 40B he-ane in mg coprostano l in standard Area reference standard RRF = x Area coprostano l in standard mg reference standard .11 8ased on formula .11, coprostanol concentration in samples was determined by using following formula: RRF x Area coprostano l x mg Internal Standard mg coprostano l = Area Internal Standard .21 The response of internal standard .IS1, reference standard, and coprostanol was the area of IS, reference standard, and coprostanol, that are determined from the C output. Total Coliform Analysis 49 Journal of Coastal Development ISSN: 1410-5217 Volume 6, Number 1, October 2002 : 47-54 Accredited: 69/Di ti/Kep/2000 Co iform analyses were done only for sterile metal loop 3 mm in diameter, one water samples.