Microbial Quality of the Avon River The effect of major ... · The effect of major earthquakes and...

Microbial Quality of the Avon River

The effect of major earthquakes and

subsequent sewage discharges on the quality

of the water and sediments in the Avon River

Follow on study

Prepared as part of scientific services to

Environment Canterbury

by

Elaine Moriarty, Megan Devane

and Brent Gilpin

June 2013

Technical Report

(CSC 13006)

Microbial Quality of the Avon River

The effect of major earthquakes and

subsequent sewage discharges on the quality

of the water and sediments in the Avon River

Follow on study

Water Programme Manager

John Wright

Project Leader Peer Reviewer

Dr Elaine Moriarty Dr Louise Weaver

Client Report

(CSC 13006)

DISCLAIMER

This report or document ("the Report") is given by the Institute of Environmental Science

and Research Limited ("ESR") for the benefit of Environment Canterbury as defined in the

respective Contracts between ESR and above parties, and is strictly subject to the

conditions laid out in those Contracts.

Neither ESR nor any of its employees makes any warranty, express or implied, or assumes

any legal liability or responsibility for use of the Report or its contents by any other person

or organisation.

ACKNOWLEDGMENTS

We would like to acknowledge the cooperation of staff from Environment Canterbury and

Christchurch City Council for contributing to this project.

Avon River post Earthquake – Follow up study June 2013

TABLE OF CONTENTS

1. INTRODUCTION .................................................................................................... 7

1.1. Focus of this report ............................................................................................. 9

1.2. Microorganisms examined in this study ........................................................... 10

1.3. Faecal source tracking ....................................................................................... 10

1.4. Faecal Aging of river water .............................................................................. 11

2. Sampling locations and Sample analysis .............................................................. 11

3. Results ...................................................................................................................... 12

3.1. E. coli in Avon River water .............................................................................. 12

3.2. E. coli in Avon River Sediment ........................................................................ 13

3.3. Bacteriophage in Avon River water .................................................................. 15

3.4. Bacteriophage in Avon River sediment ............................................................ 15

3.5. Bacteriophage in estuarine sediment ................................................................. 15

3.6. Enterococci in estuarine sediment ..................................................................... 17

3.7. Campylobacter spp. .......................................................................................... 18

3.8. Cryptosporidium and Giardia spp. ................................................................... 20

3.9. Comparisons during discharge immediately post-discharge and 2013 ............. 22

3.10. Faecal Source Tracking (FST) in Avon River water and selected sediments ... 24

3.11. Faecal ageing tools for faecal source tracking .................................................. 28

4. Discussion ................................................................................................................ 29

4.1. Microorganisms in river water .......................................................................... 29

4.2. Microorganisms in Sediment ............................................................................ 30

4.3. Faecal Aging ..................................................................................................... 31

5. Conclusion ............................................................................................................... 32

6. References ................................................................................................................ 34

Appendix 3: faecal sterol analysis .................................................................................... 38


LIST OF TABLES

Table 1 Microorganisms tested for in the study................................................................... 10

Table 2 Sampling Sites and abbreviations used. .................................................................. 11

Table 3 Mean levels of microorganisms in the river water during discharge, post-discharge

and the present study ............................................................................................................ 22

Table 4 Mean levels of microorganisms in the sediment during discharge, post-discharge

and the present study ............................................................................................................ 23

Table 5: PCR Markers identified in water samples collected from Avon River March-

April, 2013 ........................................................................................................................... 25

Table 6: Faecal Sterol ratios for Avon River water and sediments ..................................... 26

Table 7: Summary of collated Faecal Source Tracking results in Avon River water and

sediments.............................................................................................................................. 27

Table 8: Specificity of PCR markers used in the Avon River study ................................... 36

Table 9: Interpretation of the presence/absence of the three Human PCR markers ............ 37

Table 10: Raw Data for Faecal Sterols identified in Avon River water and sediments ...... 38

Table 11: Key for interpretation of sterol ratios .................................................................. 39


LIST OF FIGURES

Figure 1 Location of sampling sites ..................................................................................... 12

Figure 2 Concentration of E. coli present in Avon River water, sediment and estuarine

sediment ............................................................................................................................... 14

Figure 3 Concentration of Bacteriophage present in Avon river water, sediment and

estuarine sediment ................................................................................................................ 16

Figure 4 Concentration of enterococci present in estuarine sediment ................................. 17

Figure 5 Concentration of Campylobacter spp. present in Avon river water, sediment and


Figure 6 Concentration of Cryptosporidium spp. present in Avon river sediment and


Figure 7 Concentration of Giardia spp. present in Avon river sediment and estuarine

sediment ............................................................................................................................... 21

Figure 8: Faecal aging ratio versus E. coli concentration in Avon River water (2011-2013)

.............................................................................................................................................. 28


EXECUTIVE SUMMARY

The 2010/2011 Christchurch earthquakes resulted in direct sewage discharges to the Avon

River of more than 10,000 cubic meters a day for almost a year. A study (March 2011-

March 2012) was undertaken to use this unusual situation to 1) re-evaluate the validity of

E. coli as an indicator of public health risk following contamination events; 2) to evaluate

the relationship between concentrations of indicator bacteria and pathogens in recreational

water during active sewage discharges, and in the period following cessation of sewage

discharges; and 3) to investigate the potential of riverbed and seabed sediments to act as a

sink for indicator bacteria and pathogens that might then be re-mobilized with disturbance.

The samples for this study were taken during “Active Discharges” of sewage to the Avon

(March 2011-Sept 2011) and “Post Active Discharges” (Sept 2011-Mar 2012). The

findings of this study are reported in “The effect of major earthquakes and subsequent

sewage discharges on the quality of the water and sediments in the Avon River”, Moriarty

et al 2012.

The Current Study (March - May 2013) was undertaken with the following aims:

Evaluate the current state of the water and sediments in the Avon River and

estuarine sites and compare results with those found in the previous 2011-2012

study.

Ensure public health messages are accurately reflecting recent water and sediment

microbial quality.

The key findings of this current study are:

The Boatsheds site has similar water quality to that seen during Active Discharge to

the Avon River. Faecal source tracking tools (FST) have indicated the likely

sources of pollution at the Boatsheds to be wildfowl, with intermittent human

pollution.

The water quality at Kerrs Reach is also polluted with the average E. coli

concentrations close to those seen during Active Discharge (2011). FST results

indicate fresh human inputs as well as wildfowl faeces.


Owles Terrace has seen the greatest improvement in water quality when the levels

of E. coli are compared to those seen During and Post Active Discharge. FST

analysis has reported the source of E. coli as wildfowl.

The results from this study showed a marked decrease in Giardia concentrations in

sediments, which indicates the long term quality of the water is improving.

All sites, except Kerrs Reach, had a decrease in average concentration of all

microorganisms tested for in the sediment.

The two estuarine sediment sites showed significant decrease in indicator microbial

concentrations compared with the During and Post Active Discharge samples

(2011-2012). This indicates that there is no significant health risk related to contact

with the estuarine sediments based on these results.

Recreational contact with the Avon river water, sediment and estuarine sediments,

particularly at Kerrs Reach, may continue to pose health risks, and the public

should continue to minimize ingestion of the water.


ABBREVIATIONS

AC/TC ratio Atypical Coliforms/Total Coliforms

AdV adenovirus

CCC Christchurch City Council

CFU Colony Forming Units

CV Coefficient of variance

CI Confidence interval

FST Faecal source tracking

FWA Fluorescent Whitening Agents

M Magnitude (earthquake)

MfE Ministry for the Environment

MPN Most Probable Number

ND Not detected

NT Not tested

NIWA National Institute of Water and Atmospheric Research

PCR Polymerase Chain Reaction

Avon River Post Earthquake – Follow on study June 2013

1. INTRODUCTION

The Avon River is an intrinsic feature of the urban environment of Christchurch City and

is popular for recreational and tourist activities. The Avon River traverses the city from its

source in the western suburb of Avonhead through Hagley Park and the Central Business

District to the eastern suburbs. It departs the built environment at the northern entrance to

the Avon-Heathcote Estuary before amalgamating with the Heathcote River to flow into

Pegasus Bay.

Urban rivers are susceptible to intermittent impacts from industrial and human effluents. In

addition, animal faecal scats, including wildfowl, may cause deterioration in the quality of

the water. Contamination of water from these faecal sources of pollution can result in

increased risks of microbial diseases. Microbial water quality is measured by enumerating

the presence of the bacteria Escherichia coli, which is common in faecal material. The New

Zealand Microbiological Water Quality Guidelines for Marine and Freshwater

Recreational Areas (MfE, 2003) state that freshwater containing less than 260 E. coli per

100 mL is acceptable.

The Avon River has been monitored at various locations by the Christchurch City Council

(CCC) and Environment Canterbury (ECan) over many years. A 2009 study applied faecal

source tracking tools (faecal sterol analysis, fluorescent whitening agents, and PCR

markers) to better understand contamination (Moriarty and Gilpin, 2009). This study found

that the primary sources of water quality degradation in the Avon River appeared to be

related to wildfowl and possibly dog faecal material. Rainfall resulted in significant

degradation of the microbial water quality of the Avon River due both to wildfowl and dog

faeces being washed into the river, and some low level human sewage inputs from the

sewage system.

Following the major Christchurch earthquake on 22nd

Feb 2011, a study was commissioned

to 1) re-evaluate the validity of E. coli as an indicator of public health risk following

contamination events; 2) to evaluate the relationship between concentrations of indicator

bacteria and pathogens in recreational water during active sewage discharges, and in the

period following cessation of sewage discharges; and 3) to investigate the potential of


riverbed and seabed sediments to act as a sink for indicator bacteria and pathogens that

might then be re-mobilised with disturbance. Water and sediment samples were collected

from three sites on the Avon River – Antigua Boatsheds, Kerrs Reach and Owles Terrace,

sediment samples from two estuary sites – Penguin Street and Humphreys Drive, and

beach sand from Cave Rock, Sumner. All samples were collected between April 2011 and

March 2012. Samples were tested for the presence of microbial water quality indicators

(E. coli, enterococci, bacteriophage and Clostridium perfringens), potential pathogens

(Campylobacter, Giardia and Cryptosporidium), and faecal source tracking markers (PCR

markers, faecal sterols and fluorescent whitening agents (FWAs)).

The key findings of the previous study were:

After a major faecal contamination event, measurement of E. coli in the waterway

is a suitable indicator for establishing a public health risk. The New Zealand

Microbiological Water Quality Guidelines for Marine and Freshwater

Recreational Areas (MfE, 2003) specify levels of E. coli above 550 per 100 mL as

Action/Red Mode level. In this study E. coli levels in water above 550 were

correlated with an increased likelihood of detection of potential pathogens

including Campylobacter, Giardia and Cryptosporidium. While E. coli could be

detected in sediments throughout the study, levels were generally lower than in the

water suggesting minimal impact of sediment borne E. coli on recreational water

quality monitoring.

Overall, levels of pathogens were lower than may have been expected due to high

levels of groundwater infiltration into the sewage, which acted to dilute

microorganisms present. This was evident by the decreased concentration of

microorganisms and increased volume of raw sewage entering the wastewater

treatment plant. There was also no increase in community levels of infection, which

together with the groundwater infiltration, resulted in lower levels of pathogens in

the sewage entering the Avon River. The health risks were therefore less than may

have been expected.

Giardia was the pathogen found most frequently and at the highest levels. It was

found in water samples where the faecal sources could be attributed to human

sewage and to wildfowl sources. Further study would be needed to assess the

viability and source of these protozoa.


All the microorganisms tested in this study could be recovered from sediments. The

indicator bacteria Clostridium perfringens accumulated in the sediments, and there

is evidence to support the low level persistence of Cryptosporidium and Giardia in

sediments after cessation of sewage discharges. Bacteriophage and Campylobacter

species do not appear to accumulate in the Avon river sediments. Faecal sterols

and FWAs accumulated in the sediments.

It is well documented that microorganisms can survive for long periods of time in

riverbed sediment. In the event of disturbances of the sediment, it is highly

probable that there could be re-mobilisation of microorganisms, including

pathogens into the water column. Chemical contaminants in the sediment may also

be re-mobilised. Resuspension events, therefore, increase the potential of risk to

human health for those who participate in recreational and work-related activities in

the river environment.

Faecal source tracking tools (Faecal sterols, fluorescent whitening agents and PCR

markers) are valuable for confirming when elevated levels of E. coli are due to

human sources, and when they are the result of inputs from wildfowl, dogs or other

sources. The faecal source tracking markers identified in the water column

represent a snapshot of contamination at the time of sampling. In contrast, the

contamination signature in the underlying sediment is presenting a historical picture

of the impact of pollution inputs to a river system and does not appear to be

correlated with real-time events. There remains a need for assessing the health risks

of water primarily impacted by non-human sources, in particular wildfowl.

1.1. Focus of this report

This report is a follow up to the previous Avon River studies to determine the

concentration of microorganisms in the Avon River water and sediments, and in estuarine

sediments relative to these previous studies. Public health messages relating to

recreational water quality of the city waterways since the publication of the ‘Microbial

Quality of the Avon River’ report (ESR 2012) have referred to the report findings that

indicated some of the pathogens tested had accumulated in the sediments post-quake and

could be re-suspended following disturbance from recreational activity.


It has been a year since the last sampling occasion in the post-quake study and in order to

ensure that the public health message remains relevant a further study was undertaken to

establish the on-going concentrations of indicators and pathogens present in Avon River

sediments.

1.2. Microorganisms examined in this study

This report describes microbial characterisation of water and sediments from three sites on

the Avon River and sediments from two sites in the Avon/Heathcote Estuary.

Methodological details for analyses of target organisms and full description of the

organisms are described in the previous progress report (Moriarty et al., 2012).

Table 1 Microorganisms tested for in the study

Organisms Water Sediment

E. coli

Yes Yes

enterococci No Estuarine sediment only

Campylobacter spp.

Yes Yes

Bacteriophage

Yes Yes

Cryptosporidium spp.

No Yes

Giardia spp.

No Yes

1.3. Faecal source tracking

A range of faecal source tracking (FST) tools can identify whether faecal pollution is from

human, dog or wildfowl sources (Field and Samadpour, 2007). In this study, FST tools

were applied to a limited number of samples where the concentrations of microorganism


present warranted further investigation. FST markers evaluated were faecal sterols

(indicative of human and wildfowl sources), fluorescent whitening agents (FWAs)

(indicative of human sources) and PCR-based molecular markers (indicative of human,

wildfowl and dog sources).

1.4. Faecal Aging of river water

A secondary investigation to FST in the Avon River was to explore potential tools that

provide clarity around the aging of the faecal pollution identified in the river. One of the

tools trialled in this study was a faecal aging ratio, which relies on the comparison between

stable indigenous microflora concentrations in the river system and the total coliform

concentrations that rise after a fresh faecal input to the river. The aging ratio is called

AC/TC, which is taken from the numbers of atypical colonies (AC) on the standard method

plate assay for Total Coliforms (TC). The atypical colonies represent the indigenous river

microflora. Ratios of AC/TC are low (< 1 for fresh human sewage; Brion, 2005) because

concentrations of TC are high, and the ratio increases as the faecal input ages and total

coliforms die-off.

2. Sampling locations and Sample analysis

Three sites along the Avon River and three sites within the Avon/Heathcote Estuary were

chosen for analysis (Table 2 and Figure 1) and were sampled three times between March

and April 2013.

Table 2 Sampling Sites and abbreviations used.

Abbreviation used Site Sample type

BS Antigua Boatsheds River water and sediments

KR Kerrs Reach River water and sediments

OT Owles Terrace River water and sediments

PS Penguin St Estuarine sediments

HD Humphreys Drive Estuarine sediments


Figure 1 Location of sampling sites

3. Results

3.1. E. coli in Avon River water

The concentration of E. coli in Avon river water sediment and estuarine sediment from

April 2011 to April 2013 is shown in Figure 2 and a summary of the data is displayed in

Table 3 and Table 4. At the Boatsheds, the average E. coli concentration in this study

(1,035 cfu per 100 ml) is lower in average concentration compared with the Active

Discharge average concentration (average 1,338per 100 ml) and the Post Discharge

average concentration (1,629 E. coli per 100 ml).

The concentration of E. coli in the water at Kerrs Reach during the 2013 sampling (average

3,523 E. coli per 100 ml) was lower than the average concentration while direct sewage

discharges were occurring (4,600 E. coli per 100 ml). It was significantly higher than the

level recorded following the cessation of sewage discharges at Kerrs Reach

(1,893 E. coli per 100 ml).

Antigua Boatsheds

Kerrs Reach

Owles Terrace

Penguin Street Humphreys Drive

Cave Rock

Penguin Street


On all three sampling occasions in 2013, Owles Terrace had the lowest concentration of

E. coli relative to the other two upstream sites. The first two samples (650 and 750 E. coli

per 100 ml) were similar in concentration to those seen post sewage discharge. The final

sample (240 E. coli per 100 ml) was the lowest recorded at this site over the period of our

study.

3.2. E. coli in Avon River Sediment

The concentration of E. coli in the sediment at the Boatsheds during the 2013 sampling

(average 132 E. coli per g dry weight) was significantly lower than that during known

sewage discharges (average 1,287 E. coli per g dry weight) and similar to those seen post

discharge (average 250 E. coli per g dry weight).

The concentration of E. coli at Kerrs Reach was significantly different to the other sites

with the highest concentration of E. coli recorded (91, 635 E. coli per g dry weight) over

the course of the two year study. All three 2013 samples were higher than the average

concentration seen during active sewage discharges (2,875) E. coli per g dry weight) and

following the cessation of discharges (5,119) E. coli per g dry weight). It must be noted

due to problems with access to the river at Kerrs Reach the 2nd

and 3rd

sampling of 2013

were taken approximately 50 m downstream of the other samples, close to the last pontoon

in the vicinity of Christ’s College Boat Club.

The concentration of E. coli in the sediment at Owles Terrace appears to be decreasing

over time. The average concentration seen during the 2013 sampling (144 E. coli per g

sediment dry weight), was significantly lower than those during direct discharges

(3,603 E. coli per g dry weight) and following discharges (620 E. coli per g sediment dry

weight).

The two estuarine sites sampled in this study were Penguin St and Humphreys Drive. In

this round of sampling all sediment samples at the estuarine sites contained six or less E.

coli per g of sediment dry weight. These counts were similar to those seen in estuarine

sediments since Nov 2011, when the estuary ceased to be impacted by discharges.


Figure 2 Concentration of E. coli present in Avon River water, sediment and

estuarine sediment

2011 2012 2013

2011 2012 2013

2011 2012 2013

Mar Apr May

Mar Apr May

Mar Apr May


3.3. Bacteriophage in Avon River water

Bacteriophage (phage) concentrations were determined as an indicator of the presence of

pathogenic viruses in the samples. Phage were detected on one occasion in 2013 at the

Boatsheds and was at a low level (50 Phage per 100 ml of water). At Kerrs Reach phage

were detected on the first two sampling occasions. These were at concentrations (100 per

100 ml water) similar to those seen post discharge (average 158 phage per 100 ml water)

and significantly lower than during discharges (2263 phage per 100 ml). Phage was

detected once during the 2013 sampling at Owles Terrace (150 phage per 100 ml water).

Phage was intermittently detected at Owles Terrace following cessation of active

discharges (average 33 phage per 100 ml), significantly lower than during active sewage

discharges (2200 phage per 100 ml).

3.4. Bacteriophage in Avon River sediment

Phage was not detected in any of the Avon river sediment samples during the 2013

sampling. Following the cessation of direct sewage discharges (October 2011) phage has

only been detected once at each of the river sediment sampling sites.

3.5. Bacteriophage in estuarine sediment

Phage was detected in one sample from Humphreys Drive and never from the Penguin St

samples during the current 2013 sampling. Over the course of the two year study phage

have been detected on three occasions at Penguin St and once at Humphreys drive. The

concentration detected at Humphreys drive (12.8 phage per g sediment dry weight) was

significantly lower than previously detected at Humphreys Drive during active sewage

discharge (74 phage per g sediment dry weight).


Figure 3 Concentration of Bacteriophage present in Avon river water, sediment and

estuarine sediment

2011 2012 2013

2011 2012 2013

2011 2012 2013

Mar Apr May

Mar Apr May

Mar Apr May


3.6. Enterococci in estuarine sediment

The presence of enterococci was analysed for in the estuarine sediment samples taken from

Humphreys drive and Penguin St. The concentration of enterococci present in the sediment

at Penguin St has been steadily decreasing since direct discharges to the estuary ceased.

During discharge the sediment contained an average of 566 enterococci per g of sediment

dry weight, decreasing to 99 enterococci per g dry weight following cessation of

discharges and reducing to 8 enterococci per g dry weight in 2013.

The concentration of enterococci present in the sediment at Humphreys Drive has also

been steadily decreasing since direct discharges to the estuary ceased. During discharge an

average of 330 enterococci per g of sediment dry weight were present, decreasing to 36

enterococci per g dry weight following cessation of discharges and finally 27 enterococci

per g dry weight in 2013.

Figure 4 Concentration of enterococci present in estuarine sediment

2011 2012 2013

Mar Apr May


3.7. Campylobacter spp.

All water and sediment samples were tested for the presence of Campylobacter spp.. It was

detected on all three sampling occasions at each of the river water sampling sites in 2013.

The concentration in the water ranged from 2.3 to 46 campylobacter MPN (most probable

number) per 100 ml. While numbers at The Boatsheds and Owles Terrace were low, Kerrs

Reach had the highest concentrations (46 mpn per 100 ml on date) seen since sewage

discharges ceased.

In the river sediment samples Campylobacter spp. was detected only in the sediments in

Kerrs Reach during the 2013 sampling. Previously, Campylobacter spp had been detected

in all sediment samples over the course of the study, at low concentrations. The level of

campylobacter detected in the sediment at Kerrs Reach on the second sampling occasion of

2013 (11.08 mpn per g sediment dry weight) was the highest seen throughout the study.

Campylobacter spp was detected again at Kerrs Reach on the final sampling occasion , but

was significantly lower than the previous sample (0.54 mpn per g dry weight).

Campylobacter spp was not detected at any of the estuarine sediment samples sampled in

2013.

All isolates of campylobacter were confirmed by PCR and speciated. Of the 33 isolates

analysed, two were thermotolerant campylobacter, two contained both C. jejuni and

C. coli and the remaining 29 isolates contained C. jejuni only.


Figure 5 Concentration of Campylobacter spp. present in Avon river water, sediment

and estuarine sediment

2011 2012 2013

2011 2012 2013

2011 2012 2013

Mar Apr May

Mar Apr May

Mar Apr May


3.8. Cryptosporidium and Giardia spp.

In the 2013 study, only the sediment samples were analyzed for Cryptosporidium and

Giardia spp.. Cryptosporidium spp. were detected at low levels on two occasions at

Penguin St, and all other samples were negative for Cryptosporidium spp.. Giardia spp.

which had previously been detected at very high levels (2,254 cysts per g dry weight

sediment) was detected on three occasions during the 2013 sampling; twice at Penguin St

and once at Kerrs Reach. These samples all contained low concentrations of Giardia spp.

(<1 per g dry weight sediment) and may have come from a number of environmental

sources such as birds, dogs etc.

Figure 6 Concentration of Cryptosporidium spp. present in Avon river sediment and

estuarine sediment

Mar Apr May

2011 2012 2013


Figure 7 Concentration of Giardia spp. present in Avon river sediment and estuarine

sediment

Mar Apr May Mar Apr May

2011 2012 2013


3.9. Comparisons during discharge immediately post-discharge and 2013

Table 3 Mean levels of microorganisms in the river water during discharge, post-

discharge and the present study

Organism Time Boatsheds Kerrs Reach Owles Terrace

E. coli

CFU per 100

mL

During

discharge

1,338 4,600 17,975

Post discharge 1,629 1,893 1,493

Current study 1,035 3,523 547

Bacteriophage

PFU per 100

mL

During

discharge

425 2,263 2,200

Post discharge 67 158 33

Current study 17 100 50

Campylobacter

spp.

MPN per 100

mL

During

discharge

2 32 45

Post discharge 3 5 4

Current study 5 32 6

Dates: During discharge: 1st April – 8

th Sept 2011(n=4)

Post discharge: 27th

Sept – March 2012 (n=7)

Current study: March-April 2013 (n=3)


Table 4 Mean levels of microorganisms in the sediment during discharge, post-

discharge and the present study

Organism Time Boatsheds Kerrs

Reach

Owles

Terrace

Humphreys

Drive

Penguin

St

E. coli

CFU g dw

During

discharge 1,287 2,875 3,603 134 82

Post

discharge 250 5,119 620 48 9

Current study 132 43,661 144 1 3

Bacteriophage

PFU gdw

During

discharge 16 41 41 27 19

Post

discharge 1 2 2 6 0

Current study 0 0 0 0 4

Campylobacter

spp.

g dw

During

discharge 0 2 3 2 1

Post

discharge 1 0 0 0 0

Current study 0 4 0 0 0

enterococci

During

discharge

Not tested

284 330

Post

discharge 366 36

Current study 8 27

Cryptosporidium

spp. gdw

During

discharge 1.0 0.4 0.8 0.2 0.0

Post

discharge 57.3 8.9 4.6 0.0 0.2

Current study 0.0 0.0 0.0 0.3 0.0

Giardia spp.

gdw

During

discharge 23.6 8.6 10.3 0.3 0.0

Post

discharge 431.8 47.1 21.1 0.4 2.1

Current study 0.0 0.3 0.0 0.1 0.0

Dates: During discharge: 1st April – 8

th Sept 2011

Post discharge: 27th

Sept – March 2012 Current study: March-April 2013


3.10. Faecal Source Tracking (FST) in Avon River water and selected sediments

Faecal source tracking tools (PCR markers, faecal sterols and fluorescent whitening

agents) were applied to all river water samples collected and three sediment samples from

Kerrs Reach and the Boatsheds (Table 5 and Table 6) with final FST interpretations in

Table 7. Additional information on interpretation of FST tools can be found in Appendices

2 and 3.

All of the river water samples analysed by FST tools had elevated levels of E. coli, with

the exception of a sample from Owles Terrace collected on 8th

April, which was below the

alert level for recreational water. All samples contained > 2000 parts per trillion (ppt) of

sterols which is sufficient for interpretation of sterol ratios. FST analysis of river water on

11th

March 2013 identified only wildfowl faecal contamination at the three sites, the

Boatsheds, Kerrs Reach and Owles Terrace. On 25th

March human faecal pollution was

identified in river water at the Boatsheds and Kerrs Reach. In addition, wildfowl faecal

pollution was identified at all three sites. The low level of Human PCR marker BiAdo at

Owles Terrace is suggestive of seagull contamination (Appendix 2) rather than human.

River water analysis on 8th

April identified human contamination at all three sites and

wildfowl pollution at the Boatsheds and Kerrs Reach. Very low levels of Fluorescent

Whitening Agents (FWAs) were also detected in the water samples from the Boatsheds

and Kerrs Reach on 8th

April. The canine PCR marker was not detected in water analysed

in the current study. Faecal sterol analysis of sediment samples (Table 6) reported wildfowl

and plant decay as the likely sources of sterols.


Table 5: PCR Markers identified in water samples collected from Avon River March-April, 2013

Date

2013

Location PCR markers Interpretation

Genbac3 BiAdo HumM3 BacH

Reischer

GFD

Bird

E2

Duck

DogBac

11-

Mar

Boatsheds ++++ ND ND + + ++ ND wildfowl

Kerrs

Reach

++++ ND ND ND ++ +++ ND wildfowl

Owles

Tce

++++ ND ND ND + ND ND wildfowl

25

Mar

Boatsheds ++++ +++ ND + ++ ++++ ND (aged/low level)

human, wildfowl

Kerrs

Reach

++++ +++ + + + +++ ND human, wildfowl

Owles

Tce

++++ + ND ND ++ ND ND (aged?) low level

human, wildfowl

8-Apr Boatsheds ++++ +++ ND + ++ ++++ ND human, wildfowl

Kerrs

Reach

++++ +++ ND + ND ++ ND human, wildfowl

Owles

Tce

++++ + ND ND ND ND ND aged/low level

human

+ = very weak positive

++ = weak positive

+++ = positive

++++ = strong positive


Table 6: Faecal Sterol ratios for Avon River water and sediments

Faecal Ratios Human Indicative Ruminant Indicative Wildfowl Indicative Plant

**Interpretation Location

Sample

Type

Date

2013

F1 F2 H3 H1 H2 H4 R3 R1 R2 A1 A2 P1

>0.5* >0.5 >1 >5

% >0.7

>75

% <1

>5

%

<30

% ≥0.30 ≥0.67 >4

Boatsheds water 11-Mar 0.54* 0.42 1.3 3.9 0.35 56.8 15.4 2.9 56.8 0.68 0.61 15.4 wildfowl/plant

decay

Kerrs Reach water 11-Mar 0.25 0.59 1.0 1.3 0.20 50.8 25.8 1.3 50.8 0.61 0.78 25.8 wildfowl/plant

decay

Owles

Terrace water 11-Mar 0.37 0.52 1.7 1.9 0.27 63.6 18.3 1.1 63.6 0.61 0.69 18.3

wildfowl/plant

decay

Boatsheds water 25-Mar 0 . 9 1 1.25 0.9 3.2 0.48 47.4 11.8 3.6 47.4 0.43 0.49 11.8 Wildfowl/plant

decay

Kerrs Reach water 25-Mar 0.69 1.40 1.4 3.1 0.41 58.9 11.8 2.1 58.9 0.41 0.57 11.8 Wildfowl/plant

decay

Owles

Terrace water 25-Mar 0.41 1.02 1.1 1.2 0.29 53.3 11.4 1.1 53.3 0.45 0.67 11.4

Wildfowl/plant

decay

Boatsheds water 8-Apr 1.08 2.22 0.7 4.3 0.52 41.1 5.9 6.1 41.1 0.30 0.45 5.9 #plant decay

Kerrs Reach water 8-Apr 0.28 4.07 1.0 3.4 0.22 50.8 8.8 3.3 50.8 0.19 0.77 8.8 Wildfowl/plant

decay

Owles

Terrace water 8-Apr 1.19 3.03 1.6 4.5 0.54 61.5 6.9 2.8 61.5 0.24 0.42 6.9 plant decay

Kerrs Reach sediment 25-Mar 0.28 1.04 0.5 0.5 0.22 35.4 47.1 1.0 35.4 0.49 0.74 47.1 Wildfowl/plant

decay

Boatsheds sediment 8-Apr 0.58 0.68 1.3 1.6 0.37 56.3 24.3 1.3 56.3 0.56 0.57 24.3 Wildfowl/plant

decay

Kerrs Reach sediment 8-Apr 0.22 0.34 0.6 0.5 0.18 38.5 66.1 0.7 38.5 0.70 0.78 66.1 Wildfowl/plant

decay

*Shading indicates that the sample meets the criteria for that ratio, refer to Appendix 3, Table 11 for explanation of ratio abbreviations

**words in bold suggest the major contributor to pollution as determined by these assays, however, other faecal sources may be

present/dominate that were not targeted. #some evidence for mammalian faecal contamination


Table 7: Summary of collated Faecal Source Tracking results in Avon River water and sediments

Location Sample

type

Date

2013

E. coli

CFU/100mL

/g dry wtg.

Human PCR markers Non-human PCR

markers Sterols FWAs **Interpretation

BiAdo HumM3 BacH

Reischer Wildfowl Canine

Boatsheds water

11-Mar

1,055 ND ND Weak

Positive Positive ND wildfowl/plant decay NT Wildfowl, plant decay

Kerrs Reach water 5,000 ND ND ND Positive ND wildfowl/plant decay NT Wildfowl, plant decay

Owles

Terrace water 650 ND ND ND Positive ND wildfowl/plant decay NT wildfowl/plant decay

Boatsheds water

25-Mar

900 Positive ND Weak

Positive Positive ND Wildfowl/plant decay NT Human, wildfowl

Kerrs Reach water 1,070 Positive Weak

Positive

Weak

Positive Positive ND Wildfowl/plant decay <0.01

Fresh

human,,wildfowl,

plant decay

Owles

Terrace water 750

Weak

Positive ND ND Positive ND Wildfowl/plant decay NT

wildfowl,

plant decay

Boatsheds water

8-Apr

1,150 Positive ND Weak

Positive Positive ND *plant decay 0.03

Human, wildfowl,

plant decay

Kerrs Reach water 4,500 Positive ND Weak

Positive Positive ND Wildfowl/plant decay 0.03

Human, wildfowl,

plant decay

Owles

Terrace water 240

Weak

Positive ND ND ND ND

low level human,

plant decay NT

(aged?/ low level

human), plant decay

Kerrs Reach sediment 25-Mar 91,600

NT NT NT NT NT Wildfowl/plant decay NT Wildfowl/plant decay

Boatsheds sediment 8-Apr

220 NT NT NT NT NT Wildfowl/plant decay NT Wildfowl/plant decay

Kerrs Reach sediment 19,600 NT NT NT NT NT Wildfowl/plant decay NT Wildfowl/plant decay

Grey shading indicate samples requested by ECan for FST analysis ND = sample analysed and target not detected

*some evidence for weak signal from mammalian indicators NT = sample not analysed

**words in bold suggest the major contributor to pollution as determined by these assays, however, other faecal sources may be

present/dominate that were not targeted.


3.11. Faecal ageing tools for faecal source tracking

Figure 8 presents the faecal aging ratio of AC/TC plotted against the concentration of E.

coli detected in water at the three Avon River locations over the sampling period March

2011 - April 2013. This sampling regime represents 19 samples analysed at each site.

AC/TC values at the Boatsheds varied between 0.3-5.8 with an arithmetric mean of 1305

CFU/100 mL E. coli. Less variability in AC/TC was seen at Kerrs Reach with all values

<3.0 and a higher mean of 4470 CFU/100 mL E. coli. During discharge at Owles Terrace,

E. coli concentrations were approximately 10 fold higher (mean 30,000 E. coli/100 mL)

compared with post-discharge (mean 1200 E. coli/100 mL) and AC/TC ratios were all

below 0.92 during discharge at Owles Terrace. The two circles in Figure 8 depict during

and post-discharge values for the faecal aging ratio at Owles Terrace and show that post-

discharge, the aging ratio was above 1.2, which was statistically, significantly different to

the aging ratio during discharge (p = <0.001) but only at Owles Terrace. Concentrations of

E. coli during and after discharge were also significantly different at both Kerrs Reach (p =

0.014) and Owles Terrace (p = <0.001) but not the Boatsheds. The significant differences

between faecal aging ratios and E. coli at Owles Terrace, has been accompanied by a

decrease in the faecal sterol concentrations post-discharge.

Figure 8: Faecal aging ratio versus E. coli concentration in Avon River water (2011-2013)

AC/TC faecal aging ratio

0 1 2 3 4 5 6 16

E.

co

li C

FU

/100 m

L

101

102

103

104

105

106

Boatsheds

Kerrs Reach

Owles Terrace

Action level (550 E. coli CFU/100mL)

Alert level (260 E. coli CFU/100 mL)

Samples collected from Owles Terrace prior to discharges ceasing

Samples collected from Owles Terrace post-discharge


4. Discussion This study was undertaken a year after a comprehensive study of the Avon River water,

sediment, estuarine water and sediment, coastal water and sediment was undertaken. The

aim of this study was to assess the microbial loading of the Avon River water, sediment

and estuarine sediment to ensure accurate public health messages were disseminated.

Table 3 and Table 4 in the report detail the average concentrations of microorganisms

detected in the Avon River water, sediment and estuarine sediment over the course of the

entire study (April 2011-March 2013).

4.1. Microorganisms in river water

The average E. coli concentrations at the Boatsheds have decreased in our current study

compared with during and post Active Discharge. Faecal source tracking analyses at the

Boatsheds suggest wildfowl contamination is present during all sampling periods of the

current study and human faecal pollution was identified in the river water on the last two

sampling occasions. Identification of human contamination may be related to the increased

activity in the area such as dredging of the river bed, construction of new pipelines and

contamination issues adjacent to and upstream of the Boatsheds. These factors may also

contribute to the presence of wildfowl indicators which were identified in both sediment (8

April) and overlying water (all events) at the Boatsheds. Immediately post-quake no

known direct discharges were occurring upstream of the Boatsheds. Since then various

investigations undertaken by CCC have uncovered problems particularly relating to drains

feeding into the Avon (Riccarton Main Drain, Addington Main Drain). Prior to the

September 2010 earthquake, and subsequent sewage discharges, the water quality at the

Boatsheds frequently did not comply with Recreational Water Quality. The source of the

pollution was determined in an ESR Report 2009 (Moriarty et al) to be wildfowl and dog

faeces, with no human pollution detected in the study.

At Kerrs Reach a higher average concentration of E. coli per 100 ml (3,523 cfu) was

detected compared with the post-discharge average concentration of E. coli (1, 629 cfu).

FST analysis identified wildfowl markers in the water and sediment, and human markers

were present in the water on the last two sampling events. A number of overflow events

have occurred in the area in recent times and weed cutting had been undertaken. Several

new water and wastewater pipes have also been laid in the area. All of these activities


have the potential to add to the microbial loading in the river. As this is still an area where

significant recreational activity takes place, water users should be kept up to date on the

water quality and warning signs erected where appropriate.

Owles Terrace which received significant volumes of direct discharge post-quake has the

lowest concentration of E. coli per 100 ml in the water in this current study. The FST

analyses did not identify human markers in the water at Owles Terrace, except for a weak

signal on the last sampling occasion when strong human indicators were present at both

upstream sites. This site is also tidally impacted so would be expected to have the highest

input of non-sewage impacted sea water compared with other sites.

Water and sediment samples were analysed for bacteriophage. At all three sites

concentrations have decreased significantly to very low levels at or just above our limit of

detection (50 PFU per 100 ml). In our previous report we signalled that bacteriophage

show promise as indicators of Campylobacter presence. Results from this study are

inconclusive, and it will be a potential correlation we will continue to monitor in our

studies.

Campylobacter concentrations in the river water samples were similar throughout the study

at the Boatsheds, with the current study having the highest average concentration per

100 ml. At Kerrs Reach the concentration decreased following the cessation of discharges,

but has increased significantly again in the final sampling round. This may be due to a high

number of wildfowl in the area and continual sewage pipe repairs which may lead to

discharges to the river. Owles Terrace which had the highest average concentration of

campylobacters during discharge has remained relatively low since discharges ceased.

4.2. Microorganisms in Sediment

In terms of microorganisms present in riverbed and estuarine sediment, the most

significant finding was at Kerrs Reach. Here the sediments were 15 times more

contaminated with E. coli than during active discharges. This may be a build-up of E. coli

over time in the sediments due to known (and unknown) discharges to the river. The

Boatsheds had reduced on average by 47% since the post discharge study and Owles

Terrace by 77%. Levels in the two estuarine sediment sites are low and do not warrant any

concerns.


Bacteriophage at all but one site (Penguin St.) had no detectable phage present in the

current sampling round. Penguin St was the only site found to contain phage, and this was

at a low level. Campylobacter was only detected at one site (Kerrs Reach) in the current

study and had the highest average concentration when compared to the two previous

sampling timeframes. In our previous study we noted that Bacteriophage and

Campylobacter do not appear to accumulate in the Avon River sediments and both

microorganisms have short survival times after discharge to the river system. These

findings support this hypothesis and highlight that the Campylobacter would have been

recently deposited in the sediment.

Cryptosporidium was only detected at one sediment sampling site – Humphreys Drive.

The concentrations were very low and do not warrant any public health concern. Giardia

which had previously been very high (2,254 cysts per gram) at the Boatsheds was not

detected there on this current sampling round. It was however detected at a low level at

Kerrs Reach (0.8 per gram) and Penguin St (<0.2 per gram). The concentrations seen were

significantly lower than those recorded during discharges due to the lack of fresh inputs

and in the sampling period immediately after them. The decrease may also be related to

the dredging of the riverbed which may have removed sediment where the giardia were

concentrated.

Enterococci were analysed in the two estuarine samples (Humphreys Drive and Penguin

St) and found to have decreased significantly compared with results during active

discharges. Penguin St was higher than Humphreys Drive but there was also evidence of

significant vehicular traffic on the sands and demolition activity which may have added to

the microbial loadings of the sediments.

4.3. Faecal Aging

The river water was tested for the faecal aging ratio of AC/TC (Atypical colonies/ Total

Coliforms). This ratio compares normal microflora concentrations in the river with the

total coliform concentrations from faecal inputs. Lower ratios of AC/TC with high TC

concentrations, suggest more recent faecal inputs. According to Brion (2005), fresh inputs

to river systems are observed when the AC/TC ratio is 5 or less with fresh human sewage

reporting values of less than 1.0 as observed at Owles Terrace during continuous sewage


discharges (Figure 8). Influx from stormwater drains of aged human inputs increases the

ratio up to 5.0 making it difficult to distinguish from other animal/bird inputs. AC/TC

values derived from wildfowl faecal inputs have not been studied specifically, but

agricultural inputs to river systems have been reported as having AC/TC values of

approximately 10 (Nieman and Brion et al. 2003). The ratios of AC/TC were, in general,

below 6.0 throughout the study even during the 2013 sampling, except for one sample at

Owles Terrace on 25th

March, 2013 which was 15.8. The low AC/TC values suggest that

this river system is subjected to on-going inputs of faecal material, which from the FST

data is not exclusively human derived. In conclusion, at these three sites on the Avon

River, the AC/TC values and FST results suggest that the river has been continually

impacted by recent faecal inputs from human and non-human sources. AC/TC ratio values

of approximately 20 and above indicate a healthier water quality environment (Brion,

2005), which has a reduced risk to human health and is more suitable for recreational

activities.

5. Conclusion

The results of this follow up study on the Avon River water, sediment and estuarine

sediment highlight the on-going water quality issues. The Boatsheds at Antigua St

continues to have poor water quality similar to the levels measured throughout the active

discharges and post discharges. Historically, the water quality at the Boatsheds has been

poor and this continues to be the case. FST tools have indicated the likely sources of

pollution at the Boatsheds to be wildfowl, with intermittent human pollution. The water

quality at Kerrs Reach is also unsatisfactory in terms of public health risk with the average

E. coli concentration close to those seen during active sewage discharges. FST results

indicate fresh human inputs as well as wildfowl faeces at this site. Finally, Owles Terrace

has seen the greatest improvement in water quality post-discharge with the final sample

(240 E.coli per 100 ml) below alert level for the recreational water guidelines 260 E. coli

per 100 ml), and FST reporting the source of E. coli as wildfowl.

Post cessation of direct discharges to the Avon, the sediments of the river contained a

significant number of protozoa. The results from this study showed a marked decrease in

concentration, which indicates improvements for the long term quality of the water. At


Kerrs Reach the highest concentration of E. coli per g of sediment was detected. FST

determined the source as wildfowl. It should be noted that the 2nd

and 3rd

sampling of this

study, sediment and water samples were taken further downstream at Kerrs Reach than

previously sampled due to problems accessing the original sampling location. The two

estuarine sediment sites showed significant decrease in microorganisms tested compared

with previous sampling and do not warrant any public health concern based on the

organisms analysed in this study.

As the Avon River is used year round for recreational activity, users should be kept

updated with the water quality, particularly at the Boatsheds where a number of people

unfamiliar with the water quality of the Avon have recreational contact with it. Due to the

low levels of Giardia and Campylobacter spp. present in the sediment, care should always

be advised in relation to disturbance of the sediments, especially at Kerrs Reach, due to the

high concentration of E. coli.


6. References Brion, G.M., 2005. The AC/TC bacterial ratio: a tool for watershed quality management.

Journal of Water and Environment Technology 3(2), 271- 277.

Moriarty, E.M., and Gilpin, B.J., 2009. Faecal Tracking in the Avon River, Christchurch.

CSC Report Number FW09075.

Moriarty, E.M., Devane, M.L., Williamson, W., Gilpin, B.J., 2012. Microbial Quality of

the Avon River, the effect of major earthquakes and subsequent sewage discharges

on the quality of the water and sediments in the Avon River. Progress Report. CSC

Report Number FW11043.

Nieman, J. and Brion, G.M., 2003. Novel bacterial ratio for predicting faecal age. Water

Science and Technology 47(3), 45-49.

Ministry for the Environment 2003. Microbiological Water Quality Guidelines for Marine

and Freshwater Recreational Areas. New Zealand: 155


APPENDIX 1 : REPORT DISTRIBUTION

Copies have been made and distributed to:

Participating Councils

Environment Canterbury - Michele Stevenson

Environment Canterbury - Lesley Bolton-Ritchie

Christchurch City Council - Mike Bourke

ESR

Elaine Moriarty Meg Devane Louise Weaver

Brent Gilpin

CSC Library

Further copies of this report may be obtained from: Dr Elaine Moriarty

Christchurch Science Centre

P O Box 29-181

Christchurch


Appendix 2

PCR Assays

The specificity of the assays, as tested against known faecal samples is shown below (last

updated May 2013).

Semi-quantitative results are reported on a scale from Extremely Strong Positive down to

Very Weak Positive

Table 8: Specificity of PCR markers used in the Avon River study

Assay Present in faeces from: Low level non-

specificity

Negative in faeces

from:

General

GenBac

Human, Cat, Dog, Cow,

Sheep, Deer, Horse, Goat,

Pig, Rabbit, Possum, Duck,

Swan, Seagull, Canada

Goose Goose, Chicken

(can be low in seagull and

Canada Goose faeces)

Human BiADO Human Seagull Cat, Dog, Cow, Sheep,

Deer, Horse, Goat, Pig,

Rabbit, Possum, Duck,

Swan, Canada Goose,

Chicken

Human

HumM3

Human

Possum Rabbit Cat, Dog, Cow, Sheep,


Duck, Swan, Canada

Goose, Chicken

Human BacH Human Cat, Dog, Rabbit,

Possum Chicken Goat Cow, Sheep, Deer, Horse,

Pig, Duck, Swan, Goose,

Canine DogBac Dog Some human

municipal sewage

Human, Cat, Cow, Sheep,


Rabbit, Possum, Duck,

Swan, Seagull, Canada

Goose, Chicken

Bird GFD Duck, Swan, Seagull,

Canada Goose, Chicken Human, Cat, Dog, Cow,


Pig, Rabbit, Possum

E2 wildfowl

marker

Duck, Swan, Seagull,

Canada goose, Chicken Pig Human, Cat, Dog, Cow,


Rabbit, Possum

PCR Markers: Specificity testing of sources is an ongoing activity. The detection limit of

these methods is 1.00x103/100 mL.


Interpreting human indicative assays.

BacH is more sensitive than BiADO which is more sensitive than HumM3. The table below is

a guide to interpreting presence and absence of human indicative markers.

Table 9: Interpretation of the presence/absence of the three Human PCR markers Human

BiADO

Human

HumM3

Human

BacH Interpretation

+ + + Very strong evidence of (fresh)

human faecal contamination

+ ND +

Evidence of human faecal

contamination, but may be lower

levels, aged or partially treated

ND ND +

May be human, dog, cat, rabbit,

chicken. If canine marker absent

then can exclude dog. If wildfowl

marker absent then can exclude

chicken.

ND + +

Possible human, but if ruminant

marker present, may indicate

possum faeces.

+ ND ND

Possible human, but may indicate

aged source as it is hypothesised

that BiAdo may persist longer in the

environment than the other markers.

ND ND ND No evidence of human faecal

contamination. ND = sample was analysed, but the determinant was not detected.

The level of GenBac marker should also be considered when evaluating presence of the other

markers. For example when very high levels of GenBac, but very low levels of human

markers then suggest either other sources more dominant, or that if human markers are

present they are from aged or partially treated sources.


Appendix 3: faecal sterol analysis

Table 10: Raw Data for Faecal Sterols identified in Avon River water and sediments

Location Boatsheds Kerrs

Reach

Owles

Terrace Boatsheds

Kerrs

Reach

Owles

Terrace Boatsheds

Kerrs

Reach

Owles

Terrace

Kerrs

Reach Boatsheds

Kerrs

Reach

Date (2013) 11-Mar 11-Mar 11-Mar 25-Mar 25-Mar 25-Mar 8-Apr 8-Apr 8-Apr 25-Mar 8-Apr 8-Apr

Sample Type

(Volume/mass

analysed)

Water (4L) Water (3.5L) Water (4L) Water (4L) Water (4L) Water (2.2L) Water (4L) Water (4L) Water (4L) Sediment (1.1 g)

Sediment (1.1 g)

Sediment (1.1 g)

Sterol Name

coprostanol 822 63 103 282 146 57 302 185 165 29 63 25

24-

ethylcoprostanol 625 61 59 313 102 50 433 179 103 53 49 40

epicoprostanol 152 9 24 43 10 13 47 14 23 6 19 6

cholesterol 4877 1500 3018 2555 2212 3247 2204 1680 2012 1000 1894 1266

cholestanol 1514 253 277 311 211 140 280 668 138 102 109 113

24-

methylcholesterol 1233 458 535 793 353 277 578 396 270 666 286 784

24-

ethylepicoprostanol 72 5 13 23 1 9 24 6 7 1 8 9

stigmasterol 797 656 342 459 438 168 467 615 214 1065 182 419

24-ethylcholesterol 9628 1575 1081 3708 1199 570 2575 1578 706 2495 1191 2645

24-ethylcholestanol 1491 104 113 251 73 49 195 44 34 51 72 117

total sterols (ppt) 21211 4684 5565 8738 4745 4580 7105 5365 3672 5468 3873 5424


Table 11: Key for interpretation of sterol ratios

Ratios indicative of faecal pollution (either human or animal)

F1 coprostanol/cholestanol.. >0.5 indicative of faecal source of sterols

F2 24ethylcoprostanol/ 24-ethylcholestanol. >0.5 indicative of faecal source of sterols.

Human indicative ratios (values exceeding threshold in red)

H3 coprostanol/ 24-ethylcoprostanol Ratio >1 suggests human source

H1 % coprostanol Ratio >5-6% suggests human source

H2 coprostanol/(coprostanol+cholestanol) Ratio >0.7 suggests human source

H4 coprostanol/(coprostanol+24-ethylcoprostanol) Ratio >0.75 suggests human source

Ruminant indicative ratios (values exceeding threshold in blue)

R3 24-ethylcholesterol/24-ethylcoprostanol Ratio <1 suggests ruminant source, ratio >4 suggests plant decay

R1 % 24-ethylcoprostanol Ratio >5-6% suggests ruminant source

R2 coprostanol/(coprostanol+24-ethylcoprostanol) Ratio <30% suggests ruminant source

Avian indicative ratios (values exceeding threshold in orange)

A1 24-ethylcholestanol/(24-ethylcholestanol+24-

ethylcoprostanol+24-ethylepicoprostanol) A1 Ratio >30% suggests avian source AND A2 Ratio >67% suggests avian

source A2 cholestanol/(cholestanol+coprostanol+epicoprostanol)

Plant indicative ratios (values exceeding threshold in orange) P1 24-ethylcholesterol/24-ethylcoprostanol Ratio >4 suggests plant decay

Notes: Note that ratios must be interpreted with consideration to the levels of sterols, and relative to one another. For example H1 is typically also above 5-6% in ruminant faeces. Plant sterols and

mixed sources also have differing effects on sterol interpretations which must be considered.

Conclusions are the best interpretation of sterols in our opinion. Conclusions in bold are highly supported by the sterol data, conclusions in brackets are supported by sterol data with

some variation from pure source, and conclusions in brackets indicate likely source but with lower degree of certainty

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