Appendix A - Horry County Stormwater

Appendix A - Horry County Stormwater
APPENDIX A
QUALITY ASSURANCE PROJECT PLAN
QUALITY ASSURANCE
PROJECT PLAN
FOR
BRIARCLIFF ACRES
WATER QUALITY STUDY
HORRY COUNTY, SC
PREPARED FOR
HORRY COUNTY, SC
FINAL DRAFT
JANUARY 13, 2010
J ± 22072.0000
Thomas & Hutton Engineering Co.
Savannah, Georgia Š Brunswick, Georgia
Charleston, South Carolina Š Myrtle Beach, South Carolina
Wilmington, North Carolina
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 1/13/10
By: RPK
QUALITY ASSURANCE PROJECT PLAN
FOR
BRIARCLIFF ACRES WATER QUALITY STUDY
HORRY COUNTY, SC
Prepared by:
Thomas & Hutton Engineering Co.
935 Houston Northcutt Blvd.
Mount Pleasant, Sc 29465
Richard Karkowski, P.E., P.H.
(843) 725-5208
[email protected]
Prepared for:
Horry County, South Carolina
4401 Privetts Road
P.O. Box 1236
Conway, SC 29528
Tom Garigen
(843) 915-5160
[email protected]
Approved by:
Thomas & Hutton
Project Manager:
Horry County
Stormwater Manager:
Coastal Carolina University
Environmental Quality Lab:
Virginia Polytechnic Institute and
State University, Department of Crop
and Soil Environmental Sciences:
_________________________________
Richard P. Karkowski, P.E., P.H.
_________
Date
_________________________________
Tom Garigen
_________
Date
_________________________________
Susan Libes, Ph.D.
_________
Date
_______________________________
Charles Hagedorn, Ph.D.
_________
Date
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 1/13/10
By: RPK
TABLE OF CONTENTS
1.0
Distribution List .................................................................................. Page 1
2.0
Project Organization ........................................................................... Page 1
3.0
Project Definition / Background ......................................................... Page 1
4.0
Project / Tasks Description ................................................................ Page 1
5.0
Data Quality Objectives and Criteria for Measurement Data ............ Page 2
6.0
Special Training Requirements/Certifications .................................... Page 2
7.0
Documentation and Records .............................................................. Page 2
8.0
Sampling Process Design .................................................................... Page 3
9.0
Sampling Methods Requirements ...................................................... Page 3
10.0
Sample Handling and Custody Requirements .................................... Page 4
11.0
Analytical Methods Requirements ...................................................... Page 4
12.0
Quality Control Requirements ........................................................... Page 4
13.0
Instrument/Equipment Testing, Inspection, Maintenance
Requirements ..................................................................................... Page 4
14.0
Instrument Calibration and Frequency................................................ Page 5
15.0
Inspection/Acquisition Requirements for Supplies
and Consumables ................................................................................ Page 5
16.0
Data Acquisition Requirements (Non-Direct Measurements) ........... Page 5
17.0
Data Management ............................................................................... Page 5
18.0
Assessments and Response Actions ................................................... Page 5
19.0
Reports to Management ..................................................................... Page 6
20.0
Validation and Verification Methods ................................................. Page 6
21.0
Reconciliation with User Requirements ............................................. Page 6
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 1/13/10
By: RPK
APPENDICES
Scope of Work .................................................................................................................... A
Sampling Location Map ...................................................................................................... B
CCU EQL SOP No.: 420 ± Field Measurements with Hach Ruggedized Probes ................ C
CCU EQL SOP No.: 405 ± Turbidity Measurement with Hach Pocket Turbidmeter .......... D
CCU EQL SOP No.: 406 ± Turbidity Measurement with Hach 2100N Turbidimeter...........E
CCU EQL SOP No.: 430 ± Biochemical Oxygen Demand (BOD) ....................................... F
CCU EQL SOP No.: 447 ± Ammonia (Nitrogen) by Automated Phenate Method............... G
CCU EQL SOP No.: 435 ± Total Suspended Solids ............................................................ H
CCU EQL SOP No.: 502 ± Fecal Coliform Measurement by Direct Test (A-1 Medium) ...... I
CCU EQL SOP No.: 501 ± Enterococci Measurement by
IDEXX Enterolert -Quanti-Tray Method .............................................................. J
CCU EQL SOP No.: 602 ± Optical Brightener Measurement by Fluorometry (DRAFT) .... K
VT ± Identifying Sources of Fecal Pollution in Impaired Waters ........................................L
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
1.0
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Date: 1/13/10
By: RPK
Distribution List
Horry County, South Carolina:
Tom Garigen, Stormwater Manager
Thomas & Hutton Engineering Co.:
Richard Karkowski, P.E., P.H., Project Manager
Coastal Carolina University, Environmental Quality Lab:
Susan Libes, Ph.D., Principal Investigator
Coastal Carolina University, Environmental Quality Lab:
Joe Bennett, EQL Director
Virginia Polytechnic Institute and State University, Department of Crop and Soil Environmental
Sciences:
Charles Hagedorn, Ph.D., Bacteria Source Tracking Specialist
2.0
Project Organization
Project Sponsor:
Horry County, South Carolina
Tom Garigen, Stormwater Manager
Project Coordination, Management and Report Production:
Thomas & Hutton Engineering Co.
Richard Karkowski, P.E., P.H., Project Manager
Water Quality Principal Investigator:
Coastal Carolina University, Environmental Quality Lab:
Susan Libes, Ph.D., Principal Investigator
Water Quality Sampling and Laboratory Coordination:
Coastal Carolina University, Environmental Quality Lab:
Joe Bennett, EQL Director
Bacteria Source Tracking Analysis Coordination:
Virginia Polytechnic Institute and State University, Department of Crop and Soil
Environmental Sciences:
Charles Hagedorn, Ph.D., Bacteria Source Tracking Specialist
3.0
Project Definition / Background
Refer to Appendix A.
4.0
Project / Tasks Description
Refer to Appendices A and B.
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
5.0
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Date: 1/13/10
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Data Quality Objectives and Criteria for Measurement Data
The main objective of this monitoring program and study are:
Document historic data and information related to elevated bacteria levels in the swash
adjacent the Town of Briarcliff Acres and the adjacent Atlantic Ocean.
Document the relative number and location of septic systems within the Town of Briarcliff
Acres.
Document the watershed of the swash adjacent the Town of Briarcliff Acres and describe
potential sources and characteristics related to bacterial contamination.
Collection of water samples and analyze for the specified parameters to determine the
presence and concentration of indicator substances for bacteria and pathogens
contamination.
Determine the relative contribution of human and non-human sources of bacteria loading
found in the water samples.
The ultimate goal of this monitoring program and study is to identify or disqualify septic tank
systems located in the watershed as a significant source of bacteria loading in the watershed.
Once collected, the data from this study will be used to:
Identify the relative location of bacteria loading sources in the watershed.
Identify potential sources ± human or non-human ± of bacteria loading.
6.0 Special Training Requirements/Certifications
No special training requirements and/or certifications will be required for this project.
7.0
Documentation and Records
All sampling locations (see Appendix B) were located and documented during a field orientation
conducted on November 5, 2009. All sites will be sampled per the Scope of Services (Appendix
A). Any deviation from the locations described in the Scope of Services (Appendix A), Sample
Location Map (Appendix B), or as described in the field orientation shall be identified on the
field sample collection log.
All sampling activities will be documented through the use of a field sample collection log. The
log will record weather, tidal flow, field measurements (e.g. pH, salinity, water temperature, DO,
conductivity, etc.) and data pertinent to the collection of samples such as type of sample, time
collected, samplers, and the type, number, and volume of sample containers, and preservation
technique.
Analytical results, in hard copy form, will be maintained by each laboratory performing the
analysis for a period of five (5) years. This period of time is in accordance with the requirements
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 1/13/10
By: RPK
and conditions of laboratory certification from the South Carolina Department of Health and
Environmental Control (SCDHEC) and the National Environmental Laboratory Accreditation
Program (NELAP).
8.0
Sampling Process Design
The sampling locations were initially located as part of preparing the Scope of Work (Appendix
A) and are shown in the Sampling Location Map (Appendix B). The sites were subsequently
revised following coordination with the adjacent Meher Baba Spiritual Center and during the
field orientation conducted November 5, 2009. The final sites selected for sampling are
described in the Scope of Work (Appendix A) and shown on the Sampling Location Map
(Appendix B).
9.0
Sampling Methods Requirements
At each sampling location and for each grab sample collected, the following field sampling
methods will be used:
Record the following in the field sample collection log:
Collection Date and Time
Tidal Stage
Samplers
Wind Direction
Water Depth
Water Flow (estimated)
Measure the following per the CCU EQL Standard Operating Procedure (SOP) No. 402
(Appendix C) and record in the field sample collection log:
Specific Conductance
Salinity
Temperature
Dissolved Oxygen
pH
&ROOHFWODERUDWRU\VDPSOHVIRUHDFKRIWKHIROORZLQJXWLOL]LQJWKHDSSURSULDWH623¶VOLVWHG
Turbidity ± CCU EQL SOP No. 405 (Appendix D) or 406 (Appendix E)
Biological Oxygen Demand - CCU EQL SOP No. 430 (Appendix F)
Ammonia - CCU EQL SOP No. 447 (Appendix G)
Total Suspended Solids - CCU EQL SOP No. 435 (Appendix H)
Fecal Coliform Bacteria - CCU EQL SOP No. 502 (Appendix I)
Enterococci Bacteria - CCU EQL SOP No. 501 (Appendix J)
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 1/13/10
By: RPK
Optical Brightener Measurement by Fluorometry (Appendix K)
Note that optical brightener measurements being conducted by CCU are to be considered
provisional, since their SOP is being refined and is currently in draft form. Virginia Tech
is conducting similar analyses and the CCU results shall only be used for comparison and
validation.
Collect laboratory samples for bacteria source tracking analysis as described in Appendix L for
the following:
Bacteroides DNA primer
esp Enterococcus human DNA primer
Optical Brighteners
10.0
Sample Handling and Custody Requirements
Sample handling will be in accordance with standard, accepted field collection methods and shall
meet all the requirements of the referenced SOPs. The CCU field sampling team that collects the
samples will be responsible for initiating the Chain-of-Custody (COC) documentation by starting a
COC form for samples to be transported to the CCU EQL and a separate COC form for samples to
be shipped to the Virginia Polytechnic Institute and State University (VT) laboratory.
Each laboratory is responsible for completing the COC form as samples are received. A copy of
the completed COC forms shall be provided to the Project Coordinator when analytical results
are submitted by the laboratories.
11.0
Analytical Methods Requirements
Refer to the appropriate SOP (listed in Section 9.0) of the various parameters measured for the
analytical methods requirements.
12.0
Quality Control Requirements
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
quality control requirements.
13.0
Instrument/Equipment Testing, Inspection, Maintenance Requirements
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
instrument/equipment testing, inspection, and maintenance requirements.
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
14.0
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Instrument Calibration and Frequency Requirements
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
instrument calibration and frequency requirements.
15.0
Inspection/Acquisition Requirements for Supplies and Consumables
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
inspection and acquisition requirements for supplies and consumables.
16.0
Data Acquisition Requirements (Non-Direct Measurements)
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
data acquisition requirements.
17.0
Data Management
A field sampling collection log will be maintained and will be used for documenting the
collection of field sampling measurements and the collection of grab samples for subsequent
laboratory analysis. The log will record the sampling day, time of day at each station, weather
conditions, field measurements, grab samples collected, and any special notes of potential
importance such as unusual conditions. No special data reduction or reporting will be needed for
this project. All results from field and laboratory analysis will be provided to the Project
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electronic form in a computer database (i.e. MS Excel) such that data retrieval may be organized
as necessary.
18.0
Assessments and Response Actions
After each sampling and analysis event, project personnel will review the information generated
to assess its usability to meet project objectives. Any suggested change or refinement of a
project activity to assure that project data quality and usability objectives are met will be made to
the Project Coordinator. The suggested change or refinement shall be communicated promptly
to the Project Coordinator so that the suggestion can be incorporated in the project, if
appropriate. The Project Coordinator will assess the suggestion and will communicate the
inclusion of the suggestion to the appropriate parties. Any project related documentation will be
updated by the Project Coordinator and forwarded to the project team as needed.
In the event that a problem is identified during a particular activity, a corrective action will be
formulated and implemented without the approval of the Project Coordinator. Proper
documentation will be made of the corrective action.
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
19.0
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Date: 1/13/10
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Reports to Management
At the conclusion of the project, Thomas & Hutton Engineering Co. will prepare a report for
distribution (with input from CCU and VT as needed). The report will present a narrative of all
project activities, results of all data generating activities, and an interpretation of the results,
conclusion, and other recommendations. A preliminary copy of the report will be presented to
Horry County (and other stakeholders) for review prior to the publication of the final report.
20.0
Validation and Verification Methods
Refer to the appropriate SOP (listed in Section 9.0) for the various parameters measured for the
validation and verification methods to be used.
21.0
Reconciliation with User Requirements
A review of eaFKVDPSOLQJHYHQWV¶GDWDZLOOEHFRQGXFWHGZKHQWKHGDWDEHFRPHVDYDLODEOH7KH
review will focus on the stated project goals (listed in Section 5.0).
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix A
SCOPE OF WORK
SCOPE OF SERVICES
Introduction and Background
During the past years, beach closings in Horry County have resulted due to high levels of bacterial
contaminants being detected in the surf zone of the Atlantic Ocean. In 2000, Horry County
commissioned a study by Davis & Floyd (D&F) to identify sources of contamination and to
recommend options for improvement to water quality at storm water outfalls within the study
area (Storm Water Outfall Study ± Horry County Beaches; D&F, February 2002). In this study,
it was observed:
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systems. Significant flooding was observed in this area on several occasions over
the course of the study. During the study, it was common to observe homeowners
in the Briarcliff Acres area pumping storm water from their homes, garages and
yards into the street so that it would flow unrestricted to surface tributaries of the
surf. The percentage of isolates (bacteria) from two selected samples collected
from White Point Swash in the Briarcliff Acres area resulted in 50% and 57%
human contribution, respectively. This was significantly higher than most of the
other samples taken from the study area where the percentage of human vs.
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The study went on to recommend:
Provide Sewer to Briarcliff Acres - The Briarcliff Acres community is the only
large area identified in the study that is served by septic tank and drain field
systems. Flooding was observed in portions of this community during several rain
events. Because of the potential for septic tank and drain field dispersion of
leachate during flooding, it is recommended that Briarcliff Acres be evaluated to
determine the feasibility of connecting the homes in this community to a sanitary
sewer system. (D&F 2002, pg. 3.1-3.2)
A follow-on study was conducted by D&F in 2004 (Atlantic Intracoastal Waterway/Atlantic
Ocean Surf Bacteria Investigation, D&F, December 2004) that addressed several of the
recommendations for additional study made in the first (2002) study, but did not address septic
tank/sewer service at Briarcliff Acres.
In the past few years, several homes along the Atlantic Ocean where taken off septic tanks and
provided with sanitary sewer service. The affect of this change on water quality, and more
specifically the occurrence of beach closings have not been studied. In addition, the previous
sampling implicating the existing septic tanks was limited.
Thus, this study is aimed to confirm or disprove a link between fecal coliform concentration in
the waters around Briarcliff Acres and septic tanks in the area.
Page 1
Our scope of services will include the following tasks:
Task 1 - Data Collection
Task 2 - Septic Tank System Survey
Task 3 - Watershed Assessment
Task 4 - Water Quality Sampling
Task 5 - Data Analysis and Report
Task 1 - Data Collection
Thomas & Hutton (T&H) will gather available data and mapping of the Briarcliff Acres area.
T&H maintains an extensive in-house database of geographical data (aerials, contours, sewer and
water infrastructure, roads, etc.). However, we will confirm the availability of more recent data
and obtain it for use in the study if readily available.
T&H will also make contact SCDHEC Onsite Wastewater Management Division (both locally
and in Columbia) and other agencies (Waccamaw COG, City of Myrtle Beach, Grand Strand
Water and Sewer Authority, etc.) to collect any data relative to the situation and document
information pertinent to the study (sewer system services and location of facilities, septic tank
permits and/or records, etc.)
Historical water quality and other data will be collected. At this time, the known sources of this
data include Storm Water Outfall Study ± Horry County Beaches; D&F, February 2002; Atlantic
Intracoastal Waterway/Atlantic Ocean Surf Bacteria Investigation, D&F, December 2004; data
collected by the Coastal Carolina University Environmental Quality Laboratory in 2003; and
1997 to present beach monitoring data by Coastal Carolina University. Other data, such as local
rainfall data will also be collected from readily available sources.
Task 2 - Septic Tank System Survey
T&H will meet with Briarcliff Acres officials and/or residents to inform them about the study,
gather information concerning existing septic tanks (size, location, service interval, known
problems, etc.), and gage support for potential future recommendations (connection to sewer
system). T&H will prepare materials and submit to Horry County for review and approval prior
to the meeting issuing.
Following the meeting, T&H will estimate the general location of the septic tanks in the study
area and prepare a GIS overlay to catalogue the know/suspected septic tank locations.
Task 3 - Watershed Assessment
A limited watershed assessment of the swash draining Briarcliff Acres to the Atlantic Ocean will
be conducted. A preliminary delineation of the watershed is shown in Exhibit 1. The watershed
assessment will establish the watershed limits, define principal drainage features and patterns in
the watershed (ponds/lakes, major culverts, wetlands, swashes, etc.), delineate major sub-basins,
Page 2
identify homes served by septic tanks and by sanitary sewer collection system, and describe land
uses and impervious land cover percentages.
Task 4 - Water Quality Sampling
Sampling Plan and Quality Assurance Project Plan (QAPP)
A conceptual sampling plan has been established as part of developing the scope of this study.
However, the study plan has to be finalized with the study stakeholders and details must be
finalized. In addition, a project specific QAPP must be prepared to ensure all sampling and
laboratory testing will be defensible. The conceptual sampling plan is described in the two
sections below and include dry and wet (storm event dependent) weather sampling. All
sampling and standard laboratory analyses will be conducted by Coastal Carolina University ±
Environmental Quality Lab (DHEC Certified).
The preliminary sampling locations are illustrated in Exhibit 1. The preliminary sampling
locations include:
BA-1:
BA-2:
BA-3:
BA-4:
BA-5:
Outfall of the swash to draining Briarcliff Acres to the Atlantic Ocean.
Outfall of interior Briarcliff Acres lake to the adjacent lake/wetland.
Contributing wetland from the southwest.
Outfall of second interior Briarcliff Acres lake.
Interior sampling location, located on contributing drainage feature upstream of
second interior Briarcliff Acres lake.
MSC-1: Interior lake sampling location at Meher Spiritual Center.
Dry Weather Sampling
Each station (excluding BA-5) will be sampled at periodic times (for a total of 4 samples each).
The stations will be sampled on the same day and as close in time as possible. Samples will be
collected as close to low tide as possible to minimize the effect of tidal back flow. The water at
each site will be tested for a series of field parameters including:
Water Depth
Water Flow (estimated)
Specific Conductance
Salinity
Temperature
Dissolved Oxygen
pH
Turbidity
Grab samples will be taken, preserved, and shipped for laboratory analysis. The following
laboratory parameters will be analyzed (by Coastal Carolina University - Environmental Quality
Laboratory):
Page 3
Biological Oxygen Demand
Ammonia
Total Suspended Solids
Surfactants (optical brighteners via fluorometry)
Fecal Coliform Bacteria
Enderococci Bacteria
Bacteria Source Tracking Test ± human vs. non-human via the human Bacteroides DNA
marker and the esp human Enterococcus DNA marker test. *
* Note: Dr. Chuck Hagendorn at Virginia Tech University, a known expert in the field will
conduct the tests. The tests will indicate the relative percentage of bacterial from human
and animal sources.
Wet Weather Sampling
Three (3) wet weather sampling events will be conducted. The sampling will be conducted
immediately following the start of a rainfall event and proceed during and after the event.
Rainfall events with anticipated rainfall in excess of 0.75 inch will be targeted.* Three grab
samples will be collected for each station after the start of the rain event.
The same field and laboratory parameters will be analyzed for each wet weather grab sample as
for the dry weather samples.
Task 5 - Data Analysis and Report
The data produced from the study will be summarized and analyzed. Any conclusions that can
be derived from the data as to the amount and source of fecal bacteria contamination in the
watershed will be provided.
A draft report detailing the study procedures, findings and recommendations will be prepared.
The report will include details of the data collection, septic tank survey, and water quality
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comments; a final report will be produced.
*Note: Subsequent to the development of the scope of work, it was decided that the project
director (T&H) and field sampling manager (CCU) would work collaboratively before imminent
rain events to decide to initiate a sampling event.
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Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix B
SAMPLING LOCATION MAP
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Job Number: 22072
Produced: 6/02/09
Produced By: JMN
Scale: 1 inch = 800 feet
Modified: 11/02/09
Modified By: JMN
Projection: SC State Plane
Horizontal Datum: NAD 83 Vertical Datum:
File: N:\22072\GIS\Briarcliff_SamplingLocations.mxd
Thomas & Hutton Engineering Co. compiled the map information from the following sources:
E
S
DATE
2008
2009
2006
R
E
V
SOURCE
Horry County
Thomas & Hutton (conceptual only)
Private Source
T
O
A
DATA
Parcel Boundaries, Utilities, and Road Names
Sampli ng Locations & Watershed Limits
Aerial Photography
FO
H
Myrtle Beach Office
Horry County, South Carolina
DISCLAIMER:
Where Thomas & Hutton Engineering Co. is cited as the data source, the firm has created or
verified the data. For all other sources cited, Thomas & Hutton used the data "as is," has
made no independent investigation of the data, and makes no representation as to the accuracy
or completeness of the data. Please see each source for available documentation of its
respective data sets.
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No part of this document may be reproduced without written permission from an offic er of Thomas & Hutton Engineering Co.
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48
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix C
CCU EQL SOP NO.: 420
FIELD MEASUREMENTS WITH HACH RUGGEDIZED PROBES
Environmental Quality Lab
Chemistry Procedures
SOP No.: 420
Page 1 of 15
Conductivity, Dissolved Oxygen, pH, Salinity,
Temperature Measurements in Field with
HACH Ruggedized Portable Probes
1.0
Rev. 2
10-15-09
Reference Methods:
SM 2510 A. and B. (1997 online), SM 2550 B.
(2000 online), SM4500-H+B. (2000 online),
ASTM D888-05, and Hach Method 10360
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Technician
SCOPE/APPLICATION
HACH ruggedized probes were designed for rugged field use, easy calibrations, time-savings and results
repeatability with a single HQ40d multi meter read-out. This standard operating procedure describes the
procedures used by staff of the Environmental Quality Lab (EQL) to calibrate for and to measure
specific conductivity, dissolved oxygen, pH, salinity, and temperature in the field with HACH
ruggedized probes. Dissolved oxygen is measured using luminescent sensor technology. Specific
conductance and salinity are measured by electrical conductivity. The pH is measured by the
electrometric technique. Temperature is measured by thermistor.
Dissolved oxygen (DO) levels in natural and wastewaters depend on the physical, chemical, and
biochemical activities in the water body. The analysis for DO is a key test in water pollution and waste
treatment process control. Dissolved oxygen on the HACH ruggedized Luminescent Dissolved Oxygen
(LDO) probe is determined by the luminescent measurement using luminescent sensor technology; this
is not a membrane method. The EPA approved the LDO Hach method 10360 in July 2006. The HACH
LDO probe is coated with luminescent material and works by transmitting blue light and red light. Blue
light from an LED is transmitted to the sensor surface and excites the luminescent material. As the
luminescent material relaxes, it emits a red light, and this emission time between blue and red lights is
measured and used as an internal reference. Higher oxygen levels decrease the time between blue and
red lights, and this correlates to oxygen concentration. The probe does not require a warm-up period,
but the sensor cap must be replaced annually or when the sensor surface is < 25% black luminescent
material, whichever is longer. A salinity correction must be applied when dissolved oxygen is being
measured with the HACH ruggedized LDO probe in brackish or saline waters.
Conductivity is a measure of the amount of dissolved solids in a water sample. Dissolved solids are
primarily ions. A conductivity meter detects the amount of electricity that is conducted by a water
sample. The larger the amount of ions the greater the amount of electricity conducted and hence the
greater the conductivity of the sample. Conductivity units are Pmhos/cm, PS/cm, or mS/cm.
Salinity is defined as the weight in grams of the dissolved inorganic matter in 1 kg of seawater, after all
bromide and iodide have been replaced by the equivalent amount of chloride, and all carbonate
converted to oxide. Along with temperature, the salt content of seawater determines density. Colder
water has higher density than warm and saltier water has higher density than fresh water, and density of
the ocean increases with increasing depth. So, the waters of the deep sea are cold and salty. The salt
content of the water is determined largely by the balance between water input and loss from the ocean.
Water enters the ocean via rainfall and river runoff. It leaves via evaporation. At locations where the
rate of evaporation exceeds rainfall or river runoff, the salinity of seawater is high.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 420
Page 2 of 15
Rev. 2
10-15-09
The measurement of pH is one of the most important and frequently used tests in water chemistry.
Many phases of water supply, wastewater treatment, and environmental water chemistry processes are
pH-dependent. At a given temperature the intensity of the acidic or basic character of a solution is
indicated by pH or hydrogen ion activity, where pH is defined as –log (hydrogen ion activity). In dilute
solution (i.e., ionic strength <0.1) the activity of hydrogen ion in solution is approximately equal to the
hydrogen ion molarity.
2.0
REFERENCES
2.1
2.2
2.3
2.4
2.5
3.0
Standard Methods 2510 A. and B. (1997 online), 2550 B. (2000 online), 4500-H+ B. (2000 online)
Hach Method 10360
American Society for Testing and Materials (ASTM) Method D888-05
HACH “HQ Series Portable Meters” User Manual; 2006 Sept, ed. 5 (HACH catalog number:
HQ40d18)
Hach.com (Hach Knowledge Base searching)
DEFINITIONS
None
4.0
SAFETY
4.1
4.2
4.3
5.0
This method is restricted to use by or under the supervision of trained analysts.
Gloves, safety glasses with side shields, and protective clothing should be worn to protect
against unnecessary exposure to infectious agents (i.e., pathogens), hazardous chemicals (e.g.,
acids), and contaminants in potentially hazardous samples.
All activities performed while following this procedure should utilize appropriate laboratory
safety systems (e.g., disinfectant, fume hoods, material safety data sheets).
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.1.7
5.1.8
5.1.9
HACH HQ40d Dual-Input, Multi-Parameter Digital Meter with 4 AA batteries
IntelliCAL pH Probe-Rugged (HACH product no. pH101-05, -10, -15, or -30)
IntelliCAL Conductivity Probe-Rugged (HACH product no. CDC401-05, -10,-15, or -30)
InteliCAL LDO Probe-Rugged (HACH product no. LDO101-05, -10, -15, or -30)
“HACH Ruggedized Field Probe CALIBRATION SHEET”
Deionized water
Delicate tissue wipes (Kimwipes or equivalent)
Barometer
Calibration flask for dissolved oxygen (250 mL glass or plastic Erlenmeyer flask with
mouth large enough for probe to fit)
5.1.10 Rubber o-ring to act as a probe stopper in DO calibration
5.1.11 Parafilm
Environmental Quality Lab
Chemistry Procedures
5.2
Rev. 2
10-15-09
REAGENTS
5.2.1
5.2.2
5.2.3
5.2.4
5.2.5
5.3
SOP No.: 420
Page 3 of 15
Certified pH buffer solutions from Fisher (or equivalent): pH 4.00, 7.00, and 10.00
(Fisher catalog SB101-4, SB107-4, SB115-4; respectively)
Electrode Storage Solution (Fisher catalog number SE40-1 or equivalent)
Specific conductivity standards of various conductivities, commercially prepared (e.g.,
Fisher Traceable Conductivity Calibration 1000 uS/cm Standard, Cat. No. 09-328-3) and
laboratory prepared (e.g., For preparation of conductivity standards see Standard Method
2510 A. (1997 online), “Conductivity Introduction”). Formulations for standards of
various conductivities are provided. For example, 0.5M KCl (e.g., 37.28g KCl dissolved
in deionized water and diluted to 1L) has a conductivity of 58,670 µS/cm at 25.0ºC.)
Conductivity control sample (e.g., 0.01 M KCl, which is 0.7456g KCl dissolved in
deionized water and diluted to 1L, has a conductivity of 1,412 µS/cm at 25.0ºC)
Salinity Laboratory Control Samples: IAPSO P-series (35 psu) Standard Seawater (Ocean
Scientific International Ltd., www.osil.co.uk), and filtered (0.45µm filter) and lab-tested
seawater
DISSOLVED OXYGEN PROCEDURES
5.3.1
x
x
x
x
x
x
5.3.2
LDO Settings
Push the wrench button on the HQ40d multi-meter and push the green button to
‘Select’ the “LDO101 Method”
Observe the current method on the display screen. If the current method is not
“INITIAL LAB” push the green button to select the currently displayed method.
Use the up or down arrow to select “INITIAL LAB”, then push the green button to
select that method as the current method.
To check or modify the current method, arrow down and then select “Modify Current
Method”
For the INITIAL LAB method the parameters and options should be as follows:
o Measurement Options: allows you to choose resolution, lower and upper mg/L
limits, salinity correction (enter salinity if measuring DO in brackish or
seawater), pressure units (choose in mmHg), and to turn on averaging interval
(choose OFF)
o Units: allows to choose mg/L or % (choose mg/L)
o Calibration Options: choose “User - 100%”
o Calibration Reminder: choose “On” and set for 8 hours +30 min reminders
To exit menus push blue button repeatedly until main menu is achieved
LDO Calibration Procedure
x
The probe must be calibrated daily prior to use for measurements in the field or
laboratory, and recalibrated after 8 hours for continuous use.
x
Insert batteries in the meter if they are not present or need replacement.
x
Check the condition of the probe surface. The surface should be clean and at least 25%
black luminescent material. If it is necessary to clean the probe surface just rub with a
wet Kimwipe and wipe dry with another Kimwipe. The sensor cap must be replaced
annually or when the sensor surface is < 25% black luminescent material, whichever is
longer. A count down message appears on the screen 30 days before the sensor cap
Environmental Quality Lab
Chemistry Procedures
SOP No.: 420
Page 4 of 15
Rev. 2
10-15-09
expiration date. All measurements taken after the expiration date appear with the
calibration ? icon in the top left corner.
x
Place water in the calibration flask, cover the opening with parafilm and shake the flask
vigorously for about 1 minute. Let the flask sit for 5-10 minutes to equilibrate and
create 100% DO saturation in water saturated air.
x
Remove the plastic protective bell from the LDO probe by loosening the fitting ring
and sliding the bell off. Slide the rubber o-ring onto the LDO probe at a level that will
allow the probe to fit loosely in the flask and the probe surface to sit directly above, but
not touching, the water in the calibration flask.
x
Connect LDO probe to HQ40d multi-meter (right side connection ports makes
calibration activities easier but either the right or left port can be used) and turn meter
on. After LDO probe has had time (5-10 minutes) to equilibrate in the water
saturated air:
o If the calibration screen is displayed, press cancel so you can perform a
precalibration measurement.
o Push green button to ‘Read’ and once the reading is displayed, fill in the precalibration row in the LDO probe calibration section on the “HACH
Ruggedized Field Probe CALIBRATION SHEET”
o Press blue button to ‘Calibrate’, and then green button to ‘Read’. Once
calibration reading stabilizes record calibration information on calibration
sheet, push ‘Done’ and record that information on calibration sheet, and finally
press ‘Store’ to save the calibration.
o If the calibration was successful, the display screen will show OK in top left
corner on the main menu. If “?” is displayed and the comment on the screen is
“O2 Sensor 0 Days Remaining” the calibration is acceptable as long as the QC
checks were acceptable (see Table 1). The O2 sensor is good as long as the
sensor surface is > 25% black luminescent material (Hach personal
communication on 8-20-09). If calibration was not successful, repeat the
calibration.
o Before removing LDO probe from calibration flask, containing water saturated
air, do several (at least two) post-calibration readings. Push ‘Read’ to perform
a post-calibration reading. Wait at least 1 minute between post-calibration
readings. Record these readings on the calibration sheet.
x
The LDO probe saves the calibration within itself (intelliCAL), so it can be
disconnected and reconnected to the same or another multi-meter and not need a
recalibration.
x
Proceed with sample analyses or turn power off.
5.3.3
Sample Measurement for LDO
x
Insert batteries in the meter if they are not present or need replacement.
x
If meter power is off, turn power on. Set date and time, if needed.
x
If the salinity of the sample to be measured is greater than 1 ppt, enable a salinity
correction by pushing the wrench button on the HQ40d multi-meter, and push the
Environmental Quality Lab
Chemistry Procedures
SOP No.: 420
Page 5 of 15
Rev. 2
10-15-09
green button to ‘Select’ the “LDO101 Method”. Then arrow down to “Modify Current
Method” and then push the green button to ‘Select’. Press the green ‘Select’
“Measurement Options” and arrow down to “Salinity Correction” and push the green
button to ‘Select’. Input the salinity and arrow to the right until “OK” is selected.
Next, push the blue button several times to ‘Exit’ out to main menu and salinity
correction should appear on this main screen under the dissolved oxygen concentration.
5.4
x
For NPDES compliance analyses, analysis must be performed within 15 minutes of
sample collection.
x
Place probe into the water body or collected sample to be measured and push green
“Read” button. Wait for reading to stabilize (meter beeps). Record measurement value.
x
Switch probe, if needed, to measure another parameter.
x
After all measurements are completed, push power button to turn meter off.
x
Rinse the probes with deionized water and dry with a delicate tissue wipe. Store the
probes clean and dry.
CONDUCTIVITY PROCEDURES
5.4.1
x
x
x
x
x
x
5.4.2
Conductivity Settings
Push the wrench button on the HQ40d multi-meter and push the green button to
“select” the “CDC401 Method”.
Observe the current method displayed on the display screen. If the current method
displayed is not “CONDUCTIVITY”, push the green button to select the currently
displayed method.
On the screen that comes up, use the up or down arrow to select “CONDUCTIVITY”,
then push the green button to select that method as the current method.
To check or to modify the current method, arrow down and then select “Modify
Current Method”
For the CONDUCTIVITY method the parameters and options should be as follows:
o Parameter: Conductivity
o Measurement Options: Units - Auto or can choose µS/cm or mS/cm if desire,
Measurement Limits - 0.01 – 400,000 µS/cm, Temperature Correction –
Linear, Correction Factor - 1.90%/°C, Reference Temperature - 25°C
o Calibration Options: Custom Standard – if desire to change displayed custom
standard value, input the actual standard value by selecting “Standard Value”
then entering desired standard value in µS/cm. Preset standard concentrations
can also be selected by selecting “Std:” then selecting the desired standard
from the list.
Push the blue button several times to ‘Exit’ out to the main menu.
Conductivity Calibration Procedures
x
The probe must be calibrated daily prior to use for measurements in the field or
laboratory, and recalibrated after 8 hours for continuous use.
x
Insert batteries in the meter if they are not present or need replacement.
Environmental Quality Lab
Chemistry Procedures
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Page 6 of 15
Rev. 2
10-15-09
x
Clean the conductivity probe with deionized water and a brush if necessary. Dry the
probe with a soft cloth or paper towel.
x
Power on the meter and connect the conductivity probe to the meter (right side
connection ports makes calibration activities easier but either the right or left port can
be used). Perform a reading in air by pressing the green “Read” button and waiting for
the reading to stabilize. If the reading is ” 0.01 µS/cm the probe is clean and ready to
calibrate.
x
Note: All conductivity probe calibrations are performed using conductivity units µS/cm
or mS/cm regardless of whether conductivity, resistivity, salinity, or total dissolved
solids (TDS) is measured.
x
Specify desired conductivity settings using section 5.4.1.
x
If necessary, remove the plastic protective bell from the Conductivity probe by
loosening the fitting ring and sliding the bell off. If the bell will remain on the probe
make sure the holes in the bell line up so you can see through the bell and through the
conductivity chamber. This is to ensure free flow through the chamber.
x
Place probe in the conductivity calibration standard, tilt and jiggle the probe to remove
air bubbles which will prevent an accurate result.
x
With meter powered on and conductivity probe connected:
o If the calibration screen is displayed, press cancel so you can perform a
precalibration measurement.
o Push green button to ‘Read’ and once reading is displayed fill in the precalibration column in the conductivity probe calibration section on the “HACH
Ruggedized Field Probe CALIBRATION SHEET”
o Press blue button to ‘Calibrate’, and then green button to ‘Read’. Once
calibration reading stabilizes record calibration information on calibration
sheet, push ‘Done’ and record that information on calibration sheet, and finally
press ‘Store’ to save calibration.
o If the calibration was successful, the display screen will show OK in the top
left corner of the main menu. If it was not successful, repeat the calibration.
o Before removing conductivity probe from conductivity calibration standard,
push ‘Read’ to perform a post-calibration reading. Record this reading on
calibration sheet as well.
x
Rinse the conductivity probe and dry it. Perform a post calibration conductivity control
sample reading and fill in that section on the calibration sheet.
x
The conductivity probe saves the calibration within itself (intelliCAL), so it can be
disconnected and reconnected to the same or another multi-meter and not need a
recalibration.
x
Proceed with sample analyses or turn power off.
5.4.3
Sample Measurement for Conductivity
Environmental Quality Lab
Chemistry Procedures
5.5
SOP No.: 420
Page 7 of 15
Rev. 2
10-15-09
x
Insert batteries in the meter if they are not present or need replacement.
x
If meter power is off, turn power on. Set date and time, if needed.
x
Make sure that the holes in the bell line up so that you can see through the bell and
through the conductivity chamber. This is to make sure there is free flow of sample
through the chamber
x
Place probe into water body or collected sample to be measured, tilt and jiggle the
probe to remove air bubbles which will prevent an accurate result, and push green
“Read” button. Wait for reading to stabilize (meter beeps). Record measurement value.
x
Switch probe, if needed, to measure another parameter.
x
After all measurements are completed, push power button to turn meter off.
x
Rinse the probes with deionized water and dry with a delicate tissue wipe. Store the
probes clean and dry.
SALINITY PROCEDURES
5.5.1
x
x
x
x
x
x
5.5.2
Salinity Settings on Conductivity Probe
Push the wrench button on the HQ40d multi-meter and push the green button to
“select” the “CDC401 Method”.
Observe the current method displayed on the display screen. If the current method
displayed is not “SALINITY”, push the green button to select the currently displayed
method.
On the screen that comes up, use the up or down arrow to select “SALINITY”, then
push the green button to select that method as the current method.
To check or to modify the current method, arrow down and then select “Modify
Current Method”
For the SALINITY method the parameters and options should be as follows:
o Parameter: Salinity
o Measurement Options: Units - °/oo , Measurement Limits - 0.0 – 40.0°/oo
o Calibration Options: Custom Standard – usually 58,670, if desire to change
displayed custom standard value, input the actual standard value by selecting
“Standard Value” then entering desired standard value in µS/cm. Preset
standard concentrations can also be selected by selecting “Std:” then selecting
the desired standard from the list.
Push the blue button several times to ‘Exit’ out to the main menu.
Salinity Calibration Procedures
x
The probe must be calibrated daily prior to use for measurements in the field or
laboratory, and recalibrated after 8 hours for continuous use.
x
x
Insert batteries in the meter if they are not present.
Turn on the meter, connect the conductivity probe (right side connection ports makes
calibration activities easier but either the right or left port can be used) and check to
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Chemistry Procedures
SOP No.: 420
Page 8 of 15
Rev. 2
10-15-09
make sure the probe is set to read salinity (the main menu will show ‰ as the unit). If
needed, follow section 5.5.1 to change the parameter to salinity.
x
Clean the conductivity probe with deionized water and a brush if necessary. Dry the
probe with a soft cloth or paper towel.
x
Perform a reading in air by pressing the green “Read” button and waiting for the
reading to stabilize. If the reading is ” 0.01 µS/cm the probe is clean and ready to
calibrate.
x
Note: All conductivity probe calibrations are performed using conductivity units µS/cm
or mS/cm regardless of whether conductivity, resistivity, salinity, or total dissolved
solids (TDS) is measured.
x
If necessary, remove the plastic protective bell from the Conductivity probe by
loosening the fitting ring and sliding the bell off. If the bell will remain on the probe
make sure that the holes in the bell line up so that you can see through the bell and
through the conductivity chamber. This is to make sure there is free flow through the
chamber.
x
Place probe in the conductivity calibration standard, tilt and jiggle the probe to remove
air bubbles which will prevent an accurate result.
x
With the meter powered on and the conductivity probe connected:
o If the calibration screen is displayed, press cancel so you can perform a
precalibration measurement.
o Push the green button to ‘Read’ and once the reading is displayed fill in the
pre-calibration row in the conductivity probe calibration section on the “HACH
Ruggedized Field Probe CALIBRATION SHEET”
o Press the blue button to ‘Calibrate’, and then the green button to ‘Read’. Once
the calibration reading stabilizes record the calibration information on the
calibration sheet, push ‘Done’ and record that information on the calibration
sheet, and finally press ‘Store’ to save the calibration
o If the calibration was successful, the display screen will show OK in the top
left corner of the main menu. If it was not successful, repeat the calibration.
o Before removing the probe from the conductivity calibration standard, push
‘Read’ to perform a post-calibration reading. Record this reading on the
calibration sheet as well.
x
Rinse the conductivity probe and dry it. Perform a post calibration salinity control
sample reading and fill in that section on the calibration sheet.
x
Conductivity probe saves its calibration within itself (intelliCAL), so it can be
disconnected and reconnected to the same or another multi-meter and not need
recalibration.
x
Proceed with sample analyses or turn power off.
5.5.3
Sample Measurement for Salinity
Environmental Quality Lab
Chemistry Procedures
5.6
SOP No.: 420
Page 9 of 15
Rev. 2
10-15-09
x
Insert batteries in the meter if they are not present or need replacement.
x
If meter power is off, turn power on. Set date and time, if needed.
x
Make sure that the holes in the bell line up so that you can see through the bell and
through the conductivity chamber. This is to make sure there is free flow of sample
through the chamber
x
Place the probe into water body or collected sample to be measured, tilt and jiggle the
probe to remove air bubbles which will prevent an accurate result, and push green
“Read” button. Wait for reading to stabilize (meter beeps). Record measurement value.
x
Switch probe, if needed, to measure another parameter.
x
After all measurements are completed, push power button to turn meter off.
x
Rinse the probes with deionized water and dry with a delicate tissue wipe. Store the
probes clean and dry.
pH PROCEDURES
5.6.1
pH Settings
x
Push the wrench button on the HQ40d multi-meter and push the green button to
‘Select’ the “pHC101 Method”
x
The current method is the default method and nothing needs changed or checked.
5.6.2
pH Calibration Procedures
x
The probe must be calibrated daily prior to use for measurements in the field or
laboratory, and recalibrated after 8 hours for continuous use.
x
Insert batteries in the meter if they are not present.
x
Remove the electrode storage solution cup from the probe and rinse the probe with
deionized water. Pat the probe dry with a Kimwipe.
x
Connect the probe to the meter (right side connection ports makes calibration activities
easier but either the right or left port can be used) and power the meter on.
x
If necessary, remove the plastic protective bell from the pH probe by loosening the
fitting ring and sliding the bell off.
x
Place the pH probe in a 7 buffer. If the calibration screen is displayed, press cancel so
you can perform a precalibration measurement. Push the green button to ‘Read’.
Record this value in the pre-calibration section of the calibration sheet.
x
Rinse, blot dry and place the pH probe immediately into a 4 buffer and push the green
button to ‘Read’. Record this value in the pre-calibration section of the calibration
sheet.
Environmental Quality Lab
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Page 10 of 15
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x
Push the blue ‘Calibrate’ button, place the pH probe in the lowest pH buffer (usually
the pH 4), and press the green ‘Read’ button. Write the result on the calibration sheet.
x
When prompted, clean and place the probe in the next higher pH buffer (usually the pH
7), and press the green ‘Read’ button. Write the result on the calibration sheet.
x
Unless specifically required for the analyses being conducted, continue the calibration (
i.e., perform a three-point calibration) and when prompted, clean and place the probe in
the next high pH buffer (usually the pH 10), and press the green ‘Read’ button. Write
the result on the calibration sheet.
x
When finished reading the calibration buffers, push the up arrow under ‘Done’. Record
the rest of the calibration summary data on the calibration sheet.
x
Press the green ‘Store’ button to accept the calibration.
x
When calibration is successful, display will show OK in top left corner of main menu.
x
Place the pH probe in a buffer closest to expected sample pHs (usually the 6 or 7) and
push the green button to ‘Read’. Record this value in the post calibration check
standard section of the calibration sheet within the pH probe calibration box.
x
Rinse the probe with deionized water and blot dry with Kimwipe.
x
If storing the probe temporarily before measurements, refresh the electrode storage
solution in the cup and replace it on the probe end.
x
Replace the plastic protective bell to the probe.
x
The pH probe saves the calibration within itself (intelliCAL), so it can be disconnected
and reconnected to the same or another multi-meter and not need a recalibration for 8
hours.
x
Proceed with sample analyses or turn power off.
5.6.3
Sample Measurement for pH
x
Insert batteries in the meter if they are not present or need replacement.
x
If meter power is off, turn power on. Set date and time, if needed.
x
Remove the electrode storage solution cup from the probe and rinse the probe with
deionized water. Pat the probe dry with a Kimwipe.
x
For NPDES compliance analyses, analysis must be performed within 15 minutes of
sample collection.
x
Place the probe into water body or collected sample to be measured and push the green
“Read” button. Wait for the reading to stabilize (it beeps). Record the measurement
value.
Environmental Quality Lab
Chemistry Procedures
5.7
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Page 11 of 15
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x
After measurement, rinse the probe with deionized water, and continue to next sample
or store the probe clean and in the electrode storage solution.
x
Switch probe, if needed, to measure another parameter.
x
After all measurements are complete, push power button to turn meter off.
x
Rinse the pH probe with deionized water and blot dry with a delicate tissue wipe. Store
the pH probe clean and in the electrode storage solution
TEMPERATURE PROCEDURES
5.7.1 Temperature Calibration
x
The thermistor on each probe is calibrated annually using a NIST-Traceable
thermometer.
x
The thermistor temperature correction factor is labeled on the probes.
x
To obtain the corrected temperature apply the correction factor to the temperature read
(i.e., corrected temperature = temperature read + listed temperature correction value).
5.7.2
5.8
Sample Measurement for Temperature
x
Temperature is displayed along with the parameter result simultaneously for every
probe that is connected to the meter, so temperature is determined when any parameter
is being measured.
x
For NPDES compliance analyses, analysis must be performed within 15 minutes of
sample collection.
x
Place any probe into water body or sample collected, and push the green ‘Read’ button.
Record the temperature.
x
Apply the temperature correction factor listed for the probe to the recorded temperature
to determine the corrected temperature.
x
Clean and store the probe as specified for the probe type being used (see sections: 5.3,
5.4, 5.5, or 5.6).
QUALITY CONTROL
5.8.1
Quality control (QC) measures for analyzing samples are summarized in Table 1 and are
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as follows:
x
Prior to analyzing samples, each analyst must demonstrate the ability to generate
acceptable results (i.e., demonstration of capability).
x
The probe must be calibrated daily prior to use for measurements in the field or
laboratory, and recalibrated after every 8 hours for continuous use.
x
Acceptable calibration checks (see Table 1 for calibration check acceptance criteria) must
be performed immediately after calibration.
x
For NPDES compliance analyses, analyses for DO, pH, and temperature must be
performed within 15 minutes of sample collection.
x
Any QC sample analysis (e.g., field replicates, laboratory control samples) should be
subjected to exactly the same analytical procedures as those used on individual sample
analyses.
x
Unless otherwise specified for specific project or samples, each sampling event of up to
20 samples analyzed should include at least one field duplicate (i.e., field duplicate
sample collected same location and nearly same time as initial sample) as QC sample.
Table 1. Quality Control Requirements for Measurements with Hach Ruggedized Probes
Environmental Quality Lab
Chemistry Procedures
LCS = laboratory control sample
SOP No.: 420
Page 13 of 15
QC = quality control
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior
to any customer sample
analyses
LDO 97-104% of theoretical DO
Others 75-125% R
Others RPD < 25%
Calibration stability
monitoring
Immediately before
calibration measure
standards
Calibration
Daily prior to sample
analysis and after every
8 hours
Calibration check
Immediately following
calibration
Field duplicate (duplicate
sample collected at one
of sampling sites
One (1) per sampling
event
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
Blind PE sample
accrediting agencies and
projects
MB = method blank
Internal PE sample
5.9
Not applicable.
After calibration, measure calibration
standards (conductivity, pH, DO %
saturation of water saturated air) as sample
pH ± 0.1 of expected, others 99-101% R
Measurement of calibration standards or
LCS (conductivity, pH, DO % saturation
of LCS or of water saturated air)
Cond. 90-110% R, pH ± 0.1 of expected,
DO 97-104% sat
**LDO method requires LCS to be read
in duplicate with each calib. event**
Rev. 2
10-15-09
PE = performance evaluation
Corrective Action
Repeat until acceptable.
Not applicable. Results are
used to monitor stability of
probes and evaluate need for
maintenance.
Investigate and fix any obvious
problems. Repeat until
acceptable.
Investigate and fix any obvious
problems. Recalibrate and
repeat until acceptable.
RPD < 25%
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze all
sampling sites if possible.
75-125% R
RPD < 25%
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
%R = percent recovery
RPD = relative percent difference
CORRECTIVE ACTIONS
5.9.1
If any of the QC requirements listed in Section 5.8 (see Table 1) are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity must be
repeated, after corrective actions are taken to correct any obvious problems, until the
QC results are acceptable. If repeating the process is not possible (e.g., sample spilled),
the results report will include a discussion of the problem and the client will be
consulted.
5.9.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
Environmental Quality Lab
Chemistry Procedures
5.10
SOP No.: 420
Page 14 of 15
Rev. 2
10-15-09
EQUIPMENT MAINTENANCE
5.10.1 General
x
x
x
The probes are designed to generally be maintenance-free. If the probes become dirty
(see pH probe maintenance below), wipe their surface with a damp cloth or Kimwipe.
Use a cotton-tipped applicator to clean or dry the connectors if they get wet.
Wipe and clean probes and cables well after each field use.
Store the display meter without batteries
5.10.2 Luminescent Material on Dissolved Oxygen Probe
x
Check the condition of the probe surface. The surface should be clean and at least 25%
black luminescent material. If it is necessary to clean the probe surface just rub with a
wet Kimwipe and wipe dry with another Kimwipe. The sensor cap must be replaced
annually or when the sensor surface is < 25% black luminescent material, whichever is
longer. A count down message appears on the screen 30 days before the sensor cap
expiration date. All measurements taken after the expiration date appear with the
calibration “?” icon in the top left corner.
5.10.3 Cleaning pH electrode
x
Cleaning the electrode may improve performance when the response and stabilization
time become noticeably slower, but it should be noted that normal life expectancy for a
probe is about 2 years.
o For a mild cleaning, place the probe in a detergent solution such as Alconox for
30 minutes, and then soak in deionized water for approximately 15 minutes
before use.
o For a more vigorous cleaning, place the probe in 0.1N HCl solution for 2
minutes, rinse with deionized water, place in 0.1N NaOH solution for 2
minutes, and then rinse and place in the HCl solution again for 2 more minutes.
Rinse the probe and allow it to soak in deionized water for approximately 15
minutes before use.
o To clean organic build-up on the probe, place the probe in liquid bleach for 5
minutes, then soak in deionized water for 15 minutes before use.
5.10.4 Cleaning conductivity probe
x
6.0
If the sample contains oils, greases, or fats, the probe may become coated. If this
occurs, clean the probe with a strong detergent solution and brush. Rinse thoroughly
with DI water.
WASTE DISPOSAL
Dispose of analyzed laboratory samples and waste from calibration activities down a sink sewage
drain. Flush the sink with a large volume of tap water.
SOP No.: 420
Page 15 of 15
Environmental Quality Lab
Chemistry Procedures
Rev. 2
10-15-09
HACH Ruggedized Field Probe CALIBRATION SHEET
Probes used:
LONG / SHORT
for field sampling
date(s):
date & time of
cal:
pH probe calibration
pH 4
Std
pH 7 Std
(revised 10/15/09)
Project
name(s):
Analyst:
Post Calibration
Check Std
pH 10 Std
Std LOT #, exp date
temp
pre calibration
expected value
post calibration
measured value
calibration temperature
°C
°C
°C
°C
mV
Slope:
mV/pH
offset:
date & time of
cal:
Analyst:
mV
%:
r²:
LDO probe calibration
time
temp
(°C)
D.O. (mg/L)
D.O. %
saturation
pressure
(mmHg)
expecte
d D.O.
pre-calibration reading
cal reading
post-cal reading #1
post-cal reading #2
post-cal reading #3
slope:
Conductivity probe calibration
offset:
date & time of
cal:
Analyst:
Std LOT #, exp date
Std LOT #, exp date
ȝS/cm Cal Standard
Used
ȝS/cm LCS Used
pre calibration
temperature of cal std
cell constant
post calibration
temperature of cal std
°C
/cm
°C
LCS value after
cal.
temperature of
LCS
°C
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix D
CCU EQL SOP NO.: 405
TURBIDITY MEASUREMENT WITH HACH POCKET TURBIDMETER
Environmental Quality Lab
Chemistry Procedures
SOP No.: 405
Page 1 of 8
Turbidity Measurement in Field or Laboratory
with Hach Pocket Turbidimeter
Reference Method:
SM 2130 B. (21st ed.)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Master Technician
1.0
Rev. 2
3-14-09
SCOPE/APPLICATION
Turbidity is an expression of the optical property that causes light to be scattered and
absorbed rather than transmitted with no change in direction or flux level through the
sample. Turbidity in water is caused by suspended and colloidal matter such as clay, silt,
finely divided organic and inorganic matter, and plankton and other microscopic
organisms.
In the Environmental Quality Lab (EQL) turbidity is determined by the nephelometric
method. In this technique turbidimeters with scattered-light detectors located at 90° to
the incident beam are used. Nephelometric measurement results are reported as the
nephelometric turbidity unit (NTU).
2.0
REFERENCES
2.1
2.2
3.0
Standard Method 2130 B. (21st ed.)
Hach Pocket Turbidimeter Instrument Manual
DEFINITIONS
None
4.0
SAFETY
4.1
This method is restricted to use by or under the supervision of trained analysts.
4.2
Gloves, safety glasses with side shields, and protective clothing should be worn
to protect against unnecessary exposure to corrosive infectious agents (i.e.,
pathogens), hazardous chemicals (e.g., standards containing formazin), and
contaminants in potentially hazardous samples.
4.3
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
Environmental Quality Lab
Chemistry Procedures
5.0
SOP No.: 405
Page 2 of 8
Rev. 2
3-14-09
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.2
REAGENTS
5.2.1
5.2.2
5.3
Hach Pocket Turbidimeter
Deionized water
Delicate tissue wipes (Kimwipes or equivalent)
Hach 5 mL sample cells with caps for Pocket Turbidimeter
(Catalog 52631-00)
Silicone oil
Oiling cloth
StablCal Stabilized Formazin Standards, 1.0 NTU (Catalog 26598-42)
StablCal Stabilized Formazin Standards, 20 NTU (Catalog 26601-42)
PROCEDURE
5.3.1
2-Point Calibration
x
When the Pocket Turbidimeter is to be used for field or laboratory
measurements, a 2-point calibration is required daily within 8 hours prior
to use for measurements and recalibrated for every 8 hours of continuous
use.
x
Thoroughly rinse the Pocket Turbidimeter sample cell with deionized
water.
x
Apply a small drop of silicone oil to each of the outside four vertical
rectangular sides of the cell.
x
Spread the oil uniformly on the optical surfaces of the cell using the
oiling cloth provided. Wipe off excess oil with the oiling cloth. The cell
should appear nearly dry with little or no visible oil and no dust particles.
x
Pour 1-2 mL of properly mixed 1.0 NTU StablCal Standard into the
clean, labeled, and oiled Pocket Turbidimeter sample cell, swirl the
standard around in the cell to rinse it, then pour out the rinse.
x
Pour 5 mL of properly mixed 1.0 NTU StablCal Standard into the rinsed
sample cell.
x
Cap the cell.
x
To remove all dust particles, wipe the sample cell exterior with a delicate
tissue wipe.
x
Place the sample cell containing the 1.0 NTU standard into the
instrument sample cell compartment.
Cover the sample cell with the light shield and wait 30 seconds for the
x
Environmental Quality Lab
Chemistry Procedures
SOP No.: 405
Page 3 of 8
Rev. 2
3-14-09
standard to stabilize.
x
Press and hold the CAL key then press the READ key. Release both
keys. After a short delay, dA will flash alternating with the dark value.
x
Press and hold the READ key until the reading is stable. Release the
READ key to accept the new dark value.
x
Press the CAL key. After a short delay, the display shows C1.0
alternating with the 1.0 NTU value using the last calibration.
x
Press and hold the READ key. When the reading is stable, release the
READ key to save the new 1.0 NTU value.
x
Pour 1-2 mL of properly mixed 20 NTU StablCal Standard into the clean,
labeled, and oiled Pocket Turbidimeter sample cell, swirl the standard
around in the cell to rinse it, then pour out the rinse.
x
Pour 5 mL of properly mixed 20 NTU StablCal Standard into the rinsed
cell.
x
Cap the cell.
x
To remove all dust, wipe the sample cell exterior with a delicate tissue
wipe then insert it into the sample cell compartment.
x
Cover the sample cell with the light shield and wait 30 seconds for
instrument to stabilize.
x
Press the CAL key. After a short delay, the display shows C20 alternating
with the 20 NTU value using the last calibration.
x
Press and hold the READ key until the reading is stable. Release the
READ key to save the new 20 NTU value.
x
Press the CAL key to end the calibration. The instrument displays CLd to
indicate a new calibration has been entered. If no data points were
changed, the instrument displays OLd to show the previous calibration has
been retained.
x
Measure the turbidity of each standard following the procedure listed in
Section 5.3.3.
x
See Section 5.4.2 for acceptance criteria for standard measurements (i.e.,
within 10% of expected values). If any standard measurements do not
satisfy acceptance criteria, follow corrective actions described in Section
5.4 Table 1 and Section 5.5.
5.3.2
Calibration Check Prior to Sample Analysis
Environmental Quality Lab
Chemistry Procedures
SOP No.: 405
Page 4 of 8
Rev. 2
3-14-09
x
After calibration and prior to sample analysis a calibration check is
required by analyzing the calibration standards.
x
Thoroughly rinse the Pocket Turbidimeter sample cell with deionized
water.
x
Apply a small drop of silicone oil to each of the outside four vertical
rectangular sides of the cell.
Spread the oil uniformly on the optical surfaces of the cell using the
oiling cloth provided. Wipe off excess oil with the oiling cloth. The cell
should appear nearly dry with little or no visible oil and no dust particles.
x
x
Pour 1-2 mL of properly mixed 1.0 NTU StablCal Standard into the
clean, labeled, and oiled Pocket Turbidimeter sample cell, swirl the
standard around in the cell to rinse it, then pour out the rinse.
x
Pour 5 mL of properly mixed 1.0 NTU StablCal Standard into the rinsed
sample cell.
x
Cap the cell, then remove dust particles by wiping the cell with a delicate
tissue wipe immediately before inserting it into the sample cell
compartment.
x
Place the sample cell containing the standard into the instrument sample
cell compartment.
x
Insert the sample cell into the instrument sample compartment.
x
Cover the sample cell with the light shield and wait 30 seconds for
instrument to stabilize.
x
Press and hold the READ key until the reading stabilizes (approximately
5 seconds). Release the READ key and record the displayed reading.
x
Repeat the measurement process used for the 1.0 NTU standard for the
20 NTU standard.
x
See Section 5.4.2 for acceptance criteria for check standard measurement
(i.e., within 10% of expected value). If check standard measurement
does not satisfy acceptance criteria, follow corrective actions described
in Section 5.4 Table 1 and Section 5.5.
5.3.3
Sample Analysis
x
If samples are to be measured in the field, they should be analyzed on the
same day collected, ideally with 15 minutes of collection. Store at 1-4°C
if cannot analyze immediately, and then warm to ambient temperature
before analysis.
x
For measurements in the laboratory, samples must be stored in the dark
Environmental Quality Lab
Chemistry Procedures
SOP No.: 405
Page 5 of 8
Rev. 2
3-14-09
at 1-4°C and analyzed within 24 hours of collection. Samples should be
warmed to room temperature prior to analysis.
5.4
x
Agitate or invert the sample to be measured to uniformly distribute
particles/turbidity.
x
Rinse a clean sample cell 2-3 times with the mixed sample.
x
Pour 5 mL of sample into the Pocket Turbidimeter rinsed sample cell.
Cap the cell.
x
Wipe the outside surfaces of the cell with a delicate tissue wipe to
remove any liquid. Take care not to scratch the cell.
x
Insert the sample cell into the instrument sample compartment.
x
Cover the sample cell with the light shield and wait 30 seconds for
instrument to stabilize.
x
Press and hold the READ key until the reading stabilizes (approximately
5 seconds). Release the READ key and record the displayed reading. If
reading exceeds the concentration of the highest calibration standard, 20
NTU, the sample must be diluted with particle free water and measured
until the value is less than 20 NTU.
x
Between sample measurements in the field, rinse the sample cell
thoroughly with deionized water.
x
After completing measurements and returning to the laboratory, clean all
sample cells and caps using detergent and water. Rinse with deionized
water and dry and cap the cells for storage. Do not store samples or
standards in the plastic sample cells.
QUALITY CONTROL
Quality control (QC) measures for analyzing samples are summarized in Table 1
and are as follows:
x
When the Pocket Turbidimeter is to be used for field or laboratory
measurements, a 2-point calibration is required daily within 8 hours prior
to use for measurements and recalibrated for every 8 hours of continuous
use. The two standards used for calibration and for calibration checks of
the turbidimeter are 1.0 NTU and 20 NTU. For an acceptable 2-point
calibration, measured values of all standards must be within 10% of
expected values. For calibration checks the calibration standards are
again measured as samples, and their results must also be within 10% of
expected values.
x
Prior to analyzing samples, each analyst must demonstrate the ability to
generate acceptable results (i.e., demonstration of capability).
Environmental Quality Lab
Chemistry Procedures
x
x
5.5
SOP No.: 405
Page 6 of 8
Rev. 2
3-14-09
Samples must be analyzed within 24 hours of collection. If not analyzed
immediately after collection, samples must be stored in the dark at 1-4°C,
and then warmed to ambient or room temperature prior to analysis.
Any QC sample analysis (e.g., method blank, laboratory replicate, field
replicate) should be subjected to exactly the same analytical procedures
as those used on individual sample analyses.
x
Unless otherwise specified for specific project or samples, each batch of
up to 20 samples analyzed should include at least one method blank (i.e.,
deionized water) and one sample analyzed in duplicate (i.e., at least 5%
duplicates) as QC samples.
x
Direct turbidimeter reading (i.e., not corrected for dilution) of a sample
must fall within the range bracketed by the lowest and highest calibration
standards (i.e., 1.0 NTU and 20 NTU). Any sample reading below the
lowest calibration standard’s concentration must be reported as less than
1.0 NTU, the reporting limit (see Section 6.1). For any direct sample
reading above the highest calibration standard’s concentration, an aliquot
of the sample must be diluted with particle free water until the reading
falls within the calibration range.
CORRECTIVE ACTIONS
5.5.1
If any of the QC requirements listed in Section 5.4 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
5.5.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
SOP No.: 405
Page 7 of 8
Environmental Quality Lab
Chemistry Procedures
Rev. 2
3-14-09
Table 1. Quality Control Requirements for Turbidity by Hach Pocket Turbidimeter
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Corrective Action
Four (4) prepared
samples analyzed prior to
any customer sample
analyses
Daily prior to sample
analysis and every 8
hours for continuing
Criteria for duplicate precision
Repeat until acceptable
90-110% R (measured value of all
standards when analyzed as samples
within 10% of expected value)
Calibration check with
calibration standards
Prior to sample analysis
90-110% R
Method blank
Daily prior to sample
analysis
<1.0 NTU
Sample analysis
For all sample analyses
Direct sample reading within calibration
range (i.e., lowest and highest calibration
standard concentrations)
Sample duplicate
One (1) per preparation
batch
RPD < 25%
Investigate problem. Correct
any obvious problems. Repeat
calibration until acceptable.
Investigate problem. Correct
any obvious problems
including new 2-point
calibration if necessary.
Repeat calibration check until
acceptable.
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination.
If reading below range report
result as < RL. If result above
range dilute sample.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze entire
batch.
Capability demonstration
2-Point calibration
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
5.6
75-125% R
RPD < 25%
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
QC =
%R =
RL =
RPD =
quality control
percent recovery
reporting limit
relative percent difference
EQUIPMENT MAINTENANCE
5.6.1
Cleaning the Sample Cells and Caps
x
To maintain clean sample cells and caps, rinse with 1:1 hydrochloric
acid followed by multiple rinses with particle-free water or soak in
warm water to which a mild detergent (Liqui-nox or equivalent) has
been added; use a cotton swab to scrub the cells if necessary. Rinse
several times with turbidity-free water.
Environmental Quality Lab
Chemistry Procedures
5.6.2
Rev. 2
3-14-09
Cleaning the Sample Compartment
x
6.0
SOP No.: 405
Page 8 of 8
Clean the sample compartment and the sample cell windows with a
cotton swab dampened with isopropyl alcohol or detergent and
turbidity-free water. Rinse with turbidity-free water. Do not use
acetone or other organic solvents to clean the sample cells; damage
to the cells will result.
DATA REPORTING
6.1
REPORTING LIMIT
The reporting limit (RL) for turbidity is primarily based on the concentration
of the lowest calibration standard and the overall dilution or concentration of the
sample during sample analysis. Additionally, the RL cannot be lower than
typical method blank results or values calculated by method detection limit
determinations. The concentration of the lowest calibration standard is 1.0 NTU.
Nominally, sample analysis is done without dilution. Therefore, the nominal RL
is 1.0 NTU, and any measured sample concentration below 1.0 NTU must be
reported as less than 1.0 NTU.
6.2
MEASUREMENT RANGE REQUIREMETS
Direct turbidimeter reading (i.e., not corrected for dilution) of a sample must fall
within the range bracketed by the lowest and highest calibration standards (i.e.,
1.0 NTU and 20 NTU). Any sample reading below the lowest calibration
standard’s concentration must be reported as less than 1.0 NTU, the reporting
limit (see Section 6.1). For any direct sample reading above the highest
calibration standard’s concentration, an aliquot of the sample must be diluted
with particle free water until the reading falls within the calibration range. If it is
not possible to dilute a sample that reads greater than 20 NTU, then the result
must be reported as greater than 20 NTU.
7.0
WASTE DISPOSAL
Dispose of analyzed samples and used standards down a sink sewage drain. Flush the sink
with a large volume of tap water.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix E
CCU EQL SOP NO.: 406
TURBIDITY MEASUREMENT WITH HACH 2100N TURBIDIMETER
Environmental Quality Lab
Chemistry Procedures
SOP No.: 406
Page 1 of 9
Turbidity Measurement in Laboratory with
Hach 2100N Turbidimeter
Reference Method:
SM 2130 B. (21st ed.)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Master Technician
1.0
Rev. 3
3-15-09
SCOPE/APPLICATION
Turbidity is an expression of the optical property that causes light to be scattered and
absorbed rather than transmitted with no change in direction or flux level through the
sample. Turbidity in water is caused by suspended and colloidal matter such as clay, silt,
finely divided organic and inorganic matter, and plankton and other microscopic
organisms.
In the Environmental Quality Lab (EQL) turbidity is determined by the nephelometric
method. In this technique turbidimeters with scattered-light detectors located at 90° to
the incident beam are used. Nephelometric measurement results are reported as the
nephelometric turbidity unit (NTU).
2.0
REFERENCES
2.1
2.2
3.0
Standard Method 2130 B. (21st ed.)
Hach 2100N Laboratory Turbidimeter Instruction Manual
DEFINITIONS
None
4.0
SAFETY
4.1
This method is restricted to use by or under the supervision of trained analysts.
4.2
Gloves, safety glasses with side shields, and protective clothing should be worn
to protect against unnecessary exposure to corrosive infectious agents (i.e.,
pathogens), hazardous chemicals (e.g., standards containing formazin), and
contaminants in potentially hazardous samples.
4.3
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
Environmental Quality Lab
Chemistry Procedures
5.0
SOP No.: 406
Page 2 of 9
Rev. 3
3-15-09
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.2
REAGENTS
5.2.1
5.2.2
5.3
Hach 2100N Turbidimeter
Deionized water
Delicate tissue wipes (Kimwipes or equivalent)
Hach 30 mL sample cells with caps for 2100N Turbidimeter
Volumetric Flasks, Class A, various sizes
Volumetric Pipets, Class A, various sizes
Hach Formazin Turbidity Standard, 4000 NTU (Catalog 2461-42) or
equivalent
GELEX Secondary Standards (Catalog 25890-00, including: air, stray
light, 0-2, 0-20, 0-200, and 200-4000 ranges)
REAGENT PREPARATION
5.3.1
Preparation of Formazin Turbidity Standards for Instrument Calibration
x
Use Class A volumetric glassware for preparing each standard.
x
Use deionized water for all the following dilutions:
¾ Pipet 12.5 mL of 4000 NTU standard into 50 mL volumetric
flask and dilute to 50 mL (corresponds to 1000 NTU).
¾ Pipet 5 mL of 4000 NTU standard into 100 mL volumetric
flask and dilute to 100 mL (corresponds to 200 NTU).
¾ Pipet 1 ml of 4000 NTU standard into 200 mL volumetric
flask and dilute to 200 mL (corresponds to 20 NTU).
¾ Pipet other volumes of standard into volumetric flasks and
dilute to prepare other desired standard concentrations.
x
Record preparation of standards in Standard Preparation Log, Form
205. Record following on separate line for each standard solution:
¾ Type of Stock Standard to be Diluted or Dissolved
¾ ID of Stock Standard
¾ Conc. of Stock Standard
¾ Type of Standard Prepared
¾ Volume (mL) Stock Solution Dissolved
¾ ID of Standard Prepared
¾ Conc. of Standard Prepared
¾ Volume of Standard Prepared (mL)
¾ Date Prepared
¾ Prepared with class A glassware by: (initials)
¾ Date Discarded
Environmental Quality Lab
Chemistry Procedures
5.4
SOP No.: 406
Page 3 of 9
Rev. 3
3-15-09
PROCEDURES
5.4.1
Calibrating the Turbidimeter and Assigning Values to Gelex Standards
x
Calibrating the turbidimeter and assigning new values to the GELEX
secondary standards are done at least quarterly.
x
Press the I/O switch on the back instrument panel to turn power on, and
allow turbidimeter to warm up (minimum of 30 minutes).
x
Select automatic range selection, signal average, and ratio settings (i.e.,
confirm the corresponding indicator lights are on or select them).
x
Fill a clean sample cell to the line (approx. 30 mL) with fresh, deionized
water. Wipe the cell clean. Place it into the cell holder with the triangle
shape on the vial aligned with the index mark on the instrument sample
compartment, and close the cell cover.
x
Press the CAL key. The S0 annunciator lights. The NTU value of the
deionized water used in the previous calibration is displayed.
x
Press the ENTER key. The instrument display counts down from 60 to
0, and then makes a measurement. This result is stored and used to
calculate a correction factor for measurement of all NTU standards.
x
The instrument automatically increments to the next standard, displays
the expected NTU value (e.g., 20.00 NTU), and the S1 annunciator
flashes. Remove the sample cell from the holder.
x
Fill a clean sample cell to the line with well-mixed 20 NTU Formazin
standard. Wipe the sample cell clean. Place it into the cell holder with
the triangle shape on the vial aligned with the index mark on the
instrument sample compartment, and close the cell cover.
x
Press the ENTER key. The instrument display counts down from 60 to
0, and then displays the turbidity (compensated for deionized water
turbidity).
x
The instrument automatically increments to the next standard, displays
the expected NTU value (e.g., 200.0 NTU), and the S2 annunciator
flashes. Remove the sample cell from the holder.
x
Fill a clean sample cell to the line with well-mixed 200 NTU Formazin
standard. Wipe the sample cell clean. Place it into the cell holder with
the triangle shape on the vial aligned with the index mark on the
instrument sample compartment, and close the cell cover.
x
Press the ENTER key. The instrument display counts down from 60 to
0, and then displays the turbidity (compensated for deionized water
turbidity).
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x
The instrument automatically increments to the next standard, displays
the expected NTU value (e.g., 1000 NTU), and the S3 annunciator
flashes. Remove the sample cell from the holder.
x
Fill a clean sample cell to the line with well-mixed 1000 NTU Formazin
standard. Wipe the sample cell clean. Place it into the cell holder with
the triangle shape on the vial aligned with the index mark on the
instrument sample compartment, and close the cell cover.
x
Press the ENTER key. The instrument display counts down from 60 to
0, and then displays the turbidity (compensated for deionized water
turbidity).
x
The instrument automatically increments to the next standard, displays
the expected NTU value (e.g., 4000 NTU), and the S4 annunciator
flashes. Remove the sample cell from the holder.
x
Fill a clean sample cell to the line with well-mixed 4000 NTU Formazin
standard. Wipe the sample cell clean. Place it into the cell holder with
the triangle shape on the vial aligned with the index mark on the
instrument sample compartment, and close the cell cover.
x
Press the ENTER key. The instrument display counts down from 60 to
0, and then displays the turbidity (compensated for deionized water
turbidity).
x
The display automatically increments back to the deionized water
standard. The S0 annunciator lights, and the previously measured value
of deionized water is displayed.
x
Press the CAL key. The instrument stores the new calibration and
returns the instrument to the measurement mode.
x
To check the accuracy of the calibration, measure each standard used for
the calibration as if it were a sample (i.e., Wipe the sample cell
containing the standard clean. Place the standard in the sample
compartment with the triangle or diamond on the vial aligned with the
index mark on the instrument sample compartment. Close the samplecell cover. Record the value displayed.)
x
After the instrument is calibrated, the GELEX Secondary Turbidity
Standards can be assigned a value.
x
Confirm that automatic range selection, signal average, and ratio settings
are on.
x
Wipe the air standard sample cell clean. Place the air standard in the
sample compartment with the triangle or diamond on the vial aligned
with the index mark on the instrument sample compartment. Close the
sample-cell cover.
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x
Press the ENTER key. Record the value displayed. Remove the air
standard from the instrument, and mark this value on the vial cap.
x
Repeat these steps with the stray light, 0-2, 0-20, 0-200, and 200-4000
ranges standards.
x
After completing the calibration, continue with sample analyses or turn
instrument power off with power switch on the back panel of instrument.
5.4.2
Sample Analysis
x
Samples must be stored in the dark at 1-4°C and analyzed within 24
hours of collection. Samples should be warmed to room temperature
prior to analysis.
x
Turn turbidimeter power switch on, and allow meter to warm up
(minimum of 30 minutes).
x
Select automatic range selection, signal average, and ratio settings (i.e.,
confirm the corresponding indicator lights are on or select them).
x
For a calibration check with the GELEX Secondary Turbidity Standards,
wipe the standards clean.
x
Insert the air standard into the cell holder with the triangle or diamond
shape on the vial aligned with the index mark on the instrument sample
compartment, and close the cell cover. Record the value. This should be
within 33% of its assigned value.
x
Repeat these steps with the stray light, 0-2, 0-20, and 0-200 ranges
standards. The stray light standard should be within 33% of its assigned
value; the other standards should be within 10% of their assigned values.
o
o
If the standards are not within the acceptance criteria, identify
and fix the problem before conducting sample analyses. This
may require a new calibration and assignment of new values to
the secondary standards.
If the calibration check is acceptable, continue with analysis of
samples.
x
Fill a clean sample cell to the line (approx. 30 mL) with sample. Wipe
the cell clean. Place it into the cell holder with the triangle shape on the
vial aligned with the index mark on the instrument sample compartment,
and close the cell cover.
x
Read the digital readout for sample in NTUs and record value on the
Turbidity Determination Worksheet form, Form 505. In general, take the
reading when the digital readout stabilizes to a fixed value. In samples
which the digital readout drifts, which sometimes occurs when sample
contains numerous small swimming organisms, record the high and low
measurements for the range of measurements. Direct readings less than
Environmental Quality Lab
Chemistry Procedures
x
5.5
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1.00 NTU (i.e., report limit – see Section 6.1) must be reported as less
than 1.00 NTU. For direct readings above 4000 NTU (i.e., highest
calibration standard), an aliquot of the sample must be diluted with
particle free water and remeasured until less than 4000 NTU.
Do not leave standards or samples in instrument after test is completed.
x
Between sample measurements in the lab, rinse the sample cell
thoroughly with deionized water.
x
After completing sample measurements, measure the GELEX secondary
standards again. If the measurements do not meet the calibration check
acceptance criteria, identify the problem and fix it, if possible.
Reanalyze samples if investigation data were adversely impacted.
x
After completing all measurements, turn the instrument power off with
the power switch on the back panel of the instrument.
x
After all measurements, wash out used sample cells and caps with a
detergent solution, and gently brush them with a test tube brush. Rinse
thoroughly with tap water, and finally with deionized water. Turn tubes
upside down and allow to drain and dry.
QUALITY CONTROL
5.5.1
Quality control (QC) measures for analyzing samples are summarized in
Table 1 and are as follows:
x
Prior to analyzing samples, each analyst must demonstrate the ability to
generate acceptable results (i.e., demonstration of capability).
x
Samples must be stored in the dark at 1-4°C and analyzed within 24
hours of collection. Samples should be warmed to room temperature
prior to analysis.
x
Any QC sample analysis (e.g., method blank, laboratory replicate, field
replicate) should be subjected to exactly the same analytical procedures
as those used on individual sample analyses.
x
Unless otherwise specified for specific project or samples, each batch of
up to 20 samples analyzed should include at least one method blank (i.e.,
deionized water) and one sample analyzed in duplicate (i.e., at least 5%
duplicates) as QC samples.
x
Direct turbidimeter reading (i.e., not corrected for dilution) of a sample
must fall within the range bracketed by the lowest and highest calibration
standards (i.e., 20.0 NTU and 4000 NTU) or the lowest and highest
primary standards that were measured as samples and yielded results
within 10% of expected values. A series of low turbidity standards were
measured, and for this instrument the lowest formazin turbidity standard
that could be measured within 10% of its expected value was 1.00 NTU.
Therefore, the acceptable range for direct turbidity measurements for this
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instrument is 1.00 NTU to 4000 NTU. Any sample reading below 1.00
NTU must be reported as less than 1.00 NTU. For any direct sample
reading above 4000 NTU, an aliquot of the sample must be diluted with
particle free water until the reading falls within the acceptable
measurement range.
5.6
5.7
x
Calibrating the turbidimeter and assigning new values to the GELEX
secondary standards are done at least quarterly. Primary formazin
turbidity standards of 20.0 NTU, 200 NTU, 1000 NTU, and 4000 NTU
are used for these purposes. Step-by-step procedures are listed in Section
5.4.1.
x
Each day of sample analyses, a calibration check is done before and after
sample analyses with the GELEX Secondary Turbidity Standards.
CORRECTIVE ACTIONS
5.6.1
If any of the QC requirements listed in Section 5.5 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
5.6.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
EQUIPMENT MAINTENANCE
5.7.1
Cleaning the Sample Cells and Caps
x
Because of the extreme sensitivity of the instrument, all sample cells
must be absolutely clean inside and out. Do not allow previous test
samples to dry out in cells. All residue must be removed because
any matter remaining in the cell or on its outside surfaces will cause
error in subsequent tests. Wash out used sample cells and caps with
a detergent solution, and gently brush them with a test tube brush.
Rinse thoroughly with tap water, and finally with deionized water.
x
Turn tubes upside down and allow them to drain and dry.
x
Any cells which are scratched or permanently stained must be
discarded. To check for scratches, place the empty sample cell in the
test well while the instrument is on in a dimly lighted room. With
the cell cap and well cap off, rotate the tube slowly while looking
down into the opening. Any scratches on the tube will be visible
when the instrument’s light strikes them.
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Table 1. Quality Control Requirements for Turbidity by Hach 2100N Turbidimeter
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior to
any customer sample
analyses
Criteria for duplicate precision
4-Point calibration plus
deionized water blank
At least quarterly
90-110% R (measured value of primary
standards when analyzed as samples
within 10% of expected values)
Assign values to
permanent transfer
standards using formazin
primary standards
At least quarterly
Measurement after acceptable 4-point
calibration and values within 10% of
previous established values
Daily calibration check
Immediately prior to and
after sample analysis
GELEX Secondary Turbidity Standards
should read within 10% of assigned values
Method blank
Daily prior to sample
analysis
<1.0 NTU (i.e., < RL)
Sample analysis
For all sample analyses
Direct sample reading within acceptable
measurement range (i.e., 1.00 NTU to
4000 NTU)
Sample duplicate
One (1) per preparation
batch
RPD < 25%
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
QC =
%R =
RL =
RPD =
Corrective Action
Repeat until acceptable
Investigate problem. Correct
any obvious problems. Repeat
calibration until acceptable.
Investigate problem. Correct
any obvious problems
including replacing transfer
standards if necessary. Repeat
until acceptable.
Investigate problem. Correct
any obvious problems. If
necessary reassignment of
GELEX values and reanalyze
samples. Repeat calibration
check until acceptable.
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination.
If reading below range report
result as < RL. If result above
range dilute sample.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze entire
batch.
75-125% R
RPD < 25%
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
quality control
percent recovery
reporting limit
relative percent difference
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DATA REPORTING
6.1
REPORTING LIMIT
The reporting limit (RL) for turbidity is primarily based on the concentration of
the lowest calibration standard, or the lowest primary standard measured as a
sample that yielded a result within 10% of expected value, and the overall
dilution or concentration of the sample during sample analysis. Additionally, the
RL cannot be lower than typical method blank results or values calculated by
method detection limit determinations. A series of low turbidity standards were
measured, and for this instrument (Hach 2100N Laboratory Turbidimeter) the
lowest formazin turbidity standard that could be measured within 10% of its
expected value was 1.00 NTU. Nominally, sample analysis is done without
dilution. Therefore, the nominal RL is 1.00 NTU, and any measured sample
concentration below 1.00 NTU must be reported as less than 1.00 NTU.
6.2
MEASUREMENT RANGE REQUIREMETS
Direct turbidimeter reading (i.e., not corrected for dilution) of a sample must fall
within the range bracketed by the RL (see Section 6.1) and highest calibration
standard (i.e., acceptable measurement range of 1.00 NTU to 4000 NTU). Any
sample reading below the 1.00 NTU must be reported as less than 1.00 NTU.
For any direct sample reading above the highest calibration standard’s
concentration (i.e., 4000 NTU), an aliquot of the sample must be diluted with
particle free water until the reading falls within the calibration range. If it is not
possible to dilute a sample that reads greater than 4000 NTU, then the result must
be reported as greater than 4000 NTU.
7.0
WASTE DISPOSAL
Dispose of analyzed samples and used standards down a sink sewage drain. Flush the sink
with a large volume of tap water.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix F
CCU EQL SOP NO.: 430
BIOCHEMICAL OXYGEN DEMAND (BOD)
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Page 1 of 14
Biochemical Oxygen Demand (BOD)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Technician
1.0
Rev.1
09-17-09
Reference Method:
SM 5210 B. (2001 online)
SCOPE/APPLICATION
General
The biochemical oxygen demand (BOD) determination is an empirical test in which
standardized laboratory procedures are used to determine the relative oxygen
requirements of waste-waters, effluents, and polluted waters. The test has its widest
application in measuring waste loadings to treatment plants and in evaluating the
BOD-removal efficiency of such treatment systems. The test measures the molecular
oxygen utilized during a specified incubation period for the biochemical degradation
of organic material (carbonaceous demand) and the oxygen used to oxidize inorganic
material such as sulfides and ferrous iron. It also may measure the amount of oxygen
used to oxidize reduced forms of nitrogen (nitrogenous demand) unless their
oxidation is prevented by an inhibitor. The seeding and dilution procedures provide
an estimate of the BOD at pH 6.5 to 7.5.
Measurements of oxygen consumed in a 5-day test period (5-day BOD or BOD5),
oxygen consumed after 60 to 90 days of incubation (ultimate BOD or UBOD) and
continuous oxygen uptake (respirometric method,) are methods used to determine
oxygen demand. Many other variations of oxygen demand measurements exist,
including using shorter and longer incubation periods and tests to determine rates of
oxygen uptake. Alternative seeding, dilution, and incubation conditions can be
chosen to mimic receiving-water conditions, thereby providing an estimate of the
environmental effects of wastewaters and effluents.
Dilution Requirements
The BOD concentration in most wastewaters and many surface waters (i.e., rivers,
ponds) exceeds the concentration of dissolved oxygen (DO) available in an airsaturated sample. Therefore, it is usually necessary to dilute the sample before
incubation to bring the oxygen demand and supply into appropriate balance. Because
bacterial growth requires nutrients such as nitrogen, phosphorus, and trace
metals, these are added to the dilution water, which is buffered to ensure that the
pH of the incubated sample remains in a range suitable for bacterial growth.
Complete stabilization of a sample may require a period of incubation too long for
practical purposes; therefore 5 days has been accepted as the standard incubation
period.
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If the dilution water is of poor quality, the BOD of the dilution water will appear as
sample BOD, and a positive bias will result. This effect will be amplified by the
dilution factor. Both a dilution-water ‘CHECK’ and a ‘BLANK’ are included within
the method. Seeded dilution waters are checked further for acceptable quality by
measuring their consumption of oxygen from a known organic mixture; this is usually
glucose and glutamic acid (contains an NH2 component) are used, and sometimes
potassium hydrogen phthalate (KHP) is used because it lacks nitrogen.
The source of dilution water is not restricted and may be distilled, tap, or receivingstream water free of biodegradable organics and bio-inhibitory substances such as
chlorine or heavy metals. Distilled water may contain ammonia or volatile organics;
deionized (DI) waters often are contaminated with soluble organics leached from the
resin bed. Use of copper lined stills or copper fittings attached to distilled water lines
will produce water containing excessive amounts of copper.
2.0
REFERENCES
2.1
2.2
2.3
3.0
Standard Methods 5210 B. (2001 online), 5-Day BOD Test.
Laboratory Testing for BOD and CBOD, Post 1995. Brake, Perry & Raynovic,
Michael. NCL of Wisconsin, Inc.
A Bug’s-Eye-View of the BOD Test, January 2007. Brake, Perry.
DEFINITIONS
None
4.0
SAFETY
4.1
4.2
4.3
5.0
This method is restricted to use by or under the supervision of trained analysts.
Gloves, safety glasses with side shields, and protective clothing should be
worn to protect against unnecessary exposure to infectious agents (i.e.,
pathogens), hazardous chemicals (e.g., acids), and contaminants in potentially
hazardous samples.
All activities performed while following this procedure should utilize
appropriate laboratory safety systems (e.g., disinfectant, fume hoods, material
safety data sheets).
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1 Glassware and containers:
x Incubation Bottles: 300-mL capacity. Clean bottles using a
brush and warm tap water or two wash cycles in the dishwasher
with no detergent, rinse with RO thoroughly, and with DI water two
times. If possible, store in incubator until needed. Drain before use.
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The bottles are designed to allow a water-seal as a precaution
against drawing air into the dilution bottle during incubation.
Obtain a satisfactory water seal by capping a full sample BOD
bottle with a ground glass stopper, adding dilution water to the
flared mouth of the BOD bottles and placing a plastic cap over the
flared mouth of bottle to reduce evaporation of the water seal during
incubation.
Standard Lab glassware: Graduated cylinders, 500 mL beakers,
1000 mL beakers, Erlenmeyer flasks, and containers large enough
for preparing dilution water. Clean all glassware with warm tap
water or two wash cycles in the dishwasher with no detergent, rinse
with RO thoroughly, and with DI water two times.
5.1.2 Air Incubator: Fisher Low Temperature BOD Incubator (model
307C is currently used in Coastal Science Bldg Room 308), thermostatically controlled at 20°C r 1ºC.
5.1.3: DO meter and Probe: YSI model 5100 Dissolved Oxygen meter
(Fisher catalog #13-298-22) and YSI model 5010 BOD probe with self
stirring capability (Fisher catalog # 15-176-18).
5.1.4
Membrane cap kit: YSI 5906 Membrane cap kit, 6/pk, contains
membrane cap assemblies, bottle O2 probe solution, sanding disk, and
instruction sheet (Fisher catalog # 14-660-225).
5.1.5 Deionized water
5.1.6 Delicate tissue wipes (Kimwipes or equivalent)
5.1.7 Barometer
5.2
REAGENTS AND CHEMICALS
5.2.1
Buffer pillows -HACH BOD Nutrient Buffer Pillows
x 300 mL pillows (HACH brand, Fisher catalog # 14160-66) to be
used for 300 mL individual bottle buffering with sample volumes
>150 mL)
x 6L pillows (HACH brand, Fisher catalog #14862-66) to be used in
the preparation of the bulk dilution water
x 19L/5gal pillow (HACH brand, Fisher Catalog # 14863-98) to be
used in the preparation of the bulk dilution water
5.2.2
Phosphate Buffer Solution.
Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4x7H2O,
and 1.7 g NH4Cl in about 500 mL distilled water and dilute to 1L. The
pH should be 7.2 without further adjustment.
Commercially prepared solution is available and acceptable.
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5.2.3
Magnesium sulfate Solution:
Dissolve 22.5g MgSO4x7H20 in distilled water and dilute to 1L.
Commercially prepared solution is available and acceptable.
5.2.4
Calcium Chloride Solution:
Dissolve 27.5g CaCl2 in distilled water and dilute to 1L.
Commercially prepared solution is available and acceptable.
5.2.5
Ferric Chloride Solution:
Dissolve 0.25g FeCl3x6H2O in distilled water and dilute to 1L.
Commercially prepared solution is available and acceptable.
Prepare buffers immediately before use and be sure to discard if there is any
sign of biological growth in the pillows.
5.2.6
Preparation of dilution water with commercial buffer pillows:
1. Place desired volume of water in a suitable clean bottle (i.e., 6L
Erlenmeyer flask), aerate for no less than 1 h with a clean hollow
glass rod hooked to a vacuum pump’s HEPA filtered outflow,
making sure to have a liquid trap flask and filter in-between the
glass rod and pump exhaust.
2. After aeration of the water, place a paper towel over the bottle’s
opening and secure it with a rubber band. Place the bottle in the
incubator at 20°C r 1ºC for >24 h to stabilize.
3. Prepare buffered dilution water fresh daily. On day of analysis, 2-3
hours prior to sample set-up, dissolve the buffers in the desired
volume of the aerated water. It is best to prepare a flask of doublestrength dilution water and a flask of unbuffered water.
x When using the 6L pillows, add 2 pillows to 6L of aerated
water, and swirl gently. This will create 6L of doublestrength nutrient buffered BOD dilution water.
x When using the 19L/5gal pillow, pour 1 pillow into 100
mL aerated water, swirl until dissolved, then pour off until
63.2 mL remains. Finally pour this into the 6L of aerated
water, and swirl gently to mix. This will create 6L of
double-strength nutrient buffered BOD dilution water.
x Other volumes of buffered double strength dilution water
can be prepared by proportionately adjusting the number or
volume of buffer pillows or volume of aerated water.
4. When adding the buffer to the bottles:
x Dilution water blanks, seed controls and GGA standards are
prepared with single strength buffered dilution water, (i.e.,
equal volumes of aerated water and double strength
buffered dilution water).
x Samples ”150 mL will receive ”150 mL of sample, the
SOP No.: 430
Page 5 of 14
Environmental Quality Lab
Chemistry Procedures
x
Rev.1
09-17-09
selected quantity of seed, unbuffered aerated water, if
necessary, to make up to 150mL total, and the remainder of
the bottle volume with double-strength buffered dilution
water.
Samples >150 mL will receive > 150 mL of desired sample,
one ‘300 mL individual buffer pillow’, the selected
quantity of seed, and remainder of the bottle volume filled
with unbuffered dilution water.
5.2.7
Preparation of dilution water without commercial buffer pillows:
1. Place desired volume of water in a suitable bottle, aerate for no
less than 1 h with a clean hollow glass rod hooked to a vacuum
pump’s HEPA filtered outflow, making sure to have a liquid trap
flask and filter in-between the tube and pump discharge.
2. After aeration of the water, place a paper towel over the bottle’s
opening and secure it with a rubber band. Place the bottle in the
incubator at 20°C r 1ºC for >24 h to stabilize.
3. Prepare buffered dilution water daily. On day of analysis, 2-3
hours prior to sample set-up, dissolve the buffers in the desired
volume of aerated dilution water. It is best to prepare a flask of
double-strength dilution water and a flask of unbuffered dilution
water. Add 1 mL each of the following buffers per 1 L of water
for single-strength dilution water and 2 mL each per L for
double-strength dilution water:
x Phosphate Buffer solution (5.2.2)
x Magnesium sulfate solution (5.2.3)
x Calcium chloride solution (5.2.4)
x Ferric chloride solution (5.2.5)
4. When adding the buffer to the bottles:
x Dilution water blanks, seed controls and GGA standards are
prepared with single strength buffered dilution water, (i.e.,
equal volumes of aerated water and double strength buffered
dilution water).
x Samples ”150 mL will receive ”150 mL of sample, the
selected quantity of seed, unbuffered aerated water, if
necessary, to make up to 150mL total, and the remainder of
the bottle volume with double-strength buffered dilution
water.
x Samples >150 mL will receive > 150 mL volume of
sample, one ‘300 mL individual buffer pillow’, the selected
quantity of seed, and remainder of the bottle volume with
unbuffered dilution water.
5.2.8
Acid and alkali solutions, 1N for neutralization of caustic or acidic
waste samples.
x Acid- Dissolve slowly, while stirring, 28 mL of concentrated
Environmental Quality Lab
Chemistry Procedures
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5.2.9
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sulfuric acid, H2SO4, to distilled water. Dilute to 1L.
Alkali- Dissolve 40g sodium hydroxide, NaOH, in distilled water.
Dilute to 1L.
Sodium Sulfite Solution:
Dissolve 1.575g Na2SO3 in 1L distilled water. This solution is not
stable; prepare daily for neutralization of chlorine.
5.2.10 Glucose-Glutamic Acid Solution (G-GA):
Dry reagent-grade glucose [Dextrose (D-Glucose) Anhydrous, CAS
50-99-7 & F.W. 180.16] and reagent-grade Glutamic Acid (L-(+)Glutamic Acid, CAS 56-86-0 & F.W. 147.13) at 103ºC for 1 h. Cool
and store in a desiccator.
For standard preparation, add 150 mg glucose and 150 mg glutamic
acid to single strength buffered dilution water and dilute to 1L, or
equivalent ratio. Prepare fresh immediately before use. This makes a
concentration of 198 mg/L when 6 mL is added into 300 mL bottle
diluted with buffered water and seeded.
5.2.11 Second-source G-GA standard: There are at least two choices for a
second standard:
x
NCL brand BOD Standard,
198 ppm Glucose-Glutamic Acid Standard (NCL catalog #B12D, call 715-449-2673 or 800-648-7836) in which 6 mL are
placed into a 300mL sample bottle for 198 mg/L concentration.
x
HACH BOD Standard Solution
300 mg/L Glucose, 300 mg/L Glutamic Acid (HACH brand,
Fisher catalog # 14865-10) in which 3 mL are placed into a 300
mL sample bottle for 198 mg/L concentration.
5.2.12 Potassium Hydrogen Phthalate (KHP):
Keep a clean vial with KHP in a desiccator. Add 150 mg to 500 mL
DI water. This makes a concentration of 300 mg/L when 3 mL are
added to a 300 mL sample bottle with dilution water, and seeded.
Discard reagents if there are any signs of biological growth in the stock bottle.
5.2.13 Seed Water:
Add one capsule Polyseed™ or NCL brand to one half liter of standard
dilution water at 20ºC and stir (deep vortex in solution while stirring)
for 60 minutes. Let settle for 30 minutes and decant off into another
container. Stir at a gentle pace, and pipet from 1-2 inches under the
surface when adding to sample with a wide-tipped pipet. Use before 6
hours after preparation.
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5.3 PROCEDURE
5.3.1
Principle:
The method consists of filling with sample, to overflowing, an airtight
bottle of the specified size and incubating it at 20.0°C r 1°C for 5 days
without light. An initial dissolved oxygen (DO) reading is measured
and again after incubation. The BOD is computed from the difference
between this initial and final DO. Because the initial DO is determined
immediately after the dilution is made, all oxygen uptake, including
that occurring during the first 5 min, is included in the BOD
measurement.
5.3.2
Sampling and storage:
Samples for BOD analysis may degrade significantly during storage
between collection and analysis, which can result in low BOD values.
To minimize this reduction of BOD samples should be analyzed
promptly or by cooling it to ” 6°C during storage. However, even at
low temperatures, holding times should be kept at a minimum.
Samples should be warmed to 20ºC before analysis, but not in direct
light. Hold time for this analysis is 48 hours, but try to begin analysis
within 6 hours.
x Grab samples:
If analysis is begun within 2 hours of collection, cold storage is
unnecessary. If analysis is not started within 2 hours of sample
collection, keep sample at ” 6ºC from the time of collection. Begin
analysis within 6 hours of collection; when this is not possible
because the sampling site is distant from the laboratory, store at ”
6ºC and report length and temperature of storage with the results.
In no case should analysis start more than 48 hours after grab
sample collection; make every effort to deliver samples for analysis
within 6 hours of collection.
x Composite samples:
Keep samples at ” 6ºC during composite collection. Limit
composite collection period to 24 hours. Use the same criteria as
for storage of grab samples, starting the measurement of holding
time from end of compositing period. State storage time and
conditions as part of the results.
5.3.3
Sample Preparation:
x pH Neutralization: If the sample has a pH lower than 6.0 or
higher than 8.0, neutralize the sample to pH of 6.5 to 7.5 with 1N
sulfuric acid (H2SO4) or 1N sodium hydroxide (NaOH).If the
quantity of the neutralizing reagent dilutes the sample more than
0.5%, a stronger solution must be used. The sample’s initial pH,
final pH and mLs of H2SO4 or NaOH added are recorded.
x DO super saturation: Samples containing more than 9 mg/L
oxygen at 20ºC may be encountered in cold waters or in waters
Environmental Quality Lab
Chemistry Procedures
x
x
x
5.3.4
SOP No.: 430
Page 8 of 14
Rev.1
09-17-09
where photosynthesis occurs. To prevent loss of oxygen during
incubation of such samples, reduce DO to saturation at 20ºC by
bringing sample to about 20ºC in a partially filled bottle while
agitating by vigorous shaking or by aerating with clean, filtered
compressed air. This may also be achieved by vigorously pouring
the sample back and forth from one large mouth container to
another. Care must be taken to reduce loss of sample due to spills.
Sample temperature adjustment: Bring samples to 20ºC before
making dilutions using gradual temperature adjustments in
incubators or water baths.
Nitrification inhibition: Nitrification inhibition is not currently
being used in this lab. If nitrification inhibition is desired, add 3 mg
2-chloro-6-(trichloro methyl) pyridine (TCMP) to each 300 mL
bottle or add sufficient amounts of sample, seed and dilution water
to fill bottle to make a final concentration of 10 mg/L TCMP. Pure
TCMP may dissolve slowly and can float on top of the sample.
Some commercial formulations dissolve more readily but are not
100% TCMP; adjust the dosage accordingly. Samples that may
require nitrification inhibition include, but are not limited to
biologically treated effluents, samples seeded with biologically
treated effluents, and river waters. Note the use of nitrogen
inhibition in reporting results.
Dechlorination: If possible, avoid samples containing residual
chlorine by sampling ahead of chlorination processes. As this
laboratory intends to test surface waters and not sewage treatment
plant discharge, chlorination is not expected to be a problem.
Calibration of YSI model 5000 meter/YSI model 5010 probe:
1. At least one hour prior to use, plug power cable into the meter,
plug the DO probe cable into the meter, and press the on-off
switch to turn the meter on. Rinse the DO membrane-end of
probe with DI water and blot any water droplets off the
membrane with a Kimwipe. Rinse the BOD bottle used for
storage of the probe and fill with 1-2 inches of fresh DI water.
Insert probe into the clean BOD bottle with 1-2 inches of water.
This provides a 100% humidity environment. Wait at least 20
minutes for temperature to equilibrate.
2. After allowing the probe to polarize and the temperature to
stabilize for at least 20 minutes, (if the calibration is performed
prematurely the values will probably drift and may be out of
specification) press the calibration key.
3. Make sure the display readings are stable, and there are no
condensation droplets on the membrane, then press the [DO Cal]
soft-key soft-key to calibrate % saturation of dissolved oxygen.
Using the [UP] [DOWN] [DIGIT] soft-keys indicate 100.0% and
then press enter. The message "D.O. CALIBRATION SAVED"
Will be displayed for a few seconds.
Environmental Quality Lab
Chemistry Procedures
4.
5.
6.
7.
8.
SOP No.: 430
Page 9 of 14
Rev.1
09-17-09
Press [MODE] to return to the main mode. If the barometric
pressure and % saturation values stabilize, the instrument is now
calibrated. Otherwise repeat calibration a 2nd or 3rd time. Wait 5
minutes after calibration to start recording calibration readings in
calibration log.
A minimum of 3 readings, at least 1 minute apart, are to be
recorded in the calibration log while the probe remains in the
100% humidity oxygen saturation air. Follow the acceptance
range on the calibration sheet for stability check.
Be sure to also record the expected DO values for the
temperature and pressure. This information is provided in the
calibration log insert.
If stability check is outside acceptable limits, recalibrate and take
a minimum of 3 more readings in 100% humidity oxygen
saturated air.
Once the 100% humidity oxygen saturated air check is
acceptable, pour 300 mL of aerated DI water at 20°C into a clean
BOD bottle. Place the recently calibrated DO probe into this
bottle and turn on the probe stir bar. After the reading stabilizes
(may take a few minutes.), record the value and check to see if it
is within acceptance limits (noted on calibration sheet).
5.3.5 BOD Bottle Set-up:
1. Select 300 mL glass BOD bottles that have been washed in the
dishwasher once on the heavy wash cycle (no soap, no drying
option) and once on the normal wash cycle (no drying option) and
then triple rinsed with distilled water and stored in the incubator.
2. Refer to Table #1, on page 11, for the layout of a typical BOD run
utilizing two dilution blanks, one dilution blank check, two GGA
standards, 5 seed dilutions, and two samples including duplicates
and a spike.
3. Prepare the bench sheet with the bottle numbers, sample ID
numbers, Seed and GGA lot numbers, date, times and analyst as
well as the mL of seed used, mL of GGA used, and mL of sample
used for each BOD bottle
4. Using the descriptions in Table #1, prepare two Dilution Blanks
and one Dilution Blank Check. With the BOD meter calibrated and
stabilized (Ref 5.3.4), insert the DO probe into the first bottle. The
meter will beep upon stabilizing. Read and record the DO,
temperature, and time for each of the two Dilution blanks. Do not
insert the DO Probe into the Dilution Blank Check bottle. The
final DO will be read and compared to the two Dilution Blanks
initial and final readings to insure that the probe is not a
contributing factor in contamination. Refill the bottles with dilution
water if necessary, stopper, and cap. Place into the incubator.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 430
Page 10 of 14
Rev.1
09-17-09
5. Fill the GGA std #1 bottles half-way with single strength dilution
water. Add 6 mL of GGA std #1 and 6mL seed (typically, but may
vary depending on experience regarding seed strength and GGA
recovery). Fill the bottle with single strength dilution water. Rinse
the DO probe between each bottle to avoid cross contamination.
Insert the DO probe into each bottle and allow to stabilize.
Immediately read the DO, temperature and time for each bottle and
record. Refill the bottles with dilution water if necessary, stopper,
and cap. Place into the incubator.
6. Fill the seed bottles half-way with single strength dilution water.
Add the designated amount of seed to each (Ref Table #1), and fill
with single strength dilution water.
7. Immediately read the DO, temperature and time for each bottle and
record. Refill the bottles with dilution water if necessary, stopper,
and cap. Place into the incubator.
8. Fill the sample #1 150 mL bottle with 150mL of sample, 4 mL of
seed and fill with double strength dilution water. Fill the 300 mL
bottle only half-way with sample, add 4 mL seed (typically, but
may vary depending on experience regarding seed strength), one
300 mL nutrient buffer pillow and fill to top with sample.
Immediately read the DO, temperature and time for each bottle and
record. Refill the bottles with dilution water if necessary, stopper
and cap. Place into the incubator.
9. Repeat the procedure in 5.3.5.8 for all other samples. A field
duplicate should be included and should be tested as an
independent sample. A lab duplicate and lab spike should be run
on one in every ten samples analyzed (Ref Table #1).
10. Incubate at 20ºC ± 1ºC in the dark for 5 days ± 6 hours.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 430
Page 11 of 14
Rev.1
09-17-09
Table #1 Typical BOD Preparation Guide
ID
1
2
3
Sample
Dilution Blank
Dilution Blank Dup
Dilution Check
4,5
2 x GGA std #1
6,7
2 x GGA std #2
8
Seed Control 4mL
9
Seed Control 8mL
10
Seed Control 10mL
11
Seed Control 12mL
12
Seed Control 15mL
13
Sample #1 150 mL
14
Sample #1 300 mL
15
Sample #2 150 mL
16
Sample #2 300 mL
17
Sample #2 Dup 150 mL
18
Sample #2 Dup 300 mL
19
Sample #2 Dup Spike
Preparation in 300 ml BOD bottle
Fill with Single Strength Dilution Water
Fill with Single Strength Dilution Water
Fill with Single Strength Dilution Water
Do not insert DO probe for initial DO
reading
Single Strength Dilution Water
6 mL prepared GGA std. (Ref 5.2.10)
6 mL seed
Single Strength Dilution Water
Correct dosage GGA std. #2 (Ref 5.2.11)
6 mL seed
Single Strength Dilution Water
4 mL seed
Single Strength Dilution Water
8 mL seed
Single Strength Dilution Water
10 mL seed
Single Strength Dilution Water
12 mL seed
Single Strength Dilution Water
15 mL seed
150 mL Sample
4 mL seed
Double Strength Dilution Water
300 mL Sample
4 mL seed
Nutrient Buffer Pillow for 300 ml bottle
150 mL Sample
4 mL seed
Double Strength Dilution Water
300 mL Sample
4 mL seed
Nutrient Buffer Pillow for 300 ml bottle
150 mL Sample
4 mL seed
Double Strength Dilution Water
300 mL Sample
4 mL seed
Nutrient Buffer Pillow for 300 ml bottle
150 mL Sample
4 mL seed
Double Strength Dilution Water
6 mL prepared GGA std.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 430
Page 12 of 14
Rev.1
09-17-09
5.3.6 Final DO Determination:
1. Repeat the DO Probe calibration (Ref. 5.3.4).
2. After 5 days ± 6 hours in the incubator at 20ºC ± 1ºC, remove
the caps and stoppers from the bottles.
3. Beginning with the Dilution blanks, and Dilution blank check,
insert the DO probe and allow to stabilize. Read and record the
final DO, temperature, and time.
4. Continue with the GGA standard bottles, the seed bottles, and the
sample bottles. Be sure to rinse the DO probe between bottles to
avoid cross contamination. Read and record the final DO,
temperature and time.
5. Continue the procedure with the seed bottles, and all of the sample
bottles. Read and record the final DO, temperature and time for
each.
6.0
CALCULATIONS
6.1
When dilution water is not seeded;
BOD5, mg/l.
D1 D 2 B1 B 2 u f
P
where:
D1 =
D2 =
P=
f =
B1=
B2 =
DO of diluted sample immediately after preparation. mg/L
DO of diluted sample after 5 days incubation at 20ºC mg/L
decimal volumetric fraction of sample used
(volume of seed in diluted sample)/ (volume of seed in seed control)
DO of seed control before incubation mg/L
DO of seed control after incubation mg/L
Report results as CBOD5 if nitrification is inhibited.
Average results all sample bottles with a residual DO of at least 1 mg/L and a
DO depletion of at least 2 mg/L.
In order to do so, at least one sample dilution must meet the criteria of a
residual DO of at least 1 mg/L and a DO depletion of at least 2 mg/L, and
there must be no evidence or toxicity at higher sample concentrations or the
existence of an obvious anomaly.
In these calculations, do not make corrections for DO uptake by the dilution
water blank during incubation. This correction is unnecessary if dilution
water depletion is less than 0.2 mg/L. If the dilution water does not meet these
criteria, proper corrections are difficult and results become questionable.
Environmental Quality Lab
Chemistry Procedures
7.0
SOP No.: 430
Page 13 of 14
Rev.1
09-17-09
6.2
Control limits:
The average BOD for the bottles containing the Glucose-Glutamic acid should
lie within the range of 198 r 30.5 mg/L. If measured BOD for a glucoseglutamic acid check is outside the accepted control limit range, reject tests
made with that seed and dilution water.
6.3
Working range and detection limit:
The working range is equal to the difference between the maximum initial DO
(7 to 9 mg/L) and minimum DO residual of 1.0 mg/L multiplied by the
dilution factor. A lower detection limit of 2 mg/L is established by the
requirement for a minimum DO depletion of 2 mg/L. If the sample has been
diluted the minimum detection limit is 2 mg/L multiplied by the dilution
factor.
QUALITY CONTROL
7.1
Quality control (QC) measures for analyzing samples are summarized in
Table 2 and are as follows:
7.1.1 Prior to analyzing samples, each analyst must demonstrate the ability
to generate acceptable results (i.e., demonstration of capability).
7.1.2 Any replicate sample analyses should be subjected to exactly the same
analytical procedures as those used on individual sample analyses.
7.1.3 Unless otherwise specified for specific project or samples, each batch
should include two dilution water blanks, five seed dilutions, a known
standard (GGA), and at least one sample analyzed in duplicate as QC
samples.
7.1.4 When matrix interference is suspected, a matrix spike should be added.
7.1.5 The minimum DO depletion of 2.0 mg/L and minimum residual DO of
1.0 mg/L is required on all measurements. The results are not
considered to be valid if these criteria are not met.
7.1.6 Annual performance tests are to be performed using blind PE samples
and results reported to accrediting agencies.
8.0 WASTE DISPOSAL
8.1
Dispose of analyzed samples and used standards down a sink sewage drain.
Flush the sink with a large volume of tap water.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 430
Page 14 of 14
Rev.1
09-17-09
Table 2. Summary of QC Requirements for 5-dayBOD Analysis
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior to
any sample analyses
Criteria for LCS recovery and duplicate
precision
Dilution water blank
(Deionized water with
buffers added)
Daily prior to sample
analysis
< 0.2 mg/L DO depletion
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination.
Minimum residual DO
and minimum DO
depletion
For all measurements
Minimum DO depletion 2.0 mg/L
Residual DO in bottle • 1.0 mg/L
Results will not be considered
valid
Seed Control
For every preparation
batch
DO uptake attributable to seed added to
each bottle generally 0.6 to 1.0 mg/L but
seed amount must provide acceptable
GGA recovery
Glucose, Glutamic-acid
Standard
Two (2) per preparation
batch of samples run
Average BOD must be 198±30.5 mg/L.
Glucose, Glutamic-acid
Standard- Second source
standard
At least one (1) per
quarter (i.e., every three
months)
Average BOD must be 198±30.5 mg/L.
Matrix Spike
When suspect matrix
interference
QCSampleorActivity
Sample duplicate
or matrix spike duplicate
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
BOD matrix spike Recovery of 75 – 125%
RPD ” 25%
One (1) per batch
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
Corrective Action
Repeat until acceptable
Investigate problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, reanalyze batch
Evaluate the cause, make
appropriate corrections. High
values indicate too much seed;
low values indicate poor
quality seed or the presence of
toxic material.
Evaluate the cause, make
appropriate corrections.
Compare with, to verify the
accuracy of, the Lab Prepared
GGA standard.
Investigate problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, reanalyze batch.
Investigate problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, reanalyze batch.
Criteria for LCS recovery and duplicate
precision
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
QC =
%R =
RL =
RPD =
quality control
percent recovery
reporting limit
relative percent difference
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix G
CCU EQL SOP NO.: 447
AMMONIA (NITROGEN) BY AUTOMATED PHENATE METHOD
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 1 of 12
Determination of Ammonia (Nitrogen)
by Automated Phenate Method
Reference Methods:
SM 4500-NH3¯ G. (1997 online)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Technician
1.0
Rev. 0
09-25-09
SCOPE/APPLICATION
In this method, ammonia (nitrogen) is determined by the reaction of alkaline phenol and
hypochlorite with ammonia to form indophenol blue that is proportional to the ammonia
concentration. The blue color formed is intensified with sodium nitroprusside. Ammonia
nitrogen can be determined in potable, surface and saline waters as well as domestic and
industrial wastewaters over a range of 5 to 600 µg NH3-N/L when photometric
measurement is made at 630 nm in a 10 mm flow cell. Higher concentrations can be
determined using dilutions.
2.0
REFERENCES
2.1
2.2
2.3
2.4
3.0
DEFINITIONS
3.1
4.0
Standard Method 4500-NH3-G. (1997 online), Automated Phenate Method.
Standard Method 4500-NH3-A. (1997 online), Introduction, Selection of Method.
Lynn, E. (2008) Lachat Instruments Inc., QuikChem Method 31-107-06-1-B,
Alkaline Phenol based method; Brackish or seawater matrix. Lachat Instruments
Inc.
U. S. Environmental Protection Agency. 1983. Nitrogen, Ammonia. Method
350.1 (Colorimetric, Automated, Phenate). (Online summary) Pp.350-1.1 -- 3501.4. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020.
U.S.E.P.A., Cincinnati, Ohio, USA.
Ammonia: Ammonia is a binary compound made up of nitrogen and hydrogen,
characterized by a pungent smell and taste. Ammonia nitrogen, by definition is
only a portion of total nitrogen. Total nitrogen is comprised of ammonia nitrogen,
organic nitrogen, nitrate and nitrite. Ammonia nitrogen and organic nitrogen
together make TKN, Total Kjeldahl nitrogen.
INTERFERENCES
x
Glassware and labware contamination can be a problem in low level nitrogen
analysis. Glassware and labware should be acid washed with 25% concentrated
HCl and rinsed three times with deionized water.
x
Reagent grade chemicals may contain trace levels of nitrogen. Using the best
quality reagents will help to avoid nitrogen contamination.
x
All reagent containers should be covered to prevent contamination from airborne
ammonia.
Environmental Quality Lab
Chemistry Procedures
Rev. 0
09-25-09
x
Calcium and magnesium ions may be present in concentration sufficient to cause
precipitation problems during analysis. An EDTA solution is added to the sample
on-line to prevent the precipitation of calcium and magnesium ions from river
water and industrial waste.
x
Sample turbidity and color may interfere with this method. Turbidity must be
removed by filtration prior to analysis. Sample color that absorbs in the
photometric range used will also interfere.
5.0
6.0
SOP No.: 446
Page 2 of 12
SAFETY
x
This method is restricted to use by or under the supervision of trained analysts.
x
Gloves, safety glasses with side shields, and protective clothing should be worn to
protect against unnecessary exposure to corrosive infectious agents (i.e.,
pathogens), hazardous chemicals (e.g., phenol), and contaminants in potentially
hazardous samples.
x
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
METHOD
6.1
APPARATUS AND MATERIALS
6.1.1
6.1.2
6.1.3
6.1.4
6.1.5
6.1.6
6.1.7
6.1.8
6.1.9
6.1.10
6.1.11
6.1.12
6.1.13
6.1.14
6.1.15
6.1.16
6.1.17
6.1.18
6.1.19
6.1.20
6.1.21
Pipettes (Class A- 0.5, 1.0, 5.0 and 10.0mL)
Deionized water
Analytical balance, capable of accurately weighing to the nearest 0.0001 g.
50 mL Graduated cylinders
500 mL Graduated cylinder
300 mL Griffin beaker
250 mL Class A volumetric flasks
500 mL Class A volumetric flasks
1000 mL Class A volumetric flasks
PVC carrier lines
PVC pump tubes (Red/Red – colors are industry standard designations for
pump tubing diameters)
PVC pump tubes (Blue/Blue)
PVC pump tubes (Black/Black)
Neoprene pump tubes (Green/Green)
PVC pump tubes (White/White)
PVC pump tubes (Orange/White)
Lachat QC8500 Series FIA+ Autoanalyzer with ASX-500 Series XYZ
Auto sampler, RP-100 Reagent Pump, 4 channels with HP injection valve,
Omnion 3.0 software, four 175CM software controlled heaters and
PDS200 Dilutor
pH meter
Sonicator bath
Analytical Manifold for method # 31-107-06-1-B, Ammonia.
Glass reagent bottles for reagent storage
6.1.21.1 500-mL plastic screw-top bottle for sodium hydroxide solution
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 3 of 12
Rev. 0
09-25-09
6.1.22 Glass reagent bottles for auto sampler use and storage
6.1.22.1 1000-mL clear glass screw-top bottle for Carrier DIW
6.1.22.2 2500-mL clear glass screw-top bottle for Auto sampler/diluter
DIW rinse water
6.1.22.3 500-mL plastic screw top bottle for reagent DIW rinse water
6.1.22.4 500-mL clear glass screw-top bottle for disodium EDTA buffer
solution
6.1.22.5 500-mL dark glass stoppered bottle for sodium phenate reagent
solution
6.1.22.6 500-mL clear glass screw-top bottle for sodium hypochlorite
solution
6.1.22.7 500-mL clear glass screw-top bottle for sodium nitroprusside
solution
6.2
REAGENTS
6.2.1
6.2.2
6.2.3
6.2.4
6.2.5
6.2.6
6.2.7
6.2.8
6.2.9
6.3
Ammonia-free distilled water
6.2.1.1 Concentrated sulfuric acid, H2SO4, certified ACS or equivalent
Phenate reagent
6.2.2.1 Crystalline phenol, certified ACS or equivalent
Sodium hydroxide, NaOH, pellets, certified ACS or equivalent
Sodium hypochlorite solution containing 4.00-6.00 % NaOCl.
EDTA buffer chelating reagent
6.2.5.1 Disodium ethylenediamine tetraacetate, certified ACS or
equivalent
6.2.5.2 Sodium hydroxide pellets, certified ACS or equivalent
Sodium nitroprusside reagent certified ACS or equivalent
Stock ammonium solution
Anhydrous ammonium chloride, NH4Cl, solid, certified ACS or equivalent
Stock ammonium solution, LCS
6.2.9.1 Ammonium sulfate, (NH4)2SO4, certified ACS or equivalent
PROCEDURES
6.3.1
x
6.3.2
x
Sample Collection, Processing, and Preservation
Samples for ammonia analysis must be collected in acid-cleaned, plastic
bottles. If samples are analyzed within 24 hours, the samples must be
stored at ”6°C. If samples are preserved by adding H2SO4 to pH< 2 and
refrigerated at ” 6°C, they can be stored for up to 28 days before analysis.
If acid preservation is used, neutralize samples with NaOH or KOH
immediately before making the determination.
Note:
Although
acidification is suitable for certain types of samples, it produces
interferences when exchangeable ammonium is present with unfiltered
solids.
Preparation of Reagents
Ammonia-free distilled water (6.2.1): Special precaution must be taken to
ensure that distilled water is free of ammonia. All solutions must be made
with the ammonia-free distilled water.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 4 of 12
Rev. 0
09-25-09
x
To prepare sodium phenate solution (6.2.2): In a 500-mL volumetric flask,
dissolve 41.5 crystalline phenol in 250 mL distilled water. In small
increments and with agitation, slowly add 32g NaOH. Cool flask under
running water and dilute to 500 mL. Invert to mix. Prepare fresh every 35 days. Caution: Minimize exposure of personnel to this compound by
wearing gloves and eye protection, and using proper ventilation. Phenol
causes skin burns and is rapidly absorbed into the body through the skin.
x
To prepare sodium hypochlorite solution (6.2.4): Dilute 125 mL sodium
hypochlorite solution containing 4.00 – 6.00% NaOCl to 250 mL distilled
water. Prepare fresh daily.
x
To prepare EDTA buffer chelating reagent (6.2.5): Dissolve 50g disodium
ethylenediamine tetraacetate and 11.0 g NaOH in l-L distilled water. Stir
to mix. Prepare fresh monthly.
x
To prepare sodium nitroprusside solution (6.2.6): Dissolve 1.75 g sodium
nitroprusside in 500-mL distilled water. Prepare fresh every 1-2 weeks.
6.3.3
Preparation of Calibration and Calibration Check Solutions:
x
General: The weights of chemicals and volumes of solutions used in the
following specific procedures for the preparation of standards can be
adjusted if different volumes or different standard concentrations are
needed.
x
Preparation of Calibration Solutions:
1. Prepare the ammonia primary standard solution by dissolving
0.1909g primary standard grade ammonia chloride (6.2.8) that has
been dried at 105°C for one hour, in about 800 mL DIW. Dilute to
1L and invert to mix. This generates the primary ammonia
standard solution that is 50.0 mg NH3 N/L and should be made
fresh weekly. Store solution in a glass bottle in the refrigerator at
” 6°C.
2. Dilute 10 mL of the primary ammonia standard solution to
500mL with distilled water. This is the secondary ammonia
standard solution equal to 1.0 mg NH3–N/L and should be prepared
fresh daily and stored in the refrigerator at ” 6°C.
3. Dilute 60 mL of the secondary ammonia standard solution to 200
mL with distilled water. This is the third ammonia standard
solution or the working ammonia standard solution equal to
300µg NH3–N/L and should be prepared fresh daily and stored in the
refrigerator at ” 6°C.
x
Preparation of ammonia laboratory control sample (LCS), which is the
calibration check solution:
1. Dry ammonium sulfate, (NH4)2SO4, (6.2.9.1), in an oven at 105°C
for 24 h., then cool to room temperature in a desiccator.
SOP No.: 446
Page 5 of 12
Environmental Quality Lab
Chemistry Procedures
Rev. 0
09-25-09
2. Dissolve 0.1910g primary standard grade ammonium sulfate that has
been dried at 105°C, in about 800 mL DIW. Dilute to 1L and invert
to mix. This generates the primary stock ammonia LCS standard
solution that is 40.49 mg NH3 - N/L and should be made fresh
weekly. Store solution in a glass bottle in the refrigerator at ”6°C.
3. Dilute 10 mL primary stock ammonia LCS standard solution to
500 mL with deionized water; giving a final concentration of
0.80988 mg NH3-N/L. This is intermediate ammonia LCS
standard solution and should be made fresh daily. Store solution in
a glass bottle in the refrigerator at ”6°C for up to 24 hours.
4. Dilute 25mL intermediate ammonia LCS standard solution
(0.80988 mg N/L) to 500 mL with deionized water; giving a final
concentration of 40.49 Pg NH3-N/L. This is the working ammonia
LCS standard solution and should be made fresh daily. Store
solution in a glass bottle in the refrigerator at ”6°C for up to 24
hours.
6.3.4
Calibration and Sample Analysis on Lachat QuikChem 8500 Auto
analyzer
x
Turn on computer interfaced to the QuikChem and logon.
further actions on this computer are in boldface font.
x
Turn on the power strip for the auto sampler, diluter, and QuikChem.
x
Select the Omnion 3.0 icon on the start-up menu.
x
At the upper left hand corner of the screen, click on the tab labeled
OPEN.
x
In the CCU methods folder, sort the data results files by date.
x
Open the most recent run by clicking on the DATE tab. The run
settings should be:
x
Properties
Values
Analyte Name
Concentration Units
Calibration fit type
Clear calibration
Calibration weighing
Auto dilutor trigger
% of high standard
QuikChem Method
Chemistry
NH3
Pg NH3-N/L
2nd order Polynomial
“check” box
1/x
“check” box
110
31-107-06-1-B
Brackish
All
A popup menu will ask if you want to change the set points of the
relevant heaters. Select “Yes”
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 6 of 12
Rev. 0
09-25-09
x
Check that the canopy hood is on the HIGH setting.
x
Press the manual/run button on the peristaltic pump. The pump should
begin pumping.
x
Snap the pump tubes into place on the peristaltic pump.
x
Put the line labeled “carrier” into the Carrier DWI bottle and leave
pumping.
x
After 5 minutes, put the line labeled “Buffer” into the EDTA NaOH
Buffer Solution bottle. Leave it pumping.
x
After 5 minutes, put the line labeled “Reagent” into the Sodium Phenate
Solution bottle. Leave it pumping for 5 minutes.
x
After 5 minutes, put the line labeled “hypochlorite” into the Sodium
Hypochlorite Solution bottle. Leave it pumping for 5 minutes.
x
After 5 minutes, put the line labeled “Nitro” into the Sodium
Nitroprusside Solution bottle. Leave it pumping for 5 minutes.
x
After all tubing is on-line; no bubbles should be visible in the tubing
around the mixing loop.
x
The typical working range for this analysis by EQL is 7.5 to 300µg NH3N/L.
Enter the following expected concentrations of the standards shown
in Table 1 into the run worksheet of the Omnion software and the
listed dilution factors needed to achieve these concentrations by
diluting the 300µg NH3-N/L standard.
Table 1. Ammonia-Nitrogen Calibration Standards
Conc.(µgNH3-N/L)
Sample ID
NH3 300
300
NH3 300
188
NH3 300
150
NH3 300
100
NH3 300
75
NH3 300
60
NH3 300
30
NH3 300
15
NH3 300
10
NH3 300
7.5
DIW Blank
0.000
ADF = auto dilutor dilution factor
x
ADF
1.60
2.00
3.00
4.00
5.00
10.00
20.00
30.00
40.00
Other working ranges can be utilized by adjusting the concentration of
the undiluted standard and the ADFs.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 7 of 12
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x
After the calibrating solutions are analyzed, the Omnion software will
compute a regression equation that is used to convert sample absorbances
to concentrations.
x
The quality of the regression will be documented by r2 and by calculation
of percent Recovery (% R). The latter is the percent recovery which is
computed by taking the determined concentration divided by the
expected concentrations. The determined concentration will be reported
in the results table and is computed by comparing sample absorbances to
the calibration regression curve.
x
Immediately after the calibrating solutions, a DIW blank is analyzed.
x
Swirl the 300µg NH3-N/L standard, uncap, and pour approximately 45
mL of the standard into a 50-mL centrifuge tube. Place the tube on the
autosampler in position #1.
x
Swirl the DIW blank, uncap, and pour approximately 45 mL of the DIW
blank into a 50-mL centrifuge tube. Place the tube on the autosampler in
position #2.
x
Refer to Table 2, A Typical Sample Analysis Set-up for the
Autoanalyzer for guidance on sample and Quality Control sequencing.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 446
Page 8 of 12
Rev. 0
09-25-09
Table 2. A Typical Ammonia (Nitrogen) Sample Analysis Set-up for the Autoanalyzer
Cell ID
1 Thru 5
Parameter Standards for Calibration
curve (NH3)
Minimum 5 standards dependent on expected
sample range; Could be up to 11 standards.
6
Rinse DIW
7
Method Blank
8
LCS
Laboratory Control Standard
9
LCS
Laboratory Control Standard
10
Rinse DIW
11
Rinse DIW
12
Sample 1
13
Sample 2
14
Sample 3
15
Sample 4
16
Sample 5
17
Sample 5 Dup.
18
Sample 5 Matrix Spike
19
Sample 6
20
Sample 7
21
Sample 8
22
Sample 9
23
Sample 10
24
Artificial Seawater with Matrix
Spike
Insert to establish peak retention time windows
for brackish water (i.e., brackish timing) if
brackish or seawater samples are analyzed
25
CCC
Continuing Calibration Check
26
Rinse DIW
27
Samples 11 - 20
28
Sample Dup.
29
Sample Matrix Spike
Duplicate to be inserted with suggested one
sample out of 20
Matrix Spike to be inserted with suggested one
sample out of 20 with a concentration close to
expected sample conc. or middle working range.
30
CCC
Continuing Calibration Check
31
Rinse DIW
Duplicate to be inserted with suggested one
sample out of 20
Matrix Spike to be inserted with suggested one
sample out of 20 with a concentration close to
expected sample conc. or middle working range.
Note: The Auto dilutor is automatically triggered if any samples are above the highest standard used in the
calibration. The auto dilutor is set up to automatically perform a 10 to 1 dilution at the end of the run. The
additional dilution will require additional tubes in the auto-sampler. Extra tubes should be added at the
beginning of each run to cover such events.
SOP No.: 446
Page 9 of 12
Environmental Quality Lab
Chemistry Procedures
Rev. 0
09-25-09
x
Swirl each sample bottle, uncap, and pour approximately 8 mL of sample
into an autosampler sample cup and place onto the autosampler tray. Do
this for all the samples to be run. Position them in the autosampler tray
in the order that they will be run. Include the QC samples specified in
Table 3 at the frequency also specified in Table 3.
x
On the computer, press the PREVIEW button located at the top of
the screen.
x
Enter each EQL sample ID into the run worksheet in the column
labeled “Sample ID”.
x
End the preview mode by selecting the STOP tab at the top of the
screen.
x
Begin the run by pressing the START button (green arrow) located
at the top of the screen. The AA will then perform the calibration
followed by the analysis of samples.
x
After the run is complete, save the results in a file with the run date in the
file name. From the Run tab on the top left, select Save As, and type
in the Parameter, Type of Run (MDL), and date.
x
To clean the system, first remove the nitrogen reagent lines (Buffer and
Reagent) from their reagent bottles and place them in the Reagent DIW
rinse bottle. Let pump for about two (2) minutes. Remove the lines from
the Reagent DIW bottle.
x
Remove the reagent and carrier lines from their DIW rinse bottles.
Leave pumping air about two (2) minutes.
x
Wrap all line ends loosely in aluminum foil.
x
Unsnap the pump tubes from the peristaltic pump to relax the tension on
the tubes.
x
Cap all reagent bottles and place in refrigerator.
x
Print the report:
o
o
o
o
o
o
o
Click on the Tools Bar
Click on Custom Report
Click on Format
Click on Charts
Click on Calibration
Under File, Click on Print
Choose printer based on Room number.
x
Close the Omnion program.
x
Turn off the computer.
Environmental Quality Lab
Chemistry Procedures
x
6.4
SOP No.: 446
Page 10 of 12
Rev. 0
09-25-09
Log the run into the Autoanalyzer Run Log book. Note any problems or
maintenance that was performed.
QUALITY CONTROL
6.4.1
Quality control (QC) measures for analyzing samples are summarized in
Table 3 and are as follows:
x
Prior to analyzing samples, each analyst must demonstrate the ability to
generate acceptable results (i.e., demonstration of capability).
x
Any replicate sample analyses should be subjected to exactly the same
analytical procedures as those used on individual sample analyses.
x
Unless otherwise specified for specific project or samples, each batch of
up to 20 samples analyzed should include a DIW blank, a method blank
(i.e., deionized water or synthetic seawater in a sample bottle that
accompanied samples and received the same processing), a laboratory
control sample (i.e., sample of known concentration of ammonia), and at
least one sample analyzed in duplicate (i.e., at least 5% duplicates) as QC
samples.
x
An artificial seawater sample should be run to establish brackish timing
if brackish or seawater samples are being analyzed.
SOP No.: 446
Page 11 of 12
Environmental Quality Lab
Chemistry Procedures
Rev. 0
09-25-09
Table 3. Summary of QC requirements for Determination of Ammonia Using the
Automated Phenate Method
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior to
any sample analyses
Criteria for LCS recovery and duplicate
precision
Daily prior to sample
analysis
Calibration standards approximately,
evenly spaced over calibration range.
Standards processed like samples with 90110% R (measured value of each standard
within 10% of expected value)
Initial NH3 calibration
with standards
(at least 5-point
calibration plus blank)
Method blank
Daily with sample batch
Laboratory control
sample
At least one (1) per
preparation batch
Matrix Spike
When suspect matrix
interference
Sample analysis
For all sample analyses
Sample duplicate or
matrix spike duplicate
At least one (1) per
preparation batch
Artificial Seawater
sample with matrix spike
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
When brackish or sea
water samples analyzed
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
<7.50 µg/L
80-120% R
Ammonia-(Nitrogen) 75-125%
Direct sample reading within calibration
range (i.e., above lowest standard and
below highest standard)
RPD ” 25%
To establish peak retention time windows
for brackish water (i.e., brackish timing)
and seawater
Corrective Action
Repeat until acceptable
Investigate problem. Correct
any obvious problems. Repeat
calibration until acceptable.
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination. If significant
adverse impact on results,
reanalyze batch.
Investigate and identify the
problem. If system accuracy is
in control, no corrective action
needed. If system is out of
control, reanalyze batch.
Investigate problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, reanalyze batch.
If reading below range report
result as < RL. If result above
range, dilute sample.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze batch.
If analyte peaks not within
integration windows, manually
adjust peak integration
windows to windows
established for seawater.
Criteria for LCS recovery and duplicate
precision
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
QC =
%R =
RL =
RPD =
quality control
percent recovery
reporting limit (i.e., conc. of lowest cal. std adjusted for dilutions)
relative percent difference
Environmental Quality Lab
Chemistry Procedures
6.5
7.0
SOP No.: 446
Page 12 of 12
Rev. 0
09-25-09
CORRECTIVE ACTIONS
6.5.1
If any of the QC requirements listed in Section 5.4 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
6.5.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
CALCULATIONS
7.1
The autoanalyzer computes sample results in concentration units of mg NH3-N/L
by converting sample absorbances to concentrations using a regression curve
determined from the calibration solutions.
7.2
The auto dilutor is automatically triggered if any samples are above the highest
concentration standard used in the calibration. The autodiluter is set up to
automatically perform a 10 to 1 dilution. This is done at the end of the run. In the
results file, the dilution factor (10) is reported in the column labeled “ADF”. The
results need to be corrected for the dilution as follows:
Undiluted sample conc. = [Diluted sample conc.] x [Dilution factor]
8.0
WASTE DISPOSAL
Phenol, a reagent used in this analysis, is hazardous. Any analysis waste solutions
containing phenol, or any other hazardous substances, must be segregated and stored in the
hazardous waste accumulation area in room 303. Waste is periodically disposed of by
shipment to a hazardous waste disposal contractor by Coastal Carolina University’s Science
Lab Manager.
Dispose of analyzed samples, used standards, and used reagents not containing phenol or
any other hazardous substances down a sink sewage drain. Flush the sink with a large
volume of tap water.
9.0
EQUIPMENT MAINTENANCE
9.1
9.2
9.3
9.4
9.5
9.6
9.7
9.8
Check pump tubes for wear and replace as needed.
Replace o-rings every 6 months.
Replace flow cell flares and O-rings once a year.
Clean surfaces of the manifolds, dilutor, and autosampler every day they are used.
Check for leaks during every run.
Replace all manifold tubing every year.
Clean pump tube adapters, ports and valve connectors monthly.
Check the cadmium column to see that air has not gotten into the column. If some
air does get into the column, run the nitrogen reagents and carrier DIW through the
column for about 15 minutes.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix H
CCU EQL SOP NO.: 435
TOTAL SUSPENDED SOLIDS
Environmental Quality Lab
Chemistry Procedures
SOP No.: 435
Page 1 of 7
Total Suspended Solids Dried at 103-105ºC
Reference Method: SM 2540 D. (1997 online)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Technician
1.0
Rev. 2
9-05-09
SCOPE/APPLICATION
This standard operating procedure describes the procedures used by staff of the Environmental
Quality Lab (EQL) to determine total suspended solids (TSS) in natural waters. The primary goal
of TSS measurement is to provide an estimate of the suspended particles present in a water sample.
TSS is measured gravimetrically as follows: a well-mixed sample is filtered through a weighed
standard glass-fiber filter. The residue retained on the filter is dried to a constant weight at 103 to
105oC. The increase in weight of the filter represents the mass of suspended solids. To obtain a
concentration in mg per liter of sample, the mass is divided by the volume of filtered sample.
Large floating particles or submerged agglomerates of nonhomogeneous materials should be
excluded from the sample if it is determined that their inclusion is not desired in the final result.
Because excessive residue on the filter may form a water-entrapping crust, limit the sample size to
that yielding no more than 200 mg residue. For samples high in dissolved solids, thoroughly wash
the filter to ensure removal of dissolved material. Prolonged filtration times resulting from filter
clogging may produce high results owing to increased colloidal materials captured on the clogged
filter.
2.0
REFERENCES
2.1
3.0
Standard Method 2540 D. (1997 online) Total Suspended Solids Dried at 103-105°C
DEFINITIONS
Total Suspended Solids: Total suspended solids (TSS) in this standard operating procedure document
is referring to the total mass of solids retained by a glass-fiber filter which remains after rinsing with
deionized water to remove dissolved solids and then drying at 103-105oC to remove water. The latter
is meant to evaporate water and not lead to the volatilization of residues. Likewise, the deionized
water rinses are not meant to solubilize particles previously retained by the filter.
4.0
SAFETY
4.1
4.2
4.3
This method is restricted to use by or under the supervision of trained analysts.
Safety glasses and protective clothing should be worn to protect against unnecessary
exposure to water samples which are potentially contaminated with infectious agents (i.e.,
pathogens) and hazardous chemicals.
All activities performed in the laboratory while following this procedure should utilize
appropriate laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
Environmental Quality Lab
Chemistry Procedures
5.0
Rev. 2
9-05-09
SAMPLE COLLECTION, HANDLING, AND PRESERVATION
5.1
5.2
6.0
SOP No.: 435
Page 2 of 7
Samples should be refrigerated at < 6qC immediately upon collection and in the laboratory
until analysis.
Filtration must take place within 7 days of sample collection.
METHOD
6.1
APPARATUS AND MATERIALS
6.1.1 Drying oven, for operation at 103 to 105 ºC
6.1.2 Analytical balance capable of weighing to 0.1 mg.
6.1.3 Desiccator: provide with a desiccant containing a color indicator of moisture content.
6.1.4 Glass filter funnel and filter stand with fritted disk for filter support.
6.1.5 1- liter Erlenmeyer filter flask for the filter stand and funnel.
6.1.6 500-ml Erlenmeyer filter flask to act as a trap for the mechanical pump.
6.1.7 Rubber vacuum hoses, three-way stopcock, and quick disconnect adapters.
6.1.8 Glass fiber filter disks: Whatman grade 934 AH 47 mm diameter or equivalent
Commercially prepared and preweighed filters for TSS analysis are available and
acceptable.
6.1.9 Aluminum weighing dishes (3” diameter) with an Awl ID# (engraved with an awl).
To clean, wipe with a kimwipe dampened with deionized water.
6.1.10 Flat-blade forceps. Clean by rinsing with deionized water and wiping with a kimwipe.
6.1.11 Mechanical vacuum pump.
6.1.12 Certified balance weights (200 mg to 100 g).
6.2
REAGENTS
6.2.1 Deionized water
6.3
PROCEDURES
6.3.1
Filter Preparation
(1)
Using the forceps, place a rinsed (three times with 20 mL deionized water),
dried (103 - 105ºC), ignited (15 minutes at 550ºC), and weighed 47-mm
Whatman GFF 934-AH filter disk with wrinkled side up (grid side down) into
filtration apparatus.
(2)
Apply vacuum (150 mm Hg) and wash the filter with three successive 20 mL
volumes of deionized water. Continue suction to remove all traces of water.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 435
Page 3 of 7
Rev. 2
9-05-09
Discard washings.
(3)
Using the forceps, remove the filter from the filtration apparatus and transfer to
a clean labeled aluminum weighing dish being careful not to tear the filter.
(4)
Dry in an oven at 103 to 105ºC for at least 2 hours.
(5)
If volatile solids are also to be measured, ignite at 550 ºC for 15 minutes in a
muffle furnace. Cool in desiccator 2 hours or more to room/balance
temperature and weigh filter only to 0.000X g.
(6)
Repeat the cycle of drying, cooling, igniting, desiccating and weighing until a
constant weight is obtained or until the weight change is less than 0.5 mg. If
filter weights are not within 0.5 mg, the repeat the cycle of drying, cooling,
igniting, desiccating and weighing until the last two weighings agree within 0.5
mg.
(7)
Record the weights on the Data Recording Worksheet for Filter Preparation
for TSS/VSS. Also record the analyst name, Awl ID, date and time of each
weighing, the filter type, lot, and filter batch. Compute the weight change in
mg between repetitive weighings.
(8)
Enter all data into the Total Suspended Solids and Volatile Suspended
Solids Worksheet: Filter Prep to obtain a computation of weight change, %
weight change, and determination that a constant weight has been met. Follow
the instructions in the template for saving and file naming.
(9)
Store the prepared filters in a desiccator until needed.
6.3.2 Selection of sample volume
Select a sample volume that will yield about 2.5 to 100 mg of dried solids and not
to exceed 200 mg. For environmental samples, this usually ranges in volume from
200 to 1000 mL. This is sufficient to provide a dark visible residue on the filter. It
is important to filter smaller volumes (20mL) if the sample has a high sediment
load as a large amount (100mL) poured in all at once may clog the filter before it
can be pulled by the vacuum through the filter and make it difficult to determine
the actual volume of analyte that has passed through the filter paper.
6.3.3 Sample analysis
(1)
Place the filtration stand on the filter flask. Wet the frit with a small amount of
deionized water. Use forceps to place a prepared filter wrinkled side up onto
the filter stand. Place the filter cup onto the filter stand with filter in place and
clamp together.
(2)
Thoroughly shake the sample bottle. Then pour an aliquot of the desired
volume of the homogenized sample into a 100 mL or smaller graduated
cylinder. It is also permissible place a magnetic sir bar in the sample bottle and
place bottle on magnetic stirrer to stir the sample with a magnetic stirrer and
while stirring, pipet a measured volume onto the seated glass fiber filter.
(3)
Pour the sample into the filtration cup. Try to pour directly onto the filter to
reduce the retention of solids on the walls of the filtration cup.
Environmental Quality Lab
Chemistry Procedures
6.4
SOP No.: 435
Page 4 of 7
Rev. 2
9-05-09
(4)
Apply vacuum (150 mm Hg).
(5)
Rinse the graduated cylinder with deionized water (about 10 mL).
(6)
After the filter cup has emptied, pour the rinse water from the graduated
cylinder into the filter cup taking care to wash any residue from the side of the
filter funnel. It is also useful to use a squirt with DI water to wet down the
inside of the filtering funnel to make sure all the solids reach the filter.
(6)
Wash with two additional 10 mL volumes of reagent grade water (NOTE:
Samples with dissolved solids (e.g., seawater) may require additional
washings.). Allow complete drainage between washings and continue suction
for about 3 minutes after filtration is complete. This serves to quantitatively
transfer any sediments retained in the 100mL graduated cylinder to the filtering
funnel.
(7)
Using the forceps, carefully remove the filter from filtration apparatus and
transfer it back into its labeled aluminum-weighing dish. If analyzing multiple
samples, wash the forceps, 100mL graduated cylinder and filter cup with DI
water between each of the samples to avoid contamination. Also, when filtering
multiple samples, pay attention to the filtrate level in the filtering flask to avoid
drawing water into the vacuum trap between the filtering flask and the pump.
Empty the filtrate as needed.
(8)
Dry the sediment laden filter for at least 1 h at 103 to 105oC in an oven, and
then cool in desiccator to room/balance temperature.
(9)
Perform a balance check with a 200 mg certified weight. Record this in the
TSS Data Recording Worksheet.
(10)
Weigh the dried and cooled filter to 0.000X g.
(11)
Repeat the cycle of drying, cooling, desiccating, and weighing until a constant
weight is obtained or until the weight change is less than 4% of the previous
weight or 0.5 mg, whichever is less. If filter weights are not within 0.5 mg, then
repeat the cycle of drying, cooling, igniting, desiccating and weighing until the
last two weighings agree within 0.5 mg.
(12)
In the TSS Data Recording Worksheet, record the sample ID, Dish ID (if
commercial preweighed filter used), Awl ID, filter weight, the volume of
sample filtered, and the repetitive weights of the filter + residue. Also record
the analyst name, analysis batch, the date and time of each weighing. Compute
the weight change in mg between repetitive weighings.
(13)
Enter all data into the Total Suspended Solids and Volatile Suspended
Solids Worksheet: TSS to obtain a computation of weight change, % weight
change and determination that a constant weight has been met. The weight of
the residue will also be computed along with the TSS concentration (mg/L).
Follow the instructions in the template for saving and file naming.
(14)
If volatile solids are to be determined, use the forceps to return the filter +
residue to the aluminum dish and treat according to the SOP for Volatile
Suspended solids (VSS).
CALCULATIONS
mg total suspend solids/L
(A B) u 1000
C
SOP No.: 435
Page 5 of 7
Environmental Quality Lab
Chemistry Procedures
Rev. 2
9-05-09
Where:
A = weight of filter + dried residue, mg
B = weight of filter, mg
C = sample volume, mL
6.5
QUALITY CONTROL
6.5.1
Quality control (QC) measures for analyzing samples are summarized in Table 1 and
are as follows:
x
Prior to analyzing samples, each analyst must demonstrate the ability to generate
acceptable results (i.e., demonstration of capability).
x
Any replicate sample analyses should be subjected to exactly the same analytical
procedures as those used on individual sample analyses.
x
Unless otherwise specified for specific project or samples, each batch of up to10
samples analyzed should include a method blank (i.e., deionized water in a sample
bottle that accompanied samples and received same processing),,and at least one
sample analyzed in duplicate (i.e., at least 10% duplicates) as QC samples.
SOP No.: 435
Page 6 of 7
Environmental Quality Lab
Chemistry Procedures
Rev. 2
9-05-09
TABLE 1. SUMMARY OF QC REQUIREMENTS FOR TSS
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior to
any customer sample
analyses
90 – 110% R
< 10% RSD
Balance Calibration
Check
Prior to weighing any
sample filters
Weight of certified 200 mg weight:
0.1998 – 0.2002 g
At least one (1) per
analysis batch of up to 10
samples
For 1.0 L blank filtered: < 1.0 mg/L
Sample analysis
For all sample analyses
Total residue on filter:
>2.5 mg to < 200 mg
Laboratory Control
Sample
At least one (1) per year
90 – 110% R
Sample duplicate
One (1) per preparation
batch of up to 10 samples
RPD ” 5%
Method Blank
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
Corrective Action
Repeat until acceptable
Investigate problem including
cleaning weight and balance. If
balance is out of calibration
attempt recalibration or use
another balance until obtain
acceptable calibration check.
Investigate, identify, and
correct the problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, correct problem
before analyzing samples
If total residue on filter < 2.5
mg report result as < RL
If total residue on filter > 200
mg filter a smaller volume of
sample.
Investigate, identify, and
correct problem. If system
accuracy is in control, qualify
results. If system accuracy is
out of control, correct problem
before analyzing samples.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze entire
batch.
Criteria for LCS recovery and duplicate
precision
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
QC =
%R =
RL =
RPD =
RSD =
quality control
percent recovery
reporting limit where RL = (2.5 mg /mL filtered) x 1000 mL
relative percent difference
relative standard deviation
Environmental Quality Lab
Chemistry Procedures
6.6
6.7
SOP No.: 435
Page 7 of 7
Rev. 2
9-05-09
CORRECTIVE ACTIONS
6.6.1
If any of the QC requirements listed in Section 6.5 are not satisfied, the analyst
must consult with the Laboratory Director. Normally the activity must be repeated,
after corrective actions are taken to correct any obvious problems, until the QC
results are acceptable. If repeating the process is not possible, the results report
will include a discussion of the problem and the client will be consulted.
6.6.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
EQUIPMENT MAINTENANCE
6.7.1
Balance Calibration Checks
x Balances are certified annually by a commercial provider
x Monthly calibration checks are performed by EQL personnel using EQL SOP
203.
7.0 WASTE DISPOSAL
7.1
Dispose of analyzed samples and used standards by discharging into a laboratory sink drain.
Flush the sink with a large volume of tap water.
7.2
Discard filters in regular trash.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix I
CCU EQL SOP NO.: 502
FECAL COLIFORM MEASUREMENT BY
DIRECT TEST (A-1 MEDIUM)
Environmental Quality Lab
Microbiology Procedures
SOP No.: 502
Page 1 of 13
Fecal Coliform Measurement by
Direct Test (A-1 Medium)
Reference Methods:
SM 9221 C. (1999 online)
SM 9221 E.2. (1999 online)
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Master Technician
1.0
Rev. 5
10-15-09
SCOPE/APPLICATION
This procedure describes an elevated-temperature test for distinguishing bacteria of the total
coliform group that also belong to the fecal coliform group. The test uses A-1 medium and
is a single-step method. Using A-1 broth the test can directly isolate fecal coliforms from
water without prior enrichment in a presumptive medium.
The test using A-1 medium is applicable to source water, seawater, and treated wastewater.
2.0
REFERENCES
2.1
2.2
3.0
Standard Method 9221 C. (1999 online)
Standard Method 9221 E.2. (1999 online)
DEFINITIONS
3.1
Bacterial Indicators: Bacterial measures commonly used to estimate the amount of
sewage in contaminated water. Although these bacteria themselves are not
pathogenic, they are used to “indicate” the presence of human pathogens. Current
federal and state recreational water quality standards include or recommend at least
one of the following bacterial indicators: total coliforms, fecal coliforms,
enterococci, and E. coli.
3.2
Colony Forming Unit (CFU): A visible mass of organisms developing from one or
a group of bacteria. The mass results from the bacteria reproducing themselves.
3.3
E. coli: A species of bacteria that occurs in the intestine of warm-blooded animals.
It is part of the fecal coliform group.
3.4
Fecal Coliforms: A group of bacteria found in the feces of various warm-blooded
animals. It is a subgroup of the total coliform group and has been used as a more
definitive indicator for fecal pollution.
3.5
Most Probable Number: When multiple tubes are used in the fermentation
technique, results of the examination of replicate tubes and dilutions are reported in
terms of the Most Probable Number (MPN) of organisms present. This number,
based on certain probability formulas, is an estimate of the mean density of
coliforms in the sample.
Environmental Quality Lab
Microbiology Procedures
4.0
5.0
SOP No.: 502
Page 2 of 13
Rev. 5
10-15-09
3.6
Pathogens: Organisms that cause disease.
3.7
Total Coliforms: A large group of bacteria that can originate from soil, plants,
human waste, and animal waste. Commonly used as a bacterial indicator.
SAFETY
4.1
This method is restricted to use by or under the supervision of trained analysts.
4.2
When using the autoclave or Bunsen burner, be careful to avoid touching hot
surfaces and to not expose yourself to steam or flames.
4.3
Gloves, safety glasses with side shields, and protective clothing should be worn
to protect against unnecessary exposure to infectious agents (i.e., pathogens),
hazardous chemicals, and contaminants in potentially hazardous samples.
4.4
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.1.7
5.1.8
5.1.9
5.1.10
5.1.11
5.1.12
5.1.13
5.1.14
5.1.15
5.1.16
5.2
Incubator at 35.0 r 0.5qC, Fisher Isotemp 600 Series Standard Incubator
or equivalent
Refrigerator at < 6qC
Water bath at 44.5 r 0.2qC with water circulation pump
Autoclave
Bunsen burner
Inoculating loop, nickel-chromium alloy
Sterile plastic, serological pipets, 10 mL size graduated in 1/10 mL (Fisher
Cat. No. 13-678-11 E or equivalent)
Sterile plastic, serological pipets, 1.0 mL size graduated in 1/100 mL
(Fisher Cat No. 13-678-11 B or equivalent)
Glass pipets, volumetric, assorted sizes
Pipet bulbs
pH electrode, Orion or equivalent
pH meter, Fisher Accumet AR25 or equivalent
Glass bottles for dilution water, 300 mL
Glass culture tubes, 10x75 mm, for inversion (Fisher Cat. No. 14-961-25 or
equivalent)
Glass culture tubes, 25 mL, with rubber lined, screw close caps (Fisher Cat.
No. 14-959-25 E or equivalent)
Thermometers for controlled temperature equipment
REAGENTS
5.2.1
A-1 dehydrated medium (BD Difco ™ or equivalent)
Environmental Quality Lab
Microbiology Procedures
5.2.2
5.2.3
5.2.4
5.2.5
5.2.6
5.2.7
5.2.8
5.2.9
5.3
SOP No.: 502
Page 3 of 13
Rev. 5
10-15-09
Tryptic soy broth dehydrated medium (BD Bacto ™ or equivalent)
Buffered dilution water
Bacteria control cultures (MicroBiologics® or equivalent)
Escherchi coli, ATCC 25922
Enterobacter aerogenes, ATCC 13048
Other acceptable bacteria depending on availability those currently used
pH buffer solutions, 4.00, 7.00, 10.00
Electrode storage solution
Electrode filling solution
Sodium hydroxide
Hydrochloric acid
REAGENT PREPARATION
5.3.1
A-1 Broth
x
This medium may be used for the direct isolation of fecal coliforms from
water. Prior enrichment in a presumptive medium is not required.
x
Prepare from dehydrated media according instructions on bottle. Heat if
necessary to dissolve media.
x
Make A-1 broth of such strength that adding the sample aliquot will not
significantly reduce the medium concentration below that of the original
medium. For 10 mL sample aliquots prepare double-strength medium.
x
Suspend 31.5 grams in 1 liter of deionized water and boil, if necessary, to
dissolve completely. For preparing double strength medium suspend
63.0 grams in 1 liter. Other volumes of medium can be prepared as long
as these proportions of medium and water are used.
x
Measure pH and adjust pH, using dilute NaOH solution if necessary, so
that the final pH after sterilization will be 6.9 r 0.1 at 25ºC. Experience
in preparing each media will determine the need to adjust pH, especially
for compensating for pH changes during sterilization. pH usually drops
slightly during sterilization (up to ~0.1), so adjust pH to close to 7.0
before sterilization.
x
Dispense 10 mL into 20-30 mL screw culture tubes then insert inverted
fermentation vials. Do not insert inverted vials into two of the culture
tubes with medium (These tubes will be used for final pH
measurements).
x
Sterilize the media in the autoclave. The total time of the sterilization
cycle cannot exceed 40 minutes and must include exposure at 121-124ºC
for 10 minutes. Promptly remove sterilized medium from the autoclave
when sterilization is complete.
x
Measure final pH in the tubes without inverted vials. Final pH must be
6.9 r 0.1 at 25ºC. Discard entire batch of medium if pH is not within
limits. Record pH in Media Preparation Log, Form 907.
Environmental Quality Lab
Microbiology Procedures
x
5.3.2
SOP No.: 502
Page 4 of 13
Rev. 5
10-15-09
Store medium in refrigerator at < 6qC in tightly closed screw-cap
containers for no more than 7 days. Ignore formation of precipitate.
Tryptic Soy Broth Medium
x
This medium is used as a general growth medium for bacteria.
x
Prepare from dehydrated media according instructions on bottle. Heat if
necessary to dissolve media.
x
Make tryptic soy broth (TSB) of such strength that adding the sample
aliquot will not significantly reduce the medium concentration below that
of the original medium. For 10-50 mL sample aliquots prepare doublestrength medium.
x
Suspend 30.0 grams in 1 liter of deionized water and warm slightly, if
necessary, to dissolve completely. For preparing double strength
medium suspend 60.0 grams in 1 liter. Other volumes of medium can be
prepared as long as these proportions of medium and water are used.
x
Measure pH and adjust pH, using dilute NaOH or HCl if necessary, so
that the final pH after sterilization will be 7.3 r 0.2 at 25ºC. Experience
in preparing TSB medium will determine the need to adjust pH,
especially for compensating for pH changes during sterilization. pH
usually drops slightly during sterilization (up to ~0.1), so adjust pH to
close to 7.4 before sterilization.
x
Dispense 10 mL into 20-30 mL culture tubes with screw close caps and
25-50 mL in 100-300 mL glass bottles with screw close caps.
x
Sterilize the media in the autoclave. The sterilization cycle must include
exposure at 121ºC for 15 minutes. Promptly remove sterilized medium
from the autoclave when sterilization cycle is complete.
x
Measure final pH using two of the sterilized tubes of TSB. Final pH
must be 7.3 r 0.2 at 25ºC. Discard entire batch of medium if pH is not
within limits. Record pH in Media Preparation Log, Form 907.
x
Store sterile TSB in refrigerator at < 6qC in tightly closed screw-cap
containers for up to 3 months.
5.3.3
Buffered Dilution Water
x
Dissolve 34.0 g of potassium dihydrogen phosphate (KH2PO4) in 500
mL of deionized water, adjust to pH 7.2 r 0.25 with 1 N sodium
hydroxide (NaOH) and dilute to 1 liter with deionized water.
x
Dissolve 81.2 g of magnesium chloride (MgCl2x6H2O) to 1 liter with
deionized water.
Environmental Quality Lab
Microbiology Procedures
5.4
SOP No.: 502
Page 5 of 13
Rev. 5
10-15-09
x
To prepare buffered dilution water: dilute 1.25 mL stock phosphate
buffer solution and 5.0 mL magnesium chloride solution in 1 liter
deionized water.
x
Dispense in amount that will provide 99 r 2.0 mL in bottles or 9 r 0.2
mL in tubes after autoclaving for 15 minutes.
x
Alternatively purchase sterile buffered dilution water (e.g., Fisher or
IDEXX).
x
Store in dark at room temperature.
PROCEDURE
5.4.1
Locate samples from cooler according to the chain of custody and analysis
request forms.
5.4.2
Arrange fermentation tubes in three rows of five each for each sample
tested. The first row should contain double strength A-1 broth and the
next two rows should contain regular strength A-1 broth. At least one
tube of each set of tubes for a sample should be labeled by sample
number and dilution on the sample tube with a permanent marker
(usually double strength tube for each sample).
5.4.3
Sample analysis should begin immediately, preferably within 2 hours of
collection. The maximum transport time to the laboratory is within 6
hours of sample collection. Samples should be processed within 2 hours
of receipt at the laboratory. Immediately prior to sample analysis shake
sample vigorously 25 times before inoculating tubes.
5.4.4
Dispense with sterile 10 ml pipet, 10 ml of sample in each tube of the
first row (double strength A-1).
5.4.5
Dispense with sterile 1.0 ml pipet, 1.0 ml of sample in each tube of the
second row.
5.4.6
Dispense with sterile 1.0 ml pipet, 0.1 ml of sample per tube in the third
row.
5.4.7
If additional dilutions are required due to anticipated large bacterial
concentration, additional rows containing 0.01 sample per tube and 0.001
ml sample per tube can also be inoculated. The following guidelines are
for using volumes less than 0.1 mL of sample to inoculate tubes:
x
For a desired sample volume of 0.01 or 0.001 mL add 1.0 mL of sample
to 99 mL of sterile, buffered dilution water.
x
Shake diluted sample vigorously 25 times.
x
If 0.01 mL sample is desired, transfer 1 mL of diluted sample.
x
If 0.001 mL of sample is desired, transfer 0.1 mL of diluted sample.
Environmental Quality Lab
Microbiology Procedures
SOP No.: 502
Page 6 of 13
Rev. 5
10-15-09
5.4.8
Mix the contents of the tubes with gentle agitation.
5.4.9
Incubate inoculated tubes for 3 hours at 35.0 r 0.5qC in incubator.
Transfer tubes to a water bath at 44.5 r 0.2qC for an additional 21 r 2
hours.
5.4.10 Remove tubes from the water bath.
5.4.11 Gas production (i.e., gas accumulation in top of inverted vial in tube) in
any A-1 broth culture tube within 24 hours or less is a positive reaction
indicating the presence of fecal coliforms. Record analysis date and
analysis data in Fecal (A-1) Run Log, Form 909.
5.5
QUALITY CONTROL
5.5.1
Quality control (QC) measures for analyzing samples are as follows:
x
x
x
x
Prior to analyzing samples, each analyst must demonstrate the ability
to generate acceptable results (i.e., demonstration of capability).
Sample analysis should begin immediately, preferably within 2 hours
of collection. The maximum transport time to the laboratory is
within 6 hours of sample collection. Samples should be processed
within 2 hours of receipt at the laboratory.
Any replicate sample analyses should be subjected to exactly the
same analytical procedures as those used on individual sample
analyses.
Unless otherwise specified for specific project or samples, each batch
of up to 20 samples analyzed should include at least one sample
analyzed in duplicate (i.e., at least 5% duplicates) as QC samples.
5.5.2
Sterility of sample bottles must be assured prior their use. Bottles are
cleaned and tested for sterility as described in QAM Section 3.4.2 and
EQL SOP 301. Bottles must pass the sterility test, and test results are
recorded on the Sterility Check Log, Form 910.
5.5.3
Sterility of blank dilution water in glass bottles must be assured prior
their use. Bottles are cleaned following the same procedures as for
sample bottles (see EQL SOP 301 and Microbiology QAM Section
3.4.2). Bottles containing blank dilution water are prepared and tested
for sterility as described in this SOPs Section 5.3.2 and Microbiology
QAM Section 3.4.4. Bottles must pass the sterility test, and test results
are recorded on the Sterility Check Log, Form 910.
5.5.4
Effectiveness of the autoclave for sterilization is tested monthly as
described in EQL SOP 205. The autoclave must pass the sterility test,
and the test results are recorded on Autoclave Sterility Check Log, Form
905.
5.5.5
All glassware that comes in contact with the sample must be sterile, and a
sterilization record must be kept on the Sterility Check Log, Form 910,
unless the items are purchased presterilized.
Environmental Quality Lab
Microbiology Procedures
5.5.6
Rev. 5
10-15-09
QC requirements for controlled temperature equipment are provided in
EQL SOP 204, “Controlled Temperature Equipment”. In summary:
x
x
x
x
5.5.7
SOP No.: 502
Page 7 of 13
Incubator temperatures must be checked to the nearest 0.1 ºC and
documented at least twice daily when in use, with at least a four-hour
separation between measurements.
Refrigerator/freezer temperatures must be checked to the nearest 1ºC
and documented at least twice daily when in use. Readings should
be at least 4 hours apart.
Thermometers used to determine temperatures must be checked
against an NIST or NIST-traceable thermometer at least annually,
and for the temperature range used the thermometers must differ
from the expected temperature by <1.0 qC and must be tagged with
the temperature correction factor.
Temperatures will be recorded on the Temperature Monitoring Log,
Form 901, attached to each device.
QC requirements for TSB medium are as follows:
x
TSB medium (purchased from MicroBiologics® or equivalent) must
be stored in the dark at 4-30qC.
x
Sterility check:
¾ Each batch of TSB medium prepared must be checked and be
acceptable for sterility before use.
¾ Place one tube from the batch into the 35qC incubator and
incubate for 24-48 hours.
¾ Acceptable result is no growth (i.e., medium clear after
incubation).
¾ Discard the entire medium batch if growth is detected.
¾ Record the results of the sterility test in the Sterility Check Log,
Form 910.
x
Positive control checks:
¾ Each batch of TSB medium prepared must be checked before use
for the ability to promote growth of bacteria for the test(s) to be
performed. Appropriate bacteria used are (appropriate others can
also be used if those currently used are not available):
Escherchi coli
Enterobacter aerogenes
ATCC # 25922
ATCC # 13048
¾ Bacteria cultures are prepared by swabbing MicroBiologics® or
equivalent in 5-10 mL TSB and then incubating for 24 hours at
35ºC. Clouding or precipitation of the medium after incubation
indicates growth of the bacteria.
¾ Acceptable results are growth of all bacteria cultures.
¾ Discard the entire medium batch if growth is not detected.
¾ Record results on Positive/Negative Culture Medium Checks,
Form 908.
SOP No.: 502
Page 8 of 13
Environmental Quality Lab
Microbiology Procedures
5.5.8
Rev. 5
10-15-09
QC requirements for A-1 medium are as follows:
x
x
x
A-1 medium (purchased from (BD Difco™ or equivalent) must be
stored in the dark at 4-30qC.
Sterility check:
¾ Each batch of TSB medium prepared must be checked and be
acceptable for sterility before use.
¾ Place one tube from the batch into the 35qC incubator and
incubate for 24-48 hours.
¾ Acceptable result is no growth (i.e., medium clear after
incubation).
¾ Discard the entire medium batch if growth is detected.
¾ Record the results of the sterility test in the Sterility Check Log,
Form 910.
Positive and negative control checks:
¾ Each batch of A-1 medium prepared must be checked before use
with a positive and negative bacteria culture. Preferred QC and
currently available bacteria are (appropriate others can also be
used if those currently used are not available):
Escherchi coli
Enterobacter aerogenes
Positive:
Negative:
ATCC # 25922
ATCC # 13048
¾ Bacteria cultures are prepared by by swabbing MicroBiologics®
or equivalent in 5-10 ml trypticase soy broth and then incubating
for 24 hours at 35ºC. If the broth had been stored refrigerated,
allow it to incubate overnight at room temperature, then only use
it if no growth is observed (i.e., it is clear after overnight in the
incubator).
¾ Clouding or precipitation of the medium with culture pill after
incubation indicates growth of the bacteria.
¾ Separate culture tubes of A-1 medium are inoculated with a
culture of each bacteria using a 3mm inoculating loop. The loop
must be flame sterilized until it glows with a Bunsen burner, then
air cooled prior to submersing in the culture-containing broth.
¾ Submerge the loop into the tube with A-1 medium to inoculate it,
then resterilize the inoculating loop.
¾ Process the inoculated A-1 medium like a normal sample (i.e.,
SOP steps 5.4.8 - 5.4.11).
¾ For A-1 medium gas formation in tubes indicates growth.
Presence of growth for the positive control culture is satisfactory
as a QC check. Absence of growth is a satisfactory check for the
negative control culture. In summary:
Culture
Positive
Negative
Positive
Negative
Growth Seen
Yes
No
No
Yes
QC Passes?
Yes
Yes
No
No
SOP No.: 502
Page 9 of 13
Environmental Quality Lab
Microbiology Procedures
Rev. 5
10-15-09
For A-1 medium record the positive and negative control tests in
the Fecal Coliform (A-1) Run Log, Form 909. Also record test
results in Media Preparation Log, Form 907. In lieu of the sample
number write “E. coli” for the positive and “Enterobacter aerogenes
or equivalent” for the negative control check. The lot number of
the media is recorded in the run log form as well. This identifies
the control culture check for that particular batch of media.
5.5.9
Storage of Dehydrated Culture Media
Store dehydrated media in tightly closed bottle at less than 30ºC. Do not
use them if they are discolored or become caked and lose the character of
a free flowing powder. Purchase media in as small as quantities as
possible so that they will be used up preferably within 6 months.
Dehydrated media may be used until the manufacturer’s expiration date.
If possible the dehydrated media should be stored in a desiccator.
5.6
6.0
CORRECTIVE ACTIONS
5.6.1
If any of the QC requirements listed in Section 5.5 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
5.6.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
CALCULATIONS
To calculate coliform density, compute in terms of the Most Probable Number (MPN).
The MPN values for a variety of dilution series and results are given in Tables 1, 2, and 3
of this section. Included in these tables are the 95% confidence limits for each MPN
value determined. If the sample used are those found in the table, report the value
corresponding to the number of positive and negative results in the dilution series as the
MPN/ 100 mL or report as fecal coliform presence or absence.
The sample volumes indicated in Tables 1 and 2 relate to finished waters. Table 3
illustrates value for combination of positive and negative results when five 10 mL, five
1.0 mL and five 0.1 mL volumes of sample are tested. When the series of decimal
dilution is different from that in the table (i.e., series differ by factor of 10 but largest
volume is not 10 mL), select the MPN value from Table 3 and for the combination of
positive tubes and then calculate according to the following formula:
Actual MPN/100 ml
MPN value (from table) u
10
largest vo lume tested in dilution series used for MPN determinat ion
SOP No.: 502
Page 10 of 13
Environmental Quality Lab
Microbiology Procedures
Rev. 5
10-15-09
For example if sample volumes of five 1.0 mL, five 0.1 mL and five 0.01 mL the actual
MPN would be calculated by multiplying the MPN value from Table 3 by 10.
When more than three dilutions are used in a decimal series of dilution use the results
from only three of them in computing the MPN. To select the three dilutions to be used
in determining the MPN index, choose the highest dilution that gives positive results in
all five portions tested (no lower dilution giving any negative results) and the two next
succeeding higher dilutions. For example, if all five tubes with 10 mL sample were
positive, three tubes with 1 mL sample were positive and 0 tubes with 1 mL sample were
positive the MPN would be calculated to be 79 CFU/100mL using Table 3.
In the case such that a positive occurs in a dilution higher than the three chosen according
to the rule, incorporate it in the result for the highest chosen dilution. For example if
dilutions of 10, 1.0, 0.1 and 0.01 are used and five positives are found in the 10ml tubes,
3 positives are found in the 1.0 mL tubes and 1 positive is found in the 0.1 and 0.01 mL
tubes each. The MPN is calculated as if 5 positives were found in the 10 mL tubes, 3
positives in the 1.0 mL tubes and 2 positives in the 0.1 ml tubes and none in the 0.01 ml
tubes. The MPN calculation would be 140 CFU/100mL.
When it is desired to summarize with a single MPN value the results from a series of
samples, use the geometric mean or the median.
The MPN for a combination not appearing in the table or for other combination of tubes or
dilutions, may be estimated using Thomas' simple formula:
MPN/100 mL =
Number of positive tubes u 100
mL sample in negative tubes u mL sample in all tubes Table 1
MPN Index and 95% Confidence limits for various combinations of positive and negative
results when five 20 mL portions are used.
Number of Tubes Giving Positive
Reaction Out of 5 of 20 mL Each
0
1
2
3
4
5
Table 2
MPN Index/
100 mL
<1.1
1.1
2.6
4.6
8.0
>8.0
95 % Confidence Limits
Lower
0
0.051
0.40
1.0
2.1
3.4
Upper
3.5
5.4
8.4
13
23
Infinite
SOP No.: 502
Page 11 of 13
Environmental Quality Lab
Microbiology Procedures
Rev. 5
10-15-09
MPN Index and 95% Confidence limits for various combinations of positive and negative
results when ten 10 mL portions are used.
Number of Tubes Giving Positive
Reaction Out of 10 of 10 mL Each
0
1
2
3
4
5
6
7
8
9
10
MPN Index/
100 mL
<1.1
1.1
2.2
3.6
5.1
6.9
9.2
12
16
23
>23
95 % Confidence Limits
Lower
0
0.051
0.37
0.91
1.6
2.5
3.3
4.8
5.8
8.1
13
Upper
3.4
5.9
8.2
9.7
13
15
19
24
34
53
Infinite
SOP No.: 502
Page 12 of 13
Environmental Quality Lab
Microbiology Procedures
Rev. 5
10-15-09
Table 3
MPN Index and 95% Confidence limits for various combinations of positive and negative
results when five tubes are used per dilution (10mL, 1.0mL and 0. 1mL).
Combination
of Positives
0-0-0
0-0-1
0-1-0
0-1-1
0-2-0
0-2-1
0-3-0
1-0-0
1-0-1
1-0-2
1-1-0
1-1-1
1-1-2
1-2-0
1-2-1
1-3-0
1-3-1
1-4-0
2-0-0
2-0-1
2-0-2
2-1-0
2-1-1
2-1-2
2-2-0
2-2-1
2-2-2
2-3-0
2-3-1
2-4-0
3-0-0
3-0-1
3-0-2
3-1-0
3-1-1
3-1-2
3-2-0
3-2-1
3-2-2
3-3-0
3-3-1
MPN
Index/
100mL
<1.8
1.8
1.8
3.6
3.7
5.5
5.6
2.0
4.0
6.0
4.0
6.1
8.1
6.1
8.2
8.3
10
10
4.5
6.8
9.1
6.8
9.2
12
9.3
12
14
12
14
15
7.8
11
13
11
14
17
14
17
20
17
21
95% Confidence
Limits
Lower
Upper

0.090
0.090
0.70
0.70
1.8
1.8
0.10
0.70
1.8
0.71
1.8
3.4
1.8
3.4
3.4
3.5
3.5
0.79
1.8
3.4
1.8
3.4
4.1
3.4
4.1
5.9
4.1
5.9
5.9
2.1
3.5
5.6
3.5
5.6
6.0
5.7
6.8
6.8
6.8
6.8
6.8
6.8
6.9
10
10
15
15
10
10
15
12
15
22
15
22
22
22
22
15
15
22
17
22
26
22
26
36
26
36
36
22
23
35
26
36
36
36
40
40
40
40
Combination
of Positives
4-0-3
4-1-0
4-1-1
4-1-2
4-1-3
4-2-0
4-2-1
4-2-2
4-2-3
4-3-0
4-3-1
4-3-2
4-4-0
4-4-1
4-4-2
4-5-0
4-5-1
5-0-0
5-0-1
5-0-2
5-0-3
5-1-0
5-1-1
5-1-2
5-1-3
5-2-0
5-2-1
5-2-2
5-2-3
5-2-4
5-3-0
5-3-1
5-3-2
5-3-3
5-3-4
5-4-0
5-4-1
5-4-2
5-4-3
5-4-4
5-4-5
MPN
Index/
100mL
25
17
21
26
31
22
26
32
38
27
33
39
34
40
47
41
48
23
31
43
58
33
46
63
84
49
70
94
120
150
79
110
140
170
210
130
170
220
280
350
430
95% Confidence
Limits
Lower
Upper
9.8
6.0
6.8
9.8
10
6.8
9.8
10
14
9.9
10
14
14
14
15
14
15
6.8
10
14
22
10
14
22
34
15
22
34
36
58
22
34
52
70
70
36
58
70
100
100
150
70
40
42
70
70
50
70
70
100
70
70
100
100
100
120
100
120
70
70
100
150
100
120
150
220
150
170
230
250
400
220
250
400
400
400
400
400
440
710
710
1100
SOP No.: 502
Page 13 of 13
Environmental Quality Lab
Microbiology Procedures
3-3-2
3-4-0
3-4-1
3-5-0
4-0-0
4-0-1
4-0-2
7.0
24
21
24
25
13
17
21
9.8
6.8
9.8
9.8
4.1
5.9
6.8
70
40
70
70
35
36
40
5-5-0
5-5-1
5-5-2
5-5-3
5-5-4
5-5-5
240
350
540
920
1600
>1600
Rev. 5
10-15-09
70
100
150
220
400
700
710
1100
1700
2600
4600

WASTE DISPOSAL
All incubated samples, used media, and control cultures must be sterilized by autoclaving
for 30 minutes at 121qC. Place test racks with used media tubes into polypropylene tubs
and autoclave for 30 minutes at 121qC. Remove tubs from autoclave. Dispose of
autoclaved media down the sink’s sewage drain. Rinse tubes and all other containers in
the sink with tap water. Flush sink with lots of water. Discard broken glass in a glass
waste container.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix J
CCU EQL SOP NO.: 501
ENTEROCOCCI MEASUREMENT BY
IDEXX ENTEROLERT¥-QUANTI-TRAY¥ METHOD
Environmental Quality Lab
Microbiology Procedures
SOP No.: 501
Page 1 of 14
Enterococci Measurement by IDEXX
Enterolert¥-Quanti-Tray¥ Method
Rev. 5
3-26-09
Reference Method:
Enterolert
Approved by: ______________________________________
Laboratory Director
Reviewed by: ______________________________________
Laboratory Master Technician
1.0
SCOPE/APPLICATION
This procedure is for the detection and quantification of enterococci bacteria such as E.
faecium and E. faecalis in fresh and marine water.
This test method is based on IDEXX Defined Substrate Technology£ (DST¥) and utilizes
a nutrient indicator that fluoresces when metabolized by enterococci. When the reagent is
added to the sample and incubated, bacteria down to one CFU in a 100 mL sample can be
detected within 24 hours.
2.0
REFERENCES
2.1
3.0
IDEXX Enterolert¥ Test Kit instructions (IDEXX 06-02150-07)
DEFINITIONS
3.1
Bacterial Indicators: Bacterial measures commonly used to estimate the amount of
sewage in contaminated water. Although these bacteria themselves are not
pathogenic, they are used to “indicate” the presence of human pathogens. Current
federal and state recreational water quality standards include or recommend at least
one of the following bacterial indicators: total coliforms, fecal coliforms,
enterococci, and E. coli.
3.2
Colony Forming Unit (CFU): A visible mass of organisms developing from one or
a group of bacteria. The mass results from the bacteria reproducing themselves.
3.3
E. coli: A species of bacteria that occurs in the intestine of warm-blooded animals.
It is part of the fecal coliform group.
3.4
Enterococci: A group of bacteria in the genus Streptococcus that occurs in the
intestine of warm-blooded animals. Commonly used as a bacterial indicator.
3.5
Fecal Coliforms: A group of bacteria found in the feces of various warm-blooded
animals. It is a subgroup of the total coliform group and has been used as a more
definitive indicator for fecal pollution.
3.6
Most Probable Number: When multiple well trays are used in the technique, the
number of positive wells on a tray is reported in terms of the Most Probable
Environmental Quality Lab
Microbiology Procedures
SOP No.: 501
Page 2 of 14
Rev. 5
3-26-09
Number (MPN) of organisms present. This number, based on certain probability
formulas, is an estimate of the mean density of Enterococci in the sample.
4.0
5.0
3.7
Pathogens: Organisms that cause disease.
3.8
Total Coliforms: A large group of bacteria that can originate from soil, plants,
human waste, and animal waste. Commonly used as a bacterial indicator.
SAFETY
4.1
This method is restricted to use by or under the supervision of trained analysts.
4.2
When using the autoclave or Bunsen burner, be careful to avoid touching hot
surfaces and to not expose yourself to steam or flames.
4.3
Gloves, safety glasses with side shields, and protective clothing should be worn
to protect against unnecessary exposure to infectious agents (i.e., pathogens),
hazardous chemicals, and contaminants in potentially hazardous samples.
4.4
When using the UV lamp be sure the light is facing away from your eyes.
4.5
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
5.1.2
5.1.3
5.1.4
5.1.5
5.1.6
5.1.7
5.1.8
5.1.9
5.1.10
5.1.11
5.1.12
5.1.13
5.1.14
5.1.15
5.1.16
5.1.17
Incubator at 35.0 r 0.5qC, Fisher Isotemp 600 Series Standard Incubator
Incubator at 41.0 r 0.5qC, Fisher Isotemp 600 Series Standard Incubator
Refrigerator at 1 – 4qC
IDEXX Quanti-Tray¥ Sealers
5.1.4.1 Original Model (back-up instrument)
5.1.4.2 Model 2X
IDEXX 51-well Quanti-Trays (catalog # WQT100)
IDEXX 97-well Quanti-Trays (catalog # WQT2K)
Autoclave
Bunsen Burner
Inoculating loop, nickel-chromium alloy
Sterile plastic, serological pipets, 10 mL size graduated in 1/10 mL
Glass pipets, volumetric, assorted sizes
Pipet bulbs
UV lamp, 6 watt, 365 nm wavelength
pH meter, Accumet AR25
Glass bottles for dilution water, 300 mL
Glass culture tubes, 25 mL, with rubber lined, screw close caps
Thermometers for controlled temperature equipment
Environmental Quality Lab
Microbiology Procedures
5.2
Rev. 5
3-26-09
REAGENTS
5.2.1
5.2.2
5.2.3
5.2.4
5.2.5
5.2.6
5.2.7
5.2.8
5.2.9
5.3
SOP No.: 501
Page 3 of 14
Enterolert for 100 mL water samples (IDEXX catalog # WENT200)
Tryptic soy broth dehydrated medium
Sterile dilution water
Bacteria control cultures (MicroBiologics® or equivalent)
Enterococcus faecalis, ATCC 29212
Escherichia coli (gram -), ATCC 25922
Seratia marcescens (gram -), ATCC 14756
Aerococcus viridans (gram +), ATCC 700406
Staphylococcus aureus (gram +), ATCC 43300
Other acceptable bacteria depending on availability those currently used
pH buffer solutions, 4.00, 7.00, 10.00
Electrode storage solution
Sodium hydroxide
Hydrochloric acid
Food color
REAGENT PREPARATION
5.3.1
Tryptic soy broth
This medium is used as a general growth medium for bacteria.
x
Prepare from dehydrated media according instructions on bottle. Heat if
necessary to dissolve media.
x
Make tryptic soy broth (TSB) of such strength that adding the sample
aliquot will not significantly reduce the medium concentration below that
of the original medium. For 10-50 mL sample aliquots prepare doublestrength medium.
x
Suspend 30.0 grams in 1 liter of deionized water and warm slightly, if
necessary, to dissolve completely. For preparing double strength
medium suspend 60.0 grams in 1 liter. Other volumes of medium can be
prepared as long as these proportions of medium and water are used.
x
Measure pH and adjust pH, if necessary, so that the final pH after
sterilization will be 7.3 r 0.2 at 25ºC. Experience in preparing TSB
medium will determine the need to adjust pH, especially for
compensating for pH changes during sterilization. pH usually drops
slightly during sterilization (up to ~0.1), so adjust pH to close to 7.4
before sterilization.
x
Dispense 10 mL into 20-30 mL culture tubes with screw close caps and
25-50 mL in 100-300 mL glass bottles with screw close caps.
x
Sterilize the media in the autoclave. The sterilization cycle must include
exposure at 121ºC for 15 minutes. Promptly remove sterilized medium
from the autoclave when sterilization cycle is complete.
Environmental Quality Lab
Microbiology Procedures
Rev. 5
3-26-09
x
Measure final pH using two of the sterilized tubes of TSB. Final pH
must be 7.3 r 0.2 at 25ºC. Discard entire batch of medium if pH is not
within limits. Record pH in Media Preparation Log, Form 907.
x
Store sterile TSB at 4qC in tightly closed screw-cap containers for up to
3 months.
5.3.2
5.4
SOP No.: 501
Page 4 of 14
Blank dilution water
x
Prepare blank dilution by placing 90 mL of fresh deionized water in a
clean, glass dilution bottle.
x
Loosely screw on the cap to the bottle.
x
Record the dilution bottle preparation in the Dilution Bottle Preparation
Log, Form 919.
x
Sterilize the blank dilution water in bottles in the autoclave for at least 15
minutes at 121qC.
x
Allow the bottles to cool to room temperature.
x
Each batch of blank dilution water should be checked for sterility by adding
to 50 mL of the dilution water, 50 mL of sterile, double strength, nonselective broth (e.g., trypticase soy broth). If the broth had been stored
refrigerated, allow it to incubate overnight at room temperature, then only
use it if no growth is observed (i.e., it is clear after overnight at room
temperature).
x
Incubate at 35.0 r 0.5qC for 24 hours and check for growth. Discard all
dilution water in batch if growth is detected.
x
Record sterility check of dilution water on Sterility Check Log, From 910.
PROCEDURE
5.4.1
Turn on the Quanti-Tray Sealer; it takes approximately 5 minutes to warmup at which time the green ready light will come on.
5.4.2
Locate samples from cooler according to the chain of custody and analysis
request forms.
5.4.3
Based on observation of the samples, site histories, and analysis
experience, segregate the samples into those expected to have low and high
enterococci concentrations. Process the samples expected to have low
concentrations first to minimize possible carryover contamination between
samples.
5.4.4
Sample analysis should begin immediately, preferably within 2 hours of
collection. The maximum transport time to the laboratory is within 6
Environmental Quality Lab
Microbiology Procedures
SOP No.: 501
Page 5 of 14
Rev. 5
3-26-09
hours of sample collection. Samples should be processed within 2 hours
of receipt at the laboratory. Immediately prior to sample analysis shake
sample bottle vigorously at least 25 times.
5.4.5
Carefully open a dilution bottle and sample bottle without contaminating
either bottle.
5.4.6
A sample dilution of at least 10-fold (one volume of sample plus nine
volumes of sterile dilution water) is used for analysis of marine water
samples. For samples suspected to have very high bacteria
concentrations, higher dilutions may be used (e.g., 5 mL sample added to
95 mL of dilution water for 20-fold dilution). For fresh water samples
dilution of the sample is not mandatory.
5.4.7
Using an individually wrapped sterile 10 ml pipet transfer 10 ml of
sample to a dilution bottle containing 90 r 2ml of sterile water or other
sample and dilution water combinations to give a final volume of 100
mL. However, no more than 10 mL of marine water can be used.
5.4.8
Carefully separate an Enterolert snap pack from its strip, taking care not
to accidentally open the next pack.
5.4.9
Tap the reagent snap pack to ensure that all of the Enterolert powder is in
the bottom of the pack.
5.4.10 Open the pack by snapping back the top at the scoreline, being careful
not to touch the opening of the pack.
5.4.11 Add the Enterolert reagent to the 100 mL of sample plus dilution water.
Because the Enterolert contains buffer, do not use dilution water
that contains buffer.
5.4.12 Aseptically cap and seal the dilution water bottle.
5.4.13 Shake to completely dissolve reagent.
5.4.14 Select either a 51-well (for suspected low enterococci concentration) or
97-well (for suspected high enterococci concentration) Quanti-Tray™.
5.4.15 Pour the sample and reagent mixture into a Quanti-Tray™ as follows:
x
Use one hand to hold a Quanti-Tray upright with the well side facing
the palm.
x
Squeeze the upper part of Quanti-Tray so that the Quanti-Tray bends
towards the palm.
x
Open the Quanti-Tray pulling the foil tab away from the well side.
Avoid touching the inside of the foil or tray.
Environmental Quality Lab
Microbiology Procedures
x
x
x
SOP No.: 501
Page 6 of 14
Rev. 5
3-26-09
Pour the reagent and sample mixture directly into the Quanti-Tray
avoiding contact with the foil tab.
Allow foam to settle.
Place the sample-filled Quanti-Tray onto the rubber tray carrier of
the Quanti-Tray Sealer with the well side (plastic) of the Quanti-Tray
facing down to fit into the carrier.
5.4.16 If the Quanti-Tray Sealer green ready light is on, slide the carrier with
the Quanti-Tray on top into the chute on the side of the sealer until they
engage the roller and are pulled through the sealer. The carrier and
sealed Quanti-Tray are ejected out of the opposite side of the sealer.
5.4.17 Place the sealed Quanti-Tray in an incubator at 41.0 r 0.5ºC.
5.4.18 Incubate the sealed Quanti-Tray for 24–28 hours at 41.0 r 0.5ºC.
5.4.19 Record the following information on the Enterococcus Analysis Log,
Form 252:
x Sequential laboratory ID assigned by the lab
x Analysis start date
x Analysis start time (time sample was placed in the incubator)
x mL of sample added to the dilution blank
x mL of dilution water used for sample analysis
x Lot identification for sample bottles, sterile pipets, dilution water
bottles, Enterolert media, and Quanti-Trays
x Initials of setup analyst
5.4.20 After 24-28 hours (as close to 24 hours as possible) remove the tray from
the incubator and read the results by placing a 6 watt 365 nm wavelength
UV light within five inches of the tray in a dark environment. Be sure
the light is facing away from your eyes and towards the vessel. Any
amount of blue fluorescence indicates the presence of enterococci and is
recorded as a positive well.
5.4.21 Record the following information on the Enterococcus Analysis Log,
Form 252:
x Date and time the sample was read
x Number of positive wells
x Enterococci colonies/100 mL for diluted sample
x Enterococci colonies/100 mL of undiluted sample
x Initials of read analyst
5.5
QUALITY CONTROL
5.5.1
Quality control (QC) measures and acceptance criteria for analyzing
samples are summarized in Table 1 and are as follows:
x Prior to analyzing samples, each analyst must demonstrate the ability
to generate acceptable results (i.e., demonstration of capability).
x Sample analysis should begin immediately, preferably within 2 hours
of collection. The maximum transport time to the laboratory is
Environmental Quality Lab
Microbiology Procedures
x
x
x
SOP No.: 501
Page 7 of 14
Rev. 5
3-26-09
within 6 hours of sample collection. Samples should be processed
within 2 hours of receipt at the laboratory.
Any QC sample analysis (e.g., method blank, laboratory replicate,
field replicate) should be subjected to exactly the same analytical
procedures as those used on individual sample analyses.
At least weekly prior to analysis of samples, a method blank (i.e.,
sterile dilution water) should be analyzed.
Unless otherwise specified for specific project or samples, at least
weekly one sample should be analyzed in duplicate and also each
large batch of approximately 20 samples analyzed should include at
least one sample analyzed in duplicate (i.e., at least 5% duplicates).
5.5.2
Sterility of sample bottles must be assured prior their use. Bottles are
cleaned and tested for sterility as described in QAM Section 3.4.4 and
EQL SOP 301. Bottles must pass the sterility test, and test results are
recorded on the Sterility Check Log, Form 910.
5.5.3
Sterility of blank dilution water in glass bottles must be assured prior
their use. Bottles are cleaned following the same procedures as for
sample bottles (see EQL SOP 301 and Microbiology QAM Section
3.4.2). Bottles containing blank dilution water are prepared and tested
for sterility as described in this SOPs Section 5.3.2 and Microbiology
QAM Section 3.4.4. Bottles must pass the sterility test, and test results
are recorded on the Sterility Check Log, Form 910.
5.5.4
Effectiveness of autoclave for sterilization is tested monthly as described
in EQL SOP 205. The autoclave must pass the sterility test, and test
results are recorded on Autoclave Sterility Check Log, Form 905.
5.5.5
All glassware that comes in contact with the sample must be sterile, and a
sterilization record must be kept on the Sterility Check Log, Form 910,
unless the items are purchased presterilized.
5.5.6
QC requirements for controlled temperature equipment are provided in
EQL SOP 204, “Controlled Temperature Equipment”. In summary:
x
x
x
x
Incubator temperatures must be checked to the nearest 0.1 ºC and
documented at least twice daily when in use. Readings should be at
least 4 hours apart.
Refrigerator/freezer temperatures must be checked to the nearest 1ºC
and documented at least twice daily when in use. Readings should
be at least 4 hours apart.
Thermometers used to determine temperatures must be checked
against an NIST or NIST-traceable thermometer at least annually,
and for the temperature range used the thermometers must differ
from the expected temperature by <1.0 qC and must be tagged with
the temperature correction factor.
Temperatures must be recorded on the Temperature Monitoring Log,
Form 901, attached to each device.
SOP No.: 501
Page 8 of 14
Environmental Quality Lab
Microbiology Procedures
Rev. 5
3-26-09
Table 1. Summary of QC Requirements for enterococci analysis by Enterolert.
QC Sample or Activity
Capability demonstration
Minimum
Frequency
Four (4) prepared
samples analyzed
prior to any customer
sample analyses
Acceptance Criteria
Corrective Action
Criteria for LCS recovery
and duplicate precision
Repeat until acceptable
Media sterility check
Prior to use of new lot
of Enterolert and
weekly
No fluorescence
Media positive check
with control culture
Prior to use of new lot
of Enterolert and
weekly
Fluorescence
Media negative checks
with control cultures
(gram+ and gram-)
Prior to use of new lot
of Enterolert
No fluorescence
Method blank
At least weekly,
prior to sample
analysis
< 20 CFU/100 mL
Sample duplicate or
matrix spike duplicate
At least one (1)
weekly, and one with
all large sample
batches (~20 samples)
RPD < 200% for <150
CFU/100 mL
RPD < 100% for > 150
CFU/100 mL
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
PE =
Samples and
frequency determined
by Lab QA Officer
Samples and
frequency determined
by accrediting
agencies and projects
laboratory control sample
method blank
method detection limit
performance evaluation
Investigate problem. Eliminate
contaminations. Obtain new lot
of Enterolert if necessary.
Repeat until successful before
using Enterolert lot.
Investigate problem. Obtain
new lot of Enterolert if
necessary. Repeat until
successful before using
Enterolert lot.
Investigate problem. Eliminate
contaminations. Obtain new lot
of Enterolert if necessary.
Repeat until successful before
using Enterolert lot.
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze entire
batch.
Criteria for LCS recovery
and duplicate precision
Investigate all unacceptable
results.
Determined by PE
provider
Investigate all unacceptable
results.
QC =
%R =
RL =
RPD =
quality control
percent recovery
reporting limit
relative percent difference
Environmental Quality Lab
Microbiology Procedures
5.5.7
Rev. 5
3-26-09
Effectiveness of the Quanti-Tray Sealer operation must be checked
monthly as follows:
x
Add a dye (e.g., food color or Enterolert reagent) to 100 mL of
water.
x
Seal the colored water in a Quanti-Tray following the normal sealing
procedure (see SOP Steps 5.4.14 - 5.4.16).
x
If the colored water is observed outside the wells of the sealed
Quanti-Tray, the sealer must be repaired or the back-up sealer used.
The results of this test must be recorded in the Quanti-Tray Sealer
Check Log, Form 912.
x
5.5.8
SOP No.: 501
Page 9 of 14
QC requirements for Enterolert media are as follows:
x
Enterolert medium (purchased from IDEXX) must be stored in the
dark at 4-30qC.
x
Initial sterility check:
¾ Before use each lot of Enterolert medium must be checked and
be acceptable for sterility.
¾ Analyze 100 mL of sterile, deionized water like a normal
sample, except a presence/absence test is sufficient (i.e., incubate
the water plus reagent in the dilution bottle not in a QuantiTray).
¾ Acceptable result is no fluorescence.
¾ Record the results of the QC sterility test in the Enterolert QC
Log, Form 913.
x
Initial positive and negative control checks:
¾ Each lot of Enterolert medium must be checked before use with a
positive control culture and negative control cultures of both
gram negative and gram positive bacteria. Preferred QC bacteria
currently available are (appropriate others can also be used if
those currently used are not available):
Positive:
Negative (gram-):
Negative (gram+):
Enterococcus faecalis
Escherichia coli
Aerococcus viridans
ATCC # 29212
ATCC # 25922
ATCC #700406
¾ Bacteria cultures are prepared by swabbing MicroBiologics®
or equivalent in 5-10 mL trypticase soy broth and then
incubating for 24 hours at 35ºC. If the broth had been stored
refrigerated, allow it to incubate overnight at room temperature,
then only use it if no growth is observed (i.e., it is clear after
overnight at room temperature). Clouding or precipitation of the
inoculated medium after incubation indicates growth of the
bacteria.
SOP No.: 501
Page 10 of 14
Environmental Quality Lab
Microbiology Procedures
Rev. 5
3-26-09
¾ Separate 100 mL bottles of sterile dilution water are inoculated
with a culture of each bacteria using a 1 µL inoculating loop.
The loop must be flame sterilized until it glows in the flame of a
Bunsen burner, then air cooled prior to submersing in the
culture-containing broth.
¾ Submerge the loop into the sterile dilution water to inoculate it,
and then resterilize the inoculating loop.
¾ Process the inoculated dilution water like a normal sample,
except a presence/absence test is sufficient (i.e., samples are
incubated in the dilution bottle).
¾ For Enterolert medium, blue fluorescence under UV light
indicates growth in medium. Presence of growth for the positive
control culture is satisfactory as a QC check. Absence of growth
(i.e., no fluorescence) is a satisfactory check for the negative
control cultures. In summary:
Culture
Positive
Negative
Positive
Negative
Growth (Fluorescence)
Seen ?
Yes
No
No
Yes
QC Passes?
Yes
Yes
No
No
¾ For Enterolert medium record the results of the positive and
negative control tests in the Enterolert QC Log, Form 914.
¾ If QC data are acceptable, the medium can be used.
¾ If QC data are not acceptable, the medium cannot be used and
the problem must be investigated.
x
Weekly Enterolert sterility and positive control checks:
¾ Once per week a known positive control (enterococci pure
culture) and sterile, deionized water blank must be analyzed
using the Enterolert medium. A presence/absence test is
sufficient. These QC measurements are to be performed
following the procedures used for the initial Enterolert Positive
Control Check and the Initial Enterolert Sterility Check.
¾ Record the results of the weekly QC tests in the Enterolert QC
Log, Form 914.
¾ Acceptable results are growth (i.e., fluorescence) for the positive
control and no growth (i.e., no fluorescence) for the sterility test.
¾ If weekly QC data are acceptable, the medium may be used for
the next week or until the manufacturer’s expiration date,
whichever is sooner.
¾ If QC data are not acceptable, the medium cannot be used and
the problem must be investigated.
Environmental Quality Lab
Microbiology Procedures
5.6
6.0
SOP No.: 501
Page 11 of 14
Rev. 5
3-26-09
CORRECTIVE ACTIONS
5.6.1
If any of the QC requirements listed in Section 5.5 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
5.6.2
The problem and associated corrective actions must be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
CALCULATIONS AND DATA REPORTING
6.1
From the number of fluorescing wells on the Quanti-Tray, use appropriate QuantiTray most probable number (MPN) chart (Tables 2 and 3) to determine bacteria
colony density for the diluted sample.
6.2
The density of the undiluted sample is determined by multiplying the density of
the diluted sample by the dilution factor (i.e., 10 for 10-fold dilution, which is
100 divided by the volume of sample pipeted into the dilution bottle). The
resulting value is the enterococci density per 100 mL of sample.
For example, if 3 wells were positive (i.e., fluorescing) and the sample had been
prepared by adding 10 mL of sample to 90 mL of sterile dilution water (total
volume of 100 mL), the result from the table would be 3.1 while the reported
value would be 31 CFU/100 mL.
6.3
7.0
Report trays without positive wells as <10 when 10 mL of sample was used (i.e.,
dilution factor of 10).
WASTE DISPOSAL
All incubated samples, used media, and control cultures must be sterilized by autoclaving
for 30 minutes at 121qC. Place Trays in three to five gallon metal pots. Autoclave pot
for 30 minutes at 121 qC. Remove pot from autoclave. The autoclaving procedure will
burst most of the Quanti-Trays. Remove trays while allowing autoclaved media to drain
in a sink. Cut the paper backing of the unburst trays with a metal rod (i.e., screwdriver,
dull knife), so the trays can drain completely. Rinse the trays with tap water. The used
trays are sharp and tend to tear plastic bags. Thus, draining of trays prevents spillage.
Place used trays in a plastic garbage bag and discard. Dispose of autoclaved media down
the sink’s sewage drain. Rinse all other containers in the sink with tap water. Flush sink
with lots of water. Discard glass in a glass waste container.
SOP No.: 501
Page 12 of 14
Environmental Quality Lab
Microbiology Procedures
Table 2. 51-Well Quanti-Tray MPN Table
Number of wells giving a positive
reaction per 100 ml of sample.
Most Probable Number
(MPN/100 ml)
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
<1
1.0
2.0
3.1
4.2
5.3
6.4
7.5
8.7
9.9
11.1
12.4
13.7
15.0
16.4
17.8
19.2
20.7
22.2
23.8
25.4
27.1
28.8
30.6
32.4
34.4
36.4
38.4
40.6
42.9
45.3
47.8
50.4
53.1
56.0
59.1
62.4
65.9
69.7
73.8
78.2
83.1
88.5
94.5
101.3
109.1
118.4
129.8
144.5
165.2
200.5
>200.5
95 % Confidence Limits
Lower
Upper
0.0
3.7
0.3
5.6
0.6
7.3
1.1
9.0
1.7
10.7
2.3
12.3
3.0
13.9
3.7
15.5
4.5
17.1
5.3
18.8
6.1
20.5
7.0
22.1
7.9
23.9
8.8
25.7
9.8
27.5
10.8
29.4
11.9
31.3
13.0
33.3
14.1
35.2
15.3
37.3
16.5
39.4
17.7
41.6
19.0
43.9
20.4
46.3
21.8
48.7
23.3
51.2
24.7
53.9
26.4
56.6
28.0
59.5
29.7
62.5
31.5
65.6
33.4
69.0
35.4
72.5
37.5
76.2
39.7
80.1
42.0
84.4
44.6
88.8
47.2
93.7
50.0
99.0
53.1
104.8
56.4
111.2
59.9
118.3
63.9
126.2
68.2
135.4
73.1
146.0
78.6
158.7
85.0
174.5
92.7
195.0
102.3
224.1
115.2
272.2
135.8
387.6
146.1
infinite
Rev. 5
3-26-09
Environmental Quality Lab
Microbiology Procedures
SOP No.: 501
Page 13 of 14
Rev. 5
3-26-09
Environmental Quality Lab
Microbiology Procedures
SOP No.: 501
Page 14 of 14
Rev. 5
3-26-09
Environmental Quality Lab
Microbiology Procedures
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
Table 3. 97-Well Quanti-Tray MPN Table
SOP No.: 501
Page 15 of 14
Rev. 5
3-26-09
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix K
CCU EQL SOP NO.: 602
OPTICAL BRIGHTENER MEASUREMENT
BY FLUOROMETRY
Environmental Quality Lab
Chemistry Procedures
Optical Brightener Measurement
by Fluorometry
SOP No.: 602
Page 1 of 11
Reference Method:*
Standard Method
Approved by:
______________________________________
Laboratory Director
Reviewed by:
______________________________________
Laboratory Master Technician
1.0
Rev. 1
1-13-10
SCOPE/APPLICATION
Optical brighteners in laundry and dishwashing detergents fluoresce when exposed to certain
ultraviolet wavelengths, so water samples that fluoresce under those same wavelengths are
contaminated by residues from laundry and dishwashing detergents (human sources). There are at
least two major potential human sources of contamination that could contain optical brighteners,
and these include leachates from improperly functioning on-site wastewater systems (OWS) and
leaking pipes from community wastewater treatment systems. In rural areas where the majority of
homes are served by on-site systems, optical brighteners in water samples indicate failing
conditions within OWS in close proximity to the sampled bodies of water.
*The method used in this SOP is based on:
Cao Y, Griffith JF, Weisberg SB (2009) Evaluation of optical brightener photodecay
characteristics for detection of human fecal contamination. Water Res 43:2273±2279,
Dickerson, J.W.J., Hagedorn, C., Hassall, A., 2007. Detection and remediation of humanorigin pollution at two public beaches in Virginia using multiple source tracking methods.
Water Research 41, 3758±3770.
Hartel, P.G., Hagedorn, C., McDonald, J.L., Fisher, J.A., Suluta, M.A., Dickerson, J.R.,
Gentit, L.C., Smith, S.L., Mantripragada, N.S., Ritter, K.J., Belcher, C.N., 2007a. Exposing
water samples to ultraviolet light improves fluorometry for detecting human fecal
contamination. Water Research 41, 3629±3642.
Hartel, P.G., McDonald, J.L., Gentit, L.C., Hemmings, S.N.J., Rodgers, K., Smith, K.A.,
Belcher, C.N., Kuntz, R.L., Rivera-Torres, Y., Otero, E., Schroder, E.C., 2007b. Improving
fluorometry as a source tracking method to detect human fecal contamination. Estuaries
and Coasts 30, 551±561.
2.0
REFERENCES
2.1
3.0
Turner TD-700 Laboratory Fluorometer Operating Manual
DEFINITIONS
None
4.0
SAFETY
4.1
This method is restricted to use by or under the supervision of trained analysts.
Environmental Quality Lab
Chemistry Procedures
5.0
SOP No.: 602
Page 2 of 11
Rev. 1
1-13-10
4.2
Gloves, safety glasses with side shields, and protective clothing should be worn
to protect against unnecessary exposure to infectious agents (i.e., pathogens),
hazardous chemicals (e.g., acids, organic solvents), and contaminants in
potentially hazardous samples.
4.3
All activities performed while following this procedure should utilize appropriate
laboratory safety systems (e.g., disinfectant, fume hoods, material safety data
sheets).
METHOD
5.1
APPARATUS AND MATERIALS
5.1.1
100mL Griffin beakers (acid leached and rinsed x 10 with Type I water);
One beaker for each field sample.
5.1.2 LDPE squirt bottle
5.1.3 Aluminum foil
5.1.4 Stopwatch or suitable electronic timer
5.1.5 Refrigerator, or other means to store sample at 1-4° C in the dark
5.1.6 Aluminum foil
5.1.7 Type I water, UV irradiated
5.1.8 Refrigerator, or other means to store sample at 1-4° C in the dark
5.1.9 Polymethacrylate cuvettes (10mm x 10mm x 45mm, Turner Designs,
Sunnyvale, CA)
5.1.10 Delicate tissue wipes (Kimwipes or equivalent)
5.2
REAGENTS
5.2.1
5.2.2
5.3
Fluorescent Brightener 28 (Cat No. 158067, MP Biomedicals, Solon, OH
Liquid Tide detergent
PROCEDURE
5.3.1
Sample Preparation
5.3.1.1 Store samples in amber glass bottles at 1-4 C for up to 8 days prior
to analysis.
5.3.1.2 Under dark conditions, decant approximately 100mL of sample from
amber bottles to 100mL beakers. Completely cover beakers with
aluminum foil to prevent any light exposure during sample warming.
5.3.1.3 Transport foil-covered sample beakers to 20°C incubator and allow
samples to warm to 20°C. This process takes approximately 1 hour.
5.3.2
Pre-irradiation Sample Analysis
5.3.2.1 To ensure the Optical Brightener module is installed in the Turner
Trilogy fluorometer. If it is not installed, make sure the Trilogy is
turner off before removing the existing unit by pulling straight up
Environmental Quality Lab
Chemistry Procedures
SOP No.: 602
Page 3 of 11
Rev. 1
1-13-10
on the small handle located on top of the installed unit. Insert the
Optical Brightener module by orienting the notches on the module
with the appropriate notches on the Trilogy. The module only fits in
one orientation, so do not apply unnecessary force if resistance is
encountered. Firmly press down until the module is seated in the
Trilogy.
5.3.2.2 Work in subdued light (i.e., shades drawn, overhead lights off, only
fume hood lights on).
5.3.2.3 Immediately prior to analysis, ensure the UV light source in the
Trilogy is warm by selecting <tools>, <continuous sampling>,
<continuous sampling ON>, <total number of measurements
40>. It is not necessary for an actual sample to be in the Trilogy for
this process, which takes approximately 4 minutes. The fluorometer
is ready for use after this warm-up step, and will remain ready for 2
hours. If for any reason the fluorometer is left unused for longer
than 2 hours, repeat the warm-up procedure prior to analysis.
5.3.2.4
Reprogram the Trilogy measurement mode from continuous
sampling to single sampling by selecting <tools>, <continuous
sampling>, <continuous sampling OFF>.
5.3.2.5 Using PMMA cuvettes that have met the criteria for background
fluorescence (5.3.5), select one cuvette for each sample.
5.3.2.6 To ensure that each cuvette is clean, fill each with Type I water
and obtain a blank value. If the blank is greater than 145)8¶V,
rinse x10 with Type I water and reanalyze. Repeat this process for
each cuvette being used for the sample analysis. Do not use any
cuvette that has a blank value >145)8¶V.
5.3.2.7 For each sample, rinse each cuvette x3 with Type I water using the
squirt bottle and then x3 with sample. Thoroughly clean all sides of
the cuvette with a clean Kimwipe prior to presentation to the
fluorometer.
5.3.2.8 Insert cuvette into fluorometer with the marked, optimized cuvette
face facing the front of the instrument (5.3.5)
5.3.2.9 Press <Measure Raw Fluorescence> on the Trilogy screen and
record the raw fluorescence units (RFU) number after
measurement is completed. (Important: Each sample can only
be measured ONCE in the fluorometer since the actual
analysis exposes the sample to UV light degrading the analyte.
Pressing <Measure> a second time on the same sample already
analyzed will result in a lower reading from this degradation
and is not a valid measurement).
5.3.2.10 Remove the cuvette and discard its sample. Repeat 5.3.2.7, but DO
NOT put the sample in the fluorometer. Place the cuvette in the
light-proof sample box making sure that it is labeled in a way that
will allow you to identify the sample from other cuvettes. Each
SOP No.: 602
Page 4 of 11
Environmental Quality Lab
Chemistry Procedures
Rev. 1
1-13-10
cuvette is engraved with a unique identification number that should
be recorded in the lab book along with the sample analysis data.
5.3.2.11 Analyze the remaining samples using steps 5.3.2.7 ± 5.3.2.10 until
all samples have been run and are stored in the light-proof box.
5.3.2.12 Proceed to section 5.3.3
5.3.3
Sample UV Irradiation
5.3.3.1
30 minutes prior to irradiating the samples, turn on the 2 UV lights
in the 20°C incubator to allow them to warm up. Make sure to
wear appropriate eye protection. Ensure that the lid is closed on the
light-proof sample box before transporting the samples.
5.3.3.2
Under dark room conditions (with the exception of the UV lights
in the incubator), transfer each of the sample cuvettes from the
light-proof box to the incubator. Orient the cuvettes so that each
one is aligned directly in front of the horizontal UV light and
directly under the hanging UV light.
5.3.3.3
Set a timer to 5.0 minutes and close the incubator door. It is
important that the samples are irradiated exactly 5.0 minutes.
5.3.3.4
After 5.0 minutes of irradiation remove each sample cuvette (under
subdued light conditions), and place into the light-proof box. Close
the lid and transport the samples back to the fluorometer.
5.3.4
Post UV-Irradiation Sample Analysis
5.3.4.1 Work in subdued light (i.e., shades drawn, overhead lights off,
only fume hood lights on).
5.3.4.2 Remove the first irradiated cuvette from the light-proof box and
thoroughly wipe all sides with a Kimwipe. Present the cuvette to the
fluorometer with the marked, optimized cuvette face facing the
front of the instrument (5.3.5)
5.3.4.3 Press <Measure Raw Fluorescence> on the Trilogy screen and
record the raw fluorescence units (RFU) number after measurement
is completed.
5.3.4.4 Discard the analyte and rinse the cuvette x3 with the squirt bottle.
5.3.4.5 Repeat steps 5.3.4.1 through 5.3.4.4 for all remaining samples in the
light-proof box.
5.3.4.6 Proceed to section 5.3.6 to use the data to determine if optical
brighteners are present in the samples. Both qualitative (5.3.6.1) and
quantitative (5.3.6.2) determinations are possible.
5.3.5
PMMA Cuvette Optimization
Environmental Quality Lab
Chemistry Procedures
SOP No.: 602
Page 5 of 11
Rev. 1
1-13-10
5.3.5.1 Each polymethacrylate cuvette will have a different background
level, even when running the same sample. It is important to
determine this level and ensure it is low enough to be used for
actual sample analysis.
5.3.5.2 Fill a 2.5L glass jug with water before starting this analysis. The
water dispensed by the Milli-Q system can have varying levels of
background fluorescence at different times (depending on how
much it is being dispensed by other users), so it is critical to use the
same water for this analysis. Do not assume that water dispensed at
different times will have the same background fluorescence.
5.3.5.3 Allow the jug to come into thermal equilibrium with the room prior
to use. It is common for the water to gradually grow warmer as it is
dispensed from the Milli-Q (due to the reverse osmosis system
supplying the Miili-Q), so it is a good practice to draw water the
day prior to analysis and allow it to sit overnight in a tightly closed
2.5L glass jug.
5.3.5.4 Immediately prior to analysis, ensure the UV light source in the
Trilogy is warm by selecting <tools>, <continuous sampling>,
<continuous sampling ON>, <total number of measurements
40>. It is not necessary for an actual sample to be in the Trilogy for
this process, which takes approximately 4 minutes. The fluorometer
is ready for use after this warm-up step, and will remain ready for 2
hours. If for any reason the fluorometer is left unused for longer
than 2 hours, repeat the warm-up procedure prior to analysis.
5.3.5.5 Work in subdued light (i.e., shades drawn, overhead lights off, only
fume hood lights on).
5.3.5.6 Fill a cuvette with water from 5.3.5.2 and dry all sides with a clean
Kimwipe.
5.3.5.7 Present the cuvette to the fluorometer, close the lid and press
<Measure Raw Fluorescence>. After analysis, write this value
down. Open the fluorometer and rotate the cuvette 90° in the holder,
close the lid and re-measure. Write down the value, and repeat so
that the fluorescence values from all 4 cuvettes sides has been
measured.
5.3.5.8 The cuvette must have background fluorescence lower than
14 5)8¶V RQ DW OHDVW RQH VLGH WR EH DFFHSWDEOH IRU VDPSOH
analysis. If this criterion is met, engrave the cuvette ID number into
the top section of the cuvette (consult the lab book for the next
available ID number). Engrave a small circle in the top section of
the cuvette that identifies the optimum side determined by the
previous analysis.
5.3.5.9 Repeat 5.3.5.10 - 5.3.5.12 to optimize the remaining cuvettes.
5.3.6
Data Analysis
Environmental Quality Lab
Chemistry Procedures
SOP No.: 602
Page 6 of 11
Rev. 1
1-13-10
5.3.6.1 Qualitative Data Analysis
5.3.6.1.1 The presence of optical brighteners in a sample can be
inferred by the decrease of D VDPSOH¶V fluorescence after
H[SRVXUH WR 89 OLJKW 2SWLFDO EULJKWHQHUV ³EXUQ RXW´ YHU\
quickly, and a fluorescence decrease of 15% or greater after
a sample irradiation is indicative of their presence.
5.3.6.1.2 Use the following formula to calculate the % decrease in
fluorescence:
% Decrease = [(RFUpost-irradiation) / (RFUpre-irradiation)] x 100
% Decrease = Percent fluorescence lost after irradiation
RFUpre-irradiation = Fluorescence signal before UV exposure
RFUpost-irradiation = Fluorescence signal after UV exposure
5.3.6.2 Quantitative Data Analysis
5.3.6.2.1 Measured fluorescence can be quantitatively analyzed by use
of an 8 point calibration curve constructed using FB-28
standard diluted to the appropriate concentrations detailed
below.
5.3.6.2.2 Make a primary standard by measuring 5.0 mg of FB-28
standard and bringing it to volume with Type I water in a
100mL volumetric flask (acid leached and rinsed x10 with
Type I water) for a final concentration of 50 ppm. Store
standard in an amber glass bottle at 1-4 °C for up to 14 days.
5.3.6.2.3 Make a working standard on a daily basis by bringing a
10mL aliquot of 50 ppm standard to volume in a 1000mL
volumetric flask for a final concentration of 500 ppb.
5.3.6.2.4 Make a 250 ppb standard by bringing 50mL aliquot of 500
ppb standard to volume in a 100 mL volumetric flask.
5.3.6.2.5 Make a 100 ppb standard by bringing 20mL aliquot of
500ppb standard to volume in a 100mL volumetric flask.
5.3.6.2.6 Make a 50ppb standard by bringing a 10mL aliquot of
500ppb standard to volume in a 100mL volumetric flask.
5.3.6.2.7 Make a 25ppb standard by bringing a 5mL aliquot of 500ppb
standard to volume in a 100mL volumetric flask.
5.3.6.2.8 Make a 10ppb standard by bringing a 10mL aliquot of
500ppb standard to volume in a 500mL volumetric flask.
5.3.6.2.9 Make a 5ppb standard by bringing a 5mL aliquot of 500ppb
standard to volume in a 500mL volumetric flask.
Environmental Quality Lab
Chemistry Procedures
SOP No.: 602
Page 7 of 11
Rev. 1
1-13-10
5.3.6.2.10 Starting with a Type I water blank, run all of the
concentrations (up to 500ppb) using the same cuvette,
ensuring that the cuvette is rinsed x3 with Type I water and
x3 with sample between each analysis.
5.3.6.2.11 Obtain the regression line equation E\ HQWHULQJ WKH 5)8¶V
for each standard into a spreadsheet program (Excel or
equivalent).
5.3.6.2.12 To determine the concentration of optical brighteners in each
of the previously analyzed samples, subtract the postLUUDGLDWLRQ 5)8¶V IURP WKH 3UH-LUUDGLDWLRQ 5)8¶V and use
regression line analysis to determine the concentration of
optical brighteners in each sample.
5.3.6.2.13 Conversely, since many current publications report analysis
of field samples in units of specific detergents, it may also be
desirable to report the optical brightener concentration in
³7LGH XQLWV´ 7KLV FDQ EH DFFRPSOLVKHG E\ VXEWUDFWLQJ WKH
post-LUUDGLDWLRQ 5)8¶V IURP WKH SUH-LUUDGLDWLRQ 5)8¶V DQG
setting it up in a ratio where 100ppb liquid Tide detergent =
5)8¶V
5.4
QUALITY CONTROL
5.4.1
Quality control (QC) measures for analyzing samples are summarized in
Table 1 and are as follows:
Prior to analyzing samples, each analyst must demonstrate the ability to
generate acceptable results (i.e., demonstration of capability).
Store samples in dark at 1-4 C prior to filtering. Analyze no longer than 8
days after collection.
Any QC sample analysis (eg., method blank, laboratory replicate, field
replicate) should be subjected to exactly the same analytical procedures
as those used on individual sample analyses.
Unless otherwise specified for specific project or samples, each batch of
up to 20 samples analyzed should include at least one method blank (i.e.,
filtered and extracted deionized water, which preferably had been placed
in an empty sample bottle and stored and transported with samples) and
one sample analyzed in duplicate (i.e., at least 5% duplicates) as QC
samples.
Direct fluorometer reading (i.e., not corrected for dilution) of a sample
must fall within the range bracketed by the lowest and highest calibration
standards. Any sample reading below the lowest calibration standard¶V
concentration must be reported as less than the reporting limit. For any
GLUHFW VDPSOH UHDGLQJ DERYH WKH KLJKHVW FDOLEUDWLRQ VWDQGDUG¶V
concentration, an aliquot of the sample must be diluted until the reading
SOP No.: 602
Page 8 of 11
Environmental Quality Lab
Chemistry Procedures
Rev. 1
1-13-10
falls within the calibration range.
Table 1. Determination of Optical Brighteners by Turner Fluorometer
QC Sample or Activity
Minimum Frequency
Acceptance Criteria
Capability demonstration
Four (4) prepared
samples analyzed prior to
any customer sample
analyses
Criteria for duplicate precision
5-Point calibration
Quarterly
90-110% R (measured value of all
standards when analyzed as samples
within 10% of expected value)
Solid secondary standard
value establishment
Quarterly
Measurement after acceptable 5-point
calibration and value within 10% of
previous established value
Calibration check with
solid secondary standards
Daily prior to sample
analysis
90-110% R
Method blank
Daily prior to sample
analysis
< RL
Sample analysis
For all sample analyses
Direct sample reading within calibration
range (i.e., lowest and highest calibration
standard concentrations)
Sample duplicate
One (1) per preparation
batch
RPD < 25%
Internal PE sample
Blind PE sample
LCS =
MB =
MDL =
MS =
PE =
Samples and frequency
determined by Lab QA
Officer
Samples and frequency
determined by
accrediting agencies and
projects
laboratory control sample
method blank
method detection limit
matrix spike
performance evaluation
QC =
%R =
RL =
RPD =
Corrective Action
Repeat until acceptable
Investigate problem. Correct
any obvious problems. Repeat
calibration until acceptable.
Investigate problem. Correct
any obvious problems
including obtain new solid
secondary standards if
necessary
Investigate problem. Correct
any obvious problems
including new 5-point
calibration if necessary. Repeat
calibration check until
acceptable.
Clean analytical system and
repeat MB analysis. Identify
and eliminate source of
contamination.
If reading below range report
result as < RL. If result above
range dilute sample.
Investigate problem. If system
precision is in control, qualify
results. If system precision is
out of control, reanalyze entire
batch.
75-125% R
RPD < 25%
Investigate all unacceptable
results.
Determined by PE provider
Investigate all unacceptable
results.
quality control
percent recovery
reporting limit
relative percent difference
Environmental Quality Lab
Chemistry Procedures
5.5
6.0
SOP No.: 602
Page 9 of 11
Rev. 1
1-13-10
CORRECTIVE ACTIONS
5.5.1
If any of the QC requirements listed in Table 1 are not satisfied, the
analyst must consult with the Laboratory Director. Normally the activity
must be repeated, after corrective actions are taken to correct any
obvious problems, until the QC results are acceptable. If repeating the
process is not possible (e.g., sample spilled), the results report will
include a discussion of the problem and the client will be consulted.
5.5.2
The problem and associated corrective actions will be documented on a
Nonconformance and Corrective Action Report (see EQL SOP 201).
DATA REPORTING
6.1
REPORTING LIMITS
Reporting criteria will be determined either qualitatively by a decrease in sample
fluorescence after UV irradiation (>15% being indicative of optical brightener presence)
or quantitatively (with >50 ppb indicative of optical brightener presence) dependent on
the needs of the client.
7.0
WASTE DISPOSAL
Dispose of samples in a designated and labeled beaker and place in fume hood. Allow
liquid extracted sample to evaporate and discard glass fiber filter remnants into waste
receptacle.
Quality Assurance Project Plan
Briarcliff Acres Water Quality Study
Thomas & Hutton Engineering Co.
J: 22072
Date: 12/11/09
By: RPK
Appendix L
VIRGINIA TECH
IDENTIFYING SOURCES OF FECAL POLLUTION
IN IMPAIRED WATERS
Identifying Sources of Fecal Pollution in Impaired Waters
Principal Investigator
Dr. Charles Hagedorn, Professor
Department of Crop and Soil Environmental Sciences
330 Smyth Hall
Virginia Polytechnic Institute and State University
Blacksburg, VA 24061-0404
Voice: 540-231-4895
Fax: 540-231-3431
E-mail: [email protected]
1. Critical Statewide Water Problem
Nearly 65% of the impaired stream segments in Virginia are contaminated by fecal pollution.
Fecal bacteria are the major cause of impairmentVLQ9LUJLQLD¶VZDWHUZD\VDFFRUGLQJWRWKH
Virginia Department of Environmental Quality (DEQ, 1998), with agriculture and urban runoff
listed as the primary sources of contamination (http://www.deq.state.va.us/water/98-305b.html).
This fecal contamination results in increased health risks to persons exposed to the water,
degradation of recreational and drinking water quality, and shellfish bed closures. In Virginia,
DEQ and the Department of Conservation and Recreation (DCR) face a mandate from USEPA to
complete numerous TMDLs within a short timeframe (over 600 within each 10-year period
beginning in 2000). Virginia contains approximately 49,000 miles of streams and waterways and
less than one-third of these waterways have been adequately monitored to date by the DEQ
(FORVA, 2001). Some 2,166 miles are currently listed as impaired, including large sections of
the Chesapeake Bay and adjacent shellfish-bearing waters. Based on results from those waters
that have been adequately monitored, it is believed that thousands of additional miles of
impairments will be added as monitoring is expanded in future years. Estimates vary widely
regarding the time needed to fully comply with the TMDL requirements for surface waters, but
most estimates are in the 30 to 50 year range (FORVA 2001). Regulatory agencies are slowly
coming to the realization that microbial source tracking (MST) methodology will have to be used
in virtually every fecal impairment in order to accurately determine the source(s) of that
pollution, and that watershed modeling approaches alone will not be sufficient (USEPA, 1999a).
Until recently the source(s) of fecal pollution in water could not be readily determined, but
source tracking technology has now made such determinations possible, reasonable, and
accurate. Knowledge of the type (or types) of pollution sources will aid in the restoration of
water quality, reduce the danger of infectious disease from exposure to recreational waters, and
1
with best management practices (BMP) implementation, reduce the amounts of nutrients that are
removed from land and transported into surface waters.
2. Project Benefits
:KLOHPDQ\RI9LUJLQLD¶VZDWHUVWHVWSRVLWLYHIRUIHFDOFROLIRUPVQRXVHIXOZDWershed restoration
plans or accurate TMDLs for bacteria can be developed until the source(s) of the fecal
contamination can be identified with confidence. If our procedures can reliably and accurately
separate different fecal sources (e.g. human, cattle, pets, birds, wildlife), they can provide an
essential tool to those who are responsible for public health and environmental quality and are
charged with reducing water pollution, protecting public health, and improving water quality. By
using appropriate sampling frequency protocols it should be possible to develop streammonitoring plans utilizing source tracking that will adequately characterize fecal bacteria
pollution sources for any given watershed. MST projects can provide a mechanism for non-point
source (NPS) problem identification in fecal contaminated waters and provide the necessary
information for determining TMDLs for fecal bacteria based on specific source(s) of the bacteria.
The possibility of establishing TMDLs for fecal bacteria by specific source(s) is both novel and
unique. Target audiences for BST results include the many small communities and the
agricultural industry located in watersheds, local and state officials, and regulatory agencies. The
MST community has the potential to provide agencies responsible for water quality and public
health with a mechanism to determine sources of fecal contamination and, until such sources of
pollution are identified, the risk to communities cannot be accurately assessed and water quality
improvements will remain a hit-or-miss affair.
:KLOHWKHPDLQ³GULYHU´IRUZDWHUTXDOLW\LPSURYHPHQWLQUXUDODUHDVLV70'/VLWLV
stormwater for urban areas. Regarding stormwater, runoff from storms has been identified as a
potential threat to human and ecosystem health due to the high levels of chemical and biological
contaminants it contains that have been directly linked to disease outbreaks (Curriero et al., 2001;
Gaffield et al., 2003), toxic effects in aquatic life (Bay et al., 2003; Heaney et al., 1999), and
dramatic negative impacts on water quality (Ahn et al., 2005; Makepeace et al., 1995). As
precipitation washes over land, it picks up and transports a variety of chemicals, pesticides,
metals, petroleum products, sediment, and human and animal fecal wastes. Knowledge of the
composition of the resultant runoff, as well as its delivery pathways and distribution in the
environment, is crucial in managing the overall risk associated with stormwater runoff.
Discharge of stormwater runoff onto recreational beaches in the US is particularly
problematic in terms of public health, as it is the largest known cause of beach closures and
advisories in the US (Dorfman, 2006). Many of these advisories (75% in 2005) are in response to
elevated fecal indicator bacteria (FIB) levels that exceed USEPA recommended beach water
quality standards (Dorfman, 2006; USEPA, 1986). Furthermore, US recreational waters serve as
a known route of exposure to human pathogens, with 95 documented recreational waterassociated outbreaks occurring from 1996-2000 (Arnone and Walling, 2007). Evidence from
epidemiological studies of recreational water-associated health effects suggests a causal doserelated relationship between gastrointestinal symptoms and FIB counts and strong relationships
between urban runoff and illness (Pruss, 1998). Therefore, mitigating stormwater runoff to
decrease loading rates of FIB and viral, bacterial, and protozoan pathogens to recreational
beaches is a direct way to improve beach water quality and protect public health.
2
3. Sources of Fecal Pollution
7KHUHDUHVHYHUDOGLIIHUHQWSRWHQWLDOVRXUFHVRIIHFDOFRQWDPLQDWLRQLQ9LUJLQLD¶VZDWHUV*URXQG
and surface waters (and potentially well water) can become contaminated with septage from
leaking or faulty septic drainfields or tanks. Surface waters can become contaminated from a
variety of sources, including surface run-off of livestock and poultry manure and litter that has
been applied as fertilizer, or through direct stream contamination by livestock (Alderisio and
DeLuca, 1999). Untreated poultry waste can also enter surface waters, either from surface runoff of improperly treated manure used as fertilizer, or from improperly impounded manure piles.
Fecal material from wild animals may also enter surface waters by run-off or direct deposit. In
more urban landscapes, storm runoff from impervious surfaces carries waste and fecal bacteria
from sources such as dogs and birds into surface waters, and dog wastes can be present in
substantial quantity along parks and walkways adjacent to streams. Each of these sources of
pollution are of concern, because the contamination of natural waters with untreated or partially
treated fecal material results in an increased risk of transmission of diseases to the humans who
use those waters. Many pathogenic bacteria, viruses, and protozoa are passed from one host to
WKHQH[WE\WKH³IHFDO-RUDOURXWH´RIWUDQVPLVVLRQDQGZDWHUXVXDOO\VHUYHVDVWKHFDUULHUIRUWKHVH
organisms. Of particular concern are bacterial pathogens such as Salmonella, Shigella,
diarrheagenic Escherichia coli (including E. coli 0157:H7), Campylobacter and Vibrio. Many
viruses can be transmitted by water, including poliovirus, rotaviruses, Norwalk viruses, and
hepatitis A and E viruses. Protozoan pathogens that can be transmitted by water include Giardia
and Cryptosporidium.
Different pathogens are often transmitted by different sources. For example, Giardia and
Cryptosporidium outbreaks are often associated with cattle, and E. coli 0157:H7 outbreaks are
usually tied to beef products. Giardia LVDOVRVSUHDGE\GHHUEDFNSDFNHU¶VGLVHDVHDQG&DQDGD
geese feces have been found to contain Cryptosporidium, Giardia, and Campylobacter.
Salmonellosis is commonly associated with hogs (as well as Vibrio), poultry, and waterfowl, and
Campylobacter is widespread in poultry (Holmberg et al., 1984). Most viral gastroenteritis is
caused by contact with human feces. All of these organisms pose a substantial risk to human
public health. Thus, knowledge of the type of pollution is an important factor in determining the
degree of risk. It would be desirable to be able to determine the sources of the fecal material,
both to determine the risk to the people who are exposed to the waters, and source identification
will allow development of strategies to reduce pollution levels.
Livestock and poultry producers often carry the largest share of the burden of correcting
fecal contamination problems. Livestock and poultry producers in particular, and the public, in
general, will benefit greatly from improved microbial source tracking (MST) methodologies.
More cost-effective techniques will improve modeling efforts that are used to determine how
fecal contamination problems are to be corrected. In addition, these techniques will support
stream water quality monitoring efforts, presenting a more complete picture of stream water
quality.
MST should be used in every TMDL project that contains impairments due to fecal
bacteria. When this is not done, decisions about sources can be made based on other factors. For
example, federal and state officials performed a TMDL project on the Cottonwood Creek
watershed in Idaho, without including a source-tracking component (US-EPA 1999b). At public
meetings regulatory officials reported that, based on professional judgment, livestock was a
major contributor to fecal pollution in the watershed. After ranchers raised serious objections to
this conclusion, the conclusion was changed to indicate that wildlife (probably elk) were the
major fecal contributors to the impaired stream. It did not seem to matter that elk were well
3
away from the creek while in their summer range. This is an example of what can happen when
MST is not used, and actual results from employing MST should be preferred in place of opinion
whenever possible.
4. Project Scope, Monitoring, and Source Tracking
The scope of this MST program is to use a variety of source tracking methodologies to determine
source(s) of fecal pollution in natural waters. Numerous source-tracking methods are under
development for use in determining sources of fecal pollution in watersheds. Both molecular
methods such as ribotyping, pulse-field gel electrophoresis, and randomly amplified polymorphic
DNA, and non-molecular methods such as ARA, nutritional patterning, cell wall fatty acid
analysis, and strain specific coliphages have been used (and published) in source tracking
projects (for a complete list see http://filebox.vt.edu/users/chagedor/biol_4684/BST/BST.html).
4.A. Monitoring by Membrane Filtration
Bacterial monitoring using Escherichia coli (E. coli) populations from all freshwater samples is
performed with EPA Method 1603: Escherichia coli (E.coli) in Water by Membrane Filtration Using
Modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC) (September 2002)
(PDF, 13 pp., 129 KB) - This method (EPA 821-R-02-023) combines information from a 1985
publication (Test methods for Escherichia coli and Enterococci in Water by the Membrane Filter
Procedure, EPA 600-4-85-076) and a subsequent March 2000 manual (Improved Enumeration
Methods for the Recreational Water Quality Indicators: Enterococci and Escherichia coli,
EPA/821/R-97/004). Method 1603 is a revised membrane filter (MF) procedure, a single-step
method that uses one medium, modified mTEC Agar, and does not require the transfer of the
membrane filter to another medium or other substrate. For marine water samples, Enterococcus
(EPA Method 1600) is used. EPA Method 1600 provides a direct count of enterococci in water based
on the development of colonies on the surface of a membrane filter. A water sample is filtered
through the membrane that retains the bacteria. Following filtration, the membrane containing the
bacterial cells is placed on a selective medium, mEI agar, and incubated for 24 hours at 41°C. All
colonies (regardless of color) with a blue halo are recorded as enterococci colonies. Magnification
and a small fluorescent lamp are used for counting to give maximum visibility of colonies.
4.B. Monitoring by Most-Probable Number
Colilert-18® is used for enumeration of total coliforms and E. coli DQG(QWHUROHUWŒLVXVHGIRU
Enterococcus (IDEXX Laboratories, Westbrook, ME). Duplicate samples are typically diluted to
10 ml, 1 ml, or 0.1 ml per 100 ml with deionized water. The reagents are added to each sample,
dissolved by shaking 10 times, poured into a Quanti®-Tray/2000 tray, and incubated overnight at
35°C for Colilert-ŠDQGƒ&IRU(QWHUROHUWŒDVUHTXLUHGE\PDQXIDFWXUHU¶VLQVWUXFWLRQV
7UD\VDUHFRXQWHGDFFRUGLQJWRPDQXIDFWXUHU¶VLQVWUXFWLRQVDQGPRVWSUREDEOHQXPEHU031
per 100 ml of samplHLVGHWHUPLQHGXVLQJWKHPDQXIDFWXUHU¶V031WDEOHV)RUHDFKVHWRI
duplicate MPN values (per dilution), average MPN values are calculated using the dilution set
that yielded usable counts, with preference to the least diluted sample set if more than one set
was usable.
5. Source Tracking Protocols
Our lab at Virginia Tech uses five different MST methods, depending on the project and the
goals and specific objectives of project sponsors.
4
5.A. Gene Expression Systems in the Virginia Tech Bacterial Source Tracking Program
i. Antibiotic Resistance Analysis (ARA)
ARA has been performed on the enterococci, fecal coliforms, and E. coli (Harwood et al, 2000;
Hagedorn et al., 1999; Wiggins, 1996; Wiggins et al., 1999). This method relies on different
antibiotic resistance patterns in fecal bacteria that can be related to specific sources of fecal
pollution, and is predicated on the rationale that antibiotics exert selective pressure on the fecal
flora of the animals that ingest or are treated with the antibiotic(s), and that different types of
animals receive differential exposure to antibiotics. Resistance patterns are highest in humans,
moderate in livestock, pets, and poultry, and low in birds and wildlife. Benefits of ARA include
use of simple laboratory techniques, requiring only basic equipment, and can be performed at a
relatively low cost compared to most other methods.
Twenty-eight concentrations of seven antibiotics are used to determine antibiotic
resistance patterns in E. coli. Each of the twenty-eight antibiotic/concentrations is added
separately to flasks of autoclaved and cooled Trypticase Soy Agar (TSA, BBL) from stock
antibiotic solutions to achieve the desired concentration, and then poured into sterile 15x100mm
petri dishes. Control plates (no antibiotics) are included with each set. Isolates are transferred
from the microwell plate using a stainless steel 48-prong replica plater (Sigma). The replicator is
flame-sterilized (95% ethanol) after inoculation of each TSA plate. The inoculant is allowed to
soak into the agar and the plates are then incubated for 48 hours at 37oC. Resistance to an
antibiotic is determined by comparing each isolate to the growth of that isolate on the control
plate. A one (1) is recorded if that isolate grew (a round colony, mostly filled) and a zero (0) is
recorded for no growth.
To date, ARA has been used in more source tracking projects around the US than any
other method. In addition, high levels of separation between known source bacterial isolates
have been found comparable to those reported for molecular methods (Parveen et al., 1997;
Bernhard and Field, 2000; Dombek et al., 2000).
ii. Nutrient Utilization Patterns (NUP)
The NUP system is based on nutrient utilization profiles (or fingerprints). It is a nearly foolproof
system because it uses an electronic plate reader that removes judgment decisions by laboratory
personnel when evaluating plates (Hagedorn et al., 2003). In the NUP system, each well in a 96well microplate contains a single nutrient source (one well is a water blank) and a metabolic dye
(tetrazolium violet). Each isolate of E. coli or Enterococcus is grown for 12 to 24 hrs at 37oC on
commercial Blood Agar (BBL) and then diluted to a standardized concentration in a liquid
medium that contains all nutrients for growth except a carbon source. The isolate from the Blood
Agar plate is transferred to a liquid medium with a sterile cotton swab, and enough is transferred
to reach an optical density of 0.23 to 0.25 absorbance on a spectrophotometer. Then, 150 l of
the liquid medium is added to each of the 96 wells in a plate with an automated 8-row pipettor.
After incubation for 12 to 24 hrs at 37oC, a color forms (from the metabolic dye) in any well
where the isolate was able to use the carbon compound in that well and grow. The pattern of
positive wells, out of a total of 95, is used as a metabolic profile. Positive wells are recorded as
growth (1) and clear wells as no growth (0). The results are determined and recorded with an
electronic plate reader connected to a computer
5.B. Gene Fingerprint Systems
i. Pulsed-Field Gel Electrophoresis (PFGE)
5
The PFGE procedure is the same as that reported by Simmons (Simmons and Herbein, 1998;
Simmons et al., 1995). Pulsed field gel electrophoresis differentiates closely related isolates of
the same species by detection of variations in the position of chromosomal restriction sites. In
this technique chromosomal DNA is carefully extracted and cleavage of the DNA is carried out
XVLQJD³UDUHFXWWLQJ´UHstriction enzyme such as NotI. The discrete fragments of DNA are
separated using pulsed-field gel electrophoresis, which resolves the fragments into distinct bands.
The gel is nonspecifically stained with a dye such as ethidium bromide, allowing comparison of
the banding pattern of various isolates. The molecular weight of each DNA band is then
determined by comparison with a standard DNA ladder. The banding pattern of a particular
isolate is the set of variables that is analyzed by statistical analysis. Dr. George Simmons closed
his research laboratory in the Biology Department at Virginia Tech in July 2000. His PFGE
library, E. coli culture collection (some 2,800 isolates from known sources) and BioRad PFGE
HTXLSPHQWLVQRZORFDWHGLQ'U+DJHGRUQ¶Vlaboratory. The culture collection contains a large
selection of isolates from wildlife sources plus some from humans, pets, and livestock. This
collection served as the basis for a much larger E. coli PFGE library and a new Enterococcus
library to be deYHORSHGLQ+DJHGRUQ¶VODERUDWRU\3)*(KDVQRZEHHQXVHGDVDFURVV-validation
tool in numerous source tracking projects around the US.
For source tracking purposes, the gene expression and PFGE profiles are analyzed by
discriminant analysis (DA) using JMP-In statistical software (version 7.0, SAS Inc). First,
known source isolates are analyzed and placed in various categories such as human, livestock,
wildlife, or cow, deer, horse, waterfowl, etc., depending on the level of classification desired
when developing the known source library. Discriminant analysis (DA) assigns a predicted
source to each isolate: human and animal in a 2-way split, and human, livestock, urban (dogs and
cats), or wildlife in a 4-way split, or a larger split based on specific sources (deer, cow, goose,
etc.) as a result of the fingerprints of the isolates in the library. Unknown source isolates (isolates
from water samples) are then compared against the library to classify them by source, with the
match probability set at 80% or greater correct rate.
ii. Bacteroides Human-Specific (HS) DNA-Marker
This method is one of the newer approaches and does not require either a host-origin library or
cultivation of the target microbe as DNA is extracted directly from water samples. We use the
recently developed PCR method for Bacteroides thetaiotamicron, which is an exciting candidate
for an alternative indicator of fecal contamination. Bacteroides sp. make up approximately onethird of the human fecal microflora, considerably outnumbering Enterococcus and E. coli. The
Bacteroidales group belongs to a group of nonspore forming, gram negative, obligate anaerobes,
so there is little concern over regrowth in the environment. More importantly, a range of human
and animal specific Bacteroides sp. markers have been developed, increasing the value of this
potential indicator (Field et al. 2003). The species B. thetaiotamicron is highly abundant in
human fecal waste, has been demonstrated to be tightly related to the presence of human fecal
contamination, and is typically found in very low numbers or not at all in animal feces. Finally,
bacterial markers such as Bacteroides sp. have been shown to be potentially useful source
tracking tools. In Griffith et al. (2003) the Bacteroides sp. markers correctly identified human
sources of fecal pollution when present in mixed water samples delivered blind to the laboratory.
This method does not require a library for comparison and does not require cultivation of
microbes as the DNA is extracted directly form water samples and then processed with PCR and
the correct human-origin primers to determine if complimentary DNA sequences are present in
the DNA extracted from the water.
6
5.C. Chemical Detection Systems
i. Fluorometry
Our program was the first to evaluate the use of a fluorometer in estuarine and coastal zone
environments to determine if the equipment could detect a human waste signature. The
fluorometer detects compounds that fluoresce under ultraviolet light such as fecal sterols,
detergent surfactants and optical brighteners. Optical brighteners in laundry and dishwashing
detergents fluoresce when exposed to certain ultraviolet wavelengths, so water samples that
fluoresce under those same wavelengths are contaminated by residues from laundry and
dishwashing detergents (human sources). There are at least two major potential human sources
of contamination that could contain optical brighteners, and these include leachates from
improperly functioning on-site wastewater systems (OWS) and leaking pipes from community
wastewater treatment systems. In rural areas where the majority of homes are served by on-site
systems, optical brighteners in water samples indicate failing conditions within OWS in close
proximity to the sampled bodies of water.
Detectors from different manufacturers were evaluated, and the portable fluorometer from
Turner Designs, Inc., performed the best in both laboratory and field tests. The detector located
fluorescent plumes in water samples taken in coastal rivers where a human signature was known
to exist based on microbial source-tracking results. Additionally, the detector correctly identified
samples in controlled laboratory tests that had been spiked with detergents and/or septage.
Samples without septage or detergents (or containing detergents without optical brighteners) all
failed to fluoresce. The instrument was then used successfully on a variety of waterways (both
salt and fresh) where human sources of pollution were suspected or could be confirmed with
microbial source tracking technology. In larger bodies of water, fluorescent plumes could be
identified and mapped with the fluorometer, and then traced back to the shore and directly to
locations that appeared to be the source of the pollution. The fluorescent signals appeared to be
stable over seasons, storage in refrigeration for at least four months, and over different water
conditions (Hagedorn and Weisberg, 2009). Whenever fluorescent plumes were found,
microbial source tracking tests demonstrated a human signature in every case where source
tracking was performed.
6. Molecular Analyses of Water Samples
For molecular analyses, duplicate 100 ml samples are YDFXXPILOWHUHGWKURXJKȝP
polycarbonate (PC) filters (GE Osmonics, Minnetonka, MN). The filters are placed into sterile,
DNase/RNase-free microcentrifuge tubes and stored at -80°C. DNA extractions are performed on
WKHILOWHUVXVLQJWKH8OWUD&OHDQŒ6RLO'1$,VRODWLRQ.LW0R%LR Laboratories, Inc., Solana
Beach, CA) following the protocol for maximum yield, with extracts stored at -20°C.
Quantitative PCR (QPCR) is performed on extracted DNA using the fecal Bacteroides spp.
primer-probe set (Converse et al., 2009). The DNA template in each reaction rangee from 1-50
ng, determined fluorometrically with PicoGreen (Invitrogen Carlsbad, CA) using a Turner TBS380 Fluorometer. QPCR is conducted on a SmartCycler® II (Cepheid, Sunnyvale, CA) using
TaKaRa Ex Taq version 2.1 (Mirus Bio Madison, WI), ZLWKHDFKȝOUHDFWLRQFRQWDLQLQJWKH
IROORZLQJȝPROO-1 dNTPs, 4 mmol l-1 MgCl2, 0.05 U Taq polymerase, 1X Taq buffer, and
ȝO'1$WHPSODWH5HDFWLRQ conditions are as follows: 95°C for 2 min, followed by 45 cycles of
95°C for 15 s and 60°C for 45 s. Quantification was conducted using Bacteroides
thetaiotaomicron cells (Converse et al., 2009) enumerated via epifluorescence microscopy
(Noble and Fuhrman, 1998). Quantified cells are used to establish a 4-log standard curve, with
7
reactions run in duplicate. QPCR amplification efficiencies of >90% and R2 values of >0.95
were documented for all standard curves.
Conventional PCR is performed on the DNA extracts using the human-specific
Bacteroides/Prevotella PDUNHUUHIHUUHGWRLQWKLVPDQXVFULSWDV³+6´(Bernhard and Field, 2000)
using primers targeting a segment of the 16S rRNA gene from the human feces-specific group
(HS183F and BAC708R). PCR master mix is composed of 1.25 U Hot Master polymerase
(Eppendorf, Westbury, NY), 1X Taq Polymerase self-adjusting magnesium buffer (Eppendorf,
Westbury, NY), 1 ȝPROO-HDFKSULPHUȝPROO-G173VDQGQJȝO-1 191 bovine
serum albumin. PCR is performed on a Genius thermal cycler (Techne, Burlington, NJ) using the
following cycling parameters: 2 min at 94°C, then 30 cycles of 94°C for 30 s, 58°C for 30 s, and
68°C for 30 s, followed by 5 min at 68°C. PCR products are visualized in a 1.2% agarose gel
VWDLQHGZLWKȝJPO-1 ethidium bromide and compared to a 100-bp DNA ladder (Promega,
Madison, WI).
7. Statistical Analyses
Normality tests on non-transformed data from previous projects indicate that the FIB data are not
normally distributed. Therefore, FIB measurements are log10 transformed prior to all statistical
analyses. Normality tests are conducted for the datasets to select the appropriate statistical
analyses. Independent sample t-tests examine significant differences (alpha (Į WZRtailed) between FIB concentrations for storm and baseflow samples and for seasonal
FRPSDULVRQV/HYHQH¶VWest for equality of variances is used to determine whether equal variances
ZHUHRUZHUHQRWDVVXPHGĮ WZR-tailed). Seasonal differences for FIB concentrations are
determined using the one-way ANOVA with post-hoc comparison (e.g. Abdi, 2007). A
significant relationship is determined with respect to an alpha of 0.05 (two-tailed). The percent of
samples positive for the molecular markers is calculated by dividing the number of samples with
positive detection of target by the total number of samples and multiplying by 100. Previous and
ongoing research is being conducted on fecal Bacteroides spp. QPCR data to establish
relationships among fecal contamination types. Preliminary work (Coulliette and Noble, 2008)
VXJJHVWVDQ³DFWLRQWKUHVKROG´RIFHOOVSHUPODVGHWHUPLQHG by QPCR), i.e. water
samples exhibiting concentrations of fecal Bacteroides spp. Results above this concentration
should be further examined for the potential presence of human fecal contamination using other
³WRROER[´DSSURDFKHVVXFKDVWKRVHSUHVHQWHGE\1REOHHWDO 2006 and Hagedorn et al., 2007).
Furthermore, ratios of Enterococcus spp. and E. coli and the fecal Bacteroides spp. numbers as
determined by QPCR have been suggested to potentially be predictive of source (Converse et al.,
2009).
8. Quality Assurance (QA) / Quality Control (QC)
Our MST laboratory typically will implement a formal quality control program to substantiate the
validity of the collected analytical data. Components usually include:
i. Field Duplicates
Ten percent of all field-collected samples will be duplicates. Each field duplicate will be
collected at the same time as the regular sample from that location, placed into a common
container, and then the field sample and field duplicate will be made from that single sample. If
situations should arise where the results for a given duplicate are noticeably different from its
companion sample (greater than 10% after log normalization of the plate counts), then the
statistical procedure described in Section 1010 B. Statistics, Part 1-3, (APHA, 1998) can be used
8
WRGHWHUPLQHLIWKH³RXWOLHU´UHVXOWVKRXOGEHUHMHFWHG This procedure provides a basis for
GHFLGLQJWRNHHSRUUHMHFWRXWOLHUVDVQRGDWDVKRXOGHYHUEHDUELWUDULO\UHMHFWHG$³7´VWDWLVWLFLV
calculated for the set of data from a particular site, resulting in an average and standard deviation
for every sampling site. Depending upon the desired level of accuracy, data outliers that fall
outside either a 10%, 5%, or 1% level of significance (outside of the standard deviation) can be
either accepted or rejected. It will be necessary to evaluate the results for each sampling site over
a period of time before sufficient data will be available to obtain a reliable average and standard
deviation. It may be necessary to have 2 or 3 years of results so that a reasonable average and
standard deviation for each individual site can be determined. Any data that appears to be a
possible outlier can then be compared against a meaningful average and a decision made at that
time regarding including or rejecting the apparent outlier. The same procedure is used for both
duplicate field samples and ODERUDWRU\UHSOLFDWHV2XWOLHUVDUHLGHQWLILHGRQO\DIWHUD³WUDFN
UHFRUG´IRUHDFKVDPSOLQJVLWHKDVEHHQREWDLQHGVRWKDWDUHOLDEOHVLWHDYHUDJHDQGVWDQGard
deviation can be developed.
ii. Laboratory Replicates, Standards, and Precision
Ten percent of all positive sample results will be retested. The average and range of the
cumulative retested samples will be performed so that, as each additional set is retested, it can be
used as a precision check. Any results that are outside of the cumulative range indicates loss of
precision and that a reevaluation of the retesting procedure is needed (U.S. EPA, 2005).
In microbiological analyses, reference and performance standards are usually not
incorporated into QA/QC plans since microbes are living organisms and any standards will
change as cells either die or reproduce, depending upon the conditions under which the standards
are stored or maintained. Standard Methods for the Examination of Water and Wastewater (20th
Edition, referenced under Section 4.4.1) does not recommend reference or performance standards
for microbiological analyses. The EPA Manual (1978) indicates that reference and performance
standards should be run once a year only when available. While there is no reference standard
available for Enterococcus, the positive control with a reference strain of Enterococcus
(described under Section 4.4.4), diluted to a specific number of cells before filtration, will serve
as a performance standard for each set of twice yearly samples.
For measurement of analyst precision, duplicate analyses are performed on the first 15
typical samples with positive results. Each set of duplicates is performed by the same analyst.
The counts are log normalized and the range and the mean of the range is then calculated for each
pair of transformed duplicates. After this initial test, 10% percent of all positive sample results
are retested and the range and mean of the range is calculated for each pair of transformed
duplicates. If the range of the subsequent pairs is greater than 3.27 times the range mean, then
analyst precision is out of control and all analytical results since the last precision check must be
discarded. The analytical problem must be identified and resolved before doing further analyses.
This approach is specified in:
For each set of samples, field duplicates, lab replicates, and positive controls, after
membrane filtration and incubation all plates will be counted by two analysts, consisting of the
laboratory supervisor and one assistant. Comparison of the counts obtained by the two analysts
will provide cross-validation of the counts. Ten percent of the plates will be recounted by each
analyst, and the second counts should differ by no more than 5% for the same analyst and no
more than 10% between the two analysts.
9
iii. Field Blanks
In accordance with U.S. EPA guidance (U.S. EPA, 2005), 5 percent of all field-collected samples
will be field blanks. Field blanks will consist of sterile water placed into a laboratory sample
bottle and placed into the decontaminated plunge pole or other sampling device that mimics the
collection process. Field blanks will be submitted to the laboratory as regular numbered samples.
iv. Method Controls and Validations (with Enterococcus as an example)
In order to verify that no contamination is introduced during the process of sample collection and
analysis, both positive and negative controls will be included with each sample batch.
Positive controls are performed with the reference strain Enterococcus faecalis (Andrewes and
Horder, PCI 1305, American Type Culture Collection No., 10741, ATCC, Manassas, Va). A
pure-culture suspension of the reference strain is prepared in dilution buffer equivalent to
approximately 10 viable cells/mL and is then filtered with each batch of samples. As ten mL of
this reference sample is filtered, approximately 100 Enterococcus colonies should appear on the
mEI agar plates after incubation. A suspension of the reference strain is also streaked with an
inoculating loop onto a plate of mEI agar whenever a new batch of media is either prepared in the
lab or purchased from a commercial supplier. Negative samples include the same dilution buffer,
sterilized, but no bacteria have been added to these sterile blanks so there should be no colonies
on the mEI plates after incubation. Positive and negative controls will be analyzed with each set
of samples collected twice annually. Negative controls will serve as an indicator of
contamination and will be included at the start of each set of samples and following every tenth
sample.
The U.S. EPA definition of fecal enterococci is based on hydrolysis of esculin, growth on
brain heart infusion agar in the presence of 6.5% NaCl, and a negative reaction to the catalase
test. Five percent of colonies on mEI plates from each sample will be verified for quality
assurance and for source tracking purposes. Obtaining the number of colonies from the mEI
plates needed for source tracking usually results in more that 5% of the colonies being verified as
Enterococcus. Colonies on mEI plates are confirmed as Enterococcus prior to performing
microbial source tracking by placing the colonies into wells in a microtiter plate containing
enterococcosel agar. If the suspected colony is an Enterococcus, it will hydrolyze esculin in the
enterococcoseal broth and produce a black color in the wells after incubation. As mEI is a highly
selective agar medium for the enterococci, it is very rare to select a colony that will not be
confirmed as Enterococcus. All esculin-positive cultures are transferred to brain heart infusion
agar containing 6.5% NaCl. After incubation, those colonies that are esculin positive and grew
in the presence of 6.5% NaCl are tested for catalase by adding a drop of 8.82M H2O2 to each
colony on the brain heart infusion agar plates. No reaction is a negative test and is the final step
in verification that a selected isolate is an Enterococcus.
All membrane filters will be obtained from a manufacturer who certifies that the filters
exhibit full retention and recovery of the organisms to be cultivated, stability in use, and are free
of any chemical extractables that may inhibit bacterial growth and development.
9. References
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discharges to Santa Monica Bay. Mar. Environ. Res. 56 (1-2), 205-223.
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Coastal Waters by Using Host-Specific 16S Ribosomal DNA Genetic Markers from Fecal
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precipitation and waterborne disease outbreaks in the United States, 1948-1994. American J.
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11
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methods using mixed fecal sources in aqueous test samples. J. Water Health 1:141-151.
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Pollution in a Rural Virginia Watershed. Appl. Environ. Microbiol. 65:5522-5531.
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9. Principal Investigator
Dr. Charles Hagedorn (http://fbox.vt.edu/cals/cses/chagedor/CH.html) is Professor of Soil
Microbiology in the Department of Crop and Soil Environmental Sciences at VT. He has
received awards for outstanding service from the USDA, the American Society for Microbiology,
and the USEPA. His research and scientific expertise has been recognized by awards of 67 state,
private, and federal competitive research grants; publication of 126 refereed journal articles; 17
invited review articles; 8 invited book chapters; co-editor of one book; 55 invited presentations at
international, national, and state conferences; 21 invited memberships on proposal review panels;
12 refereed bulletins; and 135 abstracts and presentation papers. Fourteen Ph.D. and twenty-one
M.S. students have completed degrees under his direction and he has generated in excess of
$4,645,000 in external grants and contracts to support his environmental microbiology program,
including public health, microbial pathogens in the environment, waste management, the impact
of releasing genetically modified organisms into the environment, and determining sources of
fecal pollution in water.
Over the past fifteen years, he has been involved in the development of microbial source
tracking methods, and has deployed these methods to determine sources of fecal pollution in 40+
projects in Virginia and 14 in other states. His research program on source tracking has been
supported by competitive awards from NSF, USDA-NRI, EPA, NOAA, and USGS. His previous
source tracking research projects included such diverse locations as Clarke County and
Northumberland County, Va., Washington, D.C., Myrtle Beach SC, Oyster Bay, NY, Huntington
Beach, CA, Nashville, TN, and Tampa Bay, FL. He participated in all three national method
comparison studies that have been performed to compare and contrast the different
methodologies being developed for source tracking. Most recently he has been involved in
development of a microbial source tracking research program on China's Yangtze River, with the
Institute of Hydroecology, Chinese Academy of Sciences, and Zhejiang University, P.R. China.
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