Buzzards Bay Action Committee Illicit Connection Detection and Stormwater Quality Monitoring in the Buzzards Bay Watershed Quality Assurance Project Plan Jeff Osuch, Stormwater Collaborative Project Manager Buzzards Bay Action Committee Joseph Costa, Executive Director Buzzards Bay National Estuary Program Prepared by Bernadette Taber, Stormwater Specialist Buzzards Bay National Estuary Program Prepared for EPA New England Regional Laboratory 11 Technology Drive North Chelmsford, MA 01863-2431 Date: March 16, 2016
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Buzzards Bay Action Committee
Illicit Connection Detection and Stormwater Quality Monitoring
in the Buzzards Bay Watershed
Quality Assurance Project Plan
Jeff Osuch, Stormwater Collaborative Project Manager
Buzzards Bay Action Committee
Joseph Costa, Executive Director
Buzzards Bay National Estuary Program
Prepared by
Bernadette Taber, Stormwater Specialist
Buzzards Bay National Estuary Program
Prepared for
EPA New England Regional Laboratory
11 Technology Drive
North Chelmsford, MA 01863-2431
Date: March 16, 2016
Buzzards Bay Action Committee
Illicit Connection Detection and Stormwater Quality Monitoring
1.3 Distribution List ...............................................................................................................................................5
1.5 Problem Definition/Background ......................................................................................................................9
1.6 Project/Task Description and Schedule............................................................................................................9
1.7 Data Quality Objectives (DQOs) ...................................................................................................................17
1.8 Special Training Requirements/Certification .................................................................................................17
1.9 Documentation and Records ..........................................................................................................................17
2.0 Data Generation and Acquisition .......................................................................................................................21
2.1 Sampling Process Design ...............................................................................................................................21
2.10 Data Management ........................................................................................................................................27
3.0 Assessment and Oversight ..................................................................................................................................27
3.1 Assessments and Response Actions ...............................................................................................................27
4.0 Data Validation and Usability ............................................................................................................................28
4.1 Data Review, Verification, and Validation ....................................................................................................28
4.2 Verification and Validation Methods .............................................................................................................28
4
Tables
Table 1. Project Organization ...............................................................................................................................9 Table 2. Monitoring Parameters .........................................................................................................................11 Table 3. Project Schedule ...................................................................................................................................18 Table 4. Laboratory Tests - Bacteria, New Bedford Health Lab and Barnstable Co. Health and Environment 18 Table 5. "In the field" Field Tests .......................................................................................................................19 Table 6. "In house" Field Tests ...........................................................................................................................19 Table 7. Other Laboratory Tests (future optional tests), Barnstable County Health and Environment ..............20 Table 8. Monitoring Events by Municipality .....................................................................................................20 Table 9. Project SOPs .........................................................................................................................................23 Table 10. Water Quality Parameters and Indicators .............................................................................................23 Table 11. Supply Inspection Requirements and Acceptance Criteria...................................................................27
Monitoring data collected by the Buzzards Bay Stormwater Collaborative's Field Sampling Teams (the Field
Teams) will meet the following water quality objectives:
The Field Teams will:
collect water quality data to characterize bacterial loading in areas closed to shellfishing and/or areas that
are impaired for bacteria (monitoring sites as depicted in Figure 3through Figure 7).
collect water quality data to identify illicit connections in the areas closed to shellfishing and/or areas that
are impaired for bacteria (monitoring sites as depicted in Figure 3 through Figure 7) that may be primary
contributors to pollutants in Buzzards Bay.
The methods and approaches utilized by the Collaborative are aimed at meeting the required levels of precision,
accuracy, field blank cleanliness, and detection limits. The minimum performance criteria for the bacteria
sampling is given in Table 4, for field tests sampling in Table 5 and Table 6 and the optional future tests in Table
7, below.
1.8 Special Training Requirements/Certification
This sampling program consists of standard field sampling techniques, field analyses, laboratory analyses, and
data validation techniques. All field personnel on the Field Sampling Teams will be required to complete a brief
training program to ensure all field sampling protocol is followed as specified in this Plan, as described in Section
B of this Plan. The Project Manager or Stormwater Specialist will maintain a signup sheet to include the name of
the trainee, date of training, and contact information
1.9 Documentation and Records
Field data will be recorded on standardized field data sheets (Appendix D) either on-site ("in the field") or at the
BBAC office ("In house").
The selected analytical laboratories are certified by MassDEP to perform all analytical procedures that will be
required during the completion of this field sampling program. All laboratory personnel are to have had training
in accordance with the procedures outlined in their QAPs. A summary of the laboratory SOPs for the required
analyses are provided in Appendix A.
The samples requiring laboratory analysis are picked up by the Stormwater Specialist (or designee) and driven to
the appropriate laboratory. Prior to releasing the samples, the onsite DPW staff member (or designee) will sign
and date the Chain of Custody (COC) on the bottom of the field data sheets. The Stormwater Specialist (or
designee) will place the "In house" samples and laboratory samples into separate coolers and deliver the
laboratory samples to the laboratory. Once the samples arrive at the laboratory, the Stormwater Specialist will
sign the COC at the bottom of the data sheet. The laboratory will make copies of all the field data sheets and COC
forms for their records with all the original data returned to the Stormwater Specialist. The Stormwater Specialist
will bring the remaining "In-house" samples to the BBAC office for testing. The results of the "In house" testing
will be placed on the original field data sheets. The original field data sheets will then be filed at the Buzzards
Bay Action Committee office with copies sent to appropriate municipal DPWs. The monitoring data will be
entered on a spreadsheet, organized by municipal name, stormdrain system, and then specific location (discharge
points) within each stormdrain system.
18
Table 3. Project Schedule
2015 2016 2017
Oct
No
v
De
c
Jan
Feb
Mar
Ap
r
May
Jun
July
Au
g
Sep
Oct
No
v
De
c
Jan
Feb
Mar
Ap
r
May
Jun
Jul
Au
g
Sep
Oct
No
v
1 Sampling Program Design
X X X X X X X
2 QAPP X X X
3 Hiring of Staff X1 X
2 X
3 X
3
4 Implementation: Field Mapping and Monitoring
X X X X X X X X X X X X X X X X
5 Reports and Analysis
X4 X
5 X
6
Notes: 1BBAC Project Manager,
2BBAC Stormwater Specialist,
3summer interns,
4annual progress meeting and interim report,
5Draft prioritization,
6Final
prioritization and data report
Table 4. Laboratory Tests - Bacteria, New Bedford Health Lab and Barnstable Co. Health and Environment
Parameter
Sample Volume/
Container
Maximum
Holding Time
Field Processing/
Preservation
Laboratory
Precision Method Units
Lower Detection
Units
MDLs
Fecal Coliform 100 ml sterilize
polyethylene
6 hours Collect, label, store on
blue ice
Logarithm range of
duplicates, PT tests for
bacterial analysis
SM9222D, 21th Edition
2005
cfu/100ml <1
Enterococci 100 ml sterilize
polyethylene
6 hours Collect, label, store on
blue ice
Logarithm range of
duplicates, PT tests for
bacterial analysis
EPA Office of Water,
Method 1600,
Membrane Filter Test
EPA 821-R-97-004.
cfu/100ml <1
19
Table 6. "In house" Field Tests
(Samples to be taken to the BBAC Office for analysis)
Analyte(s) Measured Equipment Operating Range Resolution Accuracy Holding time
Surfactants (detergents as
MBAS) CHEMetrics K-9400
1 0-3 ppm + 1 color
standard
increment
+ 30% error at 0.25 ppm, 0.63
ppm, and 1.88 ppl. 48 hours
Nitrates LaMotte Nitrate-Nitrogen
test kit (3615-01) 0.00 to 1.00 ppm 0.1ppm 0.1ppm 24 hours
1Organic sulfonates, sulfates, carboxylates, phosphates, and phenols as well as inorganic cyanates and thiocyanates may interfere. Sulfides may interfere negatively. Nitrate interferes positively; 10
ppm NO3-N may read as approximately 0.2 ppm. Isopropanol at up to 0.1% does not interfere. Chloride at up to 100 ppm does not interfere significantly. However, because higher chloride levels will
interfere positively, this test kit is not recommended for the analysis of brine or seawater samples unless additional sample manipulation is performed.
Table 5. "In the field" Field Tests
(Sample analysis to be completed onsite)
Analyte(s) Measured Equipment Operating Range Resolution Accuracy
Ammonia Hach Test Strips 0-6ppm 0.25ppm +/- one half of a color block
Conductivity YSI Model 30 0 to 200 mS/cm (auto-range) 0.0001 to 0.1 mS/cm
(range dependent)
1 m, 4 m cables:
±1.0% of the reading or 1.0 μS/cm, whichever is
greater
Salinity
Calculated from
Conductivity and Temp
YSI Model 30
0 to 70 ppt 0.01 ppt ±1% of reading or ±0.1 ppt, whichever is greater
Temperature
(°C, °F, K)
YSI Model 30 -5 to 70°C (23 to 158°F) 0.1°C ±0.2°C;
±0.3°C cables over 45-meters
Chlorine1 Hach Pocket Colorimeter II
#5870024
Range: 0.02 to 2.00 mg/L CI2
Range 2: 0.1 to 8.0 mg/L CI₂ 0.02mg/l 0.2 mg/l
1 Chlorine monitoring will be conducted as decided by the Project Manager in consultation with the Technical Advisor and Stormwater Specialist. Decision will be made based upon previous
monitoring results and available funding.
20
Table 7. Other Laboratory Tests (future optional tests), Barnstable County Health and Environment
Parameter1 Max. Holding Time Process &Storage Method (Ref) Units Lower Detection Units (MDLS)
Nitrates as Nitrogen 48 Hours Filtered and stored in the
dark at 4°C
EPA 300.0, Rev11 mg/L 0.1
Orthophosphate 28 Days 120 ml glass bottle, preserve
with H2SO4 to a pH cool to
4° C 50 ml Glass Bottle
EPA 365.1 mg/L 0.05
Total Kjeldahl Nitrogen 28 Days Sulfuric Acid to pH <2, cool
to 4° C, plastic or glass
bottle
351.2 Rev001 mg/L 0.05
Oil and Grease 28 Days Sample acidified and stored
at 4°C
EPA 1664A, Ref: US EPA-
821-R-98-002, Feb 1999 mg/L <0.5
1 The Project Manager in consultation with the QA officer and the TPA will decide on the necessity of monitoring these parameters based upon previous monitoring results and available funding.
Table 8. Monitoring Events by Municipality
Town Discharge
Pipes
Monitoring
Events
Acushnet 9 63
Dartmouth 44 308
Fairhaven 77 539
Mattapoisett 41 287
Wareham 167 1169
Total 2366
21
2.0 Data Generation and Acquisition
2.1 Sampling Process Design
The Buzzards Bays Stormwater Collaborative water quality monitoring program is designed to meet the project
quality objectives discussed in Section 1.7 ‐ Quality Objectives and Criteria. This monitoring program involves
wet and dry weather sampling of outfall pipes or roadcuts and associated stormdrain systems that discharge within
100 feet of either a shellfish closed embayment and/or bacteria impaired waters (Massachusetts 2012 Integrated
Waters List, Category 4 and 5). If future funding is available, the BBAC plans to expand this sampling program to
include other municipalities along with a more extensive “within the pipe network” monitoring program.
Approximately 338 outfall pipes/roadcuts will be sampled at least twice - one dry weather and one wet weather.
Upon the receipt of the laboratory analysis of the first wet and dry weather sampling events, the Project Manager
in consultation with the QA Officer and TPA may decide the discharge pipe and/or stormdrain system warrants
further investigation to determine potential sources of pollution. The Project Manager and/or Stormwater
Specialist will discuss the test results with the municipal collaborative representative and DPW to determine the
next sampling location. It is estimated that each discharge pipe/roadcut or associated stormdrain system will be
sampled seven times (3 wet weather and 4 dry weather).
Sampling Description:
Stormwater water quality sampling is expected to be undertaken beginning in April 2016 and will continue for
one or two years depending upon additional supplemental funding. In general, water samples will be collected
during periods of elevated bacteria survival and will be either a wet weather or dry weather event. Some samples
may be collected during cold weather for parameters less likely to be affected by temperature such as inorganic
nutrients or optical brighteners.
Wet weather defined:
Antecedent conditions where no more than 0.1inches of rainfall have occurred 72‐hours prior to the start of
the rainfall event;
During a rain event with a minimum rainfall of 0.25‐inches and a minimum duration of 1 hour before the
sample is collected
Dry weather defined:
Antecedent conditions where no more than 0.1inches of rainfall have occurred at least 48 hours prior to the
sampling event;
Currently not raining and no rainfall is predicted for that day.
All data collection will be taken as close to the discharge point as possible and as close to low tide as feasible.
Many of the pipe discharges in Buzzards Bay are partially or totally submerged even during low tides. Tides will
be monitored using data provided by the National Oceanic and Atmospheric Administration (NOAA)
https://NOAA Tide Predictions.
Antecedent rain conditions and predicted rainfall will be checked prior to an event by the Stormwater Specialist.
Precipitation forecasts will be based on meteorological forecast models provided by the National Weather Service
at the New Bedford Station. A final decision on whether or not a sample will be collected will be decided by the
Project Manager or Stormwater Specialist.
Prior to the first monitoring event, all the pre-selected discharge pipes/roadcuts will be assigned a unique site
name (i.e. Buzzards Bay Stormwater Atlas point identifier). The Stormwater Specialist (or designee) will then
conduct visual inspections of the sites, collecting GPS coordinates, photographs, and information regarding
outfall pipe /roadcut(material, size, condition, etc.). During each monitoring event, the Field Teams will confirm
This method is for determination of n-hexane extractable material (HEM; oil and grease)
and n-hexane extractable material that is not adsorbed by silica gel (SGT-HEM; non-
polar material) in surface and saline waters and industrial and domestic aqueous wastes.
Extractable materials that may be determined are relatively non-volatile hydrocarbons,
vegetable oils, animal fats, waxes, soaps, greases and related materials. The method is
based on prior Environmental Protection Agency (EPA) methods for determination of
―oil and grease‖ and ―total petroleum hydrocarbons‖.
This method is not applicable to measurement of materials that volatilize at temperatures
below approximately 85 C. Petroleum fuels from gasoline through #2 fuel oil may be
partially lost in the solvent removal operation.
Some crude oils and heavy fuel oils contain a significant percentage of materials that are
not soluble in n-hexane. Accordingly, recoveries of these materials may be low.
This method is capable of measuring HEM and SGT-HEM in the range of 5 to 1000
mg/L, and may be extended to higher levels by analysis of a smaller sample volume
collected separately.
For HEM and SGT-HEM in this method, the method detection limit (MDL) is 1.4 mg/L
and the minimum level of quantitation (ML) is 2.0 mg/L.
Summary of Method
Theory of Operation
The SPE-DEX 4750 Extractor System automates the extraction of analytes from aqueous
samples by using a 47mm SPE disk. The SPE disk offers significant advantages over
other forms of solid phase extraction.
To successfully use the SPE disk, there are three steps that must be performed:
Prewet the SPE disk with solvents
Introduce the water sample to the SPE disk
Solvent rinses the sample container and extracts the SPE disk
Prewet the SPE Disk with Solvents
The Prewet step is very important as it:
Removes manufacturing and handling impurities from the SPE disk
Activates and prepares the disk in preparation of the water sample
The necessary solvent soaking and air drying times for each prewet solvent are entered by
the user and can be changed to optimize the extraction efficiency of the disk. In no case,
3
however, during the final Prewet step, i.e., the Methanol and Reagent Water prewets,
should the SPE disk be allowed to go dry before the introduction of water sample. This is
accomplished by entering a ―0‖ time for the air dry times for the Methanol and Reagent
Water steps.
Introduce the Water Sample to the Disk
This step automatically introduces the water sample to the SPE disk. The disk extracts
the organics or Oil & Grease out of solution and onto the active chemical surface of the
disk.
Solvent Rinses the Sample Container and Extracts the SPE Disk
The solvent rinse step uses organic solvents to rinse the sample container, wash the
Extractor’s internal surfaces, and extract the analytes of interest off the SPE disk and
back into solution. The rinse solvents and analytes pass through the SPE disk and into
the collection tube.
Definitions
HEM and SGT-HEM are method-defined analytes; i.e., the definitions of both HEM and
SGT-HEM are dependent on the procedures used. The nature of the oils and/or greases,
and the presence of extractable non-oily matter in the sample will influence the material
measured and interpretation of results.
Interferences
Solvents, reagents, glassware, and other sample processing hardware may yield artifacts
that affect results. Specific selection of reagents and purification of solvents may be
required.
All materials used in the analysis shall be demonstrated to be free from interferences by
running laboratory blanks.
Safety
The toxicity or carcinogenicity of each reagent in this method has not been precisely
determined; however, each chemical should be treated as a potential health hazard.
Exposure to these chemicals should be reduced to the lowest possible level. It is
suggested that the laboratory perform personal hygiene monitoring of each analyst that
uses this method. This monitoring should be performed using Occupational Safety and
Health Administration (OSHA) or National Institute of Occupational Safety and Health
(NIOSH) approved personal hygiene monitoring methods. Results of this monitoring
should be made available to the analyst.
n-Hexane has been shown to have increased neurotoxic effects over other hexanes and
some other solvents. OSHA has proposed a time-weighted average (TWA) of 50 parts-
per-million (ppm); NIOSH concurs that an 8-hour TWA/permissible exposure limit
(PEL) of 50 ppm is appropriate for n-hexane; and the American Conference of
4
Governmental Industrial Hygienists (ACGIH) has published a threshold limit value
(TLV) of 50 ppm for n-hexane. Inhalation of n-hexane should be minimized by
performing all operations with n-hexane in an explosion-proof hood or well-ventilated
area.
N-hexane has a flash point of –23 C (-9 F), has explosive limits in air in the range of 1-7
percent, and poses a serious fire risk when heated or exposed to flame. N-hexane can
react vigorously with oxidizing materials. The laboratory should include procedures in
its operations that address the safe handling of n-hexane.
Sampling Equipment
Sample collection bottles glass, approximately 1-L, with PTFE-lined screw cap.
Equipment for calibration
Analytical Balance—Capable of weighing 0.1 mg
Volumetric flasks—Glass, 100-mL
Vials—Assorted sizes, with PTEFE-lined screw caps
Volumetric pipette—Glass, 5-mL
SPE-DEX 4700 Automated Solid Phase Extractor System (Horizon)
Speed-Vap ll 9000 Sovent Evaporation System (Horizon).
Reagents and Standards
Reagent water—Water in which HEM is not detected at or above the minimum level
(ML) of this method. Bottled distilled water or water prepared by passage of tap water
through activated carbon have been shown to be acceptable sources of reagent water.
Hydrochloric acid or sulfuric acid—ACS. Mix equal volumes of concentrated HCI and
reagent water to produce an approximately 6N solution.
n-Hexane-ACS (>85% N Hexane)
Acetone—ACS, residue less than 1 mg/L
Methanol-ACS
SPE disks (SSI #1047TL)
Fastflo Prefilters (CPI #4350-010089)
Sample Collection, Preservation and Storage
Collect approximately 250 ml, 500 ml or one liter of representative sample in a glass
bottle acidified with sulfuric acid to pH < 2 following conventional sample practices,
except that the bottle must not be pre-rinsed with sample before collection. To allow for
potential QC failures, it is recommended that additional sample aliquots be collected.
5
All samples must be refrigerated at 0-4C from the time of collection until extraction (40
CFR 136, Table II). Preservation using Sulfuric Acid is used. Before samples are
analyzed pH is performed to ensure pH is < 2. This is recorded on raw data worksheet.
All samples must be analyzed within 28 days of the date and time of collection (40 CFR
136, Table II).
Quality Control
The laboratory operates a formal quality assurance program. The program consists of an
initial demonstration of laboratory capability, ongoing analyses of standards and blanks
as a test of continued performance, and analysis of a matrix spike (MS) to assess
recovery. Laboratory performance is compared to established performance criteria to
determine if the results of analyses meet the performance characteristics of the method.
For each analytical batch the laboratory analyzes a blank, a check standard (2 mg/ml std
CPI cat# 4401-1664-2). Range 1.8-2.2 mg/L
Sample duplicates, and matrix spike duplicate (add 1.0 ml of 4 mg/ml std (NSI cat# QC-
003LSIIP to sample = 4.0 mg/ml) is performed every 10 samples (±10%).
Initial Demonstration of Laboratory Capability
Method Detection Limit (MDL)—To establish the ability to detect HEM and Sgt-HEM,
the laboratory shall determine the MDL per the procedure in 40 CFR 136, Appendix B
using the apparatus, reagents, and standards that will be used in the practice of this
method. An MDL less than or equal to the MDL in Section 1.6 or less than 1/3 the
regulatory compliance limit must be achieved prior to the practice of this method.
Matrix spikes—The laboratory must spike a minimum of 5 percent of all samples from a
given sampling site or, if for compliance monitoring, from a given discharge/waste
stream (matrix spike). The sample aliquot shall be spiked with the hexadecane/stearic
acid spiking solution. The concentration of the spike in the sample shall be determined as
follows: The concentration of the spike of HEM or SGT-HEM in the sample, shall be at
the concentration of the precision and recovery standard or at 1 to 5 times higher than the
background concentration, whichever concentration is higher.
Note: Samples containing high concentrations (> 100 mg/L) of HEM will require
a large volume of spiking solution) for the MS (and MSD). If the concentration
of HEM is expected to exceed 1000mg/L, smaller sample volumes should be
6
collected for the background measurement and MS (and MSD) so that the amount
of HEM plus the amount spiked does not exceed 1000 mg/L.
Spike the additional sample aliquot(s) with the spiking solution and analyze the
aliquot(s) to determine the concentration after spiking (A).
Calculate the percent recovery (P) of HEM or SGT-HEM in each aliquot using the
following equation:
Equation 2
P= 100(A-B)
T
Where:
A=Measured concentration of analyte after spiking
B=Measured background concentration of HEM or SGT-HEM
T=True concentration of the spike
When determining SGT-HEM, the true concentration (T) must be divided by 2 to reflect
the concentration of hexadecane that remains after removal of stearic acid.
Compare the percent recovery of the HEM or SGT-HEM with the corresponding QC
acceptance criteria in Table 1.
If the results of the spike fail the acceptance criteria, and the recovery of the QC
standard in the ongoing precision and recovery test for the analytical batch is
within the acceptance criteria in Table 1, an interference is present. In this case,
the result may not be reported or used for purposes regulatory compliance
purposes and the laboratory must assess the potential cause for the interference. If
the interference is attributable to sampling, the site or discharge/waste stream
should be resampled. If the interference is attributable to a matrix problem, the
laboratory must modify the method, repeat the tests required, and repeat the
analysis of the sample of the MS (and MSD, if performed). Most matrix
interference problems are attributable to the formation of emulsions in the
extraction.
If the results of both the spike and the ongoing precision and recovery test fail the
acceptance criteria, the analytical system is judged to be out of control, and the
problem shall be identified and corrected, and the sample batch reanalyzed. All
samples must be associated with a valid MS (and MSD, if performed).
Laboratory blanks—Laboratory reagent water blanks are analyzed to demonstrate
freedom from contamination.
Extract and concentrate a laboratory reagent water blank initially and each
analytical batch. The blank must be subjected to the same procedural steps as a
sample.
If material is detected in the blank at a concentration greater than the minimum
level, analysis of samples is halted until the source of contamination is eliminated
7
and a blank shows no evidence of contamination. All samples must be associated
with an uncontaminated method blank before the results may be reported for
regulatory compliance purposes.
Calibration verification—Verify calibration of the balance before and after each
analytical batch. If calibration is not verified after measurement of the analytical batch,
recalibrate the balance and reweigh the batch.
Ongoing precision and recovery—To demonstrate that the analysis system is in control,
and acceptable precision and accuracy is being maintained with each analytical batch, the
laboratory shall perform the following operations:
Extract and concentrate a precision and recovery standard with each analytical
batch.
Compare the recovery with the limits for ongoing precision and recovery. If the
recovery is in the range specified, the extraction, weighing processes are in
control and analysis of blanks and samples may proceed. If, however, the
recovery is not in the specified range, the analytical process is not in control. In
this event, correct the problem, re-extract the analytical batch, and repeat the
ongoing precision and recovery test.
The specifications contained in this method can be met if the apparatus used is
scrupulously cleaned and dedicated for the determination of HEM and SGT-HEM. The
standards used for initial precision and recovery, matrix spike, and ongoing precision and
recovery should be identical, so that the most precise results will be obtained.
Calibration and Standardization
Calibrate the analytical balance at 2 mg and 1000 mg using class ―S‖ weights.
Calibration shall be within 10% at 2 mg and 0.5% at 1000 mg. If values are not
within these limits, recalibrate the balance.
Procedure using SPE-DEX 4750 Extraction
1. Bring the analytical batch of samples, including the sample aliquots for the MS, to
room temperature. Use 500 ml of sample: Lower volumes may be used if high
levels are expected.
2. Place approximately 500 ml of reagent water in a clean sample bottle to serve as
the laboratory blank.
3. Prepare standard and spiked sample using identical procedure as samples
4. Verify that the pH of the sample is less than 2
5. Check the levels of all the solvent bottles and fill them if necessary
6. Empty all waste bottles (water and solvent)
7. Turn on the vacuum source (25 hg min. at pump and 15 at solvent/waste
recovery bottle)
8
8. Turn the gas supply on (check the regulator bracket and make sure there is 35 psi
on the extractor pressure side, and 8-10 psi on the solvent bottle pressure side)
9. Turn on controller (the display will show active extractors-make sure it matches
the number of extractors plugged in)
10. Free up all 3 check valves with a tie wrap (tech tip t24 in users manual)
11. Run purge method:
Place the purge gasket into the disk area, and load an empty collection
vessel and sample bottle
Select the extractor to be purged by pushing SELECT on the controller
and then entering the extractor #
Select the purge method # when prompted by controller and press ENTER
Press the PURGE key on the extractor
Repeat a second time if necessary
Select the next extractor to be purged and repeat
Remove the collection vessel, purge gasket, and sample bottle
12. Run samples:
Place the support screen and the disk in the disk area, or the O-ring and JT
Baker speedisk or CPI SPE disc.
Close the extractor mouth and snug the assembly by hand to ensure a tight
seal
Load empty collection vessel (hold by top of the adapter not the vial and
gently give a ¼ turn to ensure a good seal)
Select an extractor by pushing SELECT on the controller and then
entering the extractor # (pushing the . key will activate all extractors)
Select 1664 when prompted by the controller and press ENTER
Put foil over the top of the sample bottle and screw the adapter in place. If
larger bottles are being used, put the large adaptor on the bottle, then put
the foil over the smaller opening and screw the small cap adapter over the
foil.
Load the sample bottle onto the extractor
Press start
Repeat procedure for each sample, standard, blank, and matrix spike.
13. Sampling Handling Technique using Speed-VAP II 9000 Solvent Evaporation
System
Step 1:
Remove a clean, empty aluminum pan from the cardboard container. As
cardboard ―dust‖ can collect on the inside of the pan, use a kimwipe to
gently wipe the inside of the pan. If desired, turn the pan over, and using
a hard point pen, write the sample identification on the bottom of the pan.
This will eliminate any confusion once multiple pans are in use.
Note: From this point on only use the Pan Transfer Tongs or forceps to move or
transfer the pan. Do not touch the pan with your fingers, as finger oils
will contribute to the pan weight.
9
Step 2: Once the pan is clean and labeled, use the tongs or forceps to transfer the
pan to the analytical balance. Record the weight 3 times on the
worksheet.
Step 3: Transfer the pan to the evaporator and place the pan into any one of the
open positions.
Note: One drop of concentrated food color added to the collection vessel will
visually identify the water layer. If any water is pulled into the pipette
it can be dispensed back into the collection vessel.
Step 4: Using a polypropylene transfer pipette, carefully transfer the top, hexane
layer from the extract collection vessel into the pan. If any colored
liquid is pulled into the pipette tip, dispense the colored liquid back into
the collection vessel. Be careful not to spill any of the solvent during the
transfer to the pan.
Step 5: Using fresh n-hexane and a new transfer pipette or squeeze bottle rinse
down the walls of the collection vessel. Using the pipette originally
used to transfer the hexane into the pan, transfer the rinse hexane into
the pan. Repeat this operation a second time to ensure all of the extracted
Oil and Grease is transferred into the pan.
Step 6: Close the cover of the evaporator and turn the Vac On / Off switch to on.
Set temperature control to 40ºC.
If necessary, adjust the Control Knob on the Vacuum Generator to
achieve the desired swirling rate in the pans.
Step 7: When the solvent has visually appeared to have evaporated, press the
Cover Release button and use the tongs or forceps to transfer the pan back
to the balance. Wait 5 min. before taking weight readings. If the weight is
stable, record the new weight 3 times on worksheet. If the weight is
slowly
dropping, this may indicate solvent is still evaporating off the pan. The
pan can either be left on the balance until a stable weight is achieved, or
the pan can be returned to the evaporator for a short period of time
(approximately 1 minute).
Step 8: Repeat the above steps for all samples to be run.
Step 9: When all samples are run, open the cover by pressing and holding the
Cover Release button until the vacuum level inside the unit is vented.
If the unit is to be shut down, turn the main power off, then turn the Vac
On / Off switch to off, turn the Control Knob on the Vacuum Generator
counter-clockwise to 0 or shut off the vacuum pump/source.
Note: If the vacuum source will be turned off for more than 10 minutes, it is
10
recommended to turn off the evaporator. This will minimize any safety
issue due to solvent vapors.
14. Calculate results using equation:
HEM mg/L = weight of extractable material (mg)
SampleVolume (L)
Reporting—Report results to three significant figures for HEM and SGT-HEM found at
or above 10 mg/L, and report results to two significant figures for HEM and SGT-HEM
found below 10 mg/L.
Samples—Report results for HEM and SGT-HEM found below the MDL as <2.0
mg/L.
Blanks—Report results for HEM and SGT-HEM found below the MDL as <1.4
mg/L. Do not report results below the MDL unless required by the permitting
authority or permit.
Results from tests performed with an analytical system that is not in control must
not be reported or otherwise used for permitting or regulatory compliance
purposes but do not relieve a discharger or permittee of timely reporting.
Method Performance
The method detection limit (MDL) and minimum level of quantitation (ML) are based on
five studies conducted by EPA and described at proposal of Method 1664A and as
verified by data submitted in comments on the proposal of Method 1664A. The MDL is
performed yearly.
Waste Management
It is the laboratory’s responsibility to comply with all Federal, State, and local regulations
governing waste management, particularly the hazardous waste identification rules and
land disposal restrictions, and to protect the air, water, and land by minimizing and
controlling all releases from fume hoods and bench operations. Compliance with all
sewage discharge permits and regulations is also required.
Samples preserved with HCI or H2SO4 to pH<2 are hazardous and must be neutralized
before being disposed, or must be handled as hazardous waste.
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QUALITY ASSURANCE MANUAL
Page
Section 1 - Laboratory Facilities, Personnel and Analytical Test Procedures 2
Section 2 - Laboratory Equipment and Instrumentation 4 Section 3 - Laboratory Supplies 8 Section 4 - Media and Reagents 11 Section 5 - Sampling Procedures 13 Section 6 - Laboratory QA/QC Testing 14 Section 7 - Standard Operating Procedures 17 Section 8 - Documentation and Recordkeeping 18
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Section 1 - Laboratory Facilities and Personnel 1.1 Ventilation Central air conditioning is used to promote a well-ventilated environment which is free of dust, drafts and extreme temperature changes. Temperatures are maintained between 16 and 27oC. Air conditioning is used to reduce contamination, permit more stable operation of incubators and decrease possible moisture problems with medias or analytical balances. 1.2 Organization of Laboratory Space Entrance to the laboratory area is posted to minimize through traffic and visitors. Apart from the main laboratory area, a separate room, the chemical prep and incubation room, is used for the weighing and preparation of medias and reagents as well as various incubators and waterbaths for incubation purposes. The main laboratory area is divided into working sections which include a glassware preparation and storage area, a water purification and dishwashing area with safety shower and eyewash, and a laboratory work-bench area with analytical meters and filtration apparatus where samples are processed for analysis. The main laboratory area also has two fume hoods for working with hazardous or volatile materials. One fume hood is specific for working with acids and the other fume hood is reserved for use with organics. Sterilization is performed in the autoclave located in the Wastewater Treatment Laboratory. 1.3 Laboratory Bench Areas A minimum of 2m linear bench space is provided per analyst in addition to areas specific for preparation and support activities. The laboratory bench tops provide a smooth, impervious surface which is chemically inert, corrosion resistant and has a minimum number of seams. The bench working surfaces are illuminated with even, glare-free lighting. 1.4 Walls and Floors Walls are covered with a smooth finish that is readily cleaned and disinfected. Floors are coated with an impervious, washable surface which is textured to prevent slipping accidents. 1.5 Air Monitoring Laboratory air quality is monitored on a monthly basis using Plate Count Agar in sterile dishes,
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set on lab bench surfaces (exposed) for a 15 minute period. Following 48 hours incubation at 35oC, the number of colonies on the air density plates must not exceed 15 colonies/plate/15 minutes. Records of air quality monitoring are maintained in the lab Quality Assurance Manual. 1.6 Laboratory Cleanliness and Maintenance Laboratory rooms are cleaned regularly (weekly). Routine cleaning includes washing of benches, shelves, floors and other areas prone to dust accumulation. Floors are wet mopped and treated with a disinfectant solution. Sweeping of floors and/or accumulation of clutter is not allowed. Benchtops are treated with a disinfectant solution before and after each use. Laboratory equipment is maintained with regular inspection and servicing as necessary. Procedures specific for maintenance of equipment are outlined in Standard Operating Procedures (SOPs) and records of repair and service are kept. 1.7 Laboratory Personnel All bacteriological testing is performed by trained personnel, currently consisting of a lab director/supervisor/analyst (Leslie Aubut) and a part-time analyst (Jane Wurm). The Lab Director monitors and shares in defined bench-top work assignments to ensure that test procedures are precisely followed and that quality control measures are incorporated into the analyses. The Lab Director is responsible for ensuring the laboratory quality assurance program is followed. Job descriptions and training records for laboratory personnel are maintained on file, as a resume of pertinent experience, and are updated at least annually. Any additional training received between updates is documented in the personnel files. Laboratory personnel are subject to annual performance evaluation reviews. Copies of performance evaluations are maintained on file with the training records. Lab personnel are encouraged to take additional training for the advancement of skills and knowledge. The Lab Director is responsible for record keeping for laboratory services and archival of lab data. The Lab Director reviews all reports generated by support personnel for accuracy, and monitors record keeping procedures to ensure compliance with SOPs and appropriate regulatory guidelines. 1.8 Analytical Test Procedures For the analysis of potable waters, the analytes and methods employed are: Total Coliform by MF-SM9222B and E.coli by NA-mug -SM9222G, and for ambient and wastewaters the Enterococcus method used is EPA Method 1600.
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Section 2 - Laboratory Equipment and Instrumentation 2.1 Autoclaves The autoclave is of sufficient size to prevent crowding of the interior. It is constructed to provide uniform sterilizing temperature of 121oC within the chamber. The autoclave is equipped with digital readouts of pressure, temperature and time. Autoclave cycles must be completed within 45 minutes when a 15 minute sterilization cycle time is used, such as with sugar broths. Records are maintained in the Autoclave Logbook for each sterilization cycle and include items sterilized, date, start-up time, time temperature reached, time temperature off, time removed from autoclave, total time in autoclave, maximum registering thermometer temperature, heat sensitive tape usage and user initials. Sterilization effectiveness is monitored using spore strips weekly (Standard Methods, 20th Ed., 9020B section 3h) with results maintained in the Autoclave Logbook. The timing operation of the autoclave is checked against a stopwatch quarterly, with results recorded and maintained in the Autoclave Logbook. Periodic inspection is performed by qualified service representatives. Servicing is conducted whenever a deviation of sterilization time or effectiveness is observed and records of corrective action are maintained in the Autoclave Logbook. 2.2 Balances Operation and routine maintenance is performed in accordance with manufacturers instructions. Balances and weights are serviced and checked annually by the Sealer of Weights and Measures. Balances are calibrated monthly with a set of S weights traceable to NIST and records of calibrations are maintained in the Balance Logbook. If balance calibrations vary from true values, adjustments are made to re-center the bubble and re-calibration is performed. Continued variance from true values will result in servicing, with records maintained in the Balance Logbook. In weighing less than a gram, an analytical balance with a sensitivity less than 1 mg at a 10 g load is used. For larger quantities, a top-loading balance with sensitivity of 0.1 g at a 150 g load is used. The balance is wiped before and after each use with a soft brush. Spills are immediately cleaned-up with a damp towel. Weights are inspected with each use and are discarded if corrosion is observed. Weights are protected from manual contact, corrosion and laboratory atmosphere. 2.3 Colony Counters and Tallies A standard colony counter, Quebec model, providing magnification and good visibility is used for heterotrophic plate counts. Grid plates are inspected for scratches. Internal mirror placement
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and cleanliness is maintained to ensure optimal illumination. Hand-held colony tallies are used and checked with each use to ensure accuracy. For 50mm plates, a Spencer dissection microscope with 10X magnification and fluorescent light source is used in counting colonies. maintenance records are maintained in the Microscope Logbook. 2.4 pH Meter An electronic pH meter (Orion) graduated in 0.01 pH units with temperature compensation and reference electrode is used. Commercial buffer solutions are used to standardize the meter daily when in use. Three buffers which bracket the pH of interest are standard practice. Also included in the calibration is a record of the slope and mV readings as a check of electrode accuracy. If the slope falls outside the range of 95% to 105%, the cause is investigated, corrective action is taken and documented in the pH meter Logbook. Records of pH meter calibrations are maintained in the pH Meter Logbook. Buffer solutions are dated when opened and are used prior to the manufacturers expiration date. The lot numbers for each set of buffers in use is recorded at the end of the pH Meter Logbook. Buffer solutions are discarded after each use. Glass electrodes are stored in pH buffer solution when not in use. All samples and buffers are brought to room temperature prior to measurements. In between each sample or buffer, the electrodes are thoroughly rinsed with distilled water and wiped with a soft tissue. The maintenance of electrodes is performed as described in Standard Methods, 20th Ed., 4500-H+ section 5b. Precision and accuracy checks are conducted at least daily when the pH meter is in use and results are recorded in the Laboratory Precision and Accuracy Logbook. When unacceptable results are obtained from precision or accuracy measurements, the cause is investigated, corrective action taken and recorded in the Precision and Accuracy Logbook. 2.5 Laboratory Refrigerators The temperature of the laboratory refrigerators is checked and recorded twice daily. Each laboratory refrigerator is assigned a unique number and has its own temperature recording logbook. Refrigerators are cleaned monthly. All materials stored are identified and dated. The lab refrigerators are used to cool and maintain samples between 1 to 4oC until tested and to store prepared media, reagents and control cultures. Refrigerator temperatures are measured using two thermometers with bulbs immersed in glycerin solution, in glass vials, on upper and lower shelves. If refrigerators show unacceptable temperature ranges, a service call is made to a repair technician and servicing is recorded in the Refrigerator Logbook. 2.6 Water Distillation and Deionization Systems The laboratory water still (Barnstead MP-1) is fed from New Bedford city tap water. The still is
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drained and cleaned after each 3-5 days usage using a 10% HCl acid solution according to SOP #3.31 (1) and manufacturers instructions. Cleaning dates are recorded, with technician initials, in the system logbook. The distilled water produced is used to feed the laboratory water deionization system (Barnstead Nanopure II). The Nanopure system combines pre-filtration, mixed-bed resins, activated carbon and aseptic final filtration with a 0.22 um pore membrane to produce a reagent grade microbiologically suitable water. The water deionization system is monitored daily when in use, by observing the resistivity reading displayed on the electronic meter and recording results in the system logbook. The reagent grade water produced is analyzed annually for trace metals (Cd, Cr, Cu, Pb, Zn and Ni). The Nanopure system cartridges are replaced at intervals recommended by the manufacturer or as indicated by analytical results. The reagent grade water is tested monthly for conductivity, and each time a new lot of media is prepared (must be < 2 uS/cm at 25oC), residual chlorine (must be non-detectable), pH and heterotrophic plate count (must contain < 500 cfu/mL). All reagent grade water quality control tests are performed in accordance with 310 CMR 42.08(5)(c)12 and Standard Methods for the Examination of Water and Wastewater, 21st Ed., 2005 and pertinent laboratory SOPs. Records are maintained in the laboratory Water Systems Logbook. Any deviations from prescribed test parameter limits will result in investigation accordingly and corrective action with records maintained. 2.7 Waterbaths Each laboratory waterbath is equipped with a gable cover and is identified with a unique number. Waterbaths have an adequate capacity for the workload. The level of water in the baths is kept above the level of liquid in incubating tubes. Waterbath temperatures are maintained at 44.5oC +/- 0.2oC. Temperatures are recorded twice daily with records maintained in the Waterbaths Logbook. Deviation from prescribed temperature maintenance is investigated and rectified by repair from an authorized service representative with records maintained. The thermometers used to monitor the temperatures of the waterbaths are graduated in 0.1oC increments. Waterbath working thermometers are calibrated semi-annually against the laboratory standards thermometer (traceable to NIST) at 44.5oC and calibration records are maintained in the Thermometers Logbook. 2.8 Incubators All laboratory incubators are identified with a unique number. Incubator temperatures are checked twice daily (morning and afternoon) on the shelf areas in use (top and bottom). Records are maintained in the logbooks specific for each incubator. Thermometers are immersed in water to the stem marking. Laboratory incubators are kept in areas where the temperature is maintained at 16 to 27oC. The incubator working thermometers are calibrated annually against the laboratory
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standards thermometer (traceable to NIST) at the appropriate operational temperature, and calibration records are maintained in the Thermometers Logbook. Thermometers used in air incubators are graduated in 0.1oC increments. Incubators are loaded so that there will be at least 2.54 cm of space between stacks of plates and from incubator walls. Humidity levels of incubators are maintained such that SPC agar plates do not lose more than 15% of agar weight after 48 hours of incubation at 35oC. Agar weight loss is determined quarterly and records are maintained in the laboratory Quality Assurance Manual. Deviation from prescribed temperatures or agar weight loss is investigated and remedied by servicing if necessary, with records maintained. 2.9 Laboratory Fume Hoods Hoods are located away from high traffic areas as a fire safety and hood velocity integrity measure. Fume hood face velocity should be at least 30.5 linear meters/minute and are checked annually with an airflow measurement device. No airflow restrictive devices shall be placed in the hood. The laboratory fume hoods are made of corrosion resistant materials. 2.10 Membrane Filtration Equipment Before use, filtration units are assembled and checked for leaks. Units are discarded if inside surfaces are scratched or cracked. Filtration assemblies are washed and rinsed thoroughly after use, wrapped in non-toxic paper or foil, and sterilized by autoclaving. Membrane filter funnels are numbered and marked at appropriate graduations. Volume accuracy is determined by weight annually and documented in the Funnel Logbook. Tolerance must not exceed 2.5%. 2.11 Laboratory Thermometers Accuracy of thermometers is checked annually against a NIST traceable thermometer. The NIST traceable thermometer is checked annually for accuracy at ice-point (distilled water ice). For incubators and waterbaths, thermometers with 0.1oC increments are used. All thermometer incubation records are maintained in the laboratory Thermometers Logbook. Calibration corrections for each thermometer are marked on the thermometer identification tag, including calibration date, ID number and calibration temperature. Working thermometers are appropriately immersed in stoppered glass tubes containing water, or in waterbaths above the stem marking. Metal field thermometers are calibrated against a NIST traceable working thermometer quarterly. The calibration check is documented in the Field Thermometer Logbook. A field thermometer which has greater than a 5o correction factor is discarded. 2.12 Pipettors
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Eppendorf fixed and adjustable volume pipettors are used throughout the laboratory for dispensing of aqueous aliquots in the range of 10 uL to 1000 uL. The pipettors are assigned a unique ID number and are calibrated quarterly, using ten weighings each, on an analytical balance. Calibration results are recorded and maintained in the laboratory Pipettor Logbook. Pipet tips (Eppendorf) are placed into autoclavable trays and are sterilized prior to use. 2.13 Conductivity Meter A YSI Model 30 Handheld SCT meter, SN 04F10421, is dedicated to the use of monitoring low level conductivity of the laboratory reagent grade water. The meter is calibrated monthly, using single-use, low level calibration standard. The conductivity probe is maintained with extraordinary cleanliness by thorough cleaning with distilled/deionized water and cleaning with a mild acid solution (per manufacturer instruction manual) as dictated by any deviation observed during calibration checks. If erroneous measurements are observed despite these measures, the meter is returned to the manufacturer for servicing. Records of calibration, maintenance and servicing are maintained in the laboratory Conductivity Meter Logbook. 2.14 Dissection Microscope A Spencer Cycloptic Dissection Microscope with 10X-15X objective and Cyclospot Illuminator is used to count sheen colonies on m-Endo agar medium or enterococcus colonies on mEI medium. The microscope and cool light illuminator are maintained according to the Manufacturer Reference Manual (American Optical Co.) and serviced when necessary (ie; inability to focus) by an authorized factory representative. The microscope lenses are inspected for smears and dust particles with use and cleaned with lens cleaner and a lint free cloth. Maintenance and service records are maintained in the laboratory Microscope Logbook. Section 3 - Laboratory Supplies 3.1 Glassware and Plasticware Prior to each use, glassware is examined and items which are scratched, cracked or have chipped edges are discarded. Dilution bottles are made of borosilicate glass and are indelibly etched at the graduation level of 99 +/- 1 mL. Leakproof stopper closures are used with dilution bottles. Petri dishes are 12 mm deep and have bottoms of at least 80 mm inside diameter. Petri dishes for membrane filtration work have a 47 mm inside diameter. Petri dishes, whether sterilized glass or sterile plastic, are inspected for bubbles, scratches or cracks. Pipets are of glass or sterile plastic and are operated manually with a pipet aid. Pipets are marked to sharply contrast with pipetted
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solutions. As a general rule, for delivery of 1 mL volumes, pipets of no greater than 10 mL are used and for 0.1 mL volumes, pipets no greater than 1 mL are used. Manual use pipets have straight walls and tips are inspected for cracks to ensure accurate volume delivery. Disposable pipets and Eppendorf pipet tips are used one time only, placed in a disinfectant solution and are disposed of. Reusable glassware and plasticware is decontaminated if necessary and washed. In washing reusable sample containers, glassware and plasticware, a minimum of four fresh water rinses and a final rinse in distilled water deionized water is used to thoroughly rinse off all residual detergent. This is accomplished by running two complete wash and rinse cycles (the second cycle does not utilize detergent) in the lab dishwasher, with a final purified water rinse. To ensure glassware cleanliness, the following quality control checks for clean glassware are made: a) pH check - batches of clean glassware are spot checked for pH reaction by adding a few
drops of 0.04% bromthymol blue (BTB) and observing the color reaction. BTB should be blue-green, in the neutral range. BTB indicator solution is purchased commercially or is prepared by adding 16 mL 0.01N NaOH to 0.1 g BTB and diluting to 250 mL with distilled deionized water. Records of pH checks are maintained in the laboratory Glassware Washing Logbook. If deviation from the prescribed pH range is observed, the cause is investigated by replacing reagents and reviewing the rinse procedure.
b) Check for Inhibitory Residues - the test is performed before using a new supply of
detergent, to ensure the effectiveness of the rinsing procedure. If pre-washed, pre-sterilized plasticware is used it is also tested for inhibitory residues. The inhibitory residue test is performed according to Standard Methods for the Examination of Water and Wastewater, 20th Ed., page 9-6, and laboratory SOP #4.11(1). Records of the Inhibitory Residue Test are maintained in the Glassware Washing Logbook. If inhibitory residue results, the cause is investigated by reviewing the detergent in use and the rinse procedure, with records of corrective action maintained.
After washing, glassware is inspected for excessive water beads and rewashed if necessary. All glassware and plasticware (polypropylene), including reusable sample containers, which have been exposed to bacteriological agents are autoclaved for 30 minutes prior to washing and are re-autoclaved for 30 minutes prior to storage. The sterility of reusable sample containers is determined with each autoclaved batch by addition of Tryptic Soy Broth to a sample container followed by incubation at 35oC for 24 and 48 hours. Results of sterility checks are recorded and maintained in the laboratory Autoclave Logbook. The sterility of irradiated disposable plasticware is determined with each new manufacturer lot. Laboratory disposables are also subjected to lot comparison/productivity tests with records maintained in the Laboratory Disposables Logbook. Lack of sterility will prompt immediate investigation into the sterilization procedures and autoclave maintenance, with servicing by an authorized service technician.
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Servicing and maintenance records are maintained. 3.2 Membrane Filter Equipment Membrane filtration units are assembled and checked for leaks prior to use. The filtration units contain clearly marked graduations for measuring sample aliquots. Following use, filtration units are decontaminated by autoclaving. The filtration units are washed and re-autoclaved prior to storage and reuse. Filtration assemblies are autoclaved in non-toxic paper or aluminum foil for 30 minutes. Membrane filters are purchased through the Millipore Corp. and come provided with certification for each new lot, which includes retention, pore size, flow rate, sterility, pH, percent recovery and limits for specific inorganic chemical extractables. The lot number of the filters and usage dates is recorded in the Membrane Filters Logbook. The manufacturer’s certification report for each lot is maintained on record in the laboratory Membrane Filter Logbook.. Use of the membrane filters and apparatus is performed in accordance with laboratory SOP’s. When a new lot of membrane filters is used, comparison tests of the lot in use is made against the new lot as described in Standard Methods for the Examination of Water and Wastewater, 20th Ed., page 9-7. 3.3 Reagent Grade Water Quality The laboratory reagent grade water supply is monitored for acceptability criteria as outlined in Standard Methods, 20th Ed., Table 9020:II, page 9-6. The resistivity reading on the electronic display of the Nanopure deionization system is checked with each use to ensure a value of >16.7 megaohms/cm (per manufacturer instructions). Conductivity is monitored to ensure a value of <2 umhos/cm at 25oC. Records are maintained in the Conductivity/Salinity Logbook. The pH is checked with each pH meter calibration to be between 5.5 and 7.5 and recorded in the pH Meter Logbook. A heavy metals analysis (Cd, Cr, Cu, Ni, Pb and Zn) is performed annually and records are maintained in the Reagent Grade Lab Water Logbook. The metals analyzed must be < 0.05 mg/L. The total residual chlorine is determined monthly and records are maintained in the Reagent Grade Lab Water Logbook. The value obtained for residual chlorine must be below detectable limits. A heterotrophic plate count (HPC) is conducted monthly and records are maintained in the Reagent Grade Lab Water Logbook. HPC results must be <1000 CFU/mL. An annual Biosuitability Test (also called the Water Quality Test) is conducted annually (if conductivity tests exceed acceptability criteria) as described in Standard Methods, 19th Ed., 9020 B 3c1, page 9-5 through 9-7. The Water Quality Test is performed by NH Dept. Of Environmental Services, State of New Hampshire, 1-603-271-3503 (lab 1-603-271-3445) and results must be within the acceptable range of a 0.8 - 3.0 ratio. If water quality test results do not meet acceptability criteria, the problem is investigated and corrective measures are documented.
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3.4 Laboratory Control Cultures Laboratory control cultures are purchased commercially as inoculated swabs, or equivalent, bearing an ATCC reference number. The control cultures are revived and incubated on appropriate agar media slant tubes for 24 hours at 35oC. Control cultures are stored refrigerated and are aseptically transferred monthly to fresh agar slants. Each new set of control culture slants is assigned a lot number based on the preparation date. Records for control cultures are maintained in the Control Cultures Logbook. For each new lot of prepared culture medium, the analytical procedures are tested with known positive and negative control cultures for the organism(s) under test. Control cultures used for each type of test are as follows:
Test Positive Control Negative Control Total Coliforms E. coli or Enterobacter Staphylococcus Fecal Coliforms E. coli Enterobacter E. coli E. coli Enterobacter Fecal Streptococci Enterococcus faecalis Staphylococcus or E. coli Enterococci Streptococcus faecalis Staphylococcus or E. coli Section 4 Media and Reagents 4.1 Reagents Only reagents and chemicals of ACS grade are used for analyses of test samples. All reagents are prepared in distilled/deionized water and clean glassware, following pertinent laboratory SOP’s. All chemicals and reagents are dated when received and when first opened for use. Reagents are made to volume using Class A volumetric glassware. Preparation details are recorded in the laboratory Media and Reagents Preparation Logbook. For storage, good-quality inert plastic or borosilicate glass bottles with tight fitting lids are used. Prepared reagents are labeled with name, concentration, date prepared, lot number, preparer initials, expiration date, storage conditions, and chemical hazard rating (if applicable) according to NFPA recommendations. 4.2 Culture Media Commercially prepared media is used wherever possible for control of quality. Media is ordered in quantities to last no longer than 1 year. Upon receipt, media bottles are labeled with the date
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of receipt and the date when opened. The new media is logged into the Media Receipt Logbook with Manufacturer, lot number, amount received, date received, date opened and date discarded. Media that appears caked or exhibits signs of deterioration are discarded. The manufacturer’s lot numbers are recorded on media preparation forms (maintained as raw data in the Media Preparation Logbook). Opened bottles of media are used within 1 year of opening. Culture medias are stored in a Fisher Scientific Desiccator Cabinet containing desiccant to protect dehydrated medias from humidity. Storage of unopened medias is no longer than 2 years. Newly purchased lots of media are compared against proven lots using recovery of pure culture isolates and natural samples. a) Media Preparation - Medias are prepared in containers that are at least twice the volume
of the media being prepared. Media are stirred while heating, either on a hot plate or in a waterbath. Prepared media is identified, dated, and assigned a lot number in the Media Preparation Logbook. Media lot numbers are recorded on raw data worksheets. All medias are prepared in distilled, deionized water which has been measured with pipets or graduated cylinders conforming to NIST and APHA standards. The pH of a portion of media from each lot is checked after sterilization and recorded in the Media Preparation Logbook. Minor adjustments in pH are made with 1M NaOH or HCl solution. The media is remade if large deviations in pH are observed (> 0.5 pH units). Prepared medias are examined for unusual color, darkening or precipitation and discarded if problems are noted.
b) Media Sterilization - Media is sterilized at 121oC for the minimum time specified. A
double walled autoclave maintaining full pressure and temperature in the jacket between loads is used to reduce chances of heat damage. Manufacturer’s directions are followed for sterilization of specific medias. Autoclave times other than specified by the manufacturer are in accordance with Standard Methods, 20th Ed., pg 9-8, Table 9020:III. Sterilized media is removed from the autoclave as soon as chamber temperature reaches zero. Media is never re-autoclaved. An Autoclave Logbook is kept detailing specifics of each batch, including actual sterilization time and total autoclave time. Effectiveness of sterilization is monitored using indicator tape, an autoclave thermometer and spore strip suspensions. Weekly spore strip testing for autoclave effectiveness is documented in the Autoclave Logbook. If growth of autoclave spores occurs, then sterilization procedures are deemed inadequate and an authorized service representative is called for corrective action. As a further check of sterility, media controls are run with each series of test samples and media controls are run for each new lot of media prepared. Results of controls are recorded on raw data worksheets accompanying test samples. If growth occurs on media controls, the source of contamination is investigated, preparation of the media and analytical procedures are reviewed and new samples are requested.
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c) Media Use - Melted agars are tempered in a waterbath until use but are not held longer
than 3 hours. A control bottle of media containing a thermometer is exposed to the same heating and cooling conditions as the media to be used in pouring plates. Sterile fermentation tubes are carefully handled and examined before use to ensure that gas bubbles are absent. Fermentation tubes stored refrigerated are incubated overnight and checked for the presence of gas prior to use. Tubes showing gas are rejected and a new lot of fermentation media is prepared.
d) Media Storage - Sterile media is prepared in amounts that will be used within holding
time limits. If fermentation tube media is refrigerated, the tubes are incubated overnight before use and checked for false positives. Media that can be stored for more than one week is prepared in tightly capped screw top tubes or bottles. Pre-poured media plates are sealed in plastic bags, inverted and refrigerated to retain moisture. Holding time for pre-poured media plates is two weeks, except for NA-mug which can be held for one week. Media containing dyes are protected from light and discarded if color changes are observed. Holding times for prepared media follow those specified in Standard Methods, 20th Ed., pg 9-9, Table 9020:IV.
e) Media Quality Control - Media preparation records are maintained in a bound logbook
with the name and date of preparer, name and lot number of the medium, amount weighed, volume and solvent used, pH measurements, balance used and any other adjustments. Sterility and positive and negative control culture checks are included for each lot of media used in sample analysis. Deviations from expected results are investigated by reviewing preparation and analytical procedure, and documenting corrective action.
4.3 Dilution Water Stock phosphate buffer solutions are stored refrigerated in tightly capped 100 mL screw top bottles for up to 1 year. Working phosphate buffer solutions are prepared from the stock, in reagent grade water, and are sterilized by autoclaving 15 minutes. Each batch of dilution/rinse water is checked for sterility by adding 50 mL of sterile, double-strength non-selective broth, which is then incubated at 35 +/- 0.5oC and checked for turbidity at 24 and 48 hours and the results recorded. Dilution water is stored at room temperature in stoppered bottles for up to 6 months. Any bottles observed to have turbidity are discarded. Dilution water controls are checked with each use and the lot is discarded if any growth is observed. Dilution water is prepared in accordance with laboratory SOP # 4.13.(1).
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Section 5 - Sampling Procedures 5.1 Sampling Containers Samples are collected in clean, distilled water rinsed, sterilized bottles of borosilicate glass or non-toxic polypropylene plastic, with screw cap closures. Sodium thiosulfate (0.1 N) is added, prior to sterilization, to containers intended for the collection of water samples containing residual chlorine or other halogen requiring neutralization. Sample containers are sterilized by autoclaving for 30 minutes. The sterility of sample containers is verified, per each batch, by adding 25 mL sterile, non-selective nutritive broth to a container, rotating the media within the container and incubating for 24 and 48 hours. Results are recorded at each interval and maintained in the QA Logbook. If growth is observed, the sterilization process is reviewed, with corrective action documented in the QA Logbook. 5.2 Sample Collection Samples are collected leaving ample air space in the bottle (2.5 cm) to facilitate mixing by shaking prior to examination. Sample ports are flushed or disinfected in order to collect samples representative of the water being tested. Sample bottles are kept closed until ready to be filled. Contamination of the inner surface of the cap or neck of the bottle is avoided. Sample containers are filled and immediately capped. The volume of samples collected is sufficient to carry out all tests required. Sample containers are labeled with an identifying number or location, time and date of collection. Other pertinent sampling information is included on a field data collection form. In collecting samples from recreational areas, samples are taken at a uniform depth of approximately 1 m. Waders and shoulder length disposable gloves are available for sampling in contaminated areas. When sampling from other non-potable sources (rivers, streams, lakes or ponds) samples are taken by holding the bottle near its base in the hand and plunging it, neck downward, below the surface. The bottle is turned until the neck points slightly upward and directed towards the current, or pushing bottle forward in a direction away from the hand. A chain of custody form must accompany all samples including those shipped by mail or courier. Upon receipt of samples in the laboratory, the chain of custody is completed and signed. Samples are then logged in with a unique identification number, which follows the samples throughout the testing and reporting procedures. For this purpose, a Sample Receipt & Identification Logbook is maintained in the laboratory. The sample ID numbering system uses the month and year of sample receipt with the chronological number of samples received for the month attached.
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5.3 Sample Preservation and Storage Microbiological examination of water samples is initiated as soon as possible after collection. A iced cooler and a temperature control blank is used for transportation to the laboratory if analysis cannot be initiated within one hour. Holding times for specific water types are as follows: a) Drinking water for compliance purposes - Samples are iced in a cooler during transit to
the laboratory and refrigerated upon receipt. Samples are analyzed on the day of receipt. Maximum holding time will not exceed 24 hours from the time of collection to the time of analysis for coliform bacteria. For heterotrophic plate counts the maximum holding time is 8 hours.
b) Nonpotable water for compliance purposes - Source water, stream pollution, recreational
water and wastewater samples are kept iced in a cooler during a maximum transport time of 4 hours. Upon arrival at the laboratory, a temperature control blank is checked for adherence to the storage temperature requirement of 1 – 4oC. Samples are refrigerated upon receipt at the laboratory and processed within 2 hours.
Samples which fail to meet the preservation and storage requirements are rejected. The sampling entity is notified and new samples are requested. Section 6 - Laboratory QA/QC Testing 6.1 Microbiological Quality Control Procedures a) Plate Count Comparison - Counts on one or more positive samples is conducted
monthly and compared with those of other analysts testing the same samples. Replicate counts for the same analyst should agree within 5% and those between analysts should agree within 10%. Statistical calculation of data precision is performed in accordance with Standard Methods Section 9020B.5b. If replicate counts do not fall within prescribed limits, colony counting equipment is inspected, the plate counting procedure is reviewed and corrective action is documented.
b) Control Cultures/Positive and Negative Controls - Each new lot of medium is checked
by testing with known positive and negative control cultures for the organism(s) under test. Specific control cultures are referenced in Section 3.4 of this manual. If a new lot of medium does not perform appropriately, control cultures are replaced with fresh cultures
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and the preparation of the media employed is reviewed. Any corrective action taken is documented in the Control Culture Logbook.
c) Duplicate Analysis - Duplicate analyses are performed on 10% of samples and on at
least one sample per test run - defined as an uninterrupted series of analyses. Calculation of data precision is as described in part e) below. If duplicates do not fall within data precision criteria, the analytical procedure is reviewed and corrective action is documented in the Precision and Accuracy Logbook.
d) Sterility Checks - For each series of analyses, the sterility of media, pipets, rinse water
and glassware, as appropriate, is checked. If any contamination is indicated, the cause is determined and the analytical data is rejected.
e) Precision of Quantitative Microbial Methods - Duplicate analyses is performed on 15
positive samples undergoing quantitative microbial analyses by membrane filtration procedures. The logarithm of each duplicate result is calculated and used to determine the range (R) for each pair of transformed duplicates. The mean range of the transformed duplicates is used in the equation 3.27 (R) to establish precision criterion. If the defined precision criterion is exceeded then analytical results are discarded and the problem is identified and resolved prior to making further analyses. Precision criterion is updated for each new set of 15 duplicate results.
f) Method Verification - For membrane filtration procedures with new lots of commercial
media, positive samples may be verified by picking at least 10 representative colonies for transfer to selective mediums. Results are scored and counts are adjusted based on percentage of verification.
g) Laboratory Precision and Accuracy for Physical/Chemical Parameters - The
laboratory establishes acceptance limits for precision and accuracy and maintains quality control charts for the various physical/chemical parameters commonly analyzed in conjunction with microbial analyses (ie; pH, salinity, turbidity, chlorine, etc.) to assess the validity of the analytical process. For precision, duplicate analyses are employed as described in section c) above, for calculation of the closeness of agreement between repeated measurements. Standard deviations are applied to mean percent deviation in establishing upper and lower control limits for acceptable precision criterion. Laboratory standards are utilized to establish accuracy criterion, as the closeness of a measurement to the true value, in the assessment of possible systematic error. Standard deviations are applied to mean percent recoveries in defining upper and lower control limits. Measurements are acceptably accurate when both systematic and random errors are low.
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QC results outside the acceptability limits warrant investigative and corrective action prior to further analyses. Equipment and the analytical procedures used are examined and corrective actions are documented in the laboratory Precision and Accuracy Logbook.
h) Annual Proficiency Tests - The laboratory participates in annual Proficiency Tests with
a DEP approved Proficiency Test Provider, to demonstrate microbiological proficiency in the analysis of total coliform and fecal coliform in potable water samples and enterococcus in ambient water samples. The laboratory reports the specific analytical method and media used for each PT round to the Provider, and reports results of each sample analysis as coliform present or absent, and if coliform present, either present or absent for fecal coliform. The acceptability of results is determined by the PT Provider. Results must be sent directly from the PT Provider to the DEP Lab Certification Office (LCO). Acceptable performance for total and fecal coliform is defined as the correct analysis of at least 90% of the samples in each testing round with no false negatives. If a PT round is determined by the PT Provider to be not acceptable, the laboratory, within 30 days of receiving notification of the failure, will determine the cause of error by checking media preparation, reviewing the analytical procedures used and checking for possible contamination. Upon taking corrective action, the DEP LCO will be notified in writing describing the corrective actions taken. In this instance, the laboratory will participate in a new PT round and report results to the Provider and the DEP LCO. The follow up PT round is completed within the same calendar year, from 30-90 days after the initial failed PT notice.
Section 7 - Standard Operating Procedures 7.1 Written Standard Operating Procedures (SOPs) are maintained in a Laboratory SOP Manual, and are provided to each analyst. The SOPs describe in a detailed step-wise fashion all laboratory operations involving instrumentation, reagents, analytical methods, QA/QC, calculations and reporting requirements to assure uniform operations between analysts and analyses. The SOPs specific for recreational water testing are: SOP # 4.42 - Monitoring New Bedford Bathing Beaches: Collection, Analysis, Quality
Assurance, Regulations and Reporting. SOP # 4.48 - Bacteriological Examination of Environmental and Recreational Waters for 24
Hour Enterococci Using EPA Method 1600 and the Membrane Filtration
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Technique. SOP # 4.50 - Data Reduction, Handling and Reporting Specific To Contractual Compliance
With The MDPH Beach Program Quality Assurance Project Plan. The current complete list of Laboratory SOPs are as follows: 1.1 How to Write an SOP 1.2 Data Recording, Handling and Storage 1.3 Ensuring Compliance with GLPs 1.4 Sample Intake Procedures for Well Waters 3.15 Orion Star Meter 3 Series pH Meter 3.16 Denver Instrument Company Model XL-1800 Balance 3.17 SOP for Incubators 3.18 SOP for Waterbaths 3.21 SOP for Thermometers Used in Testing 3.31 Barnstead Mega-Pure Water Still and Barnstead Nanopure II D3077 Series 3.32 Hach DR 2010 Spectrophotometer Test for Free and Total Chlorine 3.34 Use, Calibration and Maintenance of the YSI Model 57 Dissolved Oxygen Meter 3.39 Calibration of Eppendorf Pipettors 3.45 Use of the LaMotte 2020 Turbidometer 3.48 Use of the YSI Model 30 Handheld Salinity, Conductivity & Temperature Meter 4.9 Bacteriological Examination of Shellfish Samples 4.10 Bacteriological Examination of Seawater by the Medium A-1 Method for Fecal Coliform 4.11 Washing Glassware Used in Shellfish and Waters Testing 4.12 SOP for Heterotrophic Plate Counts 4.13 Preparation of Phosphate Buffered Dilution Water 4.26 Bacteriological Examination of Potable Water for Members of the Total Coliform Group
on mEndo Agar LES, SM 9222B,Using the Membrane Filtration Technique and Rapid Confirmation of E.coli using SM 9222G on NA-Mug medium.
4.27 Bacteriological Examination of Environmental Waters for Members of the Fecal Coliform Group Using the Membrane Filtration Technique
4.28 Bacteriological Examination of Environmental, Recreational, Drinking and Ground Waters for Confirmatory Testing of Total and Fecal Coliforms for E. coli
4.29 Bacteriological Examination of Seawater and Pollution Sources for E. coli Utilizing the mTec Direct Testing Method
4.41 LYFO-DISK Microorganisms for Bacteriological Control Cultures 4.42 Monitoring New Bedford Bathing Beaches, Collection, Analysis, Quality Assurance,
Regulations and Reporting 4.43 Bacteriological Examination of Environmental Waters for the 48 hour Fecal
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Streptococci/Enterococci Using the Membrane Filtration Technique 4.44 Bacteriological Examination of Seawater Holding Tanks by the Double Strength Media
MPN Total Coliform Test 4.48 Bacteriological Examination of Environmental, Wastewater and Recreational Waters for
24 hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique 4.49 Total Suspended Solids EPA Method 160.2 4.50 Data Reduction, Handling and Reporting Specific to Contractual Compliance with the MDPH Beach program Quality Assurance Project Plan Section 8 - Documentation and Recordkeeping 8.1 The documentation and recordkeeping system provides needed information on sample collection and preservation, analytical methods, raw data, calculations through reported results, and a record of persons responsible for sampling and analyses. All records are kept in bound notebooks, with entries in ink, and corrections made by drawing a single line through any change with the correction entered next to it. Laboratory raw data worksheets are bound with copies of computer generated reports. Reports generated from laboratory raw data are checked for accuracy, and are signed and dated. All bound laboratory notebooks are archived by fiscal year and maintained on laboratory premises for a period of ten years. Electronic databases are kept secure in a locked office.
New Bedford Health Department Laboratory SOP # 4.48(13) Page 1 DATE EFFECTIVE:______February 12, 2008_____________ APPROVALS: __Leslie Aubut____________March 12, 2013__ Laboratory Director Date of Review/Update
Standard Operating Procedure for
Bacteriological Examination of Environmental and Recreational Waters for 24 Hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique
OBJECTIVE: This SOP describes the procedures used in the analysis of enterococci by membrane filtration, using EPA Method 1600. The procedure is intended for the analysis of fresh, marine and wastewater. The SOP also describes laboratory methods, reporting requirements and quality control criteria in accordance with 310 CMR 42.00 and the MDPH Beach Project QAPP. EQUIPMENT AND SUPPLIES:
1. Dissection Microscope with 10X magnification and cool, white fluorescent light for counting plates.
2. Hand Tally counting device.
New Bedford Health Department Laboratory SOP # 4.48(13) Page 2
3. Plastic pipets, bacteriological, sterile, calibrated To Deliver. 4. Graduated cylinders, 100 – 1000 mL, sterile. 5. Membrane Filtration Units, sterile, plastic. 6. Electric vacuum pump with 1L filter flask and safety trap. 7. Forceps with smooth tips. 8. Alcohol and Bunsen Burner for flame sterilization. 9. Working thermometer checked against a NIST Standards thermometer. 10. Field use thermometer. 11. Petri dishes, sterile, plastic, 9x50 mm, with tight lids. 12. Dilution bottles, sterile, glass, marked at 99 mL (1:100) and 90 mL (1:10). 13. Membrane Filters, sterile, grid marked, 47 mm with 0.45 um pore size. 14. Incubator maintained at 41 +/- 0.5oC. 15. Waterbath maintained at 45 +/- 0.5oC for tempering agar. 16. Sample Bottles, 250 mL, autoclavable polypropylene.
REAGENTS AND MEDIA: Stock phosphate buffer solution: 3.4 g phosphate dihydrogen phosphate 50 mL reagent grade water Adjust the pH of the solution to 7.2 with 1N NaOH and bring the volume to 100 mL with reagent grade water. Autoclave at 121oC for 15 minutes. Store refrigerated and discard if turbidity or evidence of contamination is observed. Stock magnesium chloride solution: 8.1 g MgCl2
.6H2O 100 mL reagent grade water Sterilize the solution by autoclaving at 121oC for 15 minutes. Store refrigerated. Discard if turbidity or contamination is observed. Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5 mL of the magnesium chloride stock per liter of reagent grade water. Dispense in appropriate amounts for dilutions or in containers for rinse water. Autoclave at 121oC for 15 minutes. Final pH should be 7.0+/-0.2. Store at room temperature and discard if turbidity or contamination is observed. Perform a sterility check with each new batch using 50 mL of double strength TSB and 50 mL of the dilution/rinse water incubated at 35+/-0.5oC for 24 and 48 hour checks for growth (G) or no growth (NG). Record the
New Bedford Health Department Laboratory SOP # 4.48(13) Page 3 results in the lab Quality Assurance Logbook. A sterility control is also run each time the dilution/rinse water is used in an analysis and results are recorded on the lab raw data worksheet. MEI Agar: Per 100 mL of media - Add 7.12 g of dehydrated basal medium (mE agar, Difco 0333) plus 0.075 g of indoxyl B-D glucoside (Sigma # I3750) to 100 mL of reagent grade water. Heat to boiling to dissolve ingredients. Autoclave at 121 C for 15 minutes and cool to 45 oC in a waterbath. After sterilization and cooling, add 0.520 mL of Nalidixic Acid solution* to the mEI medium. Add 0.002 g triphenyl tetrazolium chloride (TTC) separately to the 100 mL of prepared mEI medium and mix. Pour 4.0 ml of the mEI agar into 50 mm Petri dishes and allow to solidify. The final pH should be 7.1 +/- 0.2. *Nalidixic Acid solution - Add 0.48 g of nalidixic acid and 0.400 mL of 10N NaOH to 10 mL of sterile reagent grade water and mix. Use 0.520 mL per 100 mL of mEI medium. QUALITY CONTROL CHECK: Prior to the first use of the media, the laboratory shall test each batch with a pure culture of a known positive reaction and a known negative reaction. The results of the quality control check are recorded in the Media Preparation Logbook. STORAGE: Prepared plates are labeled with the lot# (date of preparation) and the name of the medium. Prepared mEI plates are stored inverted, in sealed bags @ 2 - 4 degrees C for up to two weeks. PROCEDURE:
1. Label Petri dishes and report forms with sample identification and volume used. Select sample amount to give preferably 20-60 colonies on the membrane surface.
2. Place a 0.45um sterile membrane filter on the filter base, with the grid side up, and attach the funnel to the base.
3. Shake the sample bottle vigorously 25 times to distribute the bacteria evenly and measure the desired volume into the filter funnel.
4. Filter the sample and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse water. Turn off the vacuum.
5. Transfer the filter to the mEI Agar Petri dish using sterile forceps. Roll the filter onto the agar surface to avoid the formation of bubbles between the membrane and the agar surface.
6. Cover the dish, invert and incubate at 41o +/- 0.5oC for 24 hours +/- 2 hours. 7. Count all colonies with a blue halo as enterococci regardless of colony color. Count
colonies using a fluorescent lamp and magnification. The positive control used for this method is Enterococcus faecalis and the negative control used is Staphylococcus
New Bedford Health Department Laboratory SOP # 4.48(13) Page 4
epidermidis. CALCULATION AND REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution: Enterococci/100 mL = 100 (number of enterococci colonies) (volume of sample filtered, in mL) Report the results as Enterococci per 100 mL. QUALITY CONTROL: Laboratory Duplicates: Analyze at least one sample in duplicate for each analytical batch of 10 samples. Calculate the logarithm of each duplicate result. If either duplicate value is <1, add 1 to both results before calculating the logarithms. Calculate the range of logarithms for each duplicate set. When 15 sets of duplicate results are obtained, calculate the precision criterion with equals 3.27 times the mean range of logarithms for the 15 duplicate sets. Thereafter, if the logarithm range of a duplicate set is greater than the precision criterion, the analytical results for this batch of samples must be reported to the sampling entity as “unacceptable precision”, and fresh samples requested for analysis. Prior to the analysis of new samples, the laboratory must derive a new precision criterion using the most recent sets of 15 duplicate results. Negative Controls (sterile blanks): At the beginning and end of the filtration series and after every 10th sample, filter sterile buffered dilution water and process as a sample to check the sterility of all components in the analytical system and to check for cross contamination. If enterococcus colonies are found in the negative control plates, the analytical results for the batch of samples must be reported to the sampling entity as “unacceptable background contamination”, and fresh samples requested for analysis. The laboratory must investigate and resolve the contamination problem prior to analysis of any new samples. Positive Controls: With each analytical batch of 10 or fewer samples, filter sterile, buffered dilution water spiked with Streptococcus faecalis and process as a sample to assess the performance of the media and check for inhibitors. If no enterococcus colonies grow on the positive control plates, the
New Bedford Health Department Laboratory SOP # 4.48(13) Page 5 analytical batch is reported to the sampling entity as “unacceptable positive control failure” and fresh samples are requested for analysis. The laboratory must investigate and resolve the failure prior to the analysis of new samples. External Proficiency (PT) Tests: To evaluate the ability of the laboratory to produce accurate and precise results within specified acceptance criteria, the laboratory will conduct enterococcus profiency testing annually on an unknown sample obtained from Environmental Resource Associates. The results of PTs are kept on file in the laboratory and sent to MADEP Lab Certification Office. WASTE MANAGEMENT: Bacterial plates, positive and negative controls and dilutions thereof, will be autoclaved prior to disposal. The bacterial plates are placed into biohazard bags prior to autoclaving at 121oC for 30 minutes. REFERENCES: Standard Methods for the Examination of Water and Wastewater, 20th Edition 1998. EPA Office of Water, Method 1600, Membrane Filter Test for Enterococci in Water , EPA 821-R-97-004. MDPH Beach project QAPP, Revision # 5, 5/15/2007.
NEW BEDFORD HEALTH DEPARTMENT LABORATORY SOP # 4.27 (12) Page 1 of 2
DATE EFFECTIVE: __December 6, 2012 APPROVALS: _Leslie Aubut 12/6/12
Authorized By Date
Standard Operating Procedure for
Bacteriological Examination of Environmental and Recreational Waters for Members of the Fecal Coliform Group Using the Membrane Filtration Technique
Objective: This SOP describes the procedures used to examine environmental and recreational waters for members of the fecal coliform bacteria group by membrane filtration using mFC medium and describes the quality control procedures for this methodology.
SAMPLE HOLDING TIME AND TEMPERATURE: Samples are held below 10oC during a maximum transport time of 6 hours. Upon receipt of samples, chain of custody and logging-in at the laboratory, samples are refrigerated and processed within 2 hours.
MEDIA PREPARATION: Prepare commercial, dehydrated mFC agar with 1% rosolic acid solution addition according to manufacturer’s instructions. Record preparation details and assigned lot number in the laboratory Media and Reagent Preparation Logbook. Pipet 5 mL of media into each sterile plastic, 60 x 15 mm Petri plate. Prepared mFC agar is stored in the refrigerator for up to two weeks, inverted, in sealed containers under refrigeration.
NEW BEDFORD HEALTH DEPARTMENT LABORATORY
SOP # 4.27 (12) Page 2 of 2
PROCEDURE: Select sample amount to give preferably 20-60 colonies on the membrane filter surface. Filter sample through a 0.45um, gridded, sterile membrane. For sample dilutions use sterile, phosphate-buffered dilution water. Rinse the sides of the filtration apparatus between successive filtrations with the dilution water. Transfer filter to the mFC agar Petri dish, avoiding air bubbles beneath the membrane. Prepared plates are sealed with laboratory parafilm, placed inverted inside a glass beaker of appropriate size and placed in a waterbath for incubation at 44.5oC for a 24+/-2 hour period. Count all various shades of blue colonies as fecal coliform. Do not count non-fecal coliform colonies which are grey to cream colored. Count colonies using a 10X magnification and calculate fecal coliform density based on dilution factors applied.
REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution, and report as fecal coliform per 100 mL of sample.
QUALITY CONTROL
Positive and Negative Controls: For each new lot of m FC broth used. Samples are analyzed concurrently with known positive and negative pure cultures. The positive control culture used is E. coli and the negative control culture is Enterobacter aerogenes. The pure cultures are purchased commercially and maintained in-house with records kept in the laboratory Control Cultures Logbook.
Sterility Checks: Check sterility of media, filters and rinse water at the start and at the end of each series of samples. Use sterile reagent water as the sample to check equipment sterility as applicable (filters, flasks, pipets, etc.). Record results of sterility checks on the laboratory raw data sheets.
Duplicate Analyses: Perform duplicate analyses on 10% of samples and on at least one sample per test run. Record results in the laboratory Precision and Accuracy Logbook. Calculate precision of duplicate analyses for the quantitative test method according to Standard Methods for the Examination of Water and Wastewater, 21th Ed., Section 9020B, pg 9-10.
REFERENCES: Standard Methods for the Examination of Water and Wastewater, Section 9222D, 21th Edition 2005.
New Bedford Health Department Laboratory SOP # 4.48(13) Page 1 DATE EFFECTIVE:______February 12, 2008_____________ APPROVALS: __Leslie Aubut____________March 12, 2013__ Laboratory Director Date of Review/Update
Standard Operating Procedure for
Bacteriological Examination of Environmental and Recreational Waters for 24 Hour Enterococci Using EPA Method 1600 and the Membrane Filtration Technique
OBJECTIVE: This SOP describes the procedures used in the analysis of enterococci by membrane filtration, using EPA Method 1600. The procedure is intended for the analysis of fresh, marine and wastewater. The SOP also describes laboratory methods, reporting requirements and quality control criteria in accordance with 310 CMR 42.00 and the MDPH Beach Project QAPP. EQUIPMENT AND SUPPLIES:
1. Dissection Microscope with 10X magnification and cool, white fluorescent light for counting plates.
2. Hand Tally counting device.
New Bedford Health Department Laboratory SOP # 4.48(13) Page 2
3. Plastic pipets, bacteriological, sterile, calibrated To Deliver. 4. Graduated cylinders, 100 – 1000 mL, sterile. 5. Membrane Filtration Units, sterile, plastic. 6. Electric vacuum pump with 1L filter flask and safety trap. 7. Forceps with smooth tips. 8. Alcohol and Bunsen Burner for flame sterilization. 9. Working thermometer checked against a NIST Standards thermometer. 10. Field use thermometer. 11. Petri dishes, sterile, plastic, 9x50 mm, with tight lids. 12. Dilution bottles, sterile, glass, marked at 99 mL (1:100) and 90 mL (1:10). 13. Membrane Filters, sterile, grid marked, 47 mm with 0.45 um pore size. 14. Incubator maintained at 41 +/- 0.5oC. 15. Waterbath maintained at 45 +/- 0.5oC for tempering agar. 16. Sample Bottles, 250 mL, autoclavable polypropylene.
REAGENTS AND MEDIA: Stock phosphate buffer solution: 3.4 g phosphate dihydrogen phosphate 50 mL reagent grade water Adjust the pH of the solution to 7.2 with 1N NaOH and bring the volume to 100 mL with reagent grade water. Autoclave at 121oC for 15 minutes. Store refrigerated and discard if turbidity or evidence of contamination is observed. Stock magnesium chloride solution: 8.1 g MgCl2
.6H2O 100 mL reagent grade water Sterilize the solution by autoclaving at 121oC for 15 minutes. Store refrigerated. Discard if turbidity or contamination is observed. Working phosphate buffered dilution water: Mix 1.25 mL of the stock phosphate buffer and 5 mL of the magnesium chloride stock per liter of reagent grade water. Dispense in appropriate amounts for dilutions or in containers for rinse water. Autoclave at 121oC for 15 minutes. Final pH should be 7.0+/-0.2. Store at room temperature and discard if turbidity or contamination is observed. Perform a sterility check with each new batch using 50 mL of double strength TSB and 50 mL of the dilution/rinse water incubated at 35+/-0.5oC for 24 and 48 hour checks for growth (G) or no growth (NG). Record the
New Bedford Health Department Laboratory SOP # 4.48(13) Page 3 results in the lab Quality Assurance Logbook. A sterility control is also run each time the dilution/rinse water is used in an analysis and results are recorded on the lab raw data worksheet. MEI Agar: Per 100 mL of media - Add 7.12 g of dehydrated basal medium (mE agar, Difco 0333) plus 0.075 g of indoxyl B-D glucoside (Sigma # I3750) to 100 mL of reagent grade water. Heat to boiling to dissolve ingredients. Autoclave at 121 C for 15 minutes and cool to 45 oC in a waterbath. After sterilization and cooling, add 0.520 mL of Nalidixic Acid solution* to the mEI medium. Add 0.002 g triphenyl tetrazolium chloride (TTC) separately to the 100 mL of prepared mEI medium and mix. Pour 4.0 ml of the mEI agar into 50 mm Petri dishes and allow to solidify. The final pH should be 7.1 +/- 0.2. *Nalidixic Acid solution - Add 0.48 g of nalidixic acid and 0.400 mL of 10N NaOH to 10 mL of sterile reagent grade water and mix. Use 0.520 mL per 100 mL of mEI medium. QUALITY CONTROL CHECK: Prior to the first use of the media, the laboratory shall test each batch with a pure culture of a known positive reaction and a known negative reaction. The results of the quality control check are recorded in the Media Preparation Logbook. STORAGE: Prepared plates are labeled with the lot# (date of preparation) and the name of the medium. Prepared mEI plates are stored inverted, in sealed bags @ 2 - 4 degrees C for up to two weeks. PROCEDURE:
1. Label Petri dishes and report forms with sample identification and volume used. Select sample amount to give preferably 20-60 colonies on the membrane surface.
2. Place a 0.45um sterile membrane filter on the filter base, with the grid side up, and attach the funnel to the base.
3. Shake the sample bottle vigorously 25 times to distribute the bacteria evenly and measure the desired volume into the filter funnel.
4. Filter the sample and rinse the sides of the funnel at least twice with 20-30 mL of sterile buffered rinse water. Turn off the vacuum.
5. Transfer the filter to the mEI Agar Petri dish using sterile forceps. Roll the filter onto the agar surface to avoid the formation of bubbles between the membrane and the agar surface.
6. Cover the dish, invert and incubate at 41o +/- 0.5oC for 24 hours +/- 2 hours. 7. Count all colonies with a blue halo as enterococci regardless of colony color. Count
colonies using a fluorescent lamp and magnification. The positive control used for this method is Enterococcus faecalis and the negative control used is Staphylococcus
New Bedford Health Department Laboratory SOP # 4.48(13) Page 4
epidermidis. CALCULATION AND REPORTING: If a dilution factor was applied, multiply the actual number of colonies counted per plate by the reciprocal of the dilution: Enterococci/100 mL = 100 (number of enterococci colonies) (volume of sample filtered, in mL) Report the results as Enterococci per 100 mL. QUALITY CONTROL: Laboratory Duplicates: Analyze at least one sample in duplicate for each analytical batch of 10 samples. Calculate the logarithm of each duplicate result. If either duplicate value is <1, add 1 to both results before calculating the logarithms. Calculate the range of logarithms for each duplicate set. When 15 sets of duplicate results are obtained, calculate the precision criterion with equals 3.27 times the mean range of logarithms for the 15 duplicate sets. Thereafter, if the logarithm range of a duplicate set is greater than the precision criterion, the analytical results for this batch of samples must be reported to the sampling entity as “unacceptable precision”, and fresh samples requested for analysis. Prior to the analysis of new samples, the laboratory must derive a new precision criterion using the most recent sets of 15 duplicate results. Negative Controls (sterile blanks): At the beginning and end of the filtration series and after every 10th sample, filter sterile buffered dilution water and process as a sample to check the sterility of all components in the analytical system and to check for cross contamination. If enterococcus colonies are found in the negative control plates, the analytical results for the batch of samples must be reported to the sampling entity as “unacceptable background contamination”, and fresh samples requested for analysis. The laboratory must investigate and resolve the contamination problem prior to analysis of any new samples. Positive Controls: With each analytical batch of 10 or fewer samples, filter sterile, buffered dilution water spiked with Streptococcus faecalis and process as a sample to assess the performance of the media and check for inhibitors. If no enterococcus colonies grow on the positive control plates, the
New Bedford Health Department Laboratory SOP # 4.48(13) Page 5 analytical batch is reported to the sampling entity as “unacceptable positive control failure” and fresh samples are requested for analysis. The laboratory must investigate and resolve the failure prior to the analysis of new samples. External Proficiency (PT) Tests: To evaluate the ability of the laboratory to produce accurate and precise results within specified acceptance criteria, the laboratory will conduct enterococcus profiency testing annually on an unknown sample obtained from Environmental Resource Associates. The results of PTs are kept on file in the laboratory and sent to MADEP Lab Certification Office. WASTE MANAGEMENT: Bacterial plates, positive and negative controls and dilutions thereof, will be autoclaved prior to disposal. The bacterial plates are placed into biohazard bags prior to autoclaving at 121oC for 30 minutes. REFERENCES: Standard Methods for the Examination of Water and Wastewater, 20th Edition 1998. EPA Office of Water, Method 1600, Membrane Filter Test for Enterococci in Water , EPA 821-R-97-004. MDPH Beach project QAPP, Revision # 5, 5/15/2007.
Appendix B - Water Quality Grab Sample Collection Standard Operating Procedure
Water Quality Grab Sample Collection Standard Operating Procedure
1.0 Scope and Application This standard operating procedure establishes the protocol to be followed for collecting water quality grab
samples for both the wet and dry weather monitoring events. Samples will be collected for bacteria (Enterococci
and Fecal Coliform) and possibly other optional parameters (Nitrates as Nitrogen, Orthophosphate, Total Kjeldahl
Nitrogen, and Oil and Grease).
2.0 Method Summary
Sample Location
When feasible, surface water samples will be collected directly from the outfall of the discharge pipe into the
laboratory analysis bottles. If the discharge pipe is inaccessible or if tidal water is back flowing into the
discharge pipe, the Field Teams will move the sampling site to the next available upstream manhole (or
catchbasin if no manholes exist in the drainage system). The sample in the manhole will be taken at the inlet
of the main stormdrain pipe. If the sample cannot be taken under free-flowing conditions, the Field Team will
take the sample as close to the inlet pipe as possible and make a notation on the comment section of field data
sheet (preferably with a photograph) as to the problems encountered at the site (tidal back flowing, inlet/outlet
elevations prevent free-flowing conditions, etc)
Sample Collection
To minimize potential sediment contamination, sample collection will begin with the bacterial samples. Once
the bacteria samples are collected, they will immediately be placed in a cooler on blue ice. Separate bottles
will then be used to collect water samples for all field tests (surfactants, ammonia, nitrates, and total chlorine)
and then any other laboratory testing (followed by appropriate storage protocol). If the sample location is not
accessible, samples will be collected using either a laboratory analysis bottle or a clean (rinsed in the field
with de-ionized water or purified water from the laboratories) sample collection container attached to a
sampling rod. If available, the Field Teams may also use a hand-held vacuum pump to collect samples. Once
collected, the container and/or tubing from the vacuum pump will be used to distribute the sample directly to
the appropriate lab bottles. The collection container and or tubing will be decontaminated (rinsing with de-
ionized water or purified laboratory water) prior to use at the next grab sample site to prevent cross
contamination.
3.0 Safety, Restrictions, and Limitations When accessing all monitoring sites, Field Teams must be aware of accessibility and safety issues, especially
during adverse weather conditions. At outfall pipes, Field Teams should not attempt collecting samples if the
gradient is too steep or slippery, water velocity too high, or water depth is over three feet. Field Teams must
follow all safety precautions as outlined by the local Department of Public Works when lifting
manholes/catchbasins grates or when entering/exiting manholes or catchbasins.
While collecting grab samples, Field Teams should wear clean latex gloves to minimize their contact with
contaminated water and also to prevent sample contamination. The Field Teams should never touch the inside of
any bottle or cap, especially the sterilized containers for the bacteria samples. If the sample is collected using a
sampling pole or vacuum pump, the sampling pole bottle or tubing must be decontaminated in the field prior to
use.
The laboratories should supply the proper size sample bottles to meet the required volume for testing. If the
containers are not adequate, multiple samples will be needed to supply the necessary volume. If possible, each
laboratory analysis bottle should be filled from a single grab sample. In addition, the QA/QC duplicate samples
should be filled from the same grab as the original sample bottles.
4.0 Sample Collection, Handling, and Preservation Samples to be analyzed for bacteria will be collected first using a sterile sampling container that has been
provided by the laboratory. Following the bacterial collection, a second sample will be taken for the analysis of
the on-site "in the field" (temperature/conductivity/salinity and ammonia) parameters. Once the "in the field"
analysis has been completed, this second bottle will be placed on ice and used for the "in house" analysis
(surfactants and nitrates). Additional samples will then be collected for any remaining laboratory parameters.
Once collected, all samples will be handled and preserved in accordance with Tables 3 through 7 in the QAPP.
The preferred method of sample collection is to fill the sample containers directly from the discharge under free-
flowing conditions. If a free-flowing sample is not feasible, the Field Team will collect the sample as close to the
discharge as possible. If the discharge is inaccessible, a sampling pole container or vacuum pump will be used to
collect the sample. Once the sample is retrieved, it will be completely mixed and then poured into the analysis
containers. If the water quality sample cannot be collected under free-flowing conditions and/or must be collected
with a sampling pole, the Field Team will document the testing conditions/methods on the field data sheets and
the chain of custody forms so that the laboratory results can be flagged for review.
5.0 Equipment and Materials .
6.0
Procedures Analysis laboratory should be contacted prior to sampling date.
Laboratory analysis bottles and equipment should be organized prior to each grab at the site.
New, clean, latex gloves should be worn at all times when handling the sample collection bottles and
obtaining samples in the field.
The caps from the lab analysis bottles should be removed just prior to collecting or receiving a sample.
They should be re-capped just after a sample is collected or received. The amount of time an opened
bottle is exposed to the environment should be minimized.
The bacteria samples should be collected first, followed by the field measurements and then any other
laboratory samples.
If the discharge pipe is free-flowing, obtain the laboratory sample directly from the pipe while minimizing
sediment disturbance. If the discharge pipe is not free-flowing, take the sample as close to the pipe as
possible. Note on the monitoring data sheet the conditions under which the sample was taken (not free-
flowing, tidal backflow, manhole, etc.) There should be a minimum of two people collecting samples at
all times, one to take all notes, fill out labels and forms, etc. while the other collects the samples.
coolers with blue ice hip-waders
pens and permanent markers manhole hook
field log books latex gloves
data collection sheet paper towels
chain of custody forms sealable bags
sampling pole or hand-held vacuum pump measuring tape
pre-labeled laboratory bottles camera
sampling pole with clean collection containers safety glasses
de-ionized water or laboratory purified water
Appendix C - Field Water Quality Measurements Standard Operating Procedure
Field Water Quality Measurements Standard Operating Procedure
1.0 Scope and Application
This standard operating procedure addresses the procedures to collect field water quality measurements using YSI
Model 30 meters (temperature, conductivity, salinity), Hach test strips (ammonia), Chemetrics K-9400
(surfactants), LaMotte Nitrate-Nitrogen test kit (nitrate) and Hach Pocket Colorimeter (chlorine).
2.0 Equipment Inspection, Maintenance, and Calibration
Prior to each sampling event, all test kits and equipment will be inspected to ensure the availability of testing
materials (Hach strips, test kits, and Chemetrics) and operability of equipment (YSI meter and Colorimeter).
Calibration of the YSI meter and Colorimeter will be in accordance with manufacturer's instructions. If any parts
of the testing equipment need to be repaired or replaced, this will be noted in the standard Equipment Inspection,
Testing and Maintenance Log Sheet.
Calibration checks on the YSI meters and colorimeter will be performed by the Field Team prior to each sampling
event with the equipment being re-adjusted as needed in accordance to manufacturer's instructions. Calibration
checks will be recorded on the standard Equipment Calibration Form and in the data log book.
3.0 Field Measurement Procedure
Field measurements will be collected at each sampling location after the filling of analysis bottles. Whenever
possible, the field measurements sample will be taken at the center of the discharge flow, at half of the depth and
upstream of the sample collector. For the YSI meter, care will be taken not to allow the probe to contact any
accumulated sediment.
As with the bacterial samples, if free-flowing conditions do not exist, the sample will be taken as close to the
discharge as possible or moved upstream at the discretion of the Stormwater Specialist (or designee). If the
discharge pipe is inaccessible, water quality samples will be collected using a sampling pole or hand held vacuum
pump. The sample collection point, collection conditions, and accessibility will be noted on the field data sheet.
All field data collections will be in accordance with manufacturer's instructions.
The Stormwater Specialist or Field Team Leader will be responsible for equipment maintenance and cleaning
following each sampling event. Any issues regarding the equipment or other testing materials will be reported
immediately to the Stormwater Specialist.
4.0 Quality Assurance
For quality assurance purposes, each Field Team will duplicate at least one sample or ten percent of all samples
collected (whichever is more) as part of each sampling event. A duplicate is a second reading of the same water
sample.
5.0 Field Water Quality Measurements Standard Operating Procedure
Prior to each sample round all of the YSI meters will be calibrated by the Field Team Leader in accordance with
the manufacturer's instructions.. The time and date of the calibration will be recorded on the standard Equipment
Quality Assurance Form.
References
YSI Pro 30 Users Manual (2011) https://www.ysi.com/File%20Library/Documents/Manuals/606082A-YSI-
Pro30-Manual-English.pdf
LaMotte Nitrate Nitrogen Test Kit (2013) http://www.lamotte.com/en/industrial/individual-test-kits/3615-01.html
Pocket Colorimeter II User Manual, Edition 1 (2014)