Date :
Title: The microbiological analysis of Sligo estuary and some of its contributing rivers and streams
Aims
· To become familiar with the preparation of various agars.
· To assess microbiologically the water quality of the Garavogue river
· To assess microbiologically the water quality of the lower Sligo eastuary
· To assess microbiologically the water quality of the entire Sligo estuary
· To become familiar with the microbiological techniques involved in routine water analysis
·To compare the final results to relevant EU leglislation
Introduction
In this experiment we will attempt to assess microbiologically the water quality of sligo estuary and some of its contributing rivers and streams. The bacteral indicators which will be used will include total aerobic psycrophiles, total aerobic mesophiles, total coliforms, faecal coliforms, faecal streptococci, and sulphite reducing clostridia. The areas to be sampled will include water of various categories, for example the water at lough gill is used as raw water and the water at cregg estuary and gibraltor is used for recreational purposes. In total nine samples will be analysed and a sketch map of these can be seen in map 1.
The main river to be analysed will be the Garavogue. This river is the principal source of freshwater in the estuary . It drains a catchment of some 360 km2 extending eastwards from sligo town to the Benbulbin mountain range. It enters the estuary in a north-westerly direction, from its source, lough Gill, which is the source for most of the sligo public water supply. The smaller streams discharging to the estuary cover a lowland catchment of 35 - 40 km2 and thus contribute less than 10% of the freshwater input from the Garavogue river. These streams include Cregg stream and Donally river, both will be analysed microbiologically in this experiment to assess their impact on the water quality of the estuary.
There are several very important point sources of pollution into sligo estuary which may be seen to effect the water quality. The main source of water pollution from sligo is the town’s raw sewage which is discharged untreated from a holding tank at the deep water quay. Up to 2.2 million gallons are discharged every day, constituting the bulk of the town’s sewage. The other main sources of pollution to the estuary include, leachate from the now closed municipal dump, milk effluent discharge from the NCF dairies which enters the estuary at Gibraltor, and domestic effluent discarge into Cregg stream. Although these may be the main sources of pollution there are numerous other sources which also affect detrimentally the water quality, the main source being agricultural runoff. One of the many unfavourable effects of municipal and agricultural wastes is the contamination of the recieving environment with bacteria, viruses, and other organisms of public health significance. Pathogenic organisms, especially those from the intestines of warm blooded animals, frequently persist for sufficient periods of time and distance to pose a threat to the health and well being of unsuspecting water users.
In this experiment samples from all the afore mentioned main pollution sources will be analysed microbiologically to help build up an in depth knowledge of the pollutional status of the estuary and its contributing rivers and streams. For the analysis the pour plate method will be used for the mesophiles, psycrophiles, and faecal coliforms. For the total coliforms, faecal coliforms, and faecal streptococci the membrane filtration method will be used.
Materials + Methods
Materials as per manual
All the various nutrient agars and broths were prepared in the first week. These were then sterilised accordingly and stored until analysis began the following week
Analysis was carried out as per manual except for DRCM broth was used instead of DRCM agar
Results
Sample used was taken from the Garavogue river behind the Bank of Ireland (site 2)
Psycrophiles
Table 1
Dilution factor / Average CFU’s / Average CFU’s/100 mls100 / 50 / 5.0*103
10-1 / 15 / 1.5*103
10-2 / 6 / 0.6*103
To be stastically correct the 100 result was taken to be the most accurate
Mesophiles
table2
Dilution factor / Average CFU’s / Average CFU’s/100 mls100 / 98 / 9.8*103
10-1 / 19 / 1.9*103
10-2 / 3 / 0.3*103
To be stastically correct the 100 result was taken to be the most accurate
Sulfite reducing Clostridia
table 3
Mls of DRCM / Mls of sample / No of tubes tested / No of positive tubes50 / 50 / 1 / 1
10 / 10 / 5 / 3
From MPN tables it was calculated that the water contained 9 presumptive SRC
Gram stain carried out resulted in large gram negative rods.
Class results
All results expressed per 100 mls
Table 4
Group number / Sample site / Psycrophiles22oC / Mesophiles
32oC / Total Coliforms
37oc / Faecal Coliforms
44.5oc / Faecal Streps
37oC / Sulphite reducing Clostridia 37oC / Faecal
coliforms (VRBA) 44.5oC
1 / The blue lagoon / ------/ 3.7*104 / 2.8*101 / 1.5*102 / 7.5*101 / 18+ PSRC / ------
2 / Behind B o I / 5.0*103 / 9.8*103 / Missing / Missing / Missing / 9 PSRC / Missing
3 / Quay Street / 5.36*104 / 8.4*102 / 3.08*104 / 3.08*104 / 1.8*102 / 9 PSRC / 6*102
4 / Deep water quay / 1.8*106 / 1*106 / TNTC / TNTC / TNTC / 18+ PSRC / 6.25*105
5 / Gibraltor / 9.0*104 / ------/ TNTC / TNTC / TNTC / 18+ PSRC / ------
6 / Carton bay / 3.2*105 / 1.83*106 / TNTC / 2.2*103 / 1.7*103 / 18+ PSRC / 6*104
7 / Donally river / 3.5*105 / 2.84*106 / TNTC / TNTC / 2.39*102 / 16 PSRC / 0.53*103
8 / Cregg stream / 1.8*106 / 3.2*106 / TNTC / TNTC / 6.36*102 / 18+ PSRC / 4.1*104
9 / Cregg Eastury / 7.21*105 / 1.17*106 / TNTC / TNTC / 3.78*102 / 18+ PSRC / 4.16*104
PSRC = Presumptive sulfite reducing clostrida
Streps = streptococci
TNTC = Too numerous to count
B O I = Bank of Ireland
Disscussion
In this experiment a water sample (sample site 2) from the river Garavogue was analysed for microbiological content. The results for these particular analysis can be seen in tables 1,2,and 3. It must however be noted that the results achieved were far from comprehensive as following incubation three of our analyses went missing, namely the total coliforms, the faecal coliforms(membrane filtration), and the faecal streptococci. The whereabouts of our results was not determined but it is however suspected that they were either thrown out accidently or mistakenly used by another group. Without these results it is impossible to obtain an accurate impression of the microbiological pollutional status of the sampling site. It was however possible from the results obtained to draw some conclusions especially when these were compared to some of the class results.
From the total psycrophiles analysis the results of which are summarised in table 1, it can be seen that the sample contained 5000cfu’s/100mls. This value which would represent the naturally present population of bacteria is lower than the value of 9800cfu’s/100mls which was obtained for the total mesophilic bacteria , therefore it is likely that the sample was contaminated faecally as the mesophilic bacteria are most likly of faecal origin. There is a raw sewage holding tank situated upstream from the sample sites which floods in periods of heavy rainfall. These elevated levels of mesophiles might indicate that the tank had flooded recently. For the sulphite reducing clostridia analysis the results of which are summarised in table 3, it was found that the sample contained a most probable number of 9 presumptive sulphite reducing clostridia . One of these positive tubes was tested using a gram stain which confirmed the analysis by resulting in large gram negative rods typical of sulphite reducing clostridia . The fact that the PSRC result was so high would indicate that the waterbody was contaminated faecally at some time in the past as clostridia are spore formers and so can persist in the environment longer than other faecal indicators thus indicating historic pollution . As the results for all the other faecal indicators were missing it is not possible to class the water from this site according to EU directives .
Although the results from site 2 did allow some conclusions about the sanitary quality of the water to be drawn it is from the class results summarised in table 4 that the most valuable information can be gathered. The first sample site analysed was one of the water as it left lough gill (The blue lagoon). This site was expected to be relatively pollution free as it is the nearest site to Lough Gill which is used for drinking water extraction. This however was not the case as there was elevated numbers of mesophiles present (37000cfu’s/100mls). There were also a large number of total coliforms (2.8 *101 cfu’s/100-ml) present. However there was an even higher number of faecal coliforms (1.5 *102 cfu’s/100-ml) present This result is most improbable as total coliform numbers should be much higher than faecal coliform numbers. Feacal Streptococci were present in larger numbers than faecal coliforms and this might indicate that the pollution was of animal origin. There were also large numbers of PSRC;s present which would again indicate pollution of faecal origin. Under E.U directives the water at the blue lagoon could be classified as being of A2 quality. It is within the requirements for bathing water, but it does not meet the criteria for water intended for human consumption, therefore treatment of this water would be necessary before it would be supplied as drinking water (as is indeed the case).
Sample site number 3 was situated at quay street which is where the incoming tide may begin to affect the rivers composition. At this site the level of mesophiles is once again elevated but it has however decreased in comparison to the previous two sites, this may be due to the increasing salinity of the water . It was also found at this site that the level of faecal coliforms was the same as the level of total coliforms (by membrane filtration), this is most likly a miss reportation of results as this is highly improbable. The levels of faecal coliforms and faecal streptococci at this site have however increased from the previous sites. A likly explanation for this would be that the incoming tide was washing up contaminated water from lower down in the estuary, thus affecting the analysis. At this site the level of faecal streptococci is less than that of the faecal coliforms, which would indicate that the pollution is still mainly of animal origin. The water at this point still meets the A2 criteria for water quality under the council directive 75/440/EEC concerning water intended for abstraction as drinking water.
The next site to be analysed was at the deep water quay. This sampling site is situated just downstream of the towns main sewer outfall, and in theory should be one of the most contaminated sites sampled. From the results following analysis it can be seen that this was indeed the case with the site having highly elevated levels of mesophiles, total coliforms, faecal coliforms, faecal streptococci, and sulphite reducing clostridia. Indeed all of the analysis carried out using membrane filtration gave colonies too numerous to count. To solve this problem and to get accurate quantitative results a smaller volume of the sample should have been filtered e.g 10mls. From the results obtained however there is no doubt that this site at the lower estuary is heavily faecally polluted and under EU directives would not be suitable for either human consumption or bathing.
Sampling site 6 which is situated in cartron bay lies downstream some 400meters from the sewage outfall. It should therefore contain significant faecal pollution. This is shown to be the case in the results where it can be seen that all the faecal indicators are present along with the mesophiles in elevated levels. The levels at which they were found were shown to be slightly less than those at the deep water quay due to dilution effects.
All of the other sampling sites tested 5,7,8,9, showed the presence of significant faecal contamination. At Gibraltor (site 5) the elevated levels of indicators was probably due to a combination of discharges from both the municipal sewer and NCF dairies .
Both of the streams tested namely Doonelly (site 7), and Cregg (site 8) showed the presence of significant elevated faecal indicators. Possible sources of this faecal contamination would include agricultural runoff and sewage discharge. Sewage discharge is known to occur into cregg stream at cregg house for example. The impact which Cregg stream is having on Cregg estuary is also significant with similar bacterial counts being made at this point in sample 9.
Conclusion
This practical has been succssful in allowing the microbiological analysis of sligo estuary and its contributing rivers and streams. It has also allowed us to perfect our technique in the isolation and examination of bacterial indicators of faecal pollution.
Recommendations
· The instalation of a sewage treatment plant to carry out secondary treatment of sligo’s sewage
· Increased public awareness about the importance of good water, to prevent the dumping of refuse e.t.c.
·Tighter control of the discharge of waste into the estuary by local industries