relationships between indicators, pathogens and water quality in an ...

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Wat. Res. Vol. 30, No. 9, pp. 2045-2054, 1996 Copyright 0 1996 Elsevier ScienceLtd Printed in Great Britain. All rights reserved

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RELATIONSHIPS BETWEEN INDICATORS, PATHOGENS AND WATER QUALITY IN AN ESTUARINE SYSTEM CHRISTOBEL

M. FERGUSON’*, BRIAN G. COOTE’, NICHOLAS J. ASHBOLT*@ and IAIN M. STEVENSON’ ‘AWT EnSight, PO Box 73, West Ryde, Sydney, Australia 2114, *School of Civil Engineering, University

of New South Wales, Sydney, Australia 2052 and ‘Faculty of Science, University of Technology, Sydney, Gore Hill, Australia 2065 (First received August 1995; accepted in revised form February 1996)

Abstract-This study examined water and sediment samples for a range of indicator and pathogenic microorganisms from six sites in an urban estuary, Sydney, Australia. Water quality was affected by rainfall and sewage overflows which were associated with significant increases in the concentration of faecal coliforms, faecal streptococci, Clostridium perfringens spores, F-RNA bacteriophage, Aeromonas spp., Giardia and Cryprosporidium spp. However, in sediments, only faecal coliform concentrations were significantly increased by rainfall, although sewage overflow again resulted in increased concentrations of faecal coliforms, faecal streptococci, C. perfringens spores and Aeromonus. Isolation of Salmonella appeared to coincide with wet weather events and occasionally identical serotypes were detected in sediments at several locations within the estuary. However, isolations of enteric virus were sporadic and did not appear to be exclusively related to wet weather events. C. perfringens was identified as the most useful indicator of faecal pollution and was the only indicator significantly correlated to the presence of pathogenic Giurdiu (r = 0.41, p i 0.05) and the opportunistic bacterial genus Aeromonas (r = 0.39, p < 0.05). F-RNA bacteriophage was not significantly correlated with any of the pathogens examined. Copyright 0 1996 Elsevier Science Ltd Key words-indicators,

pathogen, water quality, sediment, enteric virus, Giardia, Cryptosporidium

NOMENCLATURE ct = /I, = lot = I= j = k= Iriver = qriver = criver = qtriver = rvlkb =

intercept regression coefficients location locations, 1-6 dates, l-10 type, either top or bottom sediment fraction the linear polynomial for distance of location, from the sewage overtlow point the quadratic polynomial for distance of location, from the sewage overflow point the cubic polynomial for distance of location, from the sewage overflow point the quartic polynomial for distance of location, from the sewage overflow point contrast between the location in Lime Kiln Bay and those in the Georges River INTRODUCTION

Assessment of water quality has traditionally relied on the detection of faecal indicator organisms,

*Author to whom all correspondence should be addressed: c/o Burns Philp R&D Division, PO Box 219, North Ryde, Sydney, Australia 2113 [Tel.: (2) 887 191I; Fax: (2) 888 31781.

faecal coliforms particularly total coliforms, and faecal streptococci. However, these groups of microorganisms do not necessarily correlate well with the presence of pathogenic organisms. Davis et al. (1977) suggested that the total coliform group did not constitute a reliable source of information as to the pollutant content or condition of a water source, while Sayler et al. (1975) concluded that reliance on the coliform group created serious problems both in measuring environmental quality and in calculating risks to public health. The lack of significant correlations between the presence of traditional indicators and pathogens (Grabow et al., 1989; Araujo et al., 1990) and the ability of pathogens to assume a viable but non-culturable state (Grimes et al., 1986) has highlighted the inadequacy of indicator systems in predicting water quality and associated health risks. In addition, several studies have highlighted the importance of sediments as potential reservoirs for microorganisms in the aquatic environment (Hendricks, 1971; Grimes, 1975; Erkenbrecher, 1981). The prolonged survival of coliforms and possibly other faecal bacteria in sediments and the likelihood of

2045

2046

C. M. Ferguson ef al.

their being desorbed by dilution and thus resuspended indicates that sediments as well as surface waters should be examined when assessing potential health risks (Geldreich, 1970; Grimes, 1975). Alternatives to the traditional water quality indicators have been suggested. For example, C. perfringens has been used in several studies to trace sewage wastes in the marine environment (Emerson and Cabelli, 1982; Sorensen et al., 1989; Hill et al., et al. (1993) found that C. 1993) and Ashbolt perfringens was the most sensitive indicator of water quality improvement after cessation of near shore sewage disposal in Sydney, Australia. Coliphages have also been proposed as potential indicators of sewage pollution in aquatic environments because they are ubiquitous in sewage and easier to enumerate than enteric viruses (O’Keefe and Green, 1989). However, although coliphages are effective predictors of enteric virus survival in sewage and wastewater to sewage (Grabow et al., 1983), their application contaminated receiving waters has produced equivocal results (Vaughn and Metcalf, 1975; Grabow et al., 1989). This may be related to the ability of some coliphages to infect other host bacteria which replicate in the environment. To increase specificity, some studies have enumerated a subgroup of the coliphage, male-specific RNA coliphage (F-RNA bacteriophage). These coliphages are a relatively homogenous viral group, which specifically infect Escherichia coli cells that have the F or sex pilus (Havelaar et al., 1984; Rhodes and Kator, 1991). In this study both water and sediment samples from an estuary in urban Sydney, Australia were examined for a range of indicators and pathogens

including faecal coliforms, faecal streptococci, Clostridium perfringens spores, F-RNA bacteriophage, Salmonella spp., Giardia spp., Cryptosporidium spp. and the opportunistic bacterial genus Aeromonas. The aims of the study were to investigate water and sediment quality within the estuary, to identify possible correlations between indicators and pathogens and to assess the effects of rainfall and sewage inputs. MATERIALS

AND METHODS

Srudy nrea

The study encompassed the tidal regions of the Georges River which is an important recreational and economic resource within the Sydney region, used for the production of shellfish and as a popular venue for both passive and active recreation. The River receives inputs of urban runoff from surrounding metropolitan areas and may, during wet weather, receive sewage inputs from two major overflow points (Fig. I). The overflow point in Prospect Creek discharged a mixture of stormwater and untreated sewage effluent on three occasions (September and December 1992 and February 1993), while sewage overtlow of chemically disinfected effluent downstream of G1105 occurred once (December 1992). A third sewage overflow point located in Lime Kiln Bay did not operate during the course of the study. Water and sediment samples were collected on 10 occasions between September 1992 and June 1993 from four sites on the Georges River, one site in Lime Kiln Bay (G056) and one site in Prospect Creek (Gill). Three of the sampling dates (December 1992, February and March 1993) coincided with wet weather events. Fie’d samP’ing Surface water samples (0.5 m in depth) were collected in two chemically disinfected 15 litre polyethylene containers. Every second month, an additional six containers were collected to enable water testing for viruses. Containers were disinfected by washing with 12.5% sodium hypochlorite and

Fig. 1. Location of study sites in the Georges River, Sydney.

Water quality in an estuarine system subsequently rinsing first with 52% sodium thiosulfite and then with sterile distilled water. Water depth, conductivity, pH, temperature and dissolved oxygen were measured at each site using a JRC depth sounder model JFF-545, microprocessor field conductivity and pH meter and an SI model 58 DO meter, respectively. Sediment samples were collected by divers using Perspex cylinders (length 50 cm, diameter 10 cm). Four cores were collected at each site by penetrating areas of undisturbed sediment and capping both ends of the cylinder with rubber bungs. Overlying water was siphoned off and the flocculant layer was removed to a sterile 500 ml container using a sterile plastic syringe. The top 2 cm of sediment from each core was then removed using a sterile spoon and composited with the flocculant (top sediment). The next IOcm of underlying sediment was also removed using sterile spoons and placed into chemically disinfected 4 litre containers (bottom sediment). Sediment at site GO9 consisted of coarse sand with no distinguishable flocculant layer, therefore, only the top 2 cm of sediment was collected at this site. Sample storage and analysis

Large-volume water containers were kept out of direct sunlight and stored at ambient temperature. Samples for bacterial analysis (faecal coliforms, faecal streptococci, spores of Clostridium perfringens, Aeromonas spp. and Salmonella spp.) were processed within 12 h, according to procedures approved by the Standards Association of Australia. All samples were processed using membrane filtration with the exception of sediment samples analysed for Salmonella spp. Sediment samples were transported to the laboratory on ice. For bacteriological analysis, sediments were mixed thoroughly and diluted 1: 10 with sterile distilled water. This mixture was hand shaken for l-2 min and then sonicated at 100 watts for 30 s (Braun Labsonic). The sonicated solutions were left to stand for 5-10 min to allow large particles to settle. Sediment suspensions were then processed using the same methods as for water samples. During membrane filtration solutions were lightly swirled immediately before each pipetting procedure. Faecal coliforms and faecal streptococci were enumerated using m-FC agar (Difco 0883-01) and m-Enterococcus agar (Difco 0746-01) according to standard methods (APHA, 1992). Modified methods were used for the detection of C. perfringens spores and Aeromonas spp. (Ashbolt et al., 1993; AWT EnSight, 1993). Samples to be analysed for C. perfringens spores were heat shocked at 75°C for 10 min and then cooled on ice. After membrane filtration, filters were placed onto Perfringens agar (Oxoid CM543) and incubated anaerobically at 35°C for 18-24 h. Approximately 10% of presumptive C. perfringens colonies were confirmed by streaking onto Perfringens agar supplemented with methylumbelliferyl phosphate (MUP). Aeromonas spp. were estimated by placing membrane filters onto modified m-Ryans agar (Oxoid CM833, supplemented with 0.5 mg/L ampicillin, Oxoid SR136) and incubating at 30°C for 18-24 h. Typical green colonies with the appropriate morphology were counted as presumptive Aeromonas spp. Approximately 10% of presumptive colonies were confirmed by testing for anaerobic growth, oxidase reaction and resistance to the vibriostatic agent O/129. Turbidity measurements for water samples were performed according to the standard method (APHA. 1992) usina a nenhelometer (Monitek, FSE, Sydney). One litre water samples and duplicate 20 g samples of raw sediment were inoculated into 225 ml of buffered peptone water (Oxoid CM509) for the enrichment of Salmonella spp. Broths were incubated at 37°C for 18-24 h and then 0.1 ml was inoculated into Rappaport-Vassiliadis (RV) broths (Oxoid CM669) containing 0.1 ml of 0.36% novobiocin. RV broths were incubated at 42°C for 18-24 h and then streaked onto Hektoen enteric agar (Oxoid I

.

2047

CM419) and bismuth sulfite agar (Oxoid CM201). Selective agars were incubated at 37°C for 2448 h. Presumptive Salmonella spp. were purified and tested for lysine decarboxylase, urease and ONPG reactions. Strains of salmonellae were serotyped at the Institute of Medical and Veterinary Science, Adelaide. Samples for viral and parasite analysis were stored at 4°C for up to 24 h prior to sample preparation. For viral analysis, 100 g samples of raw sediment were analysed using methods described by Grohmann et al. (1993). One hundred litre water samples were concentrated to 50ml by ultrafiltration and polyethylene glycol precipitation using hollow fibre membranes (Amicon DC10 with 100,000 Da cutoff fibres). One ml aliquots were inoculated into cultures using LLC-MK2, MRCS and HEp-2 cell lines (Grohmann et al., 1993). For parasitological analysis 15-litre water samples were concentrated by a calcium flocculation method (Vesey ef al., 1993a, b), whereby NaHCOa and CaCl* were added to the sample to create a floe. Samples were left to settle overnight prior to removal of the supernatant under vacuum. The floe was dissolved in sulfamic acid and concentrated by centrifugation. The pellet was washed twice in 0.1% Tween 80, and once with 0.05% Tween 20 in phosphate-buffered saline. Bovine serum albumin was added to the sample to give a final concentration of 4%. Fluorescein isothiocyanate labelled Cryptosporidium/Giardia monoclonal antibody, capable of detecting species pathogenic to both humans and animals (Cellabs, Brookvale, Australia), was added in equal volume to the pellet, and incubated for 30 min at 37°C. Samples were sorted by flow cytometer (Coulter Epics Elite, Florida, U.S.A.) on the basis of fluorescence and particle size. Presumptive cysts and oocysts were examined for internal structures using Nomarski differential interference contrast microscopy. Samples of both water and sediment were analysed for F-RNA bacteriophage using a slight modification of the standard double agar layer plaque method (International Organisation for Standardisation, 1991). Modifications included the addition of magnesium sulfate and kanamycin to the isolation medium, and confirmation with somatic and F-specific hosts (WG45 and WG49) rather than RNase. Sediment fractions were analysed for total organic matter content by firing a 0.5-1.0 g sample in a silica crucible at 550°C to determine the weight loss. Samples were analysed for particle size by dry sieving except when the sediment had a high organic matter content, in which case the sample was wet sieved through a 0.063 mm sieve and the coarse fraction was then dry sieved. Data analysis

All data were transformed prior to statistical analysis by conversion to a logarithmiclo scale except total organic matter content which was arc sine transformed. On each sampling date, the 72 h rainfall level was determined for each site by averaging the rainfall measured at rainfall gauges which were closest to the sampling location. If sewage overflow was discharging into Prospect Creek in the 24 h prior to sampling, the logarithmic,0 volume of overflow was recorded against site Gl II (Fig. I); all other sites were assigned zero for this variable. Sewage overflow downstream of site Gl105 was not included in the regression model as this overflow discharged only treated effluent and on only one occasion during the study. A general linear model with catchment rainfall and sewage overflow at Gl 1 I as covariates was used in the comparison of sites. Total organic matter and sediment type (either top or bottom) were fitted as covariates for the analysis of sediment data only. Various particle size fractions, particularly the mud fraction (particles ~0.063 mm size) were tested as covariates for the sediment model. However, none of the particle size fractions were significant explanatory variables and were

C. M. Ferguson et al.

2048

Table I. Physico-chemical parameters for sites in the Georges River, Sydney Variable

Glll

Distance from the sewage overflow point (km) Average water depth (m) Sediment description

Conductivity (mS/m)’ Temperature (‘C)’ Dissolved oxygen (mg/l)’ DH’

3.0 4.0 Black silt, mud & clay 676 19.8 6.3 7.2

GIllW?

G1105

3.0 4.0 Black & brown silt, mud & clav 173 ’ 18.2 5.9 8.1

4.0 7.0 Brown silt, mud & sand 783 19.6 6.8 7.3

Georges River site* GO9 GO7 7.5 3.5 Coarse sand

1440 20.1 7.0 7.4

11.5 20.0 Brown silt & sand 2723 19.9 7.1 7.6

GO6

GO56

20.5 4.0 Fine sand

22.5 3.0 Brown silt & sand

4205 19.5 7.9 7.8

3616 19.5 7.9 7.7

*See Fig. 1 for locations. ‘Adjusted geometric means. ?Gl 11W indicates samples were taken from site GI 11 during wet weather

thus not used in the final model. The final form of the model was: water data y, = a + firrain72 + /J*over + BJriver, + p4qriver,

+ Pscriver, + /&qtriver, + /&rvlkb + date, + error sediment data yur = r + PIrain

+ pzover + j&tom + bdriver, + psqriver,

+ fiacriver, + PTqtriver, + pBrvlkb + date, + (lot x date),, + typer + /&(lriver x type),k + /?lo(qriver x type),r + pll(criver x type),k + /?,z(qtriver x type),r + (date x type),&+ error Geometric means and 95% confidence intervals were calculated after fitting the covariates used in the regression model, they are therefore expressed as adjusted geometric means. All statistical analyses were performed with computer programs in the Statistical Analysis System (SAS Institute Inc., Cary, NC). RESULTS

Table 1 contains descriptive information and adjusted geometric means of a range of physicochemical parameters for the Georges River sites included in this study. Adjusted geometric mean concentrations and 95% confidence intervals of the various microorganisms for water and sediment samples are given in Figs 2 and 3, respectively. The adjusted geometric means of faecal coliforms in the water column ranged from 19 cfu/lOO ml at GO7 to 8273 cfu/lOO ml at Glll during sewage overflow events. Water column geometric mean concentrations of faecal streptococci and C. perfiingens spores were generally lower than faecal coliform concentrations, while concentrations of aeromonads were generally higher. However, all followed the same general pattern, with the highest geometric mean concentrations occurring at the most upstream site Gill during wet weather events (Fig. 2). The exception was Giardia spp. for which the highest geometric mean was 0.39 cysts/l at GO56 in Lime Kiln Bay. Concentrations of F-RNA bacteriophage, Giardia and Cryptosporidium spp. were generally low and in fact, concentrations of F-RNA bacteriophage were similar at all sites @ > 0.05). Geometric mean

turbidity levels were highest at the brackish sites (G056, G1105 and Gl 1 l), particularly at Gl 11 after inputs of sewage overflow. In contrast to the water column, geometric mean concentrations of C. perfringens spores in the sediment were greater than concentrations of either faecal coliforms or faecal streptococci (Fig. 3). However, aeromonads were again the most numerous group enumerated at the brackish sites and concentrations of F-RNA bacteriophages were low throughout. The geometric mean percentage weight of total organic matter was highest at the two sites situated closest to the tidal limits of the system, Gill in Prospect Creek and GO56 in Lime Kiln Bay. Statistical models were generated to determine whether certain variables could be used as covariates to explain variation in the data. The model for water data included rainfall in the previous 72 h, sewage overflow upstream of Gl 11, site and date. The model for sediment data contained the same covariates as for the water model with additional covariates for sediment type, total organic matter content and their interactions. The percentage probabilities generated by these models for water and sediment are given in Tables 2 and 3. In the water column, rainfall was associated with significant increases in the concentration of all the microorganisms tested and turbidity. This contrasts with the sediment data where concentrations of microorganisms were not significantly increased by rainfall, with the exception of faecal coliforms (p = 0.009). However, sewage overflow was generally associated with significant increases in the concentrations of all the microorganisms tested in both water and sediment (except F-RNA bacteriophage concentrations in sediments). Faecal coliform and faecal streptococci concentrations in the sediments were also significantly higher in the presence of organic matter (p < 0.006). In addition, concentrations of faecal coliforms, faecal streptococci and Aeromonas spp. in top sediments were significantly higher than in bottom sediments (JJ < 0.001). However, concentrations of C. perfringens spores and

2049

Water quality in an estuarine system F-RNA bacteriophages in top sediments were not significantly different from those estimated in bottom sediments. In both water and sediments, adjusted geometric mean concentrations of all the variables except F-RNA bacteriophages varied significantly between sites. The partial correlation coefficients given in Table 4 were calculated after fitting the model, in order to account for variations in rainfall and sewage overflow. In the water column, concentrations of faecal coliforms, faecal streptococci, C. perfringens spores, and Aeromonas spp. were all positively correlated with each other. However, C. perfiingens spores was the only parameter which showed a positive correlation with concentrations of either of the parasitic protozoa (Giardia spp. r = 0.41, p < 0.05). There was also significant positive correlations between turbidity and all the microbiological variables measured except F-RNA bacteriophage. In sediment, the only significant correlation was a positive relationship between the concentration of

istancs ProsDect

0

fascal strepbcacci

A

faecal wliforms

d 0 WCnrseterke

0 m (km)

f ram

faecal streptococci and Aeromonas spp. (r = 0.36, p < 0.05). Salmonella spp. were isolated from seven of the 60 water samples (12%) and 20 of the 110 sediment samples (18%) collected during the study (Table 5). The majority of positive samples (74%) were collected from the two sites closest to the sewage overflows operating during the study (Gill) and G1105). Salmonella spp. were also isolated from sites in the lower reaches of the Georges River but only when there had been more than 20 mm of rainfall in the preceding 72 h. Twenty three different serotypes of Salmonella spp. were isolated, and during large wet weather events, the same serotype was occasionally isolated from several sites along the Georges River (Table 5). Also of the positive samples, 74% were recovered from either top or bottom sediment. A total of 30 water samples and 110 sediment samples were examined for the presence of enteric adenovirus and reovirus). viruses (enterovirus,

Distance ProsDec

d o wCnrseterke t

Distance Prospect

d o wCnrssterka

am f ram 5%.

and furthest from point sources of pollution, and at G09, a site of high current velocity with coarse sediment. The highest microbial concentrations were detected closest to the known point sources of sewage pollution; Gl 11, downstream from Fairfield Stormflow Treatment Plant in Prospect Creek and G1105, upstream of Chipping Norton sewage overflow. These results correlate well with the findings of Erkenbrecher (1981) who determined that sites with higher salinity water and coarser sediment showed lower overall bacterial densities than headwater sites, where freshwater runoff and decreased tidal action contributed to higher concentrations of microorganisms. In the water column the model indicated that concentrations of all the microorganisms tested as well as turbidity were significantly increased by the occurrence of rainfall in the previous 72 h and by inputs of sewage overflows. The concentration of faecal coliforms, in particular, markedly increased with inputs of sewage overflow. Concentrations of Giardia spp. in Sydney sewage are usually about two orders of magnitude higher than that of Cryptosporidium spp. (P. Hutton, personal communication); however, as observed in English rivers (C. Fricker, personal communication), densities of Giardia spp. and Cryptosporidium spp. in water from the Georges River estuary were very similar. Possible explanations for this could be that Cryptosporidium

Table 3. Percentage probabilities for covariates in the regression model of sediment data, Georges River, Sydney Variable Faecal coliforms Faecal streptococci Clostridium perfringens Aeromonas spp. F-RNA bacteriophage

Covariate Loc*Date Date

R=

Rain72

SO

TOM

Site

0.97 0.94 0.97 0.95 0.70

0.90 ns ns ns ns

0.01 0.25 0.67 0.73 ns

0.62 0.50 ns llS

0.00 0.00 0.00 0.00

oY9

IlS

ns

ns

1.82 ns

Rz = R square from regression model. Rain72 = rainfall at each site for the 72 h prior to the sampling day. SO = logarithmicto volume of sewage overflow for the 48 h prior to the sampling day. TOM = total organic matter content as a percentage of total dry weight. Type, indicates whether there is a difference between top and bottom sediment fractions. *RepreSents the interaction of two covariates. ns = indicates not significant, p value > 5%.

0.01 0.20 0.01 0.01 llS

Type

Loc’type

Date*type

0.01 0.01

0.54 0.56 ns

0::2

0.02 0.00 0.10 0.08

ns

ns

2::2

C. M. Ferguson er al.

2052

Table 4. Partial correlation coeSicients for water quality variables measured in water and sediments of the Georges River, Sydney Variable FS FS CP CP F-RNA F-RNA AERO AERO GIAR CRYP TURB

Sample tyPe water sediment water sediment water sediment water sediment water water water

Faecal streptococci (FS)

Faecal coliforms

F-RNA bacteriophage (F-FNA)

Clostridium perfringens

(CP)

Aeromonas SPP. (AERO)

Giardia

Cryptosporidium

SPP. (GIAR)

spP. (CRYP)

0.73* 0.42*

0.32*

0.69’

0.23 0.67. 0.12 0.06 - 0.25 0.549 0.16 0.07 0.03 0.57*

0.77* - 0.20 0.21 0.21 0.47’ 0.36’ 0.22 0.08 0.81’

0.16 0.09 0.39’ - 0.08 0.41* 0.27 0.76’

- 0.03 0.03 - 0.15 - 0.39 0.36

0.10 0.05 0.39*

TURB = turbidity. R values are adjusted for the covariates in the regression models: water, degrees of freedom (df) = 39 except F-RNA bacteriophage, df = 12; sediment, df = 36 except F-RNA bacteriophage, df = 27. * = R values which are significant, p < 5%.

river waters (Payment and France, 1993). In contrast, the extremely low (< 1 pfu/lOO g) concentrations of F-RNA bacteriophages in both top and bottom sediments indicate that F-RNA bacteriophage was not an effective indicator of faecal pollution in this estuarine system. The spatial distribution of Salmonella spp. isolations in both water and sediment appeared to correlate well with known point sources of faecal pollution near Gil 1 and G1105. Isolations of Salmonella spp. were most frequent during rainfall and sewage overflow events. The isolation of the same

salmonellae serotypes from sediments at several sites along the River during wet weather suggests that the top layer of sediment, in particular, may be quite mobile during large wet weather events. Of the Salmonella spp. isolated, 14% were recovered from sediments, indicating that sediments play an important role in the persistence of salmonellae in the Georges River estuary. Similarly, Hendricks (1971) reported that 90% of the Salmonella spp. recovered from the North Oconee River (Georgia, U.S.A.) were isolated from the sediment. In addition, Geldreich (1970) indicated that the relationship between the

Table 5. Source and serotype of Salmonekr spp. isolated from the Georges River, Sydney, following rainfall and sewage overflow

Date 17 September 92 28 October 92 I1 November 92 8 December 92

Rain72 (mm) 1.0 1.7 1.2 38.8

Sewage overtlow (ml)’

Salmonella serotype

Gill

G1105

Georges River site* GO9 GO7

S. typhimurium

B TB B T

P8

S. kottbus

153

S. typhimurium,

P9

S. adelaide

T B

T T

T T B

S. infantis S. kottbus

W

s. ogona

W B T

S. zanzibor s.

OS10

S. kisarawe

0.0 4.0

GO56

T

S. ohlstedt S. havana

14 January 93 17 February 93

GO6

7

16

S. typhimurium,

T

WT

P9

W

S. ohio S. typhimurium,

T

P44

S. hadar S. paratyphi

var.

TWB

Java

S. bredeney

10 March 93

23.0

s.

warragul

s.

waycross

TB T

T

S. angoda

W

S. oirchow s.

WT W

wurragul

S. Chester S. typhimurium

29 April 93 18 May 93 22 June 93

1.9 0.3 0.0

Salmonella

subsp.1

ser16:l.v.

T

*See Fig. 1 for location. iMegalitres of sewage overflow in the preceeding 48 h. SalmoneNo spp. isolated from W, water sample, T, top sediment fraction, B, bottom sediment fraction. Ram72 = average rainfall at all sites for the 72 h prior to the sampling day. P = phage type.

B

Water quality in an estuarine system

isolation of Salmonella spp. and concentrations of faecal indicator organisms varied for different types of receiving waters and stated that in estuarine waters the recovery of Salmonella spp. was approximately 60% when faecal coliform concentrations exceeded 2000 cfu/lOO ml. Results from this study support this view, with 55% of water column samples containing Salmonella spp. once faecal coliform densities exceeded 2000 cfu/ 100 ml. Enteric viruses were isolated infrequently and their presence did not appear to be related to wet weather events. The majority of enteric virus isolations were from water and top sediment samples collected during only one of the three wet weather events sampled. Given that no bottom sediments contained detectable concentrations of enteric virus, it seems doubtful that sediments act as a reservoir for enteric virus survival within the Georges River estuary, although it is likely that more efficient recovery of virus particles from samples may have increased the frequency of detection. CONCLUSION

Water quality in the Georges River estuary was significantly affected by rainfall and sewage overflows following wet weather events. Concentrations of faecal coliforms, faecal streptococci, C. perfringens spores, F-RNA bacteriophage, Aeromonas spp., Giardia and Cryptosporidium spp. in the water were all significantly increased with rainfall and sewage overflow. However, in the sediments, only faecal coliform concentrations were significantly increased by rainfall, although sewage overflow again resulted in increased concentrations of faecal coliforms, faecal streptococci, C. perfringens spores and Aeromonas spp. (but not F-RNA bacteriophage). Salmonella spp. were generally recovered from water and sediments during wet weather events and occasionally identical serotypes were detected in sediments at several locations within the estuary. However, isolations of enteric virus were sporadic and did not appear to be exclusively related to wet weather events. C. perfringens spores were identified as the best indicator of faecal pollution and were the only indicator group significantly correlated to any of the pathogen groups in the water column (Giardia spp. and Aeromonas spp.). F-RNA bacteriophage was found to be inadequate as a potential indicator of faecal pollution due to low levels of detection and lack of correlations with any of the pathogens examined. The health significance of these findings is not clear, but warrants further investigation. For example, taking the classical indicator level of median faecal coliforms > 200 cfu/lOO ml as being unacceptable, then water quality in the upper reaches of the Georges River would appear to be of concern. The recovery of salmonellae during sewage overflow

2053

would support this stance. Nonetheless, the densities of viral and protozoan pathogens were low. A way to estimate the significance of such low level pathogen presence is microbial quantitative risk assessment (MQRA). Taking the 200 cfu/lOO ml faecal coliforms as equivalent to a daily illness rate of 19/1000 swimmers in marine water (Cabelli, 1983), Haas (1995) has used MQRA to estimate that the same level of risk from rotavirus, Giardia spp. or Shigella spp., would occur at 0.03,0.86 or 3.64 organisms per litre, respectively. Hence, our findings of human viruses and protozoan cysts and oocysts in 50 and 15 litre samples, respectively, would thus appear to give no significant increase of risk of illness above the 19/ 1000 swimmers level. Acknowledgements-This work was funded by the Clean Waterways Programme. Collection of sediment cores was assisted by McLennan Diving, Sydney. We also wish to acknowledge the assistance of staff from AWT EnSight: C. Hickey, R. Pickard, Dr. C. Davies, J. Long and C. Turner for assistance with sample collection and analysis, Dr G. S. Grohmann, G. Logan, M. Logan, and P. Caragianni for virological analysis, P. Hutton and G. Rossington for enumeration of parasites and F. Souter for enumeration of F-RNA bacteriophage. REFERENCES

(1992) Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health

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