FIO’s:

Regulation,

Analysis and Result

InterpretationBy

Timothy Harris (TH-Environmental)

Contents Laboratory Regulation Overview

1. Microbiology of Recreational and Environmental Waters 2000 (Bathing Waters)

2. Microbiology of Drinking Water 2016 (River/Raw water sources)

Sample analysis

1. Common analysis methodologies (pros and cons)

2. In-situ testing – Lamotte Coliform presence/absence kit

The impact of storms on WwTW sites/network

Working Example

1. Background

2. Catchment overview

3. Results and conclusions

Laboratory Regulation Overview1. Microbiology of Recreational and Environmental Waters (2000) This covers Recreational and Environmental waters of

marine or fresh water origin which have high enough numbers of visitors or users.(Bathing water (BW) directive)

The standards apply to waters where bathing is not prohibited and is traditionally practised by large numbers of bathers

Diffuse and spot sources of pollution are responsible for the contamination of water as well as a result of activity from animals, birds and humans

Where any of these sources introduce sufficient numbers of viable, pathogenic micro-organisms into the water, the possibility of illness arising is significant

Micro-organisms that are used as indicators of faecal pollution include coliform organisms, faecal coliform organisms (Escherichia coli), enterococci/faecal streptococci, sulphite-reducing clostridia and bacteriophages.

Gastrointestinal disease is the most common disease as a result of FIO contamination but it can also irritate skin, eyes and in severe cases cause serious neurological symptoms

1. Cont.

If the water you are testing is within these BW parameters the accredited labs

would analyse it under the BW matrices

Although other parameters can be tested the most indicative is the measure

of Total Coliforms, E-coli and Enterococci numbers

The regulations recommend the analysis method of Membrane Filtration (MF)

using Chromogenic Agar particularly MLGA and S&B.

Parameter Guide Value Imperative Value

Total coliform organisms 500 cfu/100ml 10,000 cfu/100ml

Faecal coliform organisms 100 cfu/100ml 2,000 cfu/100ml

Enterococci 100 cfu/100ml -

Salmonella - 0 number/litre

Enteroviruses - 0 number/10 litres

Laboratory Regulation Overview2. Microbiology of Drinking Water (2016)

This covers all water that is potable and for

human consumption

The regulations express the requirement to

undertake a complete sample set from source to

tap and at all stages in between during the

treatment process

So within an accredited Laboratory, a normal river

sample would be covered under this regulation as

it is seen as a source water

The recommended analysis methods again

includes the use of Membrane filtration onto

Chromogenic agar but also allows a multi tube

Most Probable Number (MPN) method and

Presence or Absence

Sample Analysis1. Common analysis methodologies (Pros and Cons)

The methodologies fall into 2 main categories:

A. Membrane filtration using Chromogenic Agar – MLGA, S&B

B. Multi tube MPN using most commonly the IDEXX Colilert technique

A. Membrane Filtration

The analysis of samples for micro-organisms through the filtration of

measured volumes passed through a 0.45 micron porous nitro-cellulose

membrane filter

Chromogenic Agar “Chromogenic Culture Media is a culture medium used to

isolate, identify, and differentiate specific microorganisms. The media

contains chromogenic substrates which when utilised by the microorganisms

give coloured colonies that is specific for each microorganism. The presence

or absence of the target organism is determined and also accurately

differentiated from others based on the colour of the colony”

Most common medias used are:

MLGA – Membrane Lactose Glucuronide Agar for Total coliforms and E-coli (T/E)

Total coliforms = yellow colony (not confirmed but present)

E-coli = blue/green colony confirmed presence

S&B – Slanetz and Bartley for Enterococci/Faecal Strep

Pale pink to dark maroon colony after incubation at 44oC present but not confirmed

Where the sample allows, 100ml is commonly filtered but if the turbidity of

the sample is high, lower volumes are used and dilutions may also be used

The samples for T/E analysis are incubated at 30oC for 4hrs and then for a

further 14hrs at 37oC

The samples for Ent are incubated at 37oC for 18-24hrs and then again at 44oC

for a further 18-24hrs for a confirmed result

A. Cont

• Actual count of colonies

present

• Confirmed result within 18

hrs (E only)

• Should not get < or > results

reported

• Low waste impact of the

analysis

• >95% recovery rate of

colonies

• Increased inaccuracy with

dilutions

• Background none specific

micro organisms can

proliferate and mask the

specific micro organisms

Involves the direct inoculation of measured volumes of water into liquid media contained in a series of tubes, or bottles

The most commonly used commercially available and accredited test is the IDEXX Colilert test

The Colilert Test uses proprietary Defined Substrate Technology (DST) to simultaneously detect total coliforms and E-coli. Two nutrient-indicators, ONPG and MUG are present in Colilert and can be metabolised by the coliform enzyme β-galactosidase and the E-coli enzyme β-glucuronidase, respectively

For Rivers and Raw water samples the Quanti-tray 51 is used

100ml of sample is used and a reagent added, the combined mixture is then distributed evenly between the 51 wells of the card. The card is sealed and then incubated at 37oC for 18-24hrs

After incubation the wells are read looking for colour change and under a UV lamp looking for fluorescence:

As coliforms grow in the Colilert Test, they use β-galactosidase to metabolise ONPG and change it from colourless to yellow

E-coli use β-glucuronidase to metabolise MUG and create fluorescence

B. Multiple tube MPN

Where the sample allows 100ml is used but if the turbidity of the sample is

high lower volumes are used and dilutions can also be used, all are topped up

with sterile water as 100ml is needed to fill the wells

Testing is only available for Total coliforms and E-coli not Enterococci

The number of positive wells is interpreted using the MPN table

B. Cont

• Confirmed result within 18-

24 hrs (T+E)

• Sample fixed before analysis

no chance colonies missed

through filtration

• Clear and concise positive

indication due to the

fluorescence

• Increased inaccuracy with

dilutions

• Increased chance of < or >

results impacting modelling

of the results

• Greater waste due to the

method of analysis

The kit contains 5 glass tubes and reagents and is simply a test for the

presence or absence of Total Coliforms and E-coli

The five tubes are inoculated with 10 mL of sample water each and incubated

for 24 to 48 hours depending on the ambient temperature. A change in the

colour due to acid production and the formation of gas bubbles indicates the

presence of coliforms

The nutrient tablet contains MUG (4-methylumbelliferyl‐β‐D- glucuronide), a

fluorogenic substrate. A non-fluorescent material is acted upon by a bacterial

enzyme to produce a fluorescent compound. If E-coli is present the sample

will fluoresce with exposure to UV light (365 nm)

Same principal as Colilert but not quantifiable

Concerns over the impact of background bacteria

Sample Analysis2. In Situ Testing – Lamotte Method

Sewers

Foul – carries only sewage from households and businesses

Surface Water – surface runoff from roofs and roads

Combined – where the foul and SW combine and go on to the WwTW

Combined Sewer Overflows (CSO’s) – network pressure release valve

Network Storm Attenuation (Storm tanks associated with PS or CSO’s)

Large underground tanks placed at CSO or PS locations to capture overflows and then return to the network after the surge has passed

Last in line Pumping Stations (storm release)

not always present but the last PS before the WwTW can be included as a site asset so storm spills not included in CSO spill data

Treatment impacts of increased site flows

Primary – decreased retention time can be short circuited – may lead to septicity also

Secondary – as per the primary but can result in complete loss of treatment if biological based

Tertiary – can be flooded by river surge back up the outlet pipe happens frequently on small sites

Blanket loss across the WwTW

WwTW Storm Attenuation – Storm Tanks

WwTW pressure release valve but can be small and release direct to river when full

Maintenance issues can cause extreme impacts

The impacts of storms on WwTW sites/networks

1. Background

• I was approached by the Lake District Asset Manager to see if there was a

possibility to look at an innovative solution for a small WwTW (with bathing water

regulations) due to spiralling costs connected to maintenance and operation

• Innovative treatment solutions were put forward but deemed too high risk and no

funding was forthcoming

• So I turned to a catchment based solution firstly by looking at the status of the BW

and interrogating the regulatory permit

• I then looked at the catchment and the diffuse pollution influences that could

possibly affect the waterbody

• These include livestock and septic tanks

• A catchment sampling plan was put together to cover “The Lakes” seasonal

variations

Working Example

1

2

3

4

WwTW

2. Catchment overview

N

• To the west of the catchment was a

small B road and steep wooded

valley sides

• A small tarn is situated between SP

2 and 3 this has influences from a

farmstead to the east

• The stretch between SP3 and 4

travels through moderately farmed

livestock land and interspersed

woodland and one small farmstead

• The main village(red area below)

sits on the shores of the lake the

beck discharges into

• It is a spate river regularly bursting

its banks between SP3 and 4

3. Results and Conclusions

1. In the Lakes “winter” months the level of FIOs in the catchment

shows no variation across the stretch I suspect this is due to:

• High in river flows flushing them through quickly and

diluting the levels

• Low people population in the area due to the high numbers

of holiday lets being unoccupied

2. In the Lakes “Summer” months it is significantly different there

is vast variation across the catchment particularly:

• U/S of the WwTW shows a significant increase in

contribution from the lake particularly T

• The works discharge is having an effect on the levels as

there is a dip in the performance of the UV in the summer

• The most telling and significant headline is the big increase

between the WwTW d/s sample point and the discharge to

Lake sample point the WwTW is not the major contributor

• My suspicion is particularly septic tanks and to a lesser

extent livestock have had an impact on the watercourse

has caused that significant change