Faecal pollution indicators in the Terra Nova Bay (Ross Sea, Antarctica)

Pergamon PII: S0025-326X(97)00050-7

Marine Pollution Bulletin, Vol. 34, No. 11, pp. 908-912, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 002~326X/97 $17.00 + 0.00

Faecal Pollution Indicators in the Terra Nova Bay (Ross Sea, Antarctica) V. BRUNI*, T. L. MAUGERI* and L. MONTICELLIt

*Dipartimento di Biologia Animale ed Ecologia Marina, Salita, Sperone 31, 1-98166 Messina S. Agata, Italy ?Istituto Sperimentale Talassografico CNR, Spianata S. Raineri, 84, 1-98122 Messina, Italy

The occurrence of faecal bacteria indicators (total conforms, faecal coliforms and streptococci) in pristine waters and near Italian Base stations of the Terra Nova Bay was investigated. High bacterial densities were found at the station near to the ouffall of the sewage disposal plant and when the population at Base was more abundant. In all other stations further from the ouffall, the bacterial indicators were absent or present in very small numbers. Faecal bacteria were not detected in samples collected at pristine sites (Penguin Bay and Evans Cove) except for only 1 enterococcus per 100 ml at Evans Cove. In the seawater samples in which faecal coliforms and faecal streptococci were found, the latter were generally more abundant and in 4 samples only streptococci were isolated, although in low number. This could snggest that faecal streptococci are more suitable bacteria for investigation of the human impact on the Antarctic marine environment. © 1997 Elsevier Science Ltd

In 1985, the construction of the Italian Base in the Terra Nova Bay raised the questions of how to obtain potable water and how to dispose of wastewater and sewage. These problems were resolved by installing a seawater desalination plant with UV treatment and a biological depuration system. To begin with, the latter was sufficient to purify sewage released from a maximum of 60 users. The outfall discharged the treated water into the marine waters 50 m from the shoreline. Recently, however, this system has not been working effectively. From a health point of view, the seawater intake serving the desalination plant was situated far from the outfall system. The aims of this study were to determine the occurrence and distribution of faecal indicator bacteria in the seawater of the Terra Nova Bay, and to assess the effect of the occasional population increase at the Italian Base during the summers of 1989-1990 and 1994-1995.

Keywords: Antarctic seawater; faecal pollution; coliforms; faecal streptococci.

M a t e r i a l s and M e t h o d s

Sampling

Following the early exploratory expeditions in the Antarctic Continent, scientists have increasingly been urged to go to this uncontaminated land, for it offers a wide field for research. The presence of human settlements, initially only seasonally, but recently also permanently, has stimulated interest in determining the environmental impact of human activities, in observance of the fundamental principles laid out in the Antarctic Treaty for the preservation of this natural environment and also for the recent interest in this Continent by the media and environmental groups. It is only in the last few years that the accumulation of waste of various types has become increasingly noticeable around larger installations. At McMurdo Station--the largest human settlement in Antarctica--where the wastewaters are discharged into the cold marine environment, high densities of coliform bacteria were found along the ca 1 km shoreline and the plume extended 200-300 m seaward (Howington et al., 1992). 908

Samples were taken from the stations shown in Fig. 1. During the first sampling period (from 24 December 1989 to 1 February 1990), seawater was collected weekly only at surface ( - 3 0 cm of depth) in stations 4 and 5, using sterile 1 1 polyethylene bottles. During the second period (from 20 December 1994 to 5 February 1995) the sampling sites were distributed in a wider area and collection was carried out by Niskin bottles. These were cleaned with hot water and lowered at different depths and through holes drilled in the ice where appropriate. Some water samples and one sediment sample were also collected using sterile polyethylene bottles by SCUBA diving.

Bacterial enumeration Aliquots of the seawater samples were filtered onto membrane filters (0.45 ~tm pore diameter, Millipore Corp.) and then placed on plates according to established procedures (APHA, 1989) using the following media and incubation conditions: Bacto m-Endo Broth (Difco) incubated at 35°C for 24 h for the growth of total coliforms (TC), Bacto m-FC Agar (Difco)

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Fig. 1 Study area and sampling stations.

without rosolic acid at 44.5°C for 24 h for faecal coliforms (FC) and Bacto m-Enterococcus Agar at 35°C for 48 h for enterococci (FS). Only during the summer of 1994-1995 was a resuscitation period of the bacteria carded out onto Nutrient Agar (Difco) for 2 h at 35°C before placing the membrane filters onto Bacto m-FC Agar. Typical colonies were counted and the number of bacteria was calculated from the volume of filtered water and expressed as colony forming units (CFU per 100 ml). Microbiological analyses were immediately performed in the laboratory of the Italian Base, no more than 2 h after the sampling. An identification of E. coli was based on the assumption that ~Dglucuronidase is a selective marker for E. coll. For the enzymatic assay, typical faecal coliform colonies in Bacto m-FC Agar (Difco) were grown in E. coil Direct Agar MUG (Biolife Italiana, Milano, Italy), incubated overnight at 44.5°C and observed under Wood's lamp

(366 nm). The presence of fluorescent colony shows ~Dglucuronidase activity. The sediment sample was prepared for plating by blending in 0.1% Na4P20~. 10 H20. Serial dilutions (10 fold) were prepared from this treated sediment in sterile PBS (phosphate buffered saline, pH 7.2) and spread plated (Fredrickson et al., 1991). The physicochemical and biological characteristics of the sampling area are reported in the data reports of the Italian Project (Anonymous, 1991; Anonymous, 1992) and by Cescon (1992).

Results and discussion The temporal variations of the colimetric indexes at station 5 (in correspondence to the marine point where the outfall from the sewage disposal plant flows into the sea) are shown in Fig. 2. From this figure it is evident that the faecal indicators were always present and the 909

Marine Pollution Bulletin Station

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Fig. 2 Bacterial densities at Station 5 in December 1989-February 1990.

highest values (TC 10000 100 ml-l; FC 10000 100ml-l; FS 2700 100m1-1) occurred when the population was more numerous (173 persons on 23 January 1990). Therefore, the sewage purification system was no longer sufficient. At Station 4, at about 100 m from the outfall, the enteric bacteria were present in low density (at most FC 2 100 ml - l ) or absent. During the second sampling period, when the population of the Italian Station was approximately 80 people, the results (Table 1) emphasized the same condition, but

the bacterial densities at station 5 were still higher (FC 80000 100 ml-l). At stations 4 and 3 (at the end of the promontory) faecal bacteria were also detected, although in low abundance. However, the sediment collected at Station 4 a zone, characterized by low hydrodynamism was contaminated (TC 198 CFU 100 g - l ; FC 94 CFU 100 g - l ; FS 1160 CFU 100 g-l). Sewage purification experiments carried out in a tank at the Base, showed that coliforms are concentrated by specimens of Laternula elliptica (benthic bivalve Mollusca). This finding suggests that these allochthonous bacteria could also enter the Antarctic food web up to marine mammals (Monticelli, pers. comm.). In all other stations further from the outfall, the bacterial indicators were not present, except at the seawater intake (St. 2) for the desalination plant, where lower values (1 CFU 100 ml - l , both for total coliforms and faecal coliforms) were found. At this station, the occasional faecal bacteria occurrence would be due to low currents of the zone. Faecal bacteria were not detected in samples collected at pristine sites (Penguin Bay and Evans Cove) except for only 1 C F U enterococci per 100 ml at Evans Cove, where a small group of Weddell seals (Leptomychotes weddelli) were seen. Using I~-D-glucuronidase assay, a preliminary identification of isolated faecal coliform strains as E. coli was

TABLE 1 Date, stations and bacterial densities in December 1994-February 1995. Sample Date

St.

Type

20 December 1994 23 December 1994 26 December 1994 26 December 1994 02 January1995 03 January 1995 07 January 1995 07 January1995 08 January1995 09 January 1995 10 January 1995 15 January 1995 17 January 1995 17 January1995 18 January1995 23 January1995 23 January 1995 24 January1995 24 January1995 26 January 1995 27 January 1995 27 January1995 28 January1995 28 January 1995 02 February 1995 05February 1995 03February 1995

4 10 4 4 1 5 2 3 10 11 I 4 1 7 1 6 6 8 8 9 6 6 8 8 9 2 1

Water* Water* Water* Sediment* Water* Water Water Water Water* Water* Water* Water* Water* Water Water* Water Water Water Water Water* Water Water Water Water Water* Water Water*

nd, Not determined. * Sampled by SCUBA diving.

910

CFU 100 m l - 1

Depth Temp. (°C) 5 3 4 12 9 0.1 3 0.1 4 6 5 5 3 2 6 3 15 0.1 10 5 3 15 0.1 10 1 3 1

- 1.1 - 1.3 - 1.3 nd 0 nd nd -1.1 nd nd nd nd nd - 1.5 nd nd nd - 1.1 nd nd nd nd nd nd nd -0.7 nd

TC

FC

FS

1 0 1 198 0 nd I 6 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 nd

1 0 1 94 0 800 1 5 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9

0 0 1 1160 0 1100 0 10 0 I 5 3 0 0 0 0 0 0 0 8 0 0 0 0 12 0 102

Remarks Under ice-pack Under ice-pack CFU 100 g-1 of dry sediment Under ice-pack CFU m l - "

Under ice-pack Under ice-pack Under ice-pack

Under ice-pack Under ice-pack CFU 500 m l - l CFU 500 m l - 1 CFU 500 ml-=

Volume 34/Number l 1/November 1997 possible. This enzyme is also found in a few Salmonella and Shigella strains (Feng and Hartman, 1982) but the growth at 44.5°C in presence o f bile salts and acid production from lactose together with I]-o-glucuronidase activity support the E. coli identification and reduce false positivity. It is important to emphasize that Smith et al. (1993) also showed the persistence and expression of antibiotic resistance and conjugative plasmids in E. coli strains exposed to the Antarctic marine environment. In any case, comparison o f the antibiotic resistance between bacteria from indigenous animal populations and coliforms isolated from the outfall indicated no colonization o f indigenous fauna with human intestinal bacteria Howington et al., 1993. In the seawater samples in which faecal coliforms and faecal streptococci were found, the latter were always more abundant and in 4 samples only streptococci were isolated. Among faecal pollution indicators a higher survival of streptococci in the marine environment was shown (Barros et al., 1975). Sunlight inactivation of culturable cells o f enterococci generally required 2-3 times the insolation for culturable cells of faecal coliforms (Davies-Colley et al., 1994). A decrease of inactivation rates of enterococci at lower temperatures was also observed (Sinton et al., 1994). As is well known, the sea is not generally a favourable environment for terrigenous bacterial survival because of its natural self-purification capacity, caused by physical, chemical and biological factors. Among these factors, antibiotic production by the marine organisms has to be considered (Sieburth, 1959). The period of survival of microrganisms of human origin in seawater, particularly the pathogenic ones, has stimulated much research. This has shown that this period of time is extremely variable, ranging from fractions of one hour to days or weeks, depending on the specific characteristics of each organism and on numerous other factors (Genovese, 1977). The survival in seawater of enteric bacteria, as well as of other microrganims including enteric viruses, is favoured by cold marine temperatures and hindered by high ones (Orlob, 1956; Carlucci and Pramer, 1960; Baross et al., 1975). Furthermore, the activities and predation rates of indigenous protozoa generally appear lower at reduced temperatures. In Antarctic marine seawaters, nutrient availability rather than low temperature reduces the activity of enteric bacteria. They persist in a physiologically active, yet non recoverable, state for extended periods (Smith and McFeters, 1993). In conclusion, the Italian settlement in the Terra Nova Bay produced persistent marine faecal pollution localized in the narrow zone strictly surrounding the outfall or at the furthest 100 m from the seashore, both in ice-free waters and under the ice. This appears to be related to the density of the human population living at Base. Untreated sewage release, such as at McMurdo Station, or sewage not efficiently treated, such as at the Italian Station, is responsible for the introduction of

enteric human bacteria into the sea, which can persist for a long time. As described by McFeters et al. (1993), the patterns of the consistent current observed in the seawaters surrounding McMu'rdo Station help to explain the movement and spatial distribution of the released wastewater in the marine environment. It would, therefore, appear necessary to carry out both laboratory and field research on this specific aspect and also the other physical, chemical and biological activities of self-depuration of the Antarctic seawaters, not only for the sewage bacteria, but also for the other allocthonous bacteria. Research on the presence of enterococci in Antarctic seawater could be helpful to provide a better estimate of the human impact, on account of their larger survival in the sea and also increased resistance to chlorination. We agree with Howington et al. (1992), who reported that sewage discharges should receive full treatment prior to release in polar environments. Nevertheless, priority should be given to maintaining a constant correct relation between actual human presence and purification capacity of the plant systems, in order to reduce as much as possible anthropogenic inputs into Antarctic ecosystem. We thank Marco Nigro and Maria C. Buia for sampling by SCUBA diving and Nino Tuso for SCUBA diving assistance. This work was supported by Italian National Antarctic Research Programme, 2d.2 Project.

American Public Health Association (1989) Standard Methods for the Examination of Water and Waste Water, 17th edn. APHA, Washington, DC. Anonymous (1991) Oceanographiccampaign 1989-90. Nat. Sc. Com. Ant. Ocean Camp. 1989-90, Data Rep. I, pp. 1-409. Genova. Anonymous (1992) Oceanographiccampaign 1989-90. Nat. Sc. Com. Ant. Ocean Camp. 1989-90, Data Rep. II, pp. !-507. Genova. Baross, J. A., Hanus, F. J. and Morita, R. Y. (1975) Survival human enteric and other sewagemicroorganismsunder simulated deep-sea conditions. Applied Microbiology 30, 309-318. Carlucci, A. F. and Pramer, D. (1960) An evaluation of factors affecting the survival of Escherichia coli in seawater. Applied Microbiology $, 243-247. Cescon, P. (1992) Ricercheitaliane in Antartide: Impatto ambientale, metodologiechimiche, attivitAe risultati scientifici.In Oceanografia in Antartide, eds V. A. Gallardo, O. Ferretti and H. I. Moyano, pp. 77-115. ENEA Progetto Antartide-Italia. Davies-Colley,R. J., Bell, R. G. and Donnison, A. M. (1994) Sunlight inactivation of Enterococci and fecal coliforms in sewage effluent diluted in seawater. Applied Environmental Microbiology 60, 20492058. Feng, C. S. and Hartman, P. A. (1982) Fluorogenic assay for immediate confirmation of Escherichia coli. Applied Environmental Microbiology 43, 1320-1329. Fredrickson, J. K., Balwill, D. L., Zachara, J. M., Li, S., Brockman, F. J. and Simmon, M. A. (1991) Physiological diversity and distribution of heterotrophic bacteria in deep cretaceous sediments of the Atlantic coastal plain. Applied Environmental Microbiology 57, 402-411. Genovese, S. (1977) Inquinamento microbico e processi di autodepurazione. Atti dei convegni Lincei 31, 289-314. Howington, J. P., McFeters, G. A., Barry, J. P. and Smith, J. J. (1992) Distribution of the McMurdo Station sewage plume. Marine Pollution Bulletin 25, 324-327. Howington, J. P., Kelly, B., Smith, J. J. and McFeters, G. A. (1993) Antibiotic resistance of intestinal bacteria from the indigenous fauna of McMurdo Sound, Antarctica. Antarctic Journal of the U.S. 28, 119-120. 911

Marine Pollution Bulletin McFeters, G. A., Barry, J. P. and Howington, J. P. (1993) Distribution of enteric bacteria in Antarctic seawater surrounding a sewage outfall. Water Research 27, 645-650. Orlob, G. T. (1956) Viability of sewage bacteria in seawater. Sewage and Industrial Wastes 28, 1147-1167. Sieburth, J. McN. (1959) Antibacterial activity of antarctic marine phytoplankton. Limnology and Oceanography 4, 419--424. Sinton, L. W., Davies-Colley, R. J. and Bell, R. G. (19.94) Inactivation of enterococci and fecal coliforms from sewage and meatwoks

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effluents in seawater chambers. Applied Environmental Microbiology 60, 2040-2048. Smith, J. J. and McFeters, G. A. (1993) Survival and recoverability of enteric bacteria exposed to the Antarctic marine environment. Antarctic Journal of the U.S. 28(5), 120-123. Smith, J. J., Howington, J. P. and McFeters, G. A. (1993) Plasmid maintenance and expression in Escheriehia coil exposed to the Antarctic marine environment. Antarctic Journal of the U.S. 28, 123-124.