Swimming in Sewage by ACAP Saint John

ACAPSAI NTJOHN

SWI MMI NGI NSEWAGE

I NDI CATORSOFFAECAL WASTEONFI SHI NSAI NTJOHN, NB

Swimming in Sewage Indicators of faecal waste on fish in Saint John, New Brunswick, Canada

Heather Loomer, BSc. Dr. Karen Kidd, University of New Brunswick in Saint John Tim Vickers, Atlantic Coastal Action Program (ACAP) Saint John

Acknowledgements I would like to primarily thank my supervisor Dr. Karen Kidd, and Tim Vickers from the Atlantic Coastal Action Program Saint John for first giving me this opportunity and then for their guidance, patients, and support along the way. For technical, theoretical, and instructional advice I would like to acknowledge the contributions of Alison McAslan, Dr. John Johnson, Kelly Cummings, Dr. Sandy Wilson and the staff of the ACAP Water Quality Monitoring Program. For advice and assistance in the field I would like to thank the staff of ACAP Saint John, Methvan lab, MacLatchy lab, and Munkittric lab. For help processing samples I would like to thank Tim Barrett, Andrea Hicks, and Diana Loomer.

This initiative was made possible through a generous contribution from the

EJLB FOUNDATION

Abstract The release of raw sewage into the environment is a practice that, although has currently become less common, is still present around the world. The primary concern associated with this practice is the risk to human health through disease transmission of pathogens associated with faecal matter. Saint John N.B is one of the few cities in Canada that still releases untreated sewage into the surrounding waterways and harbour area. Water faecal coliform levels, an indicator of faecal waste and associated pathogens, are well above recreational guidelines in many of these areas. Of concern are the fishing practices that occur in the harbour area. Between August and November 2005, a series of investigations on the presence faecal coliforms on the skin of wild fish, smelt and mummichog, and caged mummichog in sewage influenced areas were done. Variables such as time and water faecal coliform level in relation to the number of faecal coliforms on the surface of the fish were investigated. The wild fish sampling demonstrated, in two species with different habitats, that faecal coliforms are present, in elevated numbers in some cases, on wild fish around the Saint John area and that the amount of faecal coliform on fish skin is influenced by the water faecal coliform level. It was also found that low levels of faecal coliforms on fish skin were associated with exposure to water considered unsafe for human contact due to a high probability of pathogen presence.

iii

Table of Contents Introduction.......................................................................................................................1 Methods............................................................................................................................11 Sampling Methods ……………………………………………………………………..11 Wild Fish…………….…………………………………………………………………...11 Smelt……………………………………………………………………………………..11 Mummichogs…………………………………………………………………………….13 Caging Exposure Experiments…………………………………………………………..14 Time Exposure…………………………………………………………………………...15 Location Exposure……………………………………………………………………….16 Water Samples…………………………….....…………………………………………..16 Quality Control…………………………………………………………………………..16 Laboratory Methods………………………………………………………………...….17 Materials………………………………………………………………………………....17 Sample Preparation………………………………………………………………...……17 Membrane Filtration Procedure…………………………………………………………18 Enumeration……………………………………………………………………………..19 Quality Control……………………………………………………………………..……20 Statistics……………………………………………………………………………..…..21 Results………………………………………………………………………….………..22 Wild Fish……………………………………………………………………………..…22 Smelt…………………………………………………………………………………..…22 Mummichogs……………………………………………………………………….........22 Caging Exposure Experiments………………………………….……………………..23 Time Exposure……………………………………………………………………….…..23 Location Exposure……………………………………………………………………….23 Discussion…………………………………………………..…………………………...24 Figures………………………………………………………………………..………….29 Tables……………………………………………………………………………………34 Literature Cited………………………………………………………………………...36 Appendices …………………………………………………………………………….. 39

List of Figures Figure 1: Scatter plot of faecal coliforms / cm2 from multiple skin samples taken from 6 smelt gillnetted from Long Wharf, Saint John on August 23, 2005. ……………………………………………………………………………………………29 Figure 2: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on wild mummichogs caught from Hazen Creek September 19, 2005, Saint’s Rest September 17 and 25, 2005, and Marsh Creek September 30, 2005. Error bars represent the standard error. The log10 plus 1 transformed water faecal coliform level (colonies / 100 ml) at the time of sampling is also shown. ……………………………………………………………………………………………30 Figure 3: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on mummichog caged at Marsh Creek Saint John, N.B. between August 5 and 13, 2005, and sampled after 12, 24 (1 day), 48 (2 days), 96 (4 days), and 192 (8 days) hrs. Error bars represent the standard error. …………………………………………………………………………………………..31 Figure 4: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on mummichog caged between October 14 and 21, 2005 at Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. Error bars represent the standard error. The average log10 plus 1 transformed water faecal coliform level (colonies / 100ml) of samples taken when the cages where put out and brought in at each location is shown. ……………………………………………………………………………………………32 Figure 5: Scatter plot by cage of faecal coliforms (colonies / cm2) on mummichog caged at Marsh Creek Saint John, N.B. during the location exposure caging experiment. ……………………………………………………………………………………………33

List of Tables Table 1: Descriptive statistics for skin samples from (faecal coliforms / cm2) wild mummichog caught from Hazen Creek, Saint’s Rest, and Marsh Creek Saint John, N.B. ……………………………………………………………………………………………34 Table 2: Descriptive statistics for skin samples from (faecal coliforms / cm2) mummichog caged at Marsh Creek Saint John, New Brunswick between August 5 and 13, 2005, sampled after12 hour, 1, 2, 4, and 8 days. ……………………………………………………………………………………………34 Table 3: Descriptive statistics for skin samples from (faecal coliforms / cm2) mummichog caged between October 14 and 21, 2005 at Tucker Park, Indian Town, Harbour Passage, and Marsh Creek Saint John, N.B. ……………………………………………………………………………………………35

List of Appendices Appendix I I 1: The percentage of sewage from different areas of the city that is treated in 1993 and 2004. ……………………………………………………………………………………………39 I 2: The areas in the City of Saint John that do not receive sewage treatment are outlined in black. ……………………………………………………………………………………………39 I 3: The faecal coliform level based on analysis of water samples taken once a week for 8 weeks around Saint John during the summer of 2005. ……………………………………………………………………………………………40

vii

Appendix II II 1: Locations where smelt were gillnetted in August, 2005. ……………………………………………………………………………………………41 II 2: Locations where wild mummichog were minnow trapped and seined in September, 2005. ……………………………………………………………………………………………41 II 3: Locations where mummichog were caged in the time and location exposure caging experiments in August and October, 2005. ……………………………………………………………………………………………42 II 4: The typical set up for a set of cages in the time and location exposure caging experiments in August and October, 2005. ……………………………………………………………………………………………43 II 5: Recipe for the buffer used in the faecal coliform testing of fish skin and water. ……………………………………………………………………………………………43 II 6: Set-up of the membrane filtration apparatus used in the faecal coliform testing of fish skin and water. ……………………………………………………………………………………………44

viii

Appendix III III 1: The faecal coliforms (colonies / cm2) found on the skin of Smelt caught from Long Wharf and Rhodney Pier Saint John, N.B between August 23 and 25, 2005. Fish fork lengths were also recorded. ……………………………………………………………………………………………45 III 2: The faecal coliform level, faecal coliforms / 100 ml, of water from the Long Wharf and Rhodney Pier Smelt sampling locations between August 23 and 25, 2005. ……………………………………………………………………………………………46 III 3: The faecal coliforms / cm2 found on the skin of wild mummichog caught between September 17 and 30, 2005 from Saint’s Rest, Hazen Creek, and Marsh Creek Saint John, N.B. ……………………………………………………………………………………………46 III 4: The faecal coliform level, faecal coliforms / 100 ml, of water from Saint’s Rest, Hazen Creek, and Marsh Creek wild mummichog sampling locations between September 17 and 30, 2005. ……………………………………………………………………………………………47 III 5: The faecal coliforms (colonies / cm2) found at different time periods on the skin of caged mummichog exposed in Marsh Creek between August 5 and 13, 2005. ……………………………………………………………………………………………47 III 6: The faecal coliform level, faecal coliforms / 100 ml, of water from Marsh Creek between August 5 and 13, 2005. ……………………………………………………………………………………………48 III 7: Scatter plots of the faecal coliforms / cm2 found at different time periods on the skin of caged mummichog exposed in Marsh Creek between August 5 and 13, 2005. ……………………………………………………………………………………………49 III 8: The faecal coliforms / cm2 found on the skin of caged mummichog exposed for four days at Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. ……………………………………………………………………………………………52 III 9: The faecal coliform level, faecal coliforms / 100 ml, of water sampled on Day 0 and Day 4 of four day caging exposures at Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. ……………………………………………………………………………………………53 III 10: Scatter plots of the faecal coliforms / cm2 found on the skin of caged mummichog exposed for four days at Indian Town, and Marsh Creek Saint John N.B (results

from Tucker Park and Harbour Passage omitted due to the absence of faecal coliforms on the mummichog). ……………………………………………………………………………………………54 III 11: Results of duplicate bacterial tests performed on randomly sampled positive faecal coliform colonies. ……………………………………………………………………………………………55

Introduction The exponential growth of the human population in the last two centuries has lead to an increasing amount of bodily waste that must be disposed off. The sewage system is the most common and sanitary disposal system. It is employed by most developed nations and is the goal of developing nations (World Health Organization and UNICEF 2000). The sewage system is also a more general disposal system for many other household cleaning and hygiene products and industrial wastes resulting in a diverse mixture of substances entering the waste stream (Servos 2001). This mixture is then released into the aquatic environment where the literature has shown it has serious effects on the health of the receiving environment and human activities. For example, sewage inputs to waterways has resulted in restrictions on water and shellfish consumption and caused the closure of beaches and restriction of other recreational activities (Kilgour 2005). One of the primary concerns with sewage is the risk of disease transmission. Various types of pathogens are found in faecal waste and contact with this waste can cause diseases such as hepatitis A, Norwalk flu, cholera and different forms of dysentery (American Public Health Association 1992). The risk of disease transmission affects the recreational and commercial use of the receiving environment. For example, beaches and shellfish harvesting closures have occurred to due to faecal contamination from leaks at sewage treatment facilities and overflowing sewers (Servos 2001).

Sewage inputs can change the structure and function of an aquatic ecosystem by increasing nitrogen and phosphorous and decreasing oxygen in the water (Servos 2001; MIAI 2006). Increased nitrogen and phosphorous stimulate algal growth, which can result in algal blooms. These blooms immediately affect the functioning of the benthic environment by preventing the sunlight from filtering down to the aquatic macrophytes. This reduces the growth of these plants, which in turn depletes the habit that serves as protection for many aquatic organisms. The death and decomposition of the algae increases the biological oxygen demand (BOD) in the aquatic environment. This high BOD reduces the amount of dissolved oxygen (in the water) available to sustain other organisms in the aquatic ecosystem (MIAI 2006). The microbial breakdown of faecal matter or other organic matter present in sewage also requires a large amount of oxygen, which increases the BOD on the aquatic environment (Servos 2001). Sewage is essentially a chemical cocktail containing mixtures of chemicals used in our everyday life. Many of these chemicals are listed as toxic substances under the Canadian Environmental Protection Act (Servos 2001). Recently there has been increasing concern over the presence of pharmaceuticals and personal care products in sewage. Of particular concern is the lack of information about the effects these chemicals may have on the aquatic organisms (Servos 2001). Due to the harmful effects related to sewage, it is typically treated before it is released. In general, there are three basic levels of treatment. Primary treatment involves removing larger solids that have settled out and layer of scum and debris formed on the surface (Servos 2001). Sewage that has received only primary treatment is more aesthetically pleasing but still contains high concentrations of microorganisms, nutrients,

and chemicals. In secondary treatment, oxygen consuming materials and finer suspended solids are broken down by bacteria within the sewage (Servos 2001). This process often uses aeration to promote bacterial activity (Sierra Legal Defence Fund 2004). The sewage then receives disinfection through chlorination, UV radiation, or ozonation (DPIWE 2006). Tertiary treatment further purifies the sewage by removing nitrogen and phosphorous. The treatment of sewage generates sludge, a mixture of extracted bio-solids and chemicals. The sludge is put in a landfill, incinerated, or applied as fertilizer to land (Servos 2001). Although the treatment of sewage prior to its release into the aquatic environment does decrease its effects in the aquatic environment even tertiary treatment does not eliminate all impacts. For this reason, new technologies are continuing to develop (Servos 2001). Sewage treatment is a costly procedure and is implemented to different degrees throughout the world. In Asia, Africa, and Latin America the struggle for sanitary sewage removal system is the primary concern. A sanitary sewage removal system is considered either a connection to a public sewage system or septic system or some form of pit latrine that does not require handling of the wastes (World Health Organization and UNICEF 2000). Less than half of the populations in the developing areas (31 to 49%) have access to a sanitary sewage removal system and only 18% of the Asian populations have household sewer connections (World Health Organization and UNICEF 2000). In developed nations 90-100% of the population is serviced by a sanitary sewage removal system and concern for sewage treatment is much higher (World Health Organization and UNICEF 2000). Many developed nations regulate sewage treatment. For example, in

1991 the council of the European Communities ruled that by December 31, 2005 all collected sewage must receive secondary treatment before release by (DESA 2004). In Canada, sewage treatment practices have improved in the past 20 years; however, there are still gains to be made (DESA 2003). In 1996, 74% of the Canadian population was served by municipal sewage systems and the remaining 26% were served by private systems (Servos 2001). A later survey in 1999 showed that of those served by municipal sewage systems, 78% were receiving secondary or tertiary treatment. The rest were releasing an estimated 3 trillion litres of untreated or primary treated sewage per year into the Canadian waterways (DESA 2003). For example, Victoria, Vancouver, Dawson City, Montreal, Saint John, Halifax, Charlottetown, and St. John’s are some of the Canadian cities known for releasing primary treated or untreated sewage (Sierra Legal Defence Fund 2004). Saint John, New Brunswick, is a city that discharges untreated sewage into its harbour and surrounding waterways. The city has been working towards “Harbour Clean Up” since 1993. At that time 60% of this city’s sewage was being released untreated into the surrounding waterways and all three levels of government, municipal, provincial, and federal, recognized that this was a problem. Recommendations were made for the upgrade and development of three sewage treatment facilities with 100% secondary sewage treatment, at a minimum, as the goal (City of Saint John 2004). Since then, the inputs of raw sewage have been reduced through investments and upgrades in the treatment facilities in the West and North sides

of the City. However, no improvements have been made to increase the treatment of sewage in the South and East ends of the City (City of Saint John 2004) (Appendix I, 1 and 2). Currently, 46% of Saint Johnâ&#x20AC;&#x2122;s sewage is released untreated into the harbour, which totals 16.3 million liters a day or 6 billion liters a year (ACAP 2005; Sierra Legal Defense Fund 2004). As the presence of untreated sewage is a risk to human health the sanitary quality of the water should be monitored. Testing for the presence of disease-causing pathogens specifically is an impractical way to monitor waterâ&#x20AC;&#x2122;s sanitary quality. First, there are many very specific and unique pathogenic micro-organisms and testing for all of them would be expensive and time consuming. Secondly, since many pathogens are found in very low densities large quantities of water would have to be processed to detect them. Therefore, a more common indicator of faecal waste (faecal coliforms) is used to determine the probability that disease causing pathogens are present (Bauman 2004). Faecal coliforms are a group of bacteria which are considered to be a good indicator of faecal waste and are commonly used in water quality analysis by governmental and commercial agencies (Havelaar 2001; Bauman 2004). Faecal coliforms are a diverse group of gram-negative bacilli that are all part of the Family Enterobacterae; however, not every member of Enterobacterae can be considered a faecal coliform (Baumann 2004). Faecal coliforms are naturally present in high densities in the intestinal tract and subsequently in the waste products of all warm blooded animals. Therefore, their presence in water or foods can be distinctly attributed to the presence of faecal waste from these animals and they are found in higher densities than any pathogens (Baumann 2004).

Most faecal coliforms do not present a large risk to human health. Within the Family Enterobacterae there are 13 out of the 41 genera contain pathogenic strains and only 6 of the genera are part of the faecal coliform group. Furthermore, these pathogens are all opportunistic and cause disease when the immune system is compromised, when reductions in neighboring bacterial populations increasing resource availability, or when they are introduced to abnormal body areas. For example, E. coli from the intestine can cause infections when introduced to the urinary tract (Baumann 2004). Tests for the presence of faecal coliforms have been developed based on the groupâ&#x20AC;&#x2122;s habitat and their alternative energy sources. Faecal coliforms are found in the intestinal tract of warm blooded animals and, unlike other bacterium, flourish at body temperature (37.5 oC) and withstand the elevated temperatures (>37.5 oC) induced by the bodyâ&#x20AC;&#x2122;s defense mechanisms (Baumann 2004). For this reason faecal coliforms are often considered thermo-tolerant. Bile from the liver acts as a surfactant to break down food and release nutrients in the digestive tract; therefore, faecal coliforms must be able to grow unaffected in the presence of surfactants. The last major defining characteristic of faecal coliforms is their ability to ferment lactose in the absence of glucose, which basically means they can convert lactose into glucose, a useable energy source (Baumann 2004). The effectiveness of faecal coliforms as indicators of the presence of diseasecausing pathogens has been investigated. It has been found that low levels of faecal coliforms accurately predict the absence of pathogens; however, high levels of faecal coliforms only predict an increased probability that enteric pathogens will be present (Hood et al 1983; Baumann 2004). This is based on the fact that there is no guarantee that

pathogens are present in faecal waste, even though this is often the case (Hood et al 1983; Baumann 2004). The major criticism of faecal coliforms as indicators of water quality is that although they behave similarly to some pathogens in the environment such as Salmonella (Hood et al 1983, Miguel 1990), they are not as resistant to environmental stressors such as varying temperature and salinity as are some pathogens, viruses and spore-forming bacteria (Craig et al 2002). Other indicators of the presence of pathogens have been investigated and used in combinations (Craig et al 2002; Golas et al 2002). However, there is no current alternative that is significantly superior to warrant changing current sanitary regulations and guidelines. Sanitary regulations and guidelines, based on faecal coliform levels, are currently in place for drinking water, recreational activities on or in water, the use of treated sewage in agriculture and aquaculture, and for shellfish consumption. Drinking water should have no faecal coliforms / 100 ml sample and water used for recreational purposes, swimming and boating should have no more than 200 faecal coliforms / 100 ml (American Public Health Association 1992). The World Health Organization guidelines for the use of treated human (faecal) waste and waste water in agriculture and aquaculture practices dictate that (faecal coliform) levels should not exceed 1000 faecal coliforms/ 100ml or 100 g (Havelaar et al 2001). For shellfish consumption faecal coliform level should not exceed 2 colonies per gram of muscle tissue (Hood 1983). For the past 12 years the Atlantic Coastal Action Program (ACAP) has been monitoring the water quality in the waterways around Saint John during the summer months. Included in the water quality monitoring program were measures such as temperature, dissolved oxygen, nitrogen, phosphorous, and faecal coliforms. All of these

measures can be affected by an influx of raw sewage; however, of major concern for human health are the faecal coliform levels. The 2005 sampling season found faecal coliform levels at sites within the Harbour and at other locations receiving untreated sewage that were well above recommended guidelines for recreational use of water (Appendix I, I 3). Although information exists on the faecal coliforms in water there is no information about the faecal coliforms on fish living in these waterways. Proper preparation of the fish would likely render them safe for consumption; however, there is concern about pathogen transmission during handling of the raw fish and contamination of preparation surfaces and utensils as well as fishing equipment (de Donne et al. 2002; Edwin et al. 2004). Overall, there is a lack of knowledge about the relationship between the microbial composition of the water and any contamination of the fish. Only a few studies have shown that sewage-related bacteria, such as faecal coliforms, have been found on the surface of fish or other aquatic organisms. Parson and Payne (2002) investigated the types, amount, and number of bacterial colonies on flounder, crab, and lobster from the St. John’s Harbour, Newfoundland. Like Saint John, St. John’s was releasing a large amount, 33.2 billion liters a year, of raw sewage into their harbour. The water faecal coliform levels were not examined; however, it was found that opportunistic pathogenic and nonpathogenic bacteria associated with human faecal waste were present on these aquatic organisms. El- Shenawy and El- Samura (1994) exposed fish to raw sewage with 21 000 faecal coliforms / 100ml for twelve hours and found an average of 20 faecal coliforms / cm2 on skin samples taken from the fish. The fish were then placed in clean water and it was determined that it took 3 days to clear the fish’s skin

of faecal coliforms. This experiment did not give justification for the use of a 12 hour exposure time so it is not known if this was sufficient time for the maximum level of faecal coliforms to be found on the fishâ&#x20AC;&#x2122;s skin. Zmyslowska et al (2003) investigated microbiological conditions of tank reared Sturgeon. The total amount thermo-tolerant bacteria in the water and in the fish mucus was compared. The level of thermo-tolerant bacteria was 7110 and 1060 colonies / cm 3 in the water and mucus, respectively. Therefore, it was concluded that bacterial counts were found in lower amount in the mucus than in the water. A more detailed investigation of the relationship between water faecal coliform concentrations or exposure time and the amount of faecal coliforms found on fish skin is needed. Saint John, New Brunswick has commercial and recreational fishing occurring in areas known to have raw sewage inputs and high faecal coliform levels. It is possible that an environment fostering the transmission of sewage-borne pathogens to humans currently exists. Fish such as Rainbow Smelt (Osmerus mordax) and American Shad (Alosa sapidissima) maybe exposed to raw sewage in the harbour and then caught and sold to the public or brought home with the fisherman. Currently there are conflicting messages regarding the regulation of the fishing practices in the Saint John Harbour. The Department of Fisheries and Oceans Canada has regulations detailing when commercial fishing of several species can occur in the Saint John Harbour; however, there are no regulations prohibiting commercial fishing in

general. Interestingly, official signs warning against recreational fishing in the harbour can be seen at public areas close to the water, for example along Harbour Passage (T. Vickers, ACAP Saint John, personal communication and H. Loomer, personal observation). The presence of these signs does not stop recreational fishing in areas of the harbour. If fish caught in the Saint John Harbour have faecal coliforms on their skin then the risk to human health would have to be assessed and changes to fishing practices might be required. In this Honourâ&#x20AC;&#x2122;s thesis, my objectives were to determine: 1) If faecal coliforms are present on wild fish in the waterways around Saint John 2) The relationship between the length of time the fish are exposed to high faecal coliform concentrations in the water and the amount on their skin. 3) The relationship between the concentration of faecal coliforms in the water and the amount of their skin. It was hypothesized that faecal coliforms would be present on wild fish caught from waters receiving raw sewage and the amount of faecal coliforms on the fish would increase with increasing water faecal coliform level. This work helped to determine if the current fishing practices present a risk to human health by potentially transmitting disease-causing pathogens associated with sewage. It also improved our understanding about the relationship between faecal coliforms in water, time of exposure and the subsequent contamination of fish.

Methods Sampling Methods Wild Fish The wild fish were caught from locations around the greater Saint John area with and without inputs of untreated sewage. The water faecal coliform levels at these locations recorded by ACAP Saint Johnâ&#x20AC;&#x2122;s Water Quality Monitoring Program show varying levels of faecal contamination. The smelt were caught from the inner harbour area and the sites were accessed by boat. The mummichog were caught from salt marshes on the east and west sites of the city and the sites were accessed by foot. Smelt: The Rainbow smelt is mainly found along the eastern coast of North America as well as some landlocked lakes and areas on the western coast of North America (Nova Scotia Department of Agriculture and Fisheries 2005). They are commonly found in the Bay of Fundy and caught from the Saint John Harbour. Smelt are an anadromous fish species, which means that they grown and mature in salt water but are born and spawn in freshwater (Nova Scotia Department of Agriculture and Fisheries 2005). Twenty-seven smelt were caught from Rhodney Pier (8 fish) Long Wharf (19 fish) in the Saint John Harbour with monofilament gillnets on August 23 and 25, 2005 (for site locations see Appendix II 1; for fish lengths see Appendix III, III 1). The locations were chosen because they are used for recreational fishing in the area. The gill nets were left in the water during the rising tide from 10 until

12 am and only live fish were collected. Fish were sacrificed by severing their spinal cord while they were still trapped in the net and then the fish were removed from the net. Care was taken to ensure that the area between the gills and the anal pore of the fish was not touched during this process to prevent transfer of bacteria to or from the site to be sampled. The fish were carefully placed on ice in a cooler for transport to the laboratory at the University of New Brunswick, Saint John (UNBSJ). At the laboratory, a 2 cm2 sample of surface tissue and some underlying muscle was taken using sterile techniques from the side of the fish that did not rest on the ice. A rectangular plastic template was used to obtain the 2 cm2 sample. Previous studies have shown that the muscle tissue of sewage-exposed fish does not contain faecal coliforms (El- Shafai et al 2004) and so the presence of muscle tissue in the sample would not affect the number of faecal coliform counted in this study. The tissue sample was stored at 4 to 5째 C in a sterile 15 ml test tube containing 5 ml of sterilized Tryptic Soy Broth (TSB). TSB is a general purpose medium that is used for cultivating most types of micro-organisms. The samples were stored at 4 to 5째 C to inhibit the growth of the bacteria during storage. All of the samples were taken within two hours of the fish being caught. Surface water samples were collected at the same time as the fish.

Mummichog (Fundulus heteroclitus): A mummichog is small bodied fish that lives in saltwater marshes or other brackish creeks along the eastern coast of North America (Paesani M. 2002). Twenty-three wild mummichog (approximately 4 to 5 cm) were caught from three locations in the Saint John area between September 17 and 30, 2005. Minnow traps were used to catch mummichog from Saintâ&#x20AC;&#x2122;s Rest (7 fish) and Marsh Creek (9 fish). A beach seine was used to catch mummichog from Hazen Creek (7 fish) (for site locations see Appendix II, II 2).

The mummichog were poured from the traps or scooped up from the net into containers that had been pre-cleaned with bleach and were full of water from the site and then transported back to the laboratory within 20 to 30 minutes of capture. At no point during their capture were the fish touched. The fish were lethally anaesthetized using CO2 gas and then their spinal cords were severed. A 2 cm2 piece of skin was removed from the upward facing side of the fish and stored as previously described. The sample was not used if the side of the fish came into contact with any surface during this procedure or if

the internal viscera were cut during sampling. Either situation could affect the bacterial count for the sample. Surface water samples were also taken at the time of fish collection.

Caging Experiments

Two caging experiments were conducted at different locations around Saint John to investigate how the length of exposure (time experiment) and water coliform concentrations (location experiment) affect bacterial counts on the skin of fish (for site locations see Appendix II, II 4). The first caging experiment was conducted at a highly contaminated site to determine whether fish become contaminated with bacteria and the amount of time it takes for maximum levels of faecal coliforms to occur on the fish. These results were then used to design the caging experiment to look at the relationship between faecal coliform counts in the water and on the surface of the fish. The locations used for both exposures were chosen based on water faecal coliform levels measured by the ACAP Water Quality Monitoring Program between July and August 2005 (Appendix I 3). Mummichog were used in both caging experiments as they are able to tolerant a wide variety of environmental conditions (Paesani M. 2002). They were collected from Hazen Creek in Saint John with a beach seine on July 19 and September 19, 2005. The fish were held at the UNBSJ in a 106 litre rectangular tub (10-15째 C, 15-20 ppt). The fish were fed once daily during the warmer months (July-September) or every second day in the cooler months (October-November) with 3.0 GR High Pro fish feed (Corey Feed Mills) or Cichlid flaked food (Nutrafin, for the smaller fish that could not eat the pellets).

The fish were held at the university prior to exposure to ensure that the fish were exposed to clean water prior to their use in the caging exposure experiments. A sub-sample (5 fish) was tested from the tanks before the fish were used to ensure that they were clear of faecal coliforms prior to use. The cages were 4 L hard plastic cylinders with two large mesh covered openings. At most sites the cages were suspended 0.5 to 1 m below the water surface during low tide and 3 m below the water surface during high tide; the exceptions were the cages at the Harbour Passage location which, at high tide, were submerged between 20 -30 m below the water surface. Cages were anchored down and arranged on a pulley-type system with ropes to increase ease during sampling and removal of the cages (Appendix II, I).

Time of Exposure Caging Experiment: Mummichog (approximately 5 to 6 cm) were put in Marsh Creek in four cages (18 fish / cage) on August 5, 2005. The fish were transported into the field in a cooler and a net was used to put them randomly into the cages. Three fish per cage were sampled as described above for the wild mummichog after 12 hours and, 1, 2, 4 and 8 days (Âą 2 hours). Surface water samples were taken at the time of sampling.

Location Exposure Caging Experiment: Changes were made to the methods used in this experiment to improve the quality control (see below) and to streamline the sample preparation procedure. Sets of 4 cages (4 fish / cage) were put in the water at four different locations around Saint John, N.B. The fish were transported into the field in 4 10 L disposable plastic bags. Each bag contained all the fish to be put in one cage and at no point during the transport procedure were the fish in contact with anything outside the bag. After 4 days at these sites the cages were taken out of the water and all the fish from one cage were transported back to the lab in a new 10 L bag in water from the site. Surface water samples were taken on days 0 and 4 of the exposure from each site.

Water Samples Water samples were collected by moving open sterile Whirl paks against the current just below the surface of the water. The water samples were transported to UNBSJ in a cooler and stored at 4 to 5째 C. Transportation time in the cooler was less than 30 minutes, and storage time did not exceed 24 hrs.

Quality Control Procedures for the Field Sampling To determine the variability of faecal coliform counts on the surface of the smelt, two or three skin samples were taken from 6 of the smelt caught from Long Wharf. Four or five fish were sampled from the holding tanks prior to the caging studies to ensure that the fish were not contaminated with faecal coliforms before they were used. To examine whether there was any faecal coliform contamination as a result of the field procedures,

four or five fish were transported out into the field at the same time as experimental fish were put in the cages and then brought back to the laboratory for sampling.

Laboratory methods Materials The magnesium phosphate buffer (Appendix II, II) was autoclaved for 15 minutes at 240째 C in two hundred refillable 100 ml amber medicine bottles. Tinfoil was used to cover the plastic caps and to prevent UV degradation of the buffer. Trypic soy broth (Difco) was prepared according to manufacturer instructions, and 5 ml was put into each 15 ml glass test tube. The test tubes were then autoclaved. The mFC (Difco) agar was plated out in 50 x 11 mm plates approved for water testing (Fisher Scientific). Approximately 300 ml of distilled water was put into 500 ml Erlenmeyer flasks and then sealed and autoclaved.

Sample preparation Wild Fish and the Time Caging Experiment: All sampling was done using sterile techniques. The tissue sample was ground up with a 5 ml glass homogenizer in the TSB. The mixture was poured from the homogenizer back into the test tube and the homogenizer was rinsed twice with 2.5 ml of magnesium phosphate buffer solution into the test tube. Buffer was used to rinse the homogenizer instead of sterile distilled water to prevent osmotic shock for the bacteria. The total volume within the test tube was 10 ml. The sample was stored at 4-5 째C for up to 12 hours until it was filtered.

Location Exposure: The skin sample that was stored in 5 ml TSB was ground up with a (5 ml) glass homogenizer. Three sub-samples of 2.5, 0.42 and 0.07 ml were taken from the homogenate and each was put in a pre-sterilized 10 ml test tube. The subsamples were then diluted with up to approximately 10 ml with sterilized buffer to ensure even dispersal of the homogenate across the membrane during filtration. The amount added did not need to be precise because it did not affect the subsequent number of faecal coliforms filtered onto the sterile filter paper. Also, due to the high viscosity of the homogenate the passage of fluid through the filter membrane was very slow. If more than 15 ml of buffer was used at this stage the time required to filter a single sample increased substantially. The total surface area of fish skin in the initial homogenate was 2 cm2. The sub-samples represented 1 cm2, 0.167 cm2, and 0.027 cm2 surface area. This was done to get a 1: 6 dilution series.

Membrane Filtration Procedure A standard vacuum filtration apparatus was used to filter the homogenate onto a pre-sterilized 47 mm Millipore filter membranes (Fisher Scientific) (Appendix II, II). The membrane was first wetted with 20 ml of the phosphate magnesium buffer before any samples were filtered. For the samples from the wild fish and from the first caging study, 0.05 to 5 ml were taken from the test tube, put into a graduated cylinder, and topped up to 15 ml with the buffer. The sample was then filtered through the membrane and the cylinder was then rinsed three times with small amounts of the buffer that was also filtered through the membrane. This same procedure was used to filter the water samples collected from the field sites except that the volume of the sub-samples varied from 0.05

to 100 ml depending on the faecal coliform levels expected at the different sites using ACAP Saint John’s Water Quality Monitoring Program data. Samples from the second caging study (location exposure) were filtered directly from the above-mentioned test tubes, and these tubes were also rinsed three times with small amounts of buffer that also was poured onto the filter. The filter membrane was then placed on an inverted 50mm mFC agar plate using sterilized tweezers. The agar plate was labelled and stored inverted on the bench top until all samples were filtered (< 1 hour). During larger sampling sessions in which the first samples filtered would have remained on the bench top for extended periods of time (up to 6 hrs) the labelled plates were stored at 4 to 5° C until the entire batch was done to ensure that colonies had equal growing time before they were enumerated. All plates were put in the incubator at 44.5 ± 0.5 °C for 24 ± 2 hrs to allow the faecal coliform colonies to grow.

Enumeration Samples were enumerated using standard methods for water samples (American Public Health Association, 1992). The plates were removed from the incubator and all of the blue colonies, a positive result for faecal coliform growth, were counted. Since all bacteria in a particular colony on the plate originated from a single bacterium trapped in the filter membrane, the number of colonies represented the number of individual bacterium in the original sample. The number of colonies was expressed as the number of faecal coliforms / cm2 tissue. Since different dilutions were done with the sample, the number of faecal coliforms / cm2 was calculated based on the dilution that yielded colony counts between 20 and 60 per plate. If no dilution yielded bacterial colony counts above

20 but more than one of the dilutions yielded counts less than 20 then the number of faecal coliforms/cm2 was based on the total colonies for these dilutions. If more than one dilution yielded between 20 and 60 faecal coliform colonies then these samples were expressed as the number of faecal coliforms/cm2 and the average of the values was used. If there were more than 60 colonies on the plate it was considered Too Numerous to Count (TNC) because crowding on the plate may inhibit the growth of some colonies and affect the accuracy of the result. A TNC count was expressed as > 60 and was recorded for some of the water samples.

Quality Control in the Laboratory Three different methods of sterilization were employed in the laboratory. Tweezers and scalpels used in the dissections and in handling the filters were sterilized by dipping them in 95% ethanol and passing them through a flame. Pipettes, graduated cylinders, and empty test tubes used to hold or measure portions of the sample were sealed and autoclaved prior to use. The filtration apparatus and the homogenizer were sterilized between uses with two rises of sterile distilled water, to osmotically shock any micro-organisms, followed by a rinse with 95% ethanol and another rinse with sterile distilled water. Buffer, distilled water and broth were autoclaved in the appropriate containers prior to use. A sterile environment was maintained within these vessels during the experiment by flaming the opening of the vessels during processing. Blanks using buffer and the pre-sterilized TSB test tubes were run at the beginning and periodically

between samples to ensure that effective sterilization and that no cross contamination occurred. Eighteen colonies were randomly selected from the plates and tested to confirm that they were faecal coliforms. Pricked colonies were streaked on TSA to produce individual colonies that did not contain the indicator chemical present in the mFC. These individual colonies were then pricked and cultured on TSA slants for 24 to 48 hrs before testing. A gram stain was done to classify the samples as a gram-negative bacillus. The samples were grown on the differential media McKonycase (Becto-Dickinson), which is selective for gram-negative lactose fermenting bacillus, and on brilliant green bile broth (Becto-Dickinson), which is selective for bacteria with the ability to grow in the presence of surfactants. Pre-prepared oxidase test slides (Becto-Dickinson) were used to ensure that the bacteria had the oxidase enzyme, an important enzyme in the metabolic pathways of all faecal coliforms. These tests confirmed the colonies as feacal coliforms (Appendix III, III 11).

Statistics Descriptive statistics were done for each experiment. Data from the first caging experiment were log10 plus 1 transformed and analysed across sampling times using ANOVA and Student Newman Keul multiple comparison tests with an alpha value set at 0.05. The data were log transformed to satisfy the ANOVAâ&#x20AC;&#x2122;s homogeneity of variance assumption and 1 was added due to the presence of 0 faecal coliforms in two samples. In the wild mummichog sampling and the second caging experiment the amount of faecal coliforms on the fish at each site did not follow a normal distribution and the variance

was not equal between locations. For this reason, a nonparametric test was used to evaluate the differences between locations. The Kruskal Wallis One-Way ANOVA was used and post hoc comparisons were done using the Mann – Whitney U test (Pett 1997). The alpha was set at 0.05 and type 1 errors were controlled for with the Holms Stepdown procedure (Pett 1997). All statistics were calculating using SPSS for Windows version 13.0 and graphs were generated with Sigmaplot 9.0.1.

Results

Wild fish Smelt On an individual fish, counts ranged from 0 to 25 coliforms / cm2 of skin and the inter-sample variability is shown in Figure 1. An average of 3.36 ± 1.4 faecal coliforms were detected on smelt caught from Long Wharf and the water faecal coliform level was 283 colonies / 100 ml. Faecal coliforms (4) were found on 1 of the smelt caught from Rhodney Pier and the water faecal coliform level was 82 colonies / 100 ml. Of all of the fish captured (27) 48.1% of the smelt had faecal coliforms on their skin.

Mummichogs Higher faecal coliform levels in the water were accompanied by higher faecal coliform levels on the fish (Figure 2). The counts on the fish from Marsh Creek (27.2 ± 9.4 faecal coliforms / cm2) were significantly higher when compared to the results for

fish from Hazen creek (0 faecal coliforms / cm2) and Saint’s Rest (0.3 ± 0.5 faecal coliforms / cm2); counts for fish the latter two sites were not significantly different (Table 1).

Caging Exposures Time Exposure at Marsh Creek Over the entire experiment, mean faecal coliform counts on the caged mummichog ranged from 7.14 ± 2.95/cm2 to 1672 ± 388 / cm2 (Table 2). The water faecal coliform concentrations ranged from 20,000 to >60,000/100ml. Faecal coliform counts on the mummichog increased significantly from 12 hours (7.14 ± 2.95 faeacal coliforms / cm2) to 4 days (1672.52 ± 388.05 faecal coliforms / cm2). After 8 days the mean faecal coliforms decreased to (13.5 ± 2.96 / cm2) and was not significantly different from what was observed at 12 hours (7.14 ± 2.95 faecal coliforms / cm2) and 1 day (20.5 ± 3.65 faecal coliforms / cm2) (Figure 3).

Location Exposure Higher faecal coliform levels in the water were accompanied by higher faecal coliform levels on the fish (Figure 4). The mean faecal coliforms on mummichog caged at Marsh Creek (116 ± 34 / cm2) and at Indian Town (1.1 ± 0.38 / cm2) were both significantly different from each other, and these two sites were significantly higher than what was observed on the fish caged at the Tucker Park (0 / cm2) and Harbour Passage (0 / cm2) sites (Table 3).

Discussion

Faecal coliforms were found on the skin of smelt, a local recreationally caught fish, and mummichog caught from different locations in the Saint John Harbour area. To understand the relevance of this, it must first be understood that these bacteria are not normally present in high concentrations in the aquatic environment. Faecal coliforms are in the aquatic environment only when faecal waste from a warm blooded animal is present. Low concentrations of faecal coliforms are common in many bodies of water due to the presence of wild life; however, only when a body of water is contaminated with faecal waste do high concentrations of faecal coliforms occur. Therefore, the presence of these bacteria on fish is not a likely occurrence in the absence of faecal contamination due to the low density of faecal coliforms in the water. The results from the wild mummichog study and the location exposure caging experiment supports this, as faecal coliforms where only detected on the skin of fish only caught from water with elevated faecal coliform levels. In contrast, the results from the smelt study show that faecal coliforms were present on one of the fish caught from Rhodney Pier, where water faecal coliform level was lower. However, unlike the mummichog study the smelt caught at the different locations were from the same population of fish and were living in an environment contaminated with raw sewage. There was no barrier between the two locations and as smelt are a migratory species it is possible that fish from Rhodney Pier had previously been feeding at Long Wharf.

The number of smelt with faecal coliforms present was higher in water where faecal coliform concentration was higher; however, a relationship between water faecal coliform level and the presence of faecal coliforms on the smelt cannot be concluded. The smaller sample size collected from Rhodney Pier (8 compared to 19) and the ability of the fish to travel between the two sites limits the ability to make comparisons. This in combination with the conclusion that the amount of faecal coliforms present on the smelt is an underestimate (discussed in following paragraph) leaves doubt about the validity to the apparent distinction between the smelt caught at the two sample sites. Even though I found that 48% of the smelt had faecal coliforms on their skin, it is possible that this study has underestimated the proportion of fish in the Saint John Harbour that are contaminated. When multiple samples were taken from the same fish, faecal coliforms were not consistently present in all samples. This suggests that faecal coliforms may still be present on the surface of the fish even when no faecal coliforms were detected using a single tissue sample. It is less likely that this is true for the mummichog tested because the mummichog were smaller than the smelt and a greater area of their skin was sampled (approximately 30-40%) compared to the area of smelt skin sampled (approximately 2-5%). The presence of faecal coliforms on fish is not unique to one fish species or to one type of aquatic environment. In this study, faecal coliforms were present on two fish species from different habitat. Previous studies have shown faecal coliforms to be present on different fish species sampled from the field or the lab (Parson and Payne 2002; ElShenawy and El- Samura 1994). This suggests that the presence of faecal coliforms on fish skin may be a widespread effect due to faecal waste contamination of the aquatic

environment and that fish from such environments may also be contaminated with faecal coliforms, which should be investigated. The caging experiments provided a more controlled design to investigate factors affecting faecal coliform counts on fish skin. The results from the time exposure experiment done at Marsh Creek were unexpected because the counts were highest after 4 days and decreased on day 8. It is interesting to note that the faecal coliform counts on the fish were similar at times when the water faecal coliform levels were quite different. For example, the faecal coliform levels on the fish were not statistically different between 12 hrs and 8 days; however, the water faecal coliform levels at 12 hrs and 8 days were >60,000 colonies / 100ml and 20,000 colonies / 100ml, respectively. This discrepancy could be due to rapid fluctuations in water faecal coliform levels, the length of exposure time to a particular water faecal coliform level, or a combination of both factors. In future studies more frequent measurements of the water faecal coliform levels should be done to have a more accurate record of what the fish were exposed to. Faecal coliform counts on the skin of fish caged at different locations around Saint John were generally higher on individuals from the more contaminated sites, and agreed with the results from the wild fish sampling. The major limitation in this study was the variability in the water faecal coliform levels over time. There were large fluctuations between the water faecal coliform levels on days 0 and 4 at both the Indian Town and the Marsh Creek sites. Therefore, the exact water faecal coliform levels that resulted in the amount of faecal coliforms on the fish cannot be determined. As stated previously, more frequent water faecal coliform measurements would improve our ability to understand the exposure history of the fish.

During the caging study it was noted that the mummichog at the Marsh Creek location were discoloured and appeared ill (personal observation). These mummichog were from the 1st, 2nd, and 3rd cage and had between 48 and 294 colonies / cm2. In contrast, the mummichog from the 4th cage placed at this site appeared healthy and had between 6 and 21 colonies / cm2 (Figure 5). No conclusions can be drawn from this; however, future work might investigate the relationship between indicators of fish health and the amount of faecal coliforms on the fish. Marsh Creek is heavily polluted with the toxic chemicals found in sewage and with creosote, a chemical used to preserve wood (ACAP 2005) These chemicals could affect the fishâ&#x20AC;&#x2122;s ability to protect itself from faecal contamination. A previous study has shown that exposure to creosote-contaminated sediments can adversely affect the composition and quantity of mucus produced by fish (Mezin and Hale 2000). Since mucus functions as part of the fishâ&#x20AC;&#x2122;s immune system, changes in its composition would affect the fishâ&#x20AC;&#x2122;s ability to protect itself from infection or illness (Collado et al 2000). When the sanitary quality of water is tested a specific density of faecal coliforms is used to determine the probability that disease causing pathogens are present; however, it is not known what density of faecal coliforms must be present on the fish to make similar judgements. When faecal coliforms were found in high densities on the surface of the fish, as was found on fish from Marsh Creek, it is probable that disease causing pathogens were also present on those fish. However, when faecal coliforms were found in lower densities on the fish skin, as on the smelt caught from Long Wharf or caged at Indian Town, the probability that disease causing pathogens were also present is unknown.

There is currently very little known about the presence of faecal coliforms on fish in areas receiving sewage. This study is one of the few that has examined how the length of exposure to water with different faecal coliform levels can affect the presence of faecal coliforms on fish. Since sanitary sewage disposal issues are a worldwide problem, the results here are applicable to many other areas. In fact, other studies have looked at faecal coliforms on wild fish or fishing gear in areas receiving raw sewage and have made similar conclusion regarding the risk to human health (Parsons and Payne 2002; Edwin et al 2004). It is recommend that more attention be given to the catching of wild fish in areas receiving untreated sewage as this has been overlooked as mode of disease transmission to humans in both developed and undeveloped countries.

Figure 1: Scatter plot of faecal coliforms / cm2 from multiple skin samples taken from 6 smelt gillnetted from Long Wharf, Saint John on August 23, 2005.

Faecal Coliforms/ 1cm

Smelt 1

Smelt 2

Smelt 3

Smelt 4

Fish Faecal coliforms/ cm2 of fish skin

Smelt 5

Smelt 6

Figure 2: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on wild mummichogs caught from Hazen Creek September 19, 2005, Saintâ&#x20AC;&#x2122;s Rest September 17 and 25, 2005, and Marsh Creek September 30, 2005. Error bars represent the standard error. The log10 plus 1 transformed water faecal coliform level (colonies / 100ml) at the time of sampling is also shown.

log10 (1 + number of faecal coliforms)

* 1

k 5) 5) ree 9/0 9/0 0 C 0 / / (25 (17 zen est Ha est R R s s int' int' Sa Sa

Location Faecal coliforms/ 1 cm2 of fish skin Faecal colifomrs/ 100 ml of water

* Significantly different from all other sample locations

k ree C rsh Ma

Figure 3: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on mummichog caged at Marsh Creek Saint John, N.B. between August 5 and 13, 2005, and sampled after 12, 24 (1 day), 48 (2 days), 96 (4 days), and 192 (8 days) hrs. Error bars represent the standard error.

log10 (1 + number of faecal colifomrs)

** 1

0 0

100

150

200

Hours Faecal coliforms/ cm2 of fish skin

* Significantly different from all other time periods ** Significantly different from Time 12 hrs. but not from Time 192 hrs.

250

Figure 4: The mean log10 plus 1 transformed faecal coliforms (colonies / cm2) on mummichog caged between October 14 and 21, 2005 at Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. Error bars represent the standard error. The average log10 plus 1 transformed water faecal coliform level (colonies / 100ml) of samples taken when the cages where put out and brought in at each location is shown.

log 10 ( 1 + number of faecal coliforms)

Tucker Park Harbour Passage Indian Town

Location

Faecal coliforms/ cm2 of fish skin Faecal coliforms/ 100 ml of water

* Significantly different from all other sample means

Marsh Creek

Figure 5: Scatter plot by cage of faecal coliforms (colonies / cm2) on mummichog caged at Marsh Creek Saint John, N.B. during the location exposure caging experiment.

350

Faecal Coliforms/ 1cm

300 250 200 150 100 50 0

Cage 1

Cage 2

Cage 3

Cages

Faecal coliforms/ cm2 of fish skin

Cage 4

Table 1: Descriptive statistics for skin samples from (faecal coliform / cm2) wild mummichog caught from Hazen Creek, Saintâ&#x20AC;&#x2122;s Rest, and Marsh Creek Saint John, N.B. 95% Confidence Interval for Mean N Mean

Std. Deviation

Std. Error

Upper Bound

Lower Bound

Minimum Maximum

Hazen Creek

Saint's Rest

-.4

Marsh Creek

27.2

28.1

9.35

5.65

48.8

Table 2: Descriptive statistics for skin samples from (faecal coliforms / cm2) mummichog caged at Marsh Creek Saint John, New Brunswick between August 5 and 13, 2005, sampled after12 hour, 1, 2, 4, and 8 days. 95% Confidence Interval for Mean N Mean

Std. Deviation

Std. Error

Upper Bound

Lower Bound

Minimum Maximum

12 Hours

7.0

3.0

.52

.00

1 Day

3.7

2.7

2 Days 10

120

187

59.3

-13.6

255

9.09

636

4 Days 12 1151

1522

439.4

184.2

2118

35.00

4500

3.0

6.7

1.8

8 Days 12

Table 3: Descriptive statistics for skin samples from (faecal coliforms / cm2) mummichog caged between October 14 and 21, 2005 at Tucker Park, Indian Town, Harbour Passage, and Marsh Creek Saint John, N.B. 95% Confidence Interval for Mean N Mean

Std. Std. Deviation Error

Upper Bound

Lower Bound

Minimum Maximum

Tucker Park

Harbour Passage

Indian Town

Marsh Creek

116

117

33.8

41.6

190.

6.00

294

Literature Cited American Public Health Association. 1992. Standard methods for the examination of water and wastewater (18th ed). Washington DC Atlantic Coastal Action Program Saint John (ACAPSJ). 2005. Environmental Issues: Harbour Clean Up. http://www.acapsj.com/harbour.html Bauman R.W. 2004. Microbiology. Person Education. San Francisco, USA City of Saint John. 2004. Water and wastewater Saint John in the 21st century. Collado R., Fouz B., Sanjuan E., and Amaro C. 2000.Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla. Diseases of Aquatic Organisms 43: 91-101. Craig D.L., Fallowfield H.J., and Cromar N.J. 2002. Comparison of decay rates of faecal indicator organisms in recreational coastal water and sediment. Water supply 2: 131-138. Department of Oceans and Fisheries Canada. 2004. Maritime Provinces Fishery Regulations: Schedule VIII (Sections 24 and 73) Shad close times. http://laws.justice.gc.ca/en/F-14/SOR-93-55/119615.html

Department of Primary Industries, Water & Environment. 2006. Waste water: sewage treatment. http://www.dpiwe.tas.gov.au/inter.nsf/WebPages/LBUN-4YP6U3 de Donno A., Montagna M.T., de Rinaldis A., Zonno V., Gabutti G. 2002. Microbiological parameters in brackish water-pond used for extensive and semiintensive fish-culture: Acquatina. Water Air and Soil Pollution 13: 205-214. Edwin S., Jeyasekaran G., Shakila R.J., Anand C. 2004. Sanitary status of Thoothukkudi Fishing Harbour of Tamil Nadu, India. Journal of Food Science and TechnologyMysore 41: 530-533. El- Shenawy MA and El- Samura ME.1994. Accumulation and elimination of pathogenic bacteria in tilapia fish. Bulletin of the National Institute of Oceanography and Fisheries (Egypt) 20: 59-68. Mid-Atlantic Integrated Assessment. 2006. Eutrophication. Environmental Protection Agency. http://www.epa.gov/maia/html/eutroph.html Golas I., Lewandowska D., Zmyslowska I., Teodorowicz M. 2002. Sanitary and bacteriological studies of water and European catfish (Silurus glanis) during wintering. Archives of Polish Fisheries 10: 177-186.

Havelaar A., Blumenthal U.J., Strauss M., Kay D., and Bartram J. 2001. Chapter 2 Guidelines, Standards and Health. Water Quality. World Health Organization. London, UK Kilgour B.W., Munkittrick K.R., Portt C.B., Hedley K., Culp J., Dixit S., and Pastershank G. 2005. Biological criteria for municipal wastewater effluent monitoring programs. Water Quality Resources Journal of Canada 40: 374–387. Mezin L.C. and Hale R.C. 2000. Effects of contaminated sediment on the epidermis of mummichog, Fundulus heteroclitus. Environmental Toxicology and Chemistry 19: 2779-2787. Moriñigo M.A., Córnax R., Muñoz M.A., Romero P., and Borrego J.J. 1990. Relationships between Salmonella spp and indicator microorganisms in polluted natural waters. Water Research 24: 117-120. Nova Scotia Department of Agriculture and Fisheries.2005. Rainbow Smelt. CanadaNova Scotia Cooperation Agreement on Economic Diversification, Resource Competitiveness Program. http://www.gov.ns.ca/nsaf/sportfishing/species/smel.shtml Paesani M. 2002. Fundulus heteroclitus. Animal Diversity Web. http://animaldiversity.ummz.umich.edu/site/accounts/information/Fundulus_heter oclitus.html Parsons, D.S. and Payne, J.F. 2002. A bacteriological investigation of selected flounder, crab and lobster collected from St. John=s Harbour, June 2001. (Unpublished report prepared for DFO, Newfoundland Region and St. Johns Harbour ACAP). Pett, M.A. 1997. Nonparametric Statistics for Health Care Research. Sage Publishing, California, USA. Selvakumar, A, Borst M, Boner m, Mallon P. 2004. Effects of sample holding time on concentrations of microorganisms in water samples. Water Environment Research 76: 67-72. Servos, M., Chambers, P., Macdonald, R. and, Van Der Kraak, G. 2001. Chapter 9 Municipal waste water effluent. Threats to sources of drinking water and aquatic

ecosystems in Canada. National Water Research Institute. Environment Canada Sierra Legal Defense Fund Report. 2004. The national sewage report card (Number three): Grading the Sewage Treatment of 22 Canadian cities. UN Department of Economic and Social Affairs. 2003. Freshwater country profile: Canada. United Nations http://www.un.org/esa/agenda21/natlinfo/countr/canada/canada_freshwater.pdf

UN Department of Economic and Social Affairs. 2004. Sanitation country profile: United Kingdom. United Nations http://www.un.org/esa/agenda21/natlinfo/countr/uk/UK_sanitation.pdf Water Quality Monitoring Program. 2003. Fecal Coliform Procedures. ACAP Laboratory Procedure World Health Organization and UNICEF. 2000. Ch. 2 Global Status. Global Water Supply and Sanitation Assessment 2000 Report. http://www.who.int/water_sanitation_health/monitoring/Glassessment1.pdf Zmyslowska I, Kolman R, and Krause J. 2003. Bacteriolocal evaluation of water, feed, and sturgeon. Archives of Polish Fisheries 11: 91-98.

Appendix I I1: The percentage of sewage from different areas of the city that is treated in 1993 and 2004.

90% 80% 70% 60% 50%

1993

40%

2004

30% 20% 10% 0% West

North

Sounth/ East

(City of Saint John 2004) I 2: The areas in the City of Saint John that do not receive sewage treatment are outlined in black.

(modified from Tim Vickers, ACAP Saint John)

I 3: The faecal coliform level based on analysis of water samples taken once a week for 8 weeks around Saint John during the summer of 2005. Sample Week June 27 July 3 July 10 July 17 July 24 July 31 August 7 August 14 Average

Inner Harbour Upstream (colonies /

Inner Harbour Downstream

March Creek Upstream (colonies /

Marsh Creek Downstream

100 ml)

(colonies / 100 ml)

100 ml)

(colonies / 100 ml)

240 600 1893 1900 517 1100 N/A 680

240 204 91 600 97 136 N/A 600

256 292 540 389 580 1180 580 1160

6 (10 3 ) 6 (10 6 ) 5.2 (10 7 ) 7.6(10 6 ) 6.4(10 6 ) 6.4(10 6 ) 1.4(10 7 ) 3.4(10 7 )

1218 +/- 229.8

281 +/- 79.5

622 +/- 127

2.4(10 7 )+/-9.6(10 6 )

Appendix II II 1: Locations where smelt were gillnetted in August 2005.

Long Wharf

Rhodney Pier

II 2: Locations where wild mummichog were minnow trapped and seined in September, 2005.

Marsh Creek Hazen Creek

Saintâ&#x20AC;&#x2122;s Rest

II 3: Locations where mummichog were caged in the time and location exposure caging experiments in August and October, 2005. Tucker Park Indian Town Harbour Passage

Marsh Creek

II 4: The typical set up for a set of cages in the time and location exposure caging experiments in August and October, 2005.

II 5: Recipe for the buffer used in the faecal coliform testing of fish skin and water. 1. Stock Potassium Phosphate Solution: • Dissolve 34 g of potassium dihydrogen phosphate in 500 ml of distilled water. • Adjust pH to 7.2 +- .5 with 1 N sodium hydroxide. • Dilute volumetrically to 1000 ml with distilled water and transfer to a sealed amber bottle. 2. Magnesium Chloride Solution: • Dissolve 81.1 g magnesium chloride in 500 ml of distilled water • Dilute volumetrically to 1000 ml with distilled water and transfer to a sealed amber bottle. 3. Magnesium Phosphate Buffer Solution: • Combine 1.25 ml of stock potassium phosphate solution and 5.00 ml of magnesium chloride solution. • Dilute volumetrically to 1000 ml. (Water Quality Monitoring Program, 2003)

II 6: Set-up of the membrane filtration apparatus used in the faecal coliform testing of fish skin and water.

Appendix III III 1: The faecal coliforms / cm2 found on the skin of smelt caught from Long Wharf and Rhodney Pier Saint John, N.B between August 23 and 25, 2005. Location Fork Fish Faecal Coliform Smelt Date Sampled (day/month/year) Sampled Lengths(cm) Level (FC / cm2) 1 23/08/ 2005 Long Wharf 12 5.0000 1 23/08/ 2005 Long Wharf 3.0000 1 23/08/ 2005 Long Wharf 0.0000 1(average) 23/08/ 2005 Long Wharf 2.6700 2 23/08/ 2005 Long Wharf 10 2.0000 2 23/08/ 2005 Long Wharf 19.0000 2 23/08/ 2005 Long Wharf 18.0000 2(average) 23/08/ 2005 Long Wharf 13.0000 3 23/08/ 2005 Long Wharf 9 0.0000 3 23/08/ 2005 Long Wharf 25.0000 3(average) 23/08/ 2005 Long Wharf 12.5000 4 23/08/ 2005 Long Wharf 12 0.0000 4 23/08/ 2005 Long Wharf 4.0000 4 23/08/ 2005 Long Wharf 0.0000 4(average) 23/08/ 2005 Long Wharf 1.3300 5 23/08/ 2005 Long Wharf 12 1.0000 5 23/08/ 2005 Long Wharf 0.0000 5 23/08/ 2005 Long Wharf 2.0000 5(average) 23/08/ 2005 Long Wharf 1.0000 6 23/08/ 2005 Long Wharf 12 3.0000 6 23/08/ 2005 Long Wharf 2.0000 6 23/08/ 2005 Long Wharf 1.4000 6(average) 23/08/ 2005 Long Wharf 2.3300 7 23/08/ 2005 Long Wharf 10 23.000 8 23/08/ 2005 Long Wharf 9 3.0000 9 23/08/ 2005 Long Wharf 10 0.0000 10 23/08/ 2005 Long Wharf 11.5 2.0000 11 23/08/ 2005 Long Wharf 11 1.0000 12 23/08/ 2005 Long Wharf 12 1.0000 13 23/08/ 2005 Long Wharf 10 0.0000 14 23/08/ 2005 Long Wharf 10 0.0000 15 23/08/ 2005 Long Wharf 11 1.0000 16 23/08/ 2005 Long Wharf 11 0.0000 17 23/08/ 2005 Long Wharf 10 0.0000 18 23/08/ 2005 Long Wharf 9.5 0.0000 19 23/08/ 2005 Long Wharf 10 0.0000 20 23/08/ 2005 Rhodney Pier 10 0.0000 21 23/08/ 2005 Rhodney Pier 10 0.0000 22 23/08/ 2005 Rhodney Pier 12 0.0000 23 23/08/ 2005 Rhodney Pier 10 0.0000 45

24 25 26 27

23/08/ 2005 25/08/ 2005 25/08/ 2005 25/08/ 2005

Rhodney Pier Rhodney Pier Rhodney Pier Rhodney Pier

11 10 9 10

0.0000 0.0000 0.0000 4.0000

III 2: The faecal coliform level, faecal coliforms / 100 ml, of water from the Long Wharf and Rhodney Pier Smelt sampling locations between August 23 and 25, 2005. Date Sampled

Location Sampled

(day/month/year)

Water Faecal Coliform Level (FC / 100 ml)

23/08/ 2005 23/08/ 2005 25/08/ 2005

Long Wharf Rhodney Pier Rhodney Pier

283 83.3 80

III 3: The faecal coliforms / cm2 found on the skin of wild mummichog caught between September 17 and 30, 2005 from Saint’s Rest, Hazen Creek, and Marsh Creek Saint John, N.B. Mummichog Date Sampled Location Sampled Fish Faecal Coliform Level (FC / cm2)

(day/month/year)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

17/09/2005 17/09/2005 17/09/2005 17/09/2005 19/09/2005 19/09/2005 19/09/2005 19/09/2005 19/09/2005 19/09/2005 19/09/2005 25/09/2005 25/09/2005 25/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005 30/09/2005

Saint’s Rest Saint’s Rest Saint’s Rest Saint’s Rest Hazen Creek Hazen Creek Hazen Creek Hazen Creek Hazen Creek Hazen Creek Hazen Creek Saint’s Rest Saint’s Rest Saint’s Rest Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek

0 2 0 0 0 0 0 0 0 0 0 0 0 0 1 7 48 13 90 7 40 3 17

III 4: The faecal coliform level, faecal coliforms/ 100 ml, of water from Saint’s Rest, Hazen Creek, and Marsh Creek wild mummichog sampling locations between September 17 and 30, 2005. Date Sampled Location Sampled Water Faecal Coliform Level (day/month/year)

17/09/2005 19/09/2005 25/09/2005 30/09/2005

(FC/100 ml)

Saint’s Rest Hazen Creek Saint’s Rest Marsh Creek

1840 32 20 6200

III 5: The faecal coliforms / cm2 found at different time periods on the skin of caged mummichog exposed in Marsh Creek between August 5 and 13, 2005. Exposure Time Fish Faecal Coliform Level Date Sampled Cages (FC / cm2)

(day/month/year)

05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 05/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 06/08/2005 07/08/2005 07/08/2005 07/08/2005

1 1 1 2 2 2 3 3 3 4 4 4 1 1 1 2 2 2 3 3 3 4 4 4 1 1 1

12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 12 hrs 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 2 days 2 days 2 days

8.0000 27.2700 3.0000 18.0000 0.0000 36.0000 2.7200 4.0000 36.3600 30.0000 2.0000 3.0000 35.0000 11.0000 11.0000 26.0000 0.0000 43.0000 17.1170 27.2700 28.0000 16.0000 25.0000 10.0000 45.4500 190.9000 100.0000

07/08/2005 07/08/2005 07/08/2005 07/08/2005 07/08/2005 07/08/2005 07/08/2005 09/08/2005 09/08/2005 09/08/2005 09/08/2005 09/08/2005 09/08/2005

2 2 3 3 4 4 4 1 1 1 2 2 2

2 days 2 days 2 days 2 days 2 days 2 days 2 days 4 days 4 days 4 days 4 days 4 days 4 days

46.0000 54.5400 72.7200 610.0000 45.0000 54.5400 50.0000 1500.0000 662.5000 1600.0000 22.7200 1800.0000 4150.0000

09/08/2005 09/08/2005 09/08/2005 09/08/2005 09/08/2005 09/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005 13/08/2005

3 3 3 4 4 4 1 1 1 2 2 2 3 3 3 4 4 4

4 days 4 days 4 days 4 days 4 days 4 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days 8 days

985.0000 1700.0000 750.0000 4500.0000 1500.0000 900.0000 18.1800 17.2700 15.5000 15.5000 3.0000 15.4500 36.0000 4.9500 5.4500 3.0000 2.0000 20.0000

III 6: The faecal coliform level, faecal coliforms / 100 ml, of water from Marsh Creek between August 5 and 13, 2005. Date Sampled Water Faecal Coliform (day/month/year) Level (FC/100 ml) 05/08/2005 >60 000 06/08/2005 >60 000 07/08/2005 >60 000 09/08/2005 13/08/2005 20 000

III 7: Scatter plots of the faecal coliforms/ cm2 found at different time periods on the skin of caged Mummichog exposed in Marsh Creek between August 5 and 13, 2005. 12 Hours 40

Faecal Coliforms / cm2

Cage

1 Day 50

Faecal Coliforms/ cm2

Cage

2 Days 700

Faecal Colfiorms/ cm2

600 500 400 300 200 100 0 1

Cage

4 Days 5000

Faecal Coliform/ cm2

4000

3000

2000

1000

Cage

8 Days 40 35

Faecal Coliform/ cm2

30 25 20 15 10 5 0 1

Cage

III 8: The faecal coliforms/ cm2 found on the skin of caged Mummichog exposed for four days at Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. Date Sampled

Cages

Exposure Location

1 1 2 2 2 3 3 3 4 4 4 4 1 1 1 2 2 3 3 3 4 4 4 1 1 1 2 2 2 3 3 3 4 4 4 1 1

Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Tucker Park Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Harbour Passage Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Indian Town Marsh Creek Marsh Creek

(day/month/year)

18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 18/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 19/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 20/10/2005 21/10/2005 21/10/2005

Fish Faecal Coliform Level (FC/ cm2)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 3 2 1 0 1 0 0 3 0 0 0 108 49

21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005 21/10/2005

1 1 2 2 2 3 4 4 4 4

Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek Marsh Creek

48 294 288 71 246 246 21 6 8 7

III 9. The water faecal coliform level (colonies/ 100 ml) on day 0 and day 4 at each location in the location exposure caging experiment. Samples were taken from Tucker Park, Harbour Passage, Indian Town, and Marsh Creek Saint John N.B. between October 14 and 21, 2005. Location Tucker Park Harbour Passage Indian Town Marsh Creek

Water faecal coliform level on Day 0 (colonies/ 100ml) 76 100 540 1100

Water faecal coliform level on Day 4 (colonies/ 100ml) 67 80 >1200 >12 000

III 10: Scatter plots of the faecal coliforms/ cm2 found on the skin of caged Mummichog exposed for four days at Indian Town, and Marsh Creek Saint John N.B (results from Tucker Park and Harbour Passage omitted due to the absence of faecal coliforms on the mummichog). Indian Town 3.5 3.0

Faecal Colfiorms/ cm2

2.5 2.0 1.5 1.0 0.5 0.0

Cage

Marsh Creek 350 300

Faecal Coliforms/ cm2

250 200 150 100 50 0

Cage

III 11: Results of duplicate bacterial tests performed on randomly sampled positive faecal coliform colonies.

Colony 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 E. coli Micrococcus

Cell Shape B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B B/B C/C

Gram Stain N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N N/N P/P

Growth on McK agar P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P N/N

Growth in BGB broth P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P N/N

B = bacillus C = Coccus N = negative result P = positive result

Oxidase Presence P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P P/P N/N