Evaluation of Coliform and Faecal Coliform Bacteria in the Lakes of Broknes & Grovnes Peninsula at Larsemann Hills, East Antarctica

Purpose: More than 150 lakes on different peninsulas and islands are situated in the Larsemann Hills. The Larsemann Hills are an ice-free area and are located halfway between the Vestfold Hills and the Amery Ice Shelf on the south-eastern coast of Prydz Bay, Princess Elizabeth Land, East Antarctica. Antarctic lakes water is being polluted due to anthropogenic activities caused by various research activities and tourism. Methods: During 34 th Indian Scientic Expedition to Antarctica (ISEA) in 2014 to 2015, twenty lake water samples in triplicates were collected from the Broknes & Grovnes peninsula. Coliform and faecal coliform bacteria were analyzed in these samples. Results: Out of twenty, eleven lake water samples were found to be contaminated with coliform bacteria. However, faecal coliform bacteria were absent in all the lake water samples. Coliforms are found in the lakes of Broknes peninsula (P2 Lake & P3 Lake) and Grovnes peninsula (L1C NG, L1D NG, L1E NG, L7 NG, L7A NG, L7B NG, L2 SG, L4 SG & L5 SG). Conclusion: The present study conrms the presence of coliform bacteria in the lakes of East Antarctica which indicates an alarming situation and needs to be investigated further.


Introduction
Antarctica is the world's most remote and unspoiled continent. It is the largest pristine wilderness in the world. Lenihan [1] stated that it is a continent of the pristine environment. Approx. 53 research stations are now located in Antarctica. The population in winter and summer is around 1000 to 4000 people respectively. It is increasing by time due to more researchers are taking an interest in research on Antarctica. Antarctic lakes are polluted by sewage waste released from research stations and commercial shing vessels. Sewage waste which contains food waste and human waste is discharged without untreated from research stations to the Antarctica and affecting its environment. Some local wildlife populations such as seals and sea birds are also releasing their faecal waste in Antarctica. Lenihan et al. [2] stated that human sewage can be the dominant source of faecal microorganisms in Antarctica and have a signi cant impact on its environment.
Waterhouse [3] studied the presence of coliforms and faecal coliforms bacteria in Antarctic water and stated that it is due to the activities of the local vertebrates and human populations. These bacteria are nonpathogenic, but their presence indicates the possibility of nding pathogens. Faecal coliform bacteria such as Escherichia coli (E. coli) are found in feces and their presence in drinking water indicates faecal contamination. E. coli can also be a pathogen itself, so if E. coli is found in the water then there is a chance that other pathogens will be present. Faecal coliform is a rod-shaped, gram-negative, nonsporulating, and facultative anaerobic bacterium. It is generally originated in the intestine of warmblooded animals. Coliforms are commonly used microbiological markers of sewage pollution in Antarctica [4,5]. Cowan et al.
[6] stated that some strains of coliform bacteria which were found in Antarctica are not indigenous to Antarctica and are transporting by anthropogenic activities. Green et al. [7] studied the different sewage indicators from sewage around Antarctic research stations.
Several researchers studied the different physical factors such as solar radiation, temperature, and ice condition which affects the survival and distribution of faecal coliform bacteria in Antarctica [8, 9,10]. The high level of Ultra-Violet (UV) radiation is also responsible to reduce the viability of sewage microorganisms in Antarctic water and air [11,12]. The survival rate of faecal bacteria can vary from a few minutes to many days and depends upon the environmental conditions [11]. Sewage microorganisms can remain viable for prolonged periods in the Antarctic environment while the terrestrial environment is potentially less hospitable due to desiccation stress and wide diurnal temperature ranges [10,13]. Sewage microbes have the potential to infect the people and become part of the gut of local wildlife populations in Antarctica [14,15].
In Antarctica, the amount of faecal coliform bacteria was found high in early winter due to increased faecal input by migrant wildlife and low doses of solar radiation, while in summer, in spite of the high population on the research station, the amount of faecal coliform bacteria was found low due to the biologically damaging effects from the radiation of solar. According to several scientists after the rst human expedition to Antarctica, the disposal of faecal waste has generally been into the sea and either buried in snow or discharged into the Antarctic lakes [5,16,17,18]. Parker & Martel [17] stated that once faecal waste is buried into the snow, it remained the same for a long time due to low temperature, it undergoes relatively little degradation and could become a long-term pollution problem in the future in Antarctica. The purpose of this study was to determine the occurrence of coliform and faecal coliform bacteria in the lake water samples of Broknes and Grovnes peninsula of Larsemann Hills, East Antarctica.

Study Area
Broknes & Grovnes peninsula of Larsemann Hills, East Antarctica were selected as a study area. The location map of the study area is shown in Fig. 1.

Sampling Sites & Collection
The sampling of the lake water samples was carried out in the month of Dec-Feb of 2014-2015. A total of 15 samples were collected randomly from P1 Lake, P2 Lake, P3 Lake, P4 Lake, and Reid Lake from

Enumeration of Coliform and Faecal Coliform Bacteria
The most probable number (MPN) of coliform and faecal coliform (coliform/100 mL) in lake water samples was determined as per the Indian Standard (IS):1622 [19]. The enumeration was carried out in triplicate and included three phases.

Presumptive Test
Three sets of test tubes were taken for every sample and each set contained ve test tubes. 10 mL of double-strength MacConkey broth (MB) was inoculated in each tube of the rst set. On the other hand, for the second and third set of test tubes 10 mL of single-strength MB was inoculated in each tube. Durham's tube (small tube) was placed inside each tube in an inverted position. Homogenized lake water samples (10 mL, 1 mL & 0.1 mL) were inoculated in the rst, second, and third set of test tubes respectively. All tubes were incubated at 37 ± 1 o C for 24-48 h (at 44.5°C for 24 h for faecal coliform). After incubation, the observation was recorded for the gas production (i.e. bubble formation) in Durham's tube. If bubbles were present, then consider that tube as positive. If no gas was observed in any test tube, then discontinue the test and record the result as less than 2 organisms/100 mL. This test is shown in the

Completed Test
Eosin Methylene Blue (EMB) media was prepared according to the positive tubes of the con rmative test and then poured into the petri discs. Three petri discs were taken for each positive tube. A loopful inoculum from each individual positive tube was streaked on EMB media plates and these plates were incubated at 37°C for 24 h in an inverted position. The presence of green metallic sheen colonies con rmed the presence of E. Coli. These isolates were further con rmed by Gram's staining for E. Coli as per IS:5887 [20]. This test is shown in the Fig. 7.

Quality Assurance/Quality Control (QA/QC)
Each sample was analyzed in triplicate. All dilutions and media were prepared in double-distilled water.
On the basis of the number of positive tubes as recorded in a con rmed test calculate the probable number of coliform and faecal coliform/100 mL of lake water sample by using the MPN table. MPN of organism present per 100 mL of sample is shown in Table 3.

An Occurrence of Coliform and Faecal Coliform Bacteria in the Broknes Peninsula, East Antarctica
No growth was observed of faecal coliform in all different lake water samples while coliform was present in P2 Lake and P3 Lake. No growth was observed of coliform in P1 Lake, P4 Lake, and Reid Lake. Maximum MPN coliform was found in P2 Lake followed by P3 Lake. An observation of MPN coliform and faecal coliform/100 mL in different lake water samples of Broknes peninsula is shown in Table 4.    [11] studied the effect of solar radiation on the survival of E. Coli at Davis Research Station, Antarctica, and stated that faecal bacteria were rapidly inactivated when exposed to sunlight in the Antarctic water.
Several scientists have reported the presence of coliform and faecal coliform in the sewage outfall of the Antarctic research stations [7,15,23]. Coliforms are less able to survive in Antarctic environmental conditions than spore-forming bacteria. Nedwell et al. [24] stated that coliform bacteria can survive < 50 years while spore-forming bacteria can survive > 80 years in Antarctica. The faecal Streptococcus strain was more resistant to the effects of radiation than the gram-negative strains [11]. Fox and Cooper [25] reported that in some areas of Antarctica, regional warming has caused a decrease in permanent snow cover around nunataks and coastal regions with the result that previously buried toilet pits, depots, and food dumps are now melting out. Green

Conclusion & Recommendation
Coliform and faecal coliform bacteria are mainly reached to Antarctica through anthropogenic activities and are useful indicators of sewage waste. However, there are other factors which may lead to coliform contamination in Antarctica such as migration of microbes through birds and transport of food items from ship to the research station. The presence of coliform bacteria in Antarctic lakes may be due to the human population, which is living at research stations. Our study con rms the presence of coliform bacteria in the lakes of East Antarctica, as reported earlier in other Antarctic regions. Untreated sewage waste releases from the research stations is the main cause of the presence of coliform in the Antarctic environment. Due to climate change the rate of snow and ice melting increased and have resulted in previously buried faecal material becoming exposed.
Prior to release sewage discharge should be fully treated; otherwise, it will leave the pathogenic contaminants in the pristine environment of Antarctica. We should give priority to maintain a relation between actual human presence and the puri cation capacity of the plant systems, in order to reduce as much as possible anthropogenic inputs into the Antarctic ecosystem. We need regular monitoring at  Con rmative Test