To swim or not to swim? A disagreement between microbial indicators on beach water quality assessment in Hong Kong.

MPB-07322; No of Pages 8 Marine Pollution Bulletin xxx (2015) xxx–xxx

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

To swim or not to swim? A disagreement between microbial indicators on beach water quality assessment in Hong Kong Pui Kwan Cheung, Ka Lai Yuen, Ping Fai Li, Wai Hing Lau, Chung Man Chiu, Suet Wai Yuen, David M. Baker ⁎ Faculty of Science, the University of Hong Kong, Hong Kong Special Administrative Region

a r t i c l e

i n f o

Article history: Received 4 July 2015 Received in revised form 6 November 2015 Accepted 10 November 2015 Available online xxxx Keywords: Beach water quality Faecal indicator bacteria Enterococci E. coli Sewage

a b s t r a c t The USEPA and the WHO now advocate the use of enterococci as indicators for marine water quality. This study investigated the outcomes for Hong Kong beach water quality assessment by comparing enterococcus measures with data from the HKEPD's monitoring programme. Six beaches were tested once every 2–3 months from November 2013 to June 2014 in order to identify the most contaminated sites, followed by intensive water sampling in sites found to have the highest enterococci densities (Clear Water Bay Second and Golden) every five to six days for six sampling events over a 30-day period in 2014. The geometric means of enterococci were found to be 124 and 41 cfu/100 mL at Clear Water Bay Second and Golden respectively, indicating that there may be higher risks of illness associated with swimming at both beaches than previously known. Moreover, beach sediments contained higher concentrations of enterococci than water, and warrant further study. © 2015 Published by Elsevier Ltd.

1. Introduction In 2012, Hong Kong reached a population of about seven million. Human sewage is thus a major source of marine pollution in Hong Kong, with high concentrations of microbes posing a direct threat to human health. Over 2.7 million m3 of treated sewage is discharged into Hong Kong coastal waters every day but over 80% of this volume receives only preliminary treatment or Chemically Enhanced Primary Treatment (CEPT) with disinfection (DSD, 2014). Moreover, 7% of the population (approximately 500,000) is not covered by public sewerage (DSD, 2014). Data from the Drainage Services Department (DSD) has shown that the effluents from Stonecutters Island Sewage Treatment Works (Fig. 1) contain a high concentration of Escherichia coli (E. coli) even with disinfection (chlorination). In September 2014 for example, the geometric mean (GM) of E. coli in treated effluent was approximately 50,000 cfu/100 mL with single measures sometimes exceeding 2 x 106 cfu/100 mL. Although the effluent is discharged through a deep tunnel, it is still believed to affect the water quality in beaches relatively close to the outfalls, such as the one in Silver Mine Bay (Fig. 1) (Thoe et al., 2012) and those in the Tsuen Wan area (Fig. 1) (EPD, 2005). The lack of high-level sewage treatment and direct discharge of untreated sewage may be compromising coastal water quality in certain areas of Hong Kong. Despite known issues with sewage pollution, 41 gazetted beaches in Hong Kong have attracted nearly 10 million visitors in 2014 (EPD, 2014). Monitoring beach water quality in Hong Kong is therefore critical ⁎ Corresponding author. E-mail address: [email protected] (D.M. Baker).

for the protection of public health, as swimming in sewagecontaminated waters increases the risk of diseases and infections, most commonly gastroenteritis, skin infections and respiratory diseases. In 1990, it was estimated that swimming in polluted water accounted for over 400,000 illness cases in Hong Kong and that swimmers were five times more likely to develop gastroenteritis than nonswimmers (Cheung et al., 1990). Therefore, a positive correlation between illness rate and bacterial load has to be established and appropriate indicator microbes must be monitored in order to accurately assess the health risk of swimming in contaminated waters. In an epidemiological research programme conducted during the 1970s, a linear relationship between enterococci density and swimming-associated highly credible gastrointestinal illness (HCGI) was reported (Cabelli et al., 1982). It was also observed in the same study that the frequency of gastroenteritis cases was associated with proximity to sewage sources. Fleisher et al. (1996) investigated the dose–response relationship in swimmers exposed to sewage-contaminated waters in the UK and also found a significant increase of acute febrile respiratory illnesses in bathers when the pollution level increased from 51 to 158 faecal streptococci per 100 mL. The correlation between illness rate and microbial indicators, such as enterococci and E. coli, thus facilitates the protection of public health by setting guideline values of these indicators. The Hong Kong Environmental Protection Department (EPD) is responsible for monitoring beach water quality in Hong Kong. During the bathing season (March to October), the EPD collects water samples at least three times a month at each of Hong Kong's 41 gazetted beaches. The beaches are graded every year based on the GM of E. coli counts throughout the bathing season. The water quality criteria for Hong

http://dx.doi.org/10.1016/j.marpolbul.2015.11.029 0025-326X/© 2015 Published by Elsevier Ltd.

Please cite this article as: Cheung, P.K., et al., To swim or not to swim? A disagreement between microbial indicators on beach water quality assessment in Hong Kong, Marine Pollution Bulletin (2015), http://dx.doi.org/10.1016/j.marpolbul.2015.11.029

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P.K. Cheung et al. / Marine Pollution Bulletin xxx (2015) xxx–xxx

Fig. 1. The locations of the beaches under study and some of the sewages treatment works (STWs) with their effluent outfall directions. BF: Butterfly; CWBS: Clear Water Bay Second; GD: Golden; Repluse Bay; Stanley Main: SL; SO: Shek O. The lack of sewage outfall directions for some STWs means the outfalls are too short and are not significant in the map.

Kong (Water Quality Objectives or WQOs) set a limit on E. coli level at 180 counts/100 mL for bathing beaches, which corresponds to a swimming-associated illness rate of 10 cases/1000 swimmers. This level is based on an epidemiological study conducted in 1987 at nine beaches in Hong Kong (Cheung et al., 1990). The study found that E. coli density correlated best with combined skin symptom and HCGI, followed by faecal streptococci and enterococci (faecal streptococci and enterococci were later considered the same bacteria by WHO (2003)). However, if HCGI was considered alone, only E. coli had a statistically significant correlation. Unexpectedly, another local epidemiological study conducted in 1992 showed no statistically significant relationship between E. coli density and gastrointestinal (GI) or HCGI symptoms (Kueh et al., 1995), which challenges the utility of E. coli as a marine water quality indicator. Yet, the use of E. coli as the sole indicator for beach water quality in Hong Kong has not been changed since the EPD was established in 1986. In 2001, the EPD initiated a feasibility study on the use of alternative faecal indicator bacteria, including faecal streptococci, enterococci and Clostridium perfringens (EPD, 2002). Thereafter, the EPD dismissed the use of alternative microbial indicators for the reason that E. coli were found to be most common in beach water samples and were in higher density than other three bacterial types (Lui et al., 2007). Soon after the EPD study was completed, the World Health Organization (WHO) published guidelines for recreational waters in 2003, which recommended using enterococci as the target indicator that measures and classifies existing levels of faecal contamination. This would be further supported by sanitary inspection in the beach hinterlands, which gauges the susceptibility to faecal contamination (WHO, 2003). The WHO microbial water quality classification was primarily based on two randomized trial studies conducted in the UK, namely Kay et al. (1994) and Fleisher et al. (1996).

The randomised trial study has three major merits over the more common retrospective case–control study and prospective cohort study (Kay et al., 1994): (1) accurate differentiation between swimmers and non-swimmers, (2) accurate measurement of the levels of exposure of each swimmer, (3) better control on disease risk factors other than exposure to contaminated water. The first study was conducted from 1989 to 1992 at four UK resorts, and recruited 1216 adults and examined the relationship between gastroenteritis and five faecal indicator bacteria, including total and faecal coliforms, faecal streptococci, total staphylococci and Pseudomonas aeruginosa. It was concluded that only faecal streptococci (i.e. enterococci) had a significant dose–response relationship with gastroenteritis and that 32 cfu/100 mL (Kay et al., 1994) was the threshold level at which significant health effects occurred. The second study also demonstrated that only enterococci had a significant relationship with acute febrile respiratory illness among the same five indicator bacteria (Fleisher et al., 1996). These two studies provide a strong basis for WHO to derive its guidelines for recreational water quality, as their study designs were superior to the traditional retrospective case–control study and prospective cohort study. In 2008, given the international trend to follow WHO (2003) guidelines and adopt enterococci for marine water quality assessment, the EPD commissioned a technical review on local conditions and overseas practices, along with other aspects in marine water quality management. However, the final report only concluded that local environmental, economic and sociocultural factors had to be taken into account before making changes in beach water quality criteria for Hong Kong (EPD, 2009). In the case of the United States, EPA conducted five prospective cohort epidemiological studies between 2003 and 2007 to obtain more recent health and water quality data for the evaluation of water quality criteria. The five studies enrolled 54,250 people at the beach and followed the health conditions of both swimmers and non-



swimmers at fresh and marine beaches across tropical and temperate zones. The definition of ‘illness’ was also broadened from GI symptoms to include diarrhea and stomachache or nausea without a fever (referred to as NGI), based on more recent studies on the etiology of viral gastroenteritis. In the marine environment, culturable enterococci and Enterococcus spp. measured by quantitative polymerase chain reaction (qPCR) were the only two indictors that had an association with NGI among the six investigated which included E. coli., Bacteroides thetaiotamicro, Bacteroidales and Clostridium spp. (measured by qPCR and male-specific coliphage by antibody assay. By this time, culturable E. coli were not even included in the studies because it was considered only suitable for freshwater environment (Prüss, 1998). The new criteria adopted enterococci as the indicator for both marine and fresh water environments and were set based on NGI illness rate. For a limit of 36 cases of NGI per 1000 primary contact recreators, the GM of enterococci is not allowed to exceed 35 cfu/100 mL in any 30-day interval (U.S. EPA, 2012). A statistical threshold value (STV) was also set at 130 cfu/100 mL, which means that no more than ten percent of the samples should exceed this level in the same 30-day interval. Except certain states that have their own sets of standards (e.g. Florida), these criteria are applicable in the U.S. The UK and U.S. studies were some of the most comprehensive studies on the epidemiology of swimming-associated illnesses for the status quo and both supported the use of enterococci as an indicator for marine water quality monitoring instead of E. coli. There is growing evidence that recontamination of bathing waters by resuspended bacteria-laden bed sediments can create a chronic source of enterococci. Numerous studies have demonstrated higher persistence of indicator bacteria and pathogens in sediment than in the overlying water column (Hanes and Fragala, 1967; Davies et al., 1995; Craig et al., 2004; Fries et al., 2008). Resuspension of the bacteria-laden sediment due to wind and tidal actions can release bacteria back into the water column (Fries et al., 2008; Roslev et al., 2008), leading to a higher levels of bacteria in water and thus possible violation of water quality standards. Therefore, it is important to improve our understanding of the sources and sinks of indicator bacteria in coastal marine waters, as well as how sediment reservoirs interact with the water column and influence management outcomes. In light of the EPA's publication of the new recreational water quality criteria and the needs for Hong Kong to review its beach water quality criteria, this study aims to investigate the implications for Hong Kong beach water quality management if enterococci levels are measured and compared against the EPA's criteria. For comparison, the E. coli data collected from the EPD's regular monitoring programme is used to determine whether there would be a difference in beach water quality evaluation. The controls of some environmental factors on enterococci density in water were also examined, namely salinity, tide level,

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cumulative rainfall in three days prior to sampling, and the amount of total suspended solids (TSS) in the water column. The following hypotheses are tested in this study: (1) the enterococci density in water at the six beaches sampled in the pilot study (stage one) will be significantly different; (2) there is a statistically significant correlation between enterococci density in sediment and in the overlying water column (stage one and two) and (3) there will be a difference in water quality assessment if results based on the EPA's criteria (i.e. the GM of enterococci not exceeding 35 cfu/100 mL in any 30-day interval) are compared to results from the EPD's regular monitoring programme (stage two). 2. Materials and methods In this study, enterococci were employed as an indicator to assess the water and sediment quality on Hong Kong beaches. The study was divided into two stages. In stage one, six beaches were selected and sampled every two to three months from November 2013 to June 2014 for a total of four sampling events. In stage two, two relatively polluted beaches among the six were sampled every five to six days between 17th September 2014 and 13th October 2014 for a total of six sampling events. The sample collection procedures and analyses were kept the same in both stages. 2.1. Stage one In stage one, we selected six popular beaches that are located in different regions of Hong Kong with a known hierarchy of pollution levels based on the 2012 EPD report (EPD, 2012) (Fig.1, Table 1). Three sites were located on Hong Kong Island: Repulse Bay (RB), Shek O (SO) and Stanley Main (SL); two were located on the western New Territories: Butterfly (BF) and Golden (GD); and the last one was in the eastern New Territories: Clear Water Bay Second (CWBS). Repulse Bay, Shek O, and Clear Water Bay Second were among the most frequently visited recreational beaches in Hong Kong with “Good” annual microbial water quality rankings in 2012 while Stanley Main, Butterfly and Golden were classified as “Fair” with occasionally “Good” weekly rankings. In other words, the former three beaches represent the (less contaminated) upper end of beach water quality whereas the latter three represented the (more contaminated) lower end. 2.2. Stage two After analyzing the water quality data in stage one, Clear Water Bay Second and Golden were found to have higher enterococci density relative to the other four. Therefore, they were selected for further study in stage two.

Table 1 Comparison of grading percentage at the six beaches under the EPD's grading system (in 2013) and WHO guidelines (in stage one of this study). Source: E. coli (EPD, 2012); enterococci: this study. Beach

Grading percentage a

FID (cfu/100 mL)

Repulse Bay Shek O Clear Water Bay Second Stanley Main Butterfly Golden a

EC ENT EC ENT EC ENT EC ENT EC ENT EC ENT

Annual ranking E grading system

≦24 Good

25–180 Fair

180–610 Poor

N610 Very poor

WHO guideline

≦40

41–200

201–500

N500

EPD WHO EPD WHO EPD WHO EPD WHO EPD WHO EPD WHO

100% 75% 65% 50% 63% 50% 25% 75% 15% 25% 9% 25%

25% 35% 50% 34% 25% 72%

3% 25% 3% 25%

85% 50% 91% 50%

25% 25%

Good N/A Good N/A Good N/A Fair N/A Fair N/A Fair N/A

FID: Faecal Indicator Bacteria, enterococci (ENT), E. coli (EC).


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2.3. Sample collection

2.7. Salinity

In each sampling event, water and sediment samples were collected within 11 am to 1 pm. For the enumeration of enterococci in the water column and in the bed sediment, one 500 mL water sample and one 50 mL sediment sample were collected from two separate locations at each beach, one at the extreme ends of the shark prevention nets giving two duplicates per beach. An extra 500 mL water sample was collected at the same two separate locations for total suspended solid analysis that also gave two duplicates per beach. Surface water was first collected with sterile plastic bottles at waist depth, i.e. about 1 m deep. Surface sediment was then collected at the same place by drilling a sterile 50 mL falcon tube into the sediment. Delivery and analysis of the samples were completed under dark and cool conditions immediately after collection. In stage one, four water and sediment samples were collected at each of the six beaches. In stage two, six water and sediment samples were collected from Clear Water Bay Second and Golden.

A refractometer was used to measure the salinity of each water sample.

2.4. Enumeration of enterococci in water samples The enumeration of enterococci in water samples followed Standard Methods 9230 C (APHA, 2012). 1, 10 and 100 mL of each sample were vacuum filtered through a 0.45 μm membrane to give about 20 to 60 colonies on each plate. Each volume was replicated three times. The membrane filter was then placed on the m-Enterococcus (mENT) agar (BD Difco™) in a culture plate and incubated at 37 °C for 48 h. All light and dark red colonies were counted as enterococci. Only the three plates of the same filtered volume with colony counts between 20 and 60 were used to calculate the GM for that water sample. The above steps were repeated for the duplicate samples. It should also be noted that the U.S. EPA (2012) recommended ‘using EPA Method 1600 (U.S. EPA, 2002a) to measure culturable enterococci, or another equivalent method that measures culturable enterococci', and that the EPA Method 1600 employs mEI agar instead of mENT. A study comparing the recovery of enterococci between mEI and mENT suggested that mEI recovers more enterococci than mENT at 35 °C (Brenner et al., 2000). In other words, the use of mENT will produce a more conservative result as the density of enterococci recovered on mENT may be lower than that on mEI.

2.5. Enumeration of enterococci in sediment samples As there is no standard method for the enumeration of indicator bacteria in sediment, the experimental procedures here are modified from Craig et al. (2004). For each sediment sample, 20 mL of sterile seawater was added to 10 and 20 mL wet sediment in separate falcon tubes. The mixtures were then sonicated in a sonication bath (Craig et al., 2004) for 8 min (model: GM-600; manufacturer: TWG). The two 20 mL supernatants were filtered and cultured using the membrane filtration method described above. The above steps were repeated for the duplicate sample and the GM of the four plates was calculated to improve accuracy. Results for the density of enterococci in sediment are expressed as cfu/100 mL (v) wet sediment.

2.8. Statistical analysis For the six beaches in stage one, the Kruskal–Wallis test was used to compare the central values of enterococci density in water between them (α = 0.05; Hypothesis 1). If the central values were significantly different, pair-wise Mann–Whitney U tests would be performed to identify the pair(s) of beaches that were different. The relationship between enterococci density in water and in sediment was evaluated using simple linear regression (Hypothesis 2). The data for water was log-transformed to meet the homoscedasticity assumption of linear regression. The data from both stages were used to construct the model, assuming that the sediment-water interaction was similar in all beach environments of Hong Kong. Stage one contributed 24 data points and stage two contributed 12, making up a total sample size of 36. As environmental factors may be important controls on the density indicator bacteria in one beach but not another (Thoe et al., 2012), Spearman's rank correlation coefficients for different environmental parameters were calculated for Clear Water Bay Second and Golden individually except for TSS because sediment suspension process was assumed to be the same in all beaches. Data in both stages were used to calculate the coefficients. Tidal information and rainfall data was obtained from the Hong Kong Observatory. Statistical analyses were performed using R 3.1.2 and SPSS Statistics 20.0. 2.9. Collection of E. coli data from the EPD's database E. coli data over the same timeframe of stage two were obtained from the EPD's database (http://www.beachwq.gov.hk/en/request_info.aspx). 3. Results 3.1. Water and sediment quality in stage one The GMs of enterococci recorded from Butterfly and Golden exceeded 35 cfu/100 mL while Clear Water Bay Second, Repulse Bay, Shek O and Stanley Main were below this level (Fig.2). Butterfly was found to have the worst water quality (56 cfu/100 mL), followed by Golden (42 cfu/100 mL), Clear Water Bay Second (32 cfu/100 mL), Stanley Main (24 cfu/100 mL), Repulse Bay (15 cfu/100 mL) and Shek O (12 cfu/100 mL). The Kruskal–Wallis test results showed that there was no overall significant difference between the enterococci density in water at the six beaches during the period of study (χ2 = 2.4843, df = 5, p = 0.7789; Hypothesis 1). In sediment samples, only the GMs of Butterfly (49 cfu/100 mL) and Clear Water Bay Second (73 cfu/100 mL) exceeded 35 cfu/100 mL. Clear Water Bay Second and Shek O (32 cfu/100 mL) had higher enterococci densities in sediment than in water on a volume basis. However, by examining the length of the whiskers of the boxplot (Fig.2), enterococci density showed a higher variability (measured by SD) in sediment than in water except for Clear Water Bay Second. 3.2. Water and sediment quality in stage two

2.6. Total suspended solids (TSS) 500 mL of each water sample was allowed to pass through preweighed 0.7 μm glass fiber filter (U.S. EPA, 2002b). The filter papers with suspended sediments were placed in a drying oven (Memmert) at 103 °C following Standard Methods 2540 D (APHA, 2012) until a constant weight was reached. The filter papers were re-weighed to 0.1 mg and the differences between initial and final weights were determined.

The enterococci density at both Clear Water Bay Second and Golden was found to be higher than the U.S. EPA's recommended level, i.e., 35 cfu/100 mL in any 30-day period (Table 2). The GMs of enterococci density at Clear Water Bay Second and Golden were measured at 124 cfu/ 100 mL and 41 cfu/100 mL respectively. The former exceeded the limit by 255% and the later by 18%. Nonetheless, the EPD's data showed that the GM of E. coli at Clear Water Bay Second was only 7 cfu/100 mL between 17th Sep. 2014 and 15th Oct. 2014 and 12 cfu/100 mL at



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Fig. 2. Box plots of enterococci density in water (left) and sediment (right) at the six selected beaches in stage one. Abbreviations as in Table 1.

Golden between 15th Sep. 2014 and 14th Oct. 2014. Under the EPD's beach grading system, both beaches were not only in compliance with the regulation, but they were also considered to have ‘Good’ water quality. This is different to our study results in which both beaches were subject to elevated levels of swimming-associated risk (Hypothesis 3). The exceptionally high density measured at Clear Water Bay Second on 17th Sep. 2014 was probably due to a heavy rainfall event within the three days prior to sampling. The EPD also recorded a high density of E. coli on the same day. Except on two occasions at Clear Water Bay Second, enterococci were always more abundant in sediment than in the overlying water column at both beaches. Overall, the GMs of enterococci in sediment were 419 and 198 cfu/100 mL at Clear Water Bay Second and at Golden respectively. They were 237% and 377% higher than those in the water column, indicating that sediment might be a reservoir of enterococci. The variability of enterococci density in sediment was also higher than that in water. The SDs for sediment at Clear Water Bay Second and Golden were 3219 and 274 cfu/100 mL respectively, both more than a double of those in water. The EPD currently does not monitor beach sand or sediment quality. In samples collected in both stages, most sediment samples had higher enterococci densities than the overlying water column. While water samples rarely exceeded 100 cfu/100 mL, at least half of the sediment samples were higher than this level. 3.3. Relationship between enterococci in water and in sediment The linear regression model (Fig. 3) revealed that there was a significant correlation between enterococci in sediment and in the overlying water column (r = 0.6742, p b 0.001).

3.4. Spearman's rank correlation coefficients for some environmental parameters Tide level was negatively correlated (r = −0.186) with enterococci at Clear Water Bay Second whereas it had a positive correlation (r = + 0.127) at Golden (Table 3). Rainfall correlated relatively strongly (r = +0.431) with enterococci at Clear Water Bay Second and correlated very weakly (r = +0.100) at Golden. Salinity also had a relatively strong relationship (r = +0.276) with enterococci at Clear Water Bay Second but literally no relationship (r = 0.000) at Golden. However, the correlations were not statistically significant (p b 0.05). The relationship between enterococci and TSS was investigated for all six beaches and a relatively weak positive correlation (r = +0.173) was found and the correlation was not significant (p b 0.05).

4. Discussion In stage one, we successfully identified two beaches with higher levels of faecal contamination, which were Clear Water Bay Second and Golden located in the east and west of the New Territories respectively (Fig. 1). Although the EPD rated the overall water quality in Clear Water Bay Second as ‘Good’ in 2012 (Table 2), nearly 40% of the weekly gradings were ‘Fair’ or ‘Poor’. Therefore, it was usual to find occasions of relatively poor water quality at Clear Water Bay Second. With over 90% of the weekly gradings were ‘Fair’ (Table 1) at Golden in 2012, the findings in stage sne confirmed that it was a more contamined beach compared to the other four. For the rest of the four beaches that were not selected to be further studied in stage two, Repluse Bay and

Table 2 Enterococci density in water and sediment in stage two and E. coli density on the closest sampling days available from the EPD. The GMs higher than the EPA criteria (35 cfu/100 mL) are in bold. Abbreviations as in Table 1. Beach

Medium

FID

CWBS CWBS GD GD

Water Sediment Water Sediment

ENT ENT ENT ENT

CWBS

Water

EC

GD

Water

EC

Date (in the year of 2014)/Density (cfu/100 mL)

Source

17/09

22/09

27/09

02/10

07/10

13/10

GM

SD

8047 3570 290 460 17/09 320 15/09 13

57 788 48 299 24/09 2 22/09 9

7 677 24 106 29/09 1 28/09 15

43 20 8 403 N/A N/A N/A N/A

895 1054 209 780 08/10 4 07/10 20

30 135 9 13 15/10 7 14/10 6

124 419 41 198

3219 1300 121 274

This study This study This study This study

7

142

EPD

12

5

EPD


6


Fig. 3. Linear regression model between log-transformed enterococci density in the water column and in sediment. Dashed lines represent 95% prediction intervals.

Shek O was indeed the best two beaches as suggested by the EPD's data (Table 1). Our findings for Butterfly and Stanley Main were also in line with the EPD's data. Despite the fact that all six beaches under study had different gradings under the EPD's system in 2012, it is not surprising to find the enterococci density was not significantly different between them because the sample size in stage one was limited and therefore not highly representative of the beach. For this reason, intensive sampling was undertaken at the Clearwater Bay and Golden beach to further investigate the high enterococci density recorded at those sites. In order to improve the water quality in Hong Kong waters, the HKSAR government has commissioned a large scale sewage collection and treatment scheme, called the Harbour Area Treatment Scheme (HATS). HATS Stage 1 was launched in 2001 and currently 75% of the sewage generated from both sides of Victoria Harbour is now collected and diverted to Stonecutters Island Sewage Treatment Works (Fig. 1) where the sewage receives CEPT and disinfection (DSD, 2014). The treated sewage is discharged through a deep tunnel to the western side of Victoria Harbour (Fig. 1). The results in stage one of this study also proved that the current sewage treatment strategy was effective in protecting water quality for beaches on the southern part of Hong Kong Island (Fig.1.). However, the findings in stage two demonstrated that the water quality (measured with enterococci) at Clear Water Bay Second and Golden were not satisfactory if the EPA's criteria were applicable in Hong Kong during the period between 17th Sep. 2014 and 13th Oct. 2014. Although it may not be suitable to directly adopt the EPA's criteria to assess beach water quality in Hong Kong due to differences in climate, endemiticity, sociocultural aspects etc., the implication of our results is totally different from the EPD's assessment using E. coli, in which both beaches were graded with ‘Good’ water quality in similar periods.

Unfortunately, since the current study only monitored enterococci and not E.coli for a single month, we cannot make a comparison of the two indicators until a long-term and simutanuous determination is conducted. In fact, the EPD has conducted a feasibilty study on the use of alternative microbial indicators in 2001, including enterococci. Enterococci were found to be at very low levels and less than half of the beaches had enterococci levels over 10 cfu/100 mL as reported by Lui et al. (2007). Since then, E. coli has remained the sole indicator for Hong Kong as it was found to be the most abundant indicator bacteria (Lui et al., 2007). Our results are consistent with a study comparing total coliform, faecal coliform (E. coli is a subgroup of faecal coliform) and enterococci response for marine recreational water quality assessment in California, USA. It was found that enterococci were responsible for 99% of noncompilance of water quality standards whereas total coliform and faecal coliform were implicated in only 40% and 56% of those events, respectively (Noble et al., 2003). Noble et al. (2003) supported the use of enterococci if only one indicator had to be chosen because it was more conservative than other two indicators. The discrepancy in the compilance of beach water quality criteria between E. coli and enterococci may be explained by the lower die-off rate of enterococci than E. coli in seawater. Hanes and Fragala (1967) reported that the die-off rate of E. coli was 3 times higher than enterococci in saline water. Enterococci were also found to be more resistant to solar radiation inactivation than E. coli (Sieracki, 1980). If enterococci are indeed a better indicator for faecal contamination in marine recreational water, our results from stage two may suggest that the current sewage collection and treatment system is insufficient to protect swimmers in certain beaches of Hong Kong, particularly those that are located near effluent outfalls from sewage treatment works (Fig. 1). In 2014, over 80% of the sewage collected in Hong Kong did not receive secondary treatment and about 500,000 citizens were not covered by public sewerage (DSD, 2014). The effluent from nearby sewage treatment works may contribute to the faecal contamination at Golden and Clear Water Bay Second. Four secondary and one preliminary sewage treatment works are located to the east and west of Golden, respectively (Fig. 1). Along with the highly contaminated Tuen Mun River (EHDCC, 2013) draining into Castle Peak Bay, these treatment works are believed to be the major sources of pollution to Golden. At Clear Water Bay Second there is only one Secondary Sewage Treatment facility that may contribute to water pollution in the Bay (Fig. 1). It is therefore surprising that the microbial water quality was found to be better at Golden than Clear Water Bay Second because the former is subject to more pollution sources than the latter. Golden is located in a more open bay than Clear Water Second, and thus better flushing may facilitate the dilution of pollutants. The epidemiological study conducted in the bathing season of 1986 and 1987 in Hong Kong showed that E. coli correlated best with combined gastroenteritis and skin symptoms than enterococci (Cheung et al., 1990). However, a later epidemiological study conducted in 1992 did not find a relationship between GI symptoms and E. coli (Kueh et al., 1995), though the sample size was equally large. Kueh et al. (1995) explained the lack of correlation by the wider coverage of contamination levels of the former study. The fact that E. coli exists

Table 3 Spearman's Rank correlation coefficients of enterococci density in water with some environmental factors. Factor

N

Clear Water Bay Second

Golden

All six beaches

Tide level

8 for CWBS, 9 for GD 10

+0.204 (p = 0.629) +0.431 (p = 0.213) −0.276 (p = 0.472) N/A

+0.050 (p = 0.898) +0.100 (p = 0.783) 0.000 (p = 1.000) N/A

N/A

Rainfall (3 days cumulative) Salinity TSS

9 36

N/A N/A +0.173 (p = 0.313)



not only in human intestines but also the intestines of many warmblooded animals (Orskov and Orskov, 1981) could have masked the true density of human-sourced E. coli and the co-existing human pathogens. It is worth noting that the Livestock Waste Control Scheme was implemented in Hong Kong in 1988, at the time between the two epidemiological studies. Although animal faeces may or may not contain pathogens, it is unlikely for them to cause enteric diseases in humans (NRC, 2004). Therefore, when the second epidemiological study was conducted it was possible that the contribution of E. coli from animal waste had been significantly reduced while the amount of human sewage has remained the same or increased, leading to a discrepancy in the findings between the two studies. We have shown that enterococci are three times more concentrated in sediment than in the water column (v/v) and the two measures were highly correlated. The strong correlation suggested that the release of bacteria from sediment to water during resuspension of sediment may be an important control on water quality. In mesocosm experiments, both enterococci and faecal coliforms decay at least two times faster in the water column than in the bed sediment (Anderson et al., 2005). Hence, enterococci can possibly be released back to the water column when the bed sediment is resuspended by wind and tidal action. Furthermore, high biochemical similarity was found between the enterococci populations in beach sediment and water in a Danish estuary (Roslev et al., 2008), meaning that the enterococci population in water was likely to be from sediment. Modelling of the tidal action in the Severn Estuary, UK, also indicated the input of enterococci from sediment was an important control on their levels in the water column during spring tides when the shear stress of tidal movements on sediment was large enough to resuspend the bed sediment (Gao et al., 2013). It was also estimated that the resuspension of the top 5 mm sediment could result in 50–2250 cfu/100 mL increase in enterococci density in the overlying water, if the original density in sediment was between 1 × 104 and 4.5 × 105 cfu/100 mL (Roslev et al., 2008). The above evidence supports the notion that monitoring of sediment quality in bathing beaches cannot be overlooked (Roslev et al., 2008). Among the four environmental parameters being studied, only rainfall and salinity were found to have a strong correlation with enterococci at Clear Water Bay Second. The relationship between high rainfall and adverse beach water quality has been widely studied (Ackerman and Weisberg, 2003; Schiff et al., 2003). Storm water runoff can bring animal faeces and bacteria-laden soil into coastal waters, and where sewage treatment plants malfunction or overflow into storm water drainage during heavy rainfall this can lead to large episodic inputs and severe deterioration of beach water quality. Storm water runoff also dilutes the salinity of seawater and therefore salinity can correlate with water quality (Thoe et al., 2012). Enterococci levels were also correlated with rainfall but not salinity at Golden. As salinity at Golden is strongly influenced by the Pearl River, it may not reflect storm water inputs in this case. Tide levels were found to have opposite correlations at Clear Water Bay and Golden. This is not contradictory because the effect of tides on water quality is highly dependent on local conditions. A local study on E. coli discovered a change in the direction of its correlation with tide level at Silver Mine Bay (Fig.1) before and after HATS was commissioned (Thoe et al., 2012). The diversion of sewage from the eastern portion of Victoria Harbour to the western side has created an impact to Silver Mine Bay during neap tides when the tidewater moves southwards from Stonecutters Island (Lee et al., 2006). Golden was found to be positively correlated with tide level. During spring tides, the tidewater moved westwards from Stonecutters Island to Tsuen Wan and to Tuen Mun (Lee et al., 2006). Apart from the three sewage treatment works on the east of Golden, flood water may also bring the contaminated effluent from Stonecutters Island to Golden (EHDCC, 2013). Counter to our speculation, a weak correlation between enterococci and TSS was observed. Indeed, it is rare to find a relationship between turbidity and enterococci levels (Fries et al., 2008) although various studies suggested over 30% of the indicator bacteria in the water column

7

were attached to suspended particles (Jin et al., 2004; Fries et al., 2006). Despite the lack of clear pathway through which resuspension of sediment can affect microbial quality in the overlying water column, it is suggested that beach water quality managers should not overlook the possibility of recontamination by baterial-laden bed sediment (Craig et al., 2004; Jin et al., 2004; Fries et al., 2008; Roslev et al., 2008; Gao et al., 2013). More research is needed to compare the die-off rates between faecal indicator bacteria and pathogens in beach water and sediment and how resuspended microbes affect health risks to recreational swimmers. Despite the long history in using faecal indicator bacteria, including both E. coli and enterococci in recreational water quality assessment, these culture-based methods have fundamental shortcomings. One of them is that both E. coli and enterococci are naturally found in various environments, which means they do not necessary come from human sewage. The former was found to persist and replicate in temperate soils (Ishii et al., 2006) and the latter was present in most beach sands surveyed along the California coast (Yamahara et al., 2007). These environmental sources of indicator bacteria may be free of pathogenic strains, rendering them inappropriate for predicting enteric diseases at recreational beaches (Desmarais et al., 2002; Yamahara et al., 2009). Other shortcomings include the lack of evidence in the correlation between indicator bacteria and human pathogens (Jiang and Chu, 2004), the paucity of relationship between indicator bacteria and illnesses at beaches that are not contaminated by human sewage (Calderon et al., 1991) and the failure for the commonly used indicator bacteria, E. coli and enterococci, to predict non-GI illnesses, e.g. respiratory illnesses and skin infections (Cheung et al., 1990). Therefore, future research should focus on the evaluation of the health risks associated with the exposure to different sources of human and non-human faecal pollution, as well as the identification and quantification method of new faecal indicator bacteria and pathogens (Boehm et al., 2009). 5. Conclusion Using enterococci as an indicator, our study has shown that the microbial water quality in two popular beaches, namely Clear Water Bay Second and Golden, in Hong Kong were not satisfactory under the EPA's criteria for recreational water. In contrast, the EPD's data on E. coli showed the water quality was in the best grade in their classification system. This discrepency raises concerns over whether the current beach water quality monitoring system in Hong Kong truly reflects the risk of swimming in sewage-contamined water. Therefore, the results of this study support the EPD's initatives to review current WQOs in Hong Kong, particularly the guidelines for beach water quality. At the same time, we found a strong correlation between indicator bacteria in sediments and the overlying water column, suggesting that sediment may be an important control on water quality and should not be overlooked in the design of water quality monitoring programmes. Bathing beaches are valuable natural resources in Hong Kong and yet the current use of E. coli for water quality assessment are not sensitive enough to reveal the true level of sewage contamination in recreational waters. While it is true that Hong Kong-specific epidemiological studies should be conducted in order to re-evaluate the predictive power of enterococci density for health risks, these data are still important for assessing the presence and severity of sewage pollution, the impacts of which can extend beyond human illness. Acknowledgments We are grateful to Maria Lo for her assistance in the preparation of the lab equipment. Also, we thank Archna Anand and, Inga ContiJerpe, and the laboratory technicians working on 3/F, Kadoorie Biological Science Building for their kind support. We thank, Alex Ye, Freda Guo, Jill Li, Chan On Tung and Cheung Wing Lam for helping with the


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sample collection and the lab work. We are thankful to Joyce Wong and Ruby Choy for their efforts in the earlier stage of this project and to Vicki Sheng for editing. This study was funded by the HKU Faculty of Science, Environmental Science program. References Ackerman, D., Weisberg, S., 2003. Relationship between rainfall and beach bacterial concentrations on Santa Monica bay beaches. J. Water Health 1, 85–89. Anderson, K.L., Whitlock, J.E., Harwood, V.J., 2005. Persistence and differential survival of fecal indicator bacteria in subtropical waters and sediments. Appl. Environ. Microbiol. 71, 3041–3048. APHA, 2012. Standard Methods for the Examination of Water and Wastewater 22th ed. American Public Health Association, Washington DC, USA. Boehm, A., Ashbolt, N., Colford, J., Dunbar, L., Fleming, L., Gold, M., Hansel, J., Hunter, P., Ichida, A., McGee, C., 2009. A sea change ahead for recreational water quality criteria. J. Water Health 7, 9–20. Brenner, K.P., Siefring, S.D., Brown, S.L., 2000. Comparison of mEnterococcus agar and the U.S. Environmental Protection Agency — Recommended Enterococci Methods, mE and mEI agar, Presented at American Society of Microbiology, Los Angeles, CA, May 21–24, 2000. Cabelli, V.J., Dufour, A.P., McCabe, L.J., Levin, M.A., 1982. Swimming-associated gastoenteritis and water quality. Am. J. Epidemiol. 115, 606–616. Calderon, R.L., Mood, E.W., Dufour, A.P., 1991. Health effects of swimmers and nonpoint sources of contaminated water. Int. J. Environ. Health Res. 1, 21–31. Cheung, W.H.S., Chang, K.C.K., Hung, R.P.S., Kleevens, J.W.L., 1990. Health effects of beach water pollution in Hong Kong. Epidemiol. Infect. 105, 139–162. Craig, D.L., Fallowfield, H.J., Cromar, N.J., 2004. Use of microcosms to determine persistence of Escherichia coli in recreational coastal water and sediment and validation with in situ measurements. J. Appl. Microbiol. 96, 922–930. Davies, C.M., Long, J.A., Donald, M., Ashbolt, N.J., 1995. Survival of fecal microorganisms in marine and freshwater sediments. Appl. Environ. Microbiol. 61, 1888–1896. Desmarais, T.R., Solo-Gabriele, H.M., Palmer, C.J., 2002. Influence of soil on fecal indicator organisms in a tidally influenced subtropical environment. Appl. Environ. Microbiol. 68, 1165–1172. DSD, 2014. Sustainability Report 2013–14. Drainage Services Department, HKSAR, Hong Kong. EHDCC, 2013. Tuen Mun Beach and River Water Quality Report. Environment, Hygiene and Development Committee (EHDCC), Tuen Mun District Council, Hong Kong, p. 5. EPD, 2002. Study report on collection of background information on the alternative faecal indicators in the environmental waters of Hong Kong. Environmental Protection Department, HKSAR. EPD, 2005. 20 Years of Beach Water Quality Monitoring in Hong Kong. Environmental Protection Department, HKSAR, Hong Kong, p. 89. EPD, 2009. Review and Development of Marine Water Quality Objectives: First Stage Public Engagement — Technical Note: Review of Local Conditions and Overseas Practices The Environmental Protection Department, HKSAR, Hong Kong. EPD, 2012. Beach water quality report in Hong Kong 2012. Environmental Protection Department, HKSAR, Hong Kong, p. 57. EPD, 2014. Beach water quality report in Hong Kong 2014. Environmental Protection Department, HKSAR, Hong Kong, p. 58. Fleisher, J.M., Kay, D., Salmon, R.L., Jones, F., Wyer, M.D., Godfree, A.F., 1996. Marine waters contaminated with domestic sewage: nonenteric illnesses associated with bather exposure in the United Kingdom. Am. J. Public Health 86, 1228–1234. Fries, J., Characklis, G., Noble, R., 2006. Attachment of fecal indicator bacteria to particles in the Neuse River Estuary, N.C. J. Environ. Eng. 132, 1338–1345. Fries, J.S., Characklis, G.W., Noble, R.T., 2008. Sediment–water exchange of vibrio sp. and fecal indicator bacteria: implications for persistence and transport in the Neuse River Estuary, North Carolina, USA. Water Res. 42, 941–950.

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