pdf 1 MB - Massachusetts Water Resources Authority

Quality Assurance Project Plan
for
Water Quality Monitoring
in Cape Cod Bay
2014 – 2016
Massachusetts Water Resources Authority
Environmental Quality Department
Report 2014-07
Citation:
Costa A, Larson E, Stamieszkin K. 2014. Quality Assurance Project Plan (QAPP) for Water Quality
Monitoring in Cape Cod Bay 2014-2016. Boston: Massachusetts Water Resources Authority. Report
2014-07. 94 p.
Citation:
Costa A, Larson E, Stamieszkin K. 2014. Quality Assurance Project Plan (QAPP) for Water Quality
Monitoring in Cape Cod Bay. Boston: Massachusetts Water Resources Authority. Report 2014-07. 94 p.
Quality Assurance Project Plan (QAPP)
for
Water Quality Monitoring in Cape Cod Bay
2014 – 2016
Prepared by
Amy Costa
Elizabeth Larson
Karen Stamieszkin
Center for Coastal Studies
Hiebert Marine Laboratory
5 Holway Avenue
Provincetown, MA 02657
(508) 487-3623
February 1, 2014
Quality Assurance Project Plan (QAPP)
for
Water Quality Monitoring in Cape Cod Bay
Prepared by: Provincetown Center for Coastal Studies, 5 Holway Ave, Provincetown, MA
02657
Program, Laboratory, and Database Manager:
___________________________________________________
Dr. Amy Costa, Director, Cape Cod Bay Monitoring Program
Provincetown Center for Coastal Studies
(508) 487-3623
__________________
Date
Field Coordinator:
___________________________________________________
Capt. Marc Costa, Marine Operations
Provincetown Center for Coastal Studies
(508) 246-1387
__________________
Date
Laboratory Quality Assurance:
__________________________________________________
Elizabeth Larson, QA Coordinator
Provincetown Center for Coastal Studies
(508) 487-3623
__________________
Date
Provincetown Center for Coastal Studies
QAPP Water Quality Monitoring in Cape Cod Bay
Distribution List
Amy Costa, CCS (Program Manager)
Richard Delaney, CCS (Executive Director)
Charles Mayo, CCS (Plankton Studies)
Elizabeth Larson, CCS (QA Coordinator)
David Taylor, MWRA (Project Manager)
Ken Keay, MWRA (Program Manager)
Michael Mickelson, MWRA (Program Manager)
Wendy Leo, MWRA (Senior Program Manager)
Revision 0
Distribution List
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TABLE OF CONTENTS
1.0 PROJECT MANAGEMENT .............................................................................................. 1
1.1
1.2
1.3
1.4
1.5
1.6
1.7
Project Organization ........................................................................................................... 1
Communication Plan .......................................................................................................... 2
Project Definition and Background .................................................................................... 2
Project Description and Schedule ....................................................................................... 2
Quality Objectives and Criteria for Measurement Data ..................................................... 6
Special Training Requirements and Certification............................................................... 9
Documentation and Records ............................................................................................... 9
2.0 MEASUREMENT/DATA AQUISITION ........................................................................ 10
2.1 Sampling Process Design (Experimental Design) ............................................................ 10
2.2 Sampling Methods Requirements ..................................................................................... 11
2.3 Sample Handling and Custody Requirements .................................................................. 14
2.4 Analytical Methods Requirements ................................................................................... 15
2.5 Quality Control Requirements .......................................................................................... 17
2.6 Preventive maintenance procedures and schedules .......................................................... 20
2.7 Corrective action contingencies ........................................................................................ 22
2.8 Inspection/Acceptance of Supplies and Consumables ..................................................... 22
2.9 Data Acquisition Requirements (non-direct measurements) ............................................ 23
2.10 Data Management ........................................................................................................... 23
3.0 ASSESSMENT/OVERSIGHT........................................................................................... 29
3.1 ASSESSMENTS AND RESPONSE ACTIONS.............................................................. 29
3.2 REPORTS TO MANAGEMENT..................................................................................... 29
4.0 DATA VALIDATION AND USABILITY ....................................................................... 29
4.1 Data Review, Validation and Verification Requirements ................................................ 29
4.2 Validation and Verification Methods ............................................................................... 30
4.3 Reconciliation with User Requirements ........................................................................... 30
5.0 REFERENCES ................................................................................................................... 31
APPENDIX A: Standard Operating Procedures ........................................................................... 32
APPENDIX B: Data Forms .......................................................................................................... 74
APPENDIX C: Guidance Protocol on the Interaction with Whales ............................................. 86
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LIST OF TABLES
Table 1-1. Contact Information for Water Quality Monitoring Program in Cape Cod Bay ........... 2
Table 1-2. Sampling locations of the water quality monitoring stations ........................................ 3
Table 1-3. Routine measurements to be conducted at the three stations ........................................ 3
Table 1-4. Sampling schedule for CCB and SBNMS water quality monitoring ............................ 4
Table 1-5. Accuracy and precision of instrument sensors .............................................................. 6
Table 1-6. Desired precision, accuracy and MDL for each parameter based on quality objectives8
Table 2-1. Sample collection and storage ..................................................................................... 12
Table 2-2. Methods of detection for analytes ............................................................................... 15
Table 2-3. Data quality objectives ................................................................................................ 18
Table 2-4. Laboratory Analytical QC: Nutrients (Nitrate+Nitrite, Ortho-Phosphate, Ammonia,
Silicate, TN, and TP) ............................................................................................................ 19
Table 2-5. Laboratory Analytical QC: Chlorophyll a .................................................................. 19
Table 2-6. Specifications for data sets ......................................................................................... 24
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LIST OF FIGURES
Figure 1-1. Organizational Chart for Water Quality Monitoring in Cape Cod Bay ....................... 1
Figure 1-2. Sampling locations in CCB and SBNMS..................................................................... 5
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1.0 PROJECT MANAGEMENT
1.1
Project Organization
Figure 1-1 presents the project management structure for the Water Quality Monitoring Program in Cape
Cod Bay for MWRA. This plan details the project organization, sample handling, sample analysis, and
data loading for this program.
Executive Director, CCS
Rich Delaney
Program Manager
Lab Manager
Database Manager
Amy Costa
QA Officer
Elizabeth Larson
Field Coordinator
Marc Costa
Field and Lab
Assistant
Jennifer Burkhardt
Figure 1-1. Organizational Chart for Water Quality Monitoring in Cape Cod Bay
Dr. Amy Costa is the CCS Director of the Cape Cod Bay Monitoring Program and will fill a number of
roles including Program Manager, Laboratory Manager, and Database Manager. As Program Manager,
she will oversee all aspects of the project that incorporate the monitoring program including: fiscal
management, project objectives, data uses, and program changes. As Laboratory Manager, she will
perform lab analyses according to QAPP and ensure correct procedures are used, holding times are met,
and adequate documentation is provided. As Database Manager, she will maintain the data systems for
the program, perform/oversee data entry, and check entries for accuracy against field and lab forms.
Capt. Marc Costa is the Field Coordinator for this project. He is responsible for the general coordination
of monitoring activities on the water. Ms. Elizabeth Larson is the QA Officer. She is responsible for
developing, directing, and coordinating the quality assurance/quality control (QA/QC) program
regarding the collection and analyzing of water samples. She is also responsible for reviewing
laboratory practices and procedures to ensure compliance with quality assurance and safety standards.
Contact information is provided in Table 1-1.
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Table 1-1. Contact Information for Water Quality Monitoring Program in Cape Cod Bay
Name
Title/Role
Location
Richard Delaney
Amy Costa
Marc Costa
Jennifer Burkhardt
Elizabeth Larson
David Taylor
Ken Keay
Executive Director
Program Manager
Field Coordinator
Research Assistant
QA Coordinator
Project Manager
Program Manager
CCS
CCS
CCS
CCS
CCS
MWRA
MWRA
1.2
Email Address
delaney@coastalstudies.org
acosta@coastalstudies.org
mcosta@capecod.com
jburkhardt08@gmail.com
elarson@coastalstudies.org
david.taylor@mwra.state.ma.us
ken.keay@mwra.state.ma.us
Phone
508.487.3622
508.247.7743
508.246.1387
585.314.8850
310.916.6634
617.788.4952
617.788.4947
Communication Plan
Amy Costa will be the primary contact for this project. Email or telephone calls will be the day-to-day
method of communication. Significant technical issues will be documented in email or memoranda,
summarizing the key discussions and actions taken. Dave Taylor will be notified immediately regarding
any issues or deviations from the project plan including if project surveys are not carried out as planned
or samples are missing. If David Taylor is not available, Ken Keay should be notified.
Annual project meetings will be held in the spring of each year to review and update the project plan,
including updating information on sample scheduling, sampling locations and frequency, analytical
methods, and staffing.
1.3
Project Definition and Background
MWRA has implemented a marine environmental monitoring program in Massachusetts and Cape Cod
Bays in support of MWRA’s outfall in Massachusetts Bay. This project will continue the ambient water
column monitoring required at three locations by adding these three stations to CCS’s ongoing Cape
Cod Bay monitoring program. Two of the stations are located in Cape Cod Bay (CCB) and one in
Stellwagen Bank National Marine Sanctuary (SBNMS).
CCS has been conducting marine mammal and habitat research in CCB and SBNMS for over 30 years.
The monitoring to be conducted as part of this project will allow CCS to better understand and protect
the ecology of CCB and SBNMS, and the whales that use the two areas. It will also provide water
quality data at the same three locations that MWRA needs to monitor in CCB and SBNMS, to meet its
NPDES permit requirements.
1.4
Project Description and Schedule
CCS will monitor MWRA’s three stations in CCB and SBNMS (Table 1-2) as part of their on-going
program to monitor for possible outfall impacts on areas downstream of the outfall. The three sites, at
locations shown in Figure 1-2, will be sampled nine times per year. Water quality monitoring at these
stations will include measurements of surface photosynthetically active radiation (PAR), temperature,
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salinity, dissolved oxygen, fluorescence, PAR, nutrient concentrations (dissolved and total nitrogen and
phosphorous, silicate), phytoplankton biomass (chlorophyll a and phaeophytin), and phytoplankton and
zooplankton identification and enumeration. The samples to be collected at each location are listed in
Table 1-3; and the proposed sampling schedule is outlined in Table 1-4.
Table 1-2. Sampling locations of the water quality monitoring stations
Station Id
Description
Target latitude
Target longitude
Average water depth (m)
F01
East CCB
41.85083
-70.4533
26.2
F02
West CCB
41.90817
-70.2283
32.8
F29
South SBNMS
42.11667
-70.29
64.7
Table 1-3. Routine measurements to be conducted at the three stations
Type of measurement
Depth
Parameter
Surface PAR
Temperature
Salinity
Dissolved oxygen
Depth of sensor
Chlorophyll fluorescence
PAR
Hydro profile
From near surface (approximately
0.5-1.5 m) to near-bottom (3-5 m
from bottom). Profiling at 0.5 m
intervals
Water chemistry
Two depths:
Near- surface
Near- bottom
Nitrate + nitrite
Ammonia
Ortho-phosphate
Silicate
Total nitrogen
Total phosphorus
Extracted chlorophyll
Near-surface
Oblique net tow
Enumeration + Identification
Enumeration + Identification
Phytoplankton
Zooplankton
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Table 1-4. Sampling schedule for CCB and SBNMS water quality monitoring
Survey
Proposed sampling
2014
WN141
2/4/2014
WN142
3/18/2014
WN143
4/8/2014
WN144
5/13/2014
WN145
6/17/2014
WN146
7/22/2104
WN147
8/19/2014
WN148
9/2/2014
WN149
10/21/2014
2015
WN151
2/3/2015
WN152
3/17/2015
WN153
4/7/2015
WN154
5/12/2015
WN155
6/16/2015
WN156
7/21/2015
WN157
8/18/2015
WN158
9/1/2015
WN159
10/20/2015
2016
WN161
2/9/2016
WN162
3/22/2016
WN163
4/12/2016
WN164
5/17/2016
WN165
6/21/2016
WN166
7/26/2016
WN167
8/23/2016
WN168
9/6/2016
WN169
10/25/2016
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Figure 1-2. Sampling locations in CCB and SBNMS
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1.5
Quality Objectives and Criteria for Measurement Data
The parameters measured and the concentration reporting units are listed in Table 2-3.
1.5.1
Quality Objectives
Data quality objectives are as follows:
•
•
•
To determine if the eutrophication status of Cape Cod Bay has a long-term response to nutrients
from the MWRA outfall and/or to other regional forcing
To ensure that the sample results are representative of the location sampled
To ensure that the sample results are accurate
1.5.2
Measurement Performance Criteria
The first objective will be met by examining data collected during these 9 surveys to measure water
column parameters, quantify nutrients and chlorophyll, and document changes in phytoplankton and
zooplankton community structure. The second objective will be met by repeated measurements
collected at the same locations over time to quantify the variability of results at each station. The third
objective will be met by analyzing laboratory replicates to ensure reproducibility of results. Definitions
of quality control samples are provided in Section 2.4.2.
1.5.2.1 Navigational and Hydrographic Data
1.5.2.1.1 Precision and Accuracy
Manufacturer precision and accuracy objectives for navigation and hydrographic sampling are presented
in Table 1-5. Navigational accuracy of 10 m is required for this program.
1.5.2.1.2 Comparability
All sampling positions will be comparable to positions obtained by previous MWRA monitoring
activities. The station locations are targets and sampling will be conducted within 300 m of the targets
as visualized on the Northstar 952XD navigation display. The electronic measurement instruments that
will be used during the water quality monitoring surveys are similar to the instruments that have been
used by MWRA contractors since 1992.
Table 1-5. Accuracy and precision of instrument sensors
Sensor
Navigation
Surface PAR
Pressure
Temperature
Model
Northstar
952XD
Biospherical
QSR-2100
Seabird SBE
19plus V2
Seabird SBE
19plus V2
Units
Degree
Range
World
Accuracy
4m
Precision
4m
μE/(m2·sec)
0.14 to 5000
10
1
Decibars
0 to 1000
0.1%
0.1
o
-5 to 35
0.001
0.01
C
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Sensor
Conductivity
Dissolved
Oxygen
Fluorometer
(Chl a)
PAR
Model
Seabird SBE
19plus V2
Seabird SBE-43
Units
mS/cm
Range
0 to 70
Accuracy
0.03
Precision
0.01
mg/L
0 to 15
0.50
0.05
WET Labs
ECO-FL
Biospherical
QSP-2300L
μg/L
0.01 to 125
0.01
0.01
μE/(m2·sec)
0.14 to 5000
10
1
1.5.2.1.3 Representativeness
The representativeness of the sampling program design is detailed in the Outfall Monitoring Plan
(MWRA 1997) and defined by the results collected since 1992. Representativeness will also be ensured
by proper handling, storage, and analysis of calibration samples so that the materials analyzed reflect the
collected material. Deviations from the data collection procedures described in this QAPP will be
documented in the survey logbook and described in the survey report.
1.5.2.1.4 Completeness
The navigation software system outputs navigation positions at an interval of 1-second. The software
system will display all position fixes and save these fixes in an electronic file during hydrocasts and
sampling operations. The project time interval requirement for obtaining positions during sampling is
one (1) minute. Thus, even if a few bad data streams from the dGPS navigation system to the computer
are experienced, the software will provide enough position fixes within each 1-minute period for 100%
data collection.
Because hydrographic data are acquired electronically and monitored in real time, no loss of data is
expected. Stations will not be occupied if CTD measurements and navigation coordinates (at a
minimum) cannot be obtained. If instrument malfunctions occur and operations are modified or
suspended during any survey day, a decision on modification of activities for that survey will be made
with consultation and agreement of MWRA, whenever possible. A 10% loss of hydrographic and
navigation data over the entire program is not expected to compromise the objectives of the program.
1.5.2.2 Water Sampling and Analysis
1.5.2.2.1 Precision and Accuracy
Precision and accuracy of laboratory procedures are ensured by the analysis of quality control (QC)
samples including procedural/filter blanks, prepared standards, standard reference samples (SRS), where
available, laboratory control samples, laboratory replicates and field replicates, as applicable. Table 1-6
lists the desired precision, accuracy, and detection limit goals for each parameter to be measured. QC
samples to be analyzed in the laboratory to assess precision and accuracy are listed in Table 2-4 and
Table 2-5. Method procedural blanks for parameters that use blank correction are the batch-average
uncorrected method procedural blanks.
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Table 1-6. Desired precision, accuracy and MDL for each parameter based on quality objectives
Parameter
Nitrate+Nitrite
Ammonia
Orthophosphate
Silicate
Total nitrogen
Total
phosphorus
Chlorophyll a
and
Phaeophytin
Field
Precision
<30% RPD2
for field
duplicates
<30% RPD
for field
duplicates
<30% RPD
for field
duplicates
<30% RPD
for field
duplicates
<30% RPD
for field
duplicates
<30% RPD
for field
duplicates
<50% RPD
for field
duplicates
Lab Precision
<10% RPD for
instrument duplicates
<10% RPD for
instrument duplicates
<10% RPD for
instrument duplicates
<10% RPD for
instrument duplicates
<10% RPD for
laboratory duplicates
<10% RPD for
laboratory duplicates
<15% RPD for
laboratory
(instrument) duplicates
Accuracy
± 15% PD3 based
on recovery of
standards
± 15% PD3 based
on recovery of
standards
± 15% PD based
on recovery of
standards
± 15% PD based
on recovery of
standards
± 15% PD based
on recovery of
standards
± 15% PD based
on recovery of
standards
± 15% PD based
on recovery of
standards
Blank
Cleanliness
Method procedural blank
≤5 x MDL
Field Blank ≤5 x MDL
Method procedural blank
≤5 x MDL
Field Blank ≤5 x MDL
Method procedural blank
≤5 x MDL
Field Blank ≤5 x MDL
Method procedural blank
≤5 x MDL
Field Blank ≤5 x MDL
MDL1
0.05 uM
0.05 uM
0.02 uM
0.05 uM
Field Blank ≤5 x MDL
1.07 uM
Field Blank ≤5 x MDL
0.23 uM
Filter Blank ≤5 x MDL
0.02 ug/L
1
MDL = method detection limit. The actual MDL may be updated periodically. MDLs are based on the target sample volumes shown
in Table 2-1
2
Relative Percent Difference (RPD)% =  (replicate 1 - replicate 2) x 2/(replicate 1 + replicate 2) x 100.
3
Percent Difference (PD) % = [(true concentration – measured concentration)/true concentration] x 100.
1.5.2.2.2 Comparability
Data will be directly comparable to results obtained previously at the same or similar sites in
Massachusetts and Cape Cod Bay by CCS and/or MWRA because field program design and analytical
procedures are similar or identical. In addition, use of written standardized procedures ensures that
sample preparation and analyses will be comparable throughout the project and with other projects.
1.5.2.2.3 Representativeness
Representativeness is addressed in sampling design. The sampling practices and laboratory
measurements that will be performed during the water quality monitoring have already been used in
many systems to characterize eutrophication and/or microbiological effects on the water column and are,
therefore, expected to yield data representative of the study area. Representativeness will also be
ensured by proper handling, storage (including appropriate preservation and holding times), and analysis
of samples so that the material analyzed reflects the material collected as accurately as possible.
Deviations from the analytical scheme described in this QAPP will be noted in the laboratory records
associated with analytical batches in the QA statements.
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1.5.2.2.4 Sensitivity
Sensitivity is the capability of methodology or instrumentation to discriminate among measurement
responses for quantitative differences of a parameter of interest. The method detection limits (MDLs)
provide the sensitivity goals for the procedures as outlined in Table 1-6.
Data users should be aware that precision and accuracy generally degrade as analyte concentrations
decrease. While numerical results are being reported down to the MDL, results below the lowest
calibration standard will often have precision and accuracy that don’t meet the data quality objectives
for the project.
1.5.2.2.5 Completeness
It is expected that 100% of samples collected for analysis will in fact be analyzed. However, a sample
loss of <10% will not compromise the objectives of the project.
1.6
Special Training Requirements and Certification
Field Monitoring. Sample collection requires no non-routine field sampling techniques, field analyses,
laboratory analyses, or data validation. Specialized training is therefore not required. Field personnel
are experienced in using the equipment identified within this QAPP.
Laboratory Analyses. Nutrient and chlorophyll measurements use routine laboratory analyses, or data
validation, therefore specialized training is not required. Lab personnel are experienced in standard
protocols specified in CCS’s Laboratory Quality Assurance Plan for handling, storing, and preparing
samples for analysis. Laboratory personnel are also experienced in using the equipment identified
within this QAPP.
1.7
Documentation and Records
CCS will maintain all documents relevant to sampling, laboratory analysis, and data analysis activities.
All data will be archived electronically and backed up online. Hard copies of field and lab notebooks
will be archived in the vault located in the basement of the Hiebert Marine Laboratory. Hard copies of
data will be kept for at least one year following the termination of the contract.
1.7.1
Document Control
CCS will maintain documents relevant to laboratory analysis activities and data entry.
A copy of the most current analyses SOP is kept in the lab area where the analysis is being performed.
This document references the SOP number without the revision number. All members of the project
team will inform the CCS QA Officer of the need for SOP revisions.
Document Control is the responsibility of the Program Manager.
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1.7.2
Laboratory Analyses Records
All data will be recorded initially into bound laboratory logbooks, onto established data forms
(Appendix B) or onto electronic file, where applicable.
1.7.3
Records Retention and Storage
All data will be archived electronically and backed up online. Recent hard copies of field and lab
notebooks will be kept in the designated file cabinet in the Coastal Ecology Lab in the Hiebert Marine
Laboratory. Archived records will be stored in the data vault located in the basement of the Hiebert
Marine Laboratory for at least one year following the termination of the contract.
1.7.4
Technical Workshop
The results of the year’s monitoring will be presented at the MWRA’s annual technical workshop
conducted in the spring. The technical workshops are typically scheduled for March or April, and
conducted in Duxbury, Woods Hole or Boston. Following the completion of the workshop, MWRA will
be provided with a digital copy of the Power Point slides, and a two page abstract describing the major
results of the year.
2.0 MEASUREMENT/DATA AQUISITION
2.1
Sampling Process Design (Experimental Design)
2.1.1
Scheduled Project Activities, Including Measurement Activities
The CCB and SBNMS surveys will be performed on an ongoing basis as specified in this QAPP. Nine
surveys will be undertaken per year between February and November.
2.1.2
Design Rationale
The objective of this project is to continue to monitor for changes in water quality in CCB and SBNMS
since the transfer of wastewater discharges offshore to Massachusetts Bay. The evaluation of water
quality changes due to the transfer of discharges offshore will be assessed through the measurement of
nutrient and chlorophyll concentration, and changes in plankton assemblages, among others.
2.1.3
Design Assumptions
Since Cape Cod Bay is generally well-mixed in cold months and has a well defined pycnocline
throughout the warmer months, samples collected near surface and near bottom will accurately
characterize the vertical variation. It is assumed that the spatial scales of variation are large enough that
the sampling locations selected for this region are representative of water quality for this region. It is
also assumed that, since surveys are conducted independent of tidal influence and weather, that the
annual survey frequency is high enough that fluctuations in conditions due to weather or tide will not
result in biased results.
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2.1.4
Procedures for Locating and Selecting Environmental Samples
All sample locations are identified using GPS coordinates. Error of +/- 10 m is considered acceptable to
allow for error in the GPS readout.
2.1.5
Classification of Measurements as Critical or Non-critical
All measurements collected as part of this survey are considered critical. All measurements are required
by the Ambient Monitoring Plan attached to MWRA’s discharge permit.
2.2
Sampling Methods Requirements
2.2.1
Sample Collection, Preparation, Decontamination Procedures
2.2.1.1 Hydrocasts and Sensor Measurements
At each station, a hydrocast will be conducted with a SBE 19plus V2 conductivity-temperature-depth
(CTD) system equipped with various sensors (dissolved oxygen, chlorophyll fluorescence, PAR).
Sensor measurements will be collected during the downcast from near surface (approximately 0.5-1.5
m) to near bottom (3-5 m from seafloor). Salinity and density (sigma-t) will be calculated from the
conductivity, temperature, and depth data. Sea surface PAR and time will be recorded concurrently with
the hydrocast measurements.
2.2.1.2 Water Collection and Plankton Net Tows
Near surface (0.5-1.5 m from surface) and near-bottom (3-5 m from seafloor) samples for each suite of
analytes are collected in PVC Niskin bottles. The Niskin bottle will be lowered by hand to the
designated depth. On deck, water from the Niskin bottle will be subsampled for analysis of dissolved
inorganic nutrients, total nutrients, chlorophyll. All sample bottles are acid washed prior to use in the
field and rinsed three times with sample water before filling. The sample bottles and analytes are shown
in Table 2-1. Surface water will be collected for phytoplankton identification and enumeration (whole
water) and an oblique net tow will be conducted to collect zooplankton for identification and
enumeration. Because we will be following similar protocols as carried out in previous years for
phytoplankton identification and enumeration, all information in this QAPP pertaining to phytoplankton
collection, analysis and quality control was taken from Libby et al. (2010) with only slight
modifications.
The following describes the optimal order of operations for water and plankton collections.
• CTD will be secured at the surface to let sensors equilibrate.
• Niskin will be lowered to the near-surface depth, targeting 1 m with a window of 0.5 to 1.5 m
depending on sea conditions, and triggered to collect the water sample.
• CTD will be lowered at a velocity of approximately 0.5 m per second to within 3-5 m of the
seafloor where it will remain for 5-10 seconds and then retrieved.
• Niskin will be lowered to within 3-5 m of seafloor and triggered to collect water sample.
• Zooplankton tow will be initiated
• Water samples from Niskin bottles will be processed.
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Table 2-1. Sample collection and storage
Parameter
Sample Container
Total Nitrogen
Total Phosphorus
Analytical Sample
Volume per analyte
Decant into 30 mL
polypropylene
bottle and freeze
until analysis.
28 days
Pass through
Nucleopore filter,
freeze filtrate in 100
ml whirl pak until
analysis
28 days
Pass sample through
Whatman GF/F.
Wrap filter in foil
and freeze until
analysis.
28 days
800 mL
Preserve with
Utermöhl’s solution
6 months
Dependent on zooplankton
density
Wash into jar. Fix
with formalin to
10% solution
6 months
500 mL
Nitrate/Nitrite
Ammonia
Silicate
500 mL
Total phosphorous
Ortho-phosphate
1-L wide-mouth
HDPE bottle
Chlorophyll a
960 mL
Phaeophytin
Phytoplankton
(Whole Water)
Zooplankton
(Net Tow)
Wide-mouth HDPE
bottle (volume
dependent on
zooplankton density)
Sample Processing
Maximum
Holding
Time to
Analysis
•
2.2.1.2.1 Dissolved Inorganic Nutrients
Water will be drawn up from a transfer bottle (1 L polypropylene container) using a 60-mL syringe.
The syringe will then be used to push the sample water through an in-line filter (Nuclepore 47–mm–
diameter, 0.4-µm-membrane filter) and into a 100-mL pre-labeled Whirl Pak. At the start of each survey
day the 60-ml syringe is rinsed with 10% HCl solution then with ultrapure de-ionized water (Milli-Q).
Additionally, the syringe is rinsed with Milli-Q between each station. The sample processing begins
with the syringe receiving a triple rinse with site water. The sample will be stored in a cooler until it is
transferred to the lab and frozen within 8 hours.
2.2.1.2.2 Total Nutrients
Water from a transfer jar will be decanted into a sterile, pre-labeled 30 ml polypropylene container.
This container will receive a triple rinse with site water before being filled with sample water. The
container will be stored in a cooler until it can be transferred to the lab and frozen within 8 hours.
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2.2.1.2.3 Chlorophyll a and Phaeophytin
Samples for chlorophyll a/phaeophytin determination will be processed according to CCS SOP for
chlorophyll a/phaeophytin. 1000 ml of sample water will be filtered through Whatman 4.7-cm-diameter
GF/F using a vacuum pump at a vacuum no greater than 6 in. Hg. The final volume will result in a light
green/brown residue on the filter. Using forceps, the filter will be removed from the filter holder, folded
in half, and blotted on acid-free blotting paper to remove excess moisture. The folded filter will then be
placed in a foil packet, and stored in a cooler in a pre-labeled whirl pak until it can be transferred to the
lab and frozen within 8 hours.
2.2.1.2.4 Whole-Water Phytoplankton
Water from the near surface Niskin sampling bottle will be poured into a graduated cylinder that has
been cut at the 850 mL mark. Before filling the cylinder to 800 ml, it will be rinsed three times with
water from the Niskin sampling bottle. The filled cylinder will then be poured into a 1-L bottle
containing 8 mL of Utermöhl's solution preservative. The preserved samples will be stored at ambient
temperature and in the dark until analysis. The Utermöhl's solution will be prepared as described in
Guillard (1973): 100 g potassium iodide, 50 g iodine, and 50 g sodium acetate each are dissolved
incrementally in distilled water to a final volume of 1 L.
2.2.1.2.5 Zooplankton
Zooplankton samples will be collected using a standard 60-cm diameter, 333-µm mesh conical net fitted
with a General Oceanics helical flow meter. A vertical-oblique net tow will be conducted at each station
to sample for zooplankton. Collections will be initiated by vertically dropping the net on-station. When
the net has dropped the full 19 meters, the net will be pulled obliquely through the water column by the
boat until a mark on the rope reaches the surface, indicating that the net is now horizontal at the surface
of the water column. At this point, the net will be retrieved. Once on board, the samples will be washed
from the nets carefully with a sea-water hose, concentrating the sample into the cod end of the net.
From there it will be concentrated further into a 6” long, 2” diameter PVC pipe capped with 333-µm
mesh. This concentrated sample will be rinsed into a sample jar and preserved with 10% buffered
formalin. Samples will be placed in a cooler until transferred to the lab.
2.2.2
Sampling/Measurement System Failure Response and Corrective Action Process
Corrective action in the field may be necessary when the sampling schedule is disrupted due to weather
or other logistical difficulties, or when sampling procedures or field analytical procedures require
modification due to unforeseen circumstances. Any corrective measures taken must be approved by the
Center’s Program Manager. It is the responsibility of the Program Manager to ensure that the corrective
measure has been implemented. Corrective actions will be documented in the field logbook.
Corrective action in the laboratory may occur at one of several phases of the analytical process.
Conditions such as broken or contaminated sample containers may be identified during sample login or
prior to analysis. The Laboratory Manager will identify the need for corrective action and consult with
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the Laboratory QA/QC officer. These corrective actions are performed prior to the release of the data
from the laboratory. The action will be documented in the laboratory notebook.
The occurrence of a practice or incident that is inconsistent with the established quality assurance and
quality control procedures of the laboratory must be formally addressed with a corrective action
response. Examples of situations requiring initiation of the corrective action process include mishandling of a
sample or its documentation or use of unapproved modifications to an analytical method.
Upon the initiation of a corrective action, the problem is documented, and a corrective action plan is
developed and then approved by the Laboratory Manager and QA manager. After required corrective
action has been taken, the information is documented and verified to be effective and sufficient by the
Laboratory Manager and QA Manager. All information is maintained in the Corrective Action
Logbook.
2.3
Sample Handling and Custody Requirements
2.3.1
Sample Custody Procedure
Field logbooks will be used to record field activities performed during the survey. Upon arriving at each
station, date and time of sampling, sample depth, Secchi depth, wind conditions, sampler’s initials and
any other relevant information will be documented (such as site-specific environmental conditions
including presence of wildlife, floatables, algae, etc.). When not in use, logbooks will be stored in the
laboratory. Each logbook cover must be labeled with the project name and the range of survey dates
included in the logbook.
Information will be entered in the logbook in pencil or waterproof ink, initialed and dated with no
erasures made. If corrections are required, the information will be crossed out with a single line and
initialed by the sampler. Sample containers will be pre-labeled with the following information: Survey
number (vessel designation and trip number), site, and depth (S for near surface, D for near bottom).
SW765 [F01] S
All other information relevant to the sample (time, date, depth, etc.) can be cross-referenced in the field
logbook.
All samples except phytoplankton samples will be collected and analyzed by the CCS Hiebert Marine
Laboratory. Phytoplankton samples will be shipped directly to David Borkman for analysis. All other
samples will be hand delivered from the boat via cooler to the laboratory for processing. All
information specific to the samples or errors made during sample collection or delivery (e.g. sample
spilled, flowmeter broken, etc.) will be written in the field logbook.
All samples covered by this QAPP will be analyzed by CCS following its SOPs (Appendix A).
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2.4
Analytical Methods Requirements
2.4.1
Preparation of Samples
All analytes will be pre-processed (filtered and/or frozen in subsample containers, depending on the
analyte) for storage until analysis. Plankton samples will be preserved on board the vessel. See Table
2-1 for a summary of storage methods and holding times.
2.4.2
Analytical Methods
Table 2-2 summarizes the methods used for sample analysis. The analyses will be conducted as described in
the SOPs listed, with are based on literature references or EPA methods.
Table 2-2. Methods of detection for analytes
Parameter
Units
Instrument
SOP 004/USGS 03-4174
Total nitrogen
SOP 001/EPA 353.4
Nitrate/Nitrite
Ammonia
Total phosphorous
SOP/Analysis Method
µmol/l
Astoria 2
Autoanalyzer
SOP 003/EPA 350.1
SOP 004/USGS 03-4174
Ortho-phosphate
SOP 002/EPA 365.5
Silicate
SOP 010/Modified EPA 366
Chlorophyll a
Phaeophytin
µg/L
Phytoplankton (Whole Water)
cells/L
Zooplankton
Organisms/m3
Turner Trilogy
Olympus BH-2
compound
microscope with
phase contrast
optics
Leica L2
Stereomicroscope
SOP 005/Modified EPA 445.0
Borkman (1994), Borkman et al.
(1993), Turner et al. (1995)
Leeney et al. 2008
2.4.2.1 Dissolved and Total Inorganic Nutrients
The analyses of dissolved inorganic nutrients are based on the cited EPA methods. Dissolved inorganic
nutrient concentrations are determined for samples that have been passed through a 0.4-µm pore size
membrane filter in the field. The concentrations of nitrate/nitrite, ortho-phosphate, ammonia, silicate,
total nitrogen and total phosphorous are measured colorimetrically on an Astoria 2 Autoanalyzer. This
instrument automates standard manual techniques for analysis of nutrients.
• For nitrate/nitrite analysis, nitrate in the sample is reduced quantitatively to nitrite by cadmium
metal in the form of an open tubular cadmium reactor (OTCR). The nitrite thus formed plus any
originally present in the sample is determined as an azo dye at 540 nm following its diazotization
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•
•
•
•
with sulfanilamide and subsequent coupling with N-1-naphthylethylenediamine. These reactions
take place in acidic solution.
For analysis of ortho-phosphate, the ortho-phosphate in the sample reacts with molybdenum (VI)
and antimony (III) in an acidic medium to form a phosphoantimonylmolybdenum complex. This
complex is subsequently reduced by ascorbic acid to a heteropolyblue with an absorbance
maximum at 880 nm.
For analysis of ammonia, The sample is mixed with o-phthaldialdehyde and sodium sulfite in a
borate-buffered solution at 75°C. After sufficient mixing, the sample concentration is measured
by fluorescence spectroscopy using 360nm excitation and 420-470nm emission wavelengths.
The increase in fluorescence is directly proportional to the ammonia concentration.
For analysis of silicate, silicomolybdic acid is formed by the reaction of silicate with molybdic
acid. The silicomolybdic acid is reduced by stannous chloride to form molybdenum blue with an
absorbance maximum at 820 nm.(1-4)
For analysis of total nitrogen and total phosphorous, an alkaline persulfate digestion oxidizes all
forms of inorganic and organic nitrogen to nitrate and hydrolyzes all forms of inorganic and
organic phosphorous to ortho-phosphate. After digestion, samples are analyzed as described for
nitrate/nitrite and ortho-phosphate.
2.4.2.2 Chlorophyll a and Pheophytin
Samples for chlorophyll a/phaeophytin are processed according to EPA method 445.0 using a Turner
Trilogy Fluorometer. Samples are filtered in the field as soon as possible after collection and the filters
stored at -10ºC. All handling steps are performed in subdued light. The chlorophyll a/phaeophytin is
extracted from the cells retained on the GF/F filter by a 16-24 hour steep in 90% acetone at 4ºC. The
extract is analyzed using a fluorometer. 150 µL of 0.1 N HCl is added to the extract and the extract is
remeasured after 90 seconds to determine phaeophytin concentrations.
2.4.2.3 Whole-Water Phytoplankton
Utermöhl's-preserved whole seawater samples will be prepared for analysis by concentrating the sample
by gravitational settling as described by Borkman (1994), Borkman et al. (1993), and Turner et al.
(1995). Samples will be settled in graduated cylinders with no more than a 5-to-1 height-to-width ratio.
Phytoplankton abundance is calculated by dividing the number of cells counted by the volume examined
in a gridded Sedgwick-Rafter chamber. The theoretical maximum possible volume that would be
examined would be an entire Sedgwick-Rafter chamber (1 ml). The grid subdivides the chamber into µl
divisions so that if an entire chamber is not counted, an exact volume can still be determined. Typical
volumes counted are one row of the chamber (50 1-µl cells or 1/20 of 1 ml). The volume of sample
examined is dependent on number of cells encountered and how long it takes to reach cut-offs of 75
entities (unicellular forms, colonies, or chains) of each of the top 3 taxa, and 400 entities total.
Calculation of abundance also accounts for the concentration factor used in the settling process.
Normally, the volume processed is 800 ml of whole-water sample, settled to 50 ml of concentrate, for a
16:1 ratio.
The following equation results in the abundance estimate for cells counted:
C * [VS / VC] [ 1000 / VTOT] = cells/ L.
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where C = cells counted
VS = Volume of concentrated sample
VC = Volume of sample examined
VTOT = Original volume
2.4.2.4 Zooplankton
The zooplankton are identified and counted by trained individuals. Sub-samples to be counted are taken
by 1) suspending the sample in a known volume of water, and taking a subsample of at least 250
organisms to count and identify using a Wildco Hensen-Stemple pipette with plunger, 2) first splitting
the sample with a Folsom plankton splitter and then continuing with the steps in (1), or 3) counting and
identifying all organisms in the sample, should sub-sampling be impossible. The results of the counts
are expressed in organisms per cubic meter (organisms/m3), derived from the flow meter correction
constant, flow meter change during the tow, the area of the mouth of the net, volume of the sample, and
volume of the sub-sample that was counted.
2.5
Quality Control Requirements
2.5.1
Calibration Procedures
2.5.1.1 Hydrographic Instruments
All hydrographic instruments and sensors are sent to the respective manufacturers (Seabird, WET Labs,
Biospherical) annually for calibration.
2.5.1.2 Nutrients (nitrate+nitrite, ortho-phosphate, ammonia, TN, and TP)
At least 6 working calibrants for each chemistry will be prepared from certified standards to cover the
concentration range of the samples to be analyzed. The calibrants are run at the beginning of the
analyses, and a calibration curve is fitted. If the correlation <0.995, new calibrants will be prepared, and
calibration will be re-done. See SOPs for more detail. Standards are supplied from Astoria Pacific.
Each standard is labeled with concentration and expiration date. Standards are stored at room
temperature. Working calibrants of concentrations >100 µM are prepared weekly and stored at 4°C.
Working calibrants of concentrations <100 µM are prepared daily.
2.5.1.3 Chlorophyll a and Pheophytin
The laboratory fluorometer is calibrated at the beginning of each monitoring season with 2 liquid pure
chlorophyll a standards and reagent. At the time of calibration a solid secondary standard is also
analyzed and the formula for calculating chlorophyll a in samples is determined. The solid secondary
standard is analyzed with each batch of samples. Blanks of 90% acetone, and an unused filter extracted
with 90% acetone are set up with each rack of samples.
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2.5.1.4 Net and Flowmeter
The net used for zooplankton collection and the flowmeter will be rinsed with fresh water and inspected
for damage following each survey. Additionally, the flowmeter will be calibrated annually to attain the
most accurate correction constant possible.
2.5.2
Data validation, reporting and verification
2.5.2.1 Analytical Methods
Data Evaluation: Both the Laboratory Manager and the QA Officer will review data to determine if it
meets the quality assurance objectives (Table 2-3). Decisions to qualify or reject the data will be made
by the Laboratory Manager and the QA Officer and if required, corrective actions will be implemented
as outlined in Table 2-4 and Table 2-5.
Table 2-3. Data quality objectives
Expected
Range
Parameter
Units MDL
Nitrite/Nitrate
µM
0.05 0 – 10
OrthoPhosphate
µM
0.02 0 – 3
Ammonia
µM
0.1
0–5
Silicate
µM
0.1
0–5
Total Nitrogen
µM
0.5
0 – 30
Total
Phosphorous
µM
0.1
0–6
Chlorophyll a
µg/l 0.02 0 – 50
Accuracy (+/-)
Precision
80-120 % recovery for QC std. and lab
fortified matrix
80-120 % recovery for QC std. and lab
fortified matrix
80-120 % recovery for QC std. and lab
fortified matrix
80-120 % recovery for QC std. and lab
fortified matrix
80-120 % recovery for QC std. and lab
fortified matrix
80-120 % recovery for QC std. and lab
fortified matrix
+ 0.1 µM if less than 0.5 µM or 20%
RPD if more than 0.5 µM
+ 0.05 µM if less than 0.1 µM or 20%
RPD if more than 0.1µM
+ 0.1 µM if less than 0.5 µM or 20%
RPD if more than 0.5 µM
+ 0.05 µM if less than 0.1 µM or 20%
RPD if more than 0.1 µM
75-125% recovery for QC std.
20% RPD
20% RPD
+ 2.0 µM if less than 15 µg/l or 25%
RPD if more than 15 µg/l
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Table 2-4. Laboratory Analytical QC: Nutrients (Nitrate+Nitrite, Ortho-Phosphate, Ammonia,
TN, and TP)
QC
Method
Blank
Reagent
Blank
Laboratory
Duplicate
Internal
Standards*
External
Standards**
Frequency/
Number
Method/SOP
QC Acceptance
Limits
Corrective
Action
Person
Responsible for
Corrective
Action
Measurement
Performance
Criteria
1 per set of 20
< MDL
Re-run
Lab Manager
< MDL
1 per set of 20
< MDL
Re-run
Lab Manager
< MDL
10% of
samples
<20%RPD
Re-run
Lab Manager
<20%RPD
Re-run
Lab Manager
Re-run
Lab Manager
90-110%
recovery
90-110%
recovery
1 per set of 20
1 per set of 20
90-110%
recovery
90-110%
recovery
*Internal standard: a known amount of a standard added to a test portion of a sample and carried through the entire determination procedure as a reference
for calibrating and controlling the precision and bias of the applied analytical method.
**External standard: USGS Standard Reference Nutrient Samples
Table 2-5. Laboratory Analytical QC: Chlorophyll a
QC
Method
Blank
Instrument
Blank
Laboratory
Duplicate
External
Standards**
Frequency/
Number
Method/SOP
QC Acceptance
Limits
1 per set of 20
< MDL
1 per set of 20
< MDL
10% of
samples
<20%RPD
90-110%
recovery
1 per set of 20
Person
Responsible for
Corrective
Action
Measurement
Performance
Criteria
Lab Manager
< MDL
Lab Manager
< MDL
Qualify
Lab Manager
<20%RPD
Qualify
Lab Manager
90-110%
recovery
Corrective
Action
Re-clean, rerun
Re-clean, rerun
*Internal standard: a known amount of a standard added to a test portion of a sample and carried through the entire determination procedure as a reference
for calibrating and controlling the precision and bias of the applied analytical method.
**External standard: either a liquid primary chlorophyll a standard provided by Turner Designs or a solid secondary standard.
2.5.2.2 Plankton Analyses
2.5.2.2.1 Whole-Water Phytoplankton
Counts of 400 phytoplankton cells will provide a precision of ±10% of the mean (Guillard 1973).
Therefore, a minimum of 400 entities (solitary single cells, chains, or colonies) will be tallied for each
sample. Unicellular forms (e.g., Cryptomonas spp., microflagellates), aggregate forms (e.g., Phaeocystis
pouchetii), and chained forms (e.g., Skeletonema spp.) will each count as one entity towards the 400entities-counted-per-sample minimum tally. To increase precision of the abundance estimates for the
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most abundant taxa, when practical at least 75 entities of each of the three most abundant taxa will be
counted in each sample. The overall goal then is to enumerate a minimum of 400 entities total and the 3
most abundant taxa to at least 75 entities each. An additional data quality procedure will be performed
on the whole water phytoplankton samples. A subset of samples will be counted in duplicate by a
different taxonomist or as a blind recount by the same taxonomist to provide an estimate of the
variability in the analysis and ensure the accuracy and comparability of the results. One whole water
sample from the surveys in February, April, June, August and October will be analyzed in duplicate.
This range of samples should cover the major taxonomic groupings and various levels of abundance.
The results, as relative percent difference (RPD), will be included in the data submission as an estimate
of the variability in the analysis. The RPD for total and the most dominant species should be ≤20%. If
the RPD is greater than 20 a second aliquot will be counted and the three results used to calculate the
relative standard deviation (RSD), which should be ≤20%.
2.5.2.2.2 Zooplankton
Zooplankton samples will be either sub-sampled with a Wildco Henson-Stemple pipette or split with a
Folsom plankton splitter, and an aliquot of at least 250 animals will be counted. If the total count in an
aliquot is less than 250 animals, additional aliquots will be counted until either the targeted number of
organisms is reached or the entire sample is counted. One sample from each survey in February, April,
June, August and October will be analyzed in duplicate. The results, as RPD, will be included in the
data submission. The RPD for total and the most dominant species should be ≤20%. If the RPD is
greater than 20 a second aliquot will be counted and the three results used to calculate the relative
standard deviation (RSD), which should be ≤20%.
2.6
Preventive maintenance procedures and schedules
2.6.1
Maintenance for Astoria 2 Autoanalyzer
The Astoria 2 Autoanalyzer will be cleaned and maintained no less than once a month as described in
the following procedure:
1. Place all lines (including autosampler wash line) in de-ionized (DI) water and pump for 10
minutes.
2. Place all lines in CHEMWASH (or equivalent) for 5 to 10 minutes.
3. Place all lines in a clean beaker of DI water for 5 to 10 minutes.
4. Place all lines in a 5 to 10% Bleach solution for 5 to 10 minutes.
5. Place all lines in a clean beaker of DI water for 5 to 10 minutes.
6. Place all lines in a 2% Neutrad solution for 5 to 10 minutes.
7. Place all lines in a clean beaker of DI water for 5 to 10 minutes.
8. Place all lines in 1N HCl for 5 to 10 minutes.
9. Place all lines in a clean beaker of DI water for 10 minutes.
10. Place reagent lines in Startup/Shutdown solution, sample probe in sample wash pot and wash
line into DI water and allow to pump for at least 20 minutes.
11. Change all pump tubes and polyflow tubing.
12. Inspect all injection fittings, sample splitter(s), debubblers, sample probe and reagent lines on
the cartridge for debris. If necessary, clean appropriately or replace with clean parts.
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13. Clean all reagent bottles with the bleach solution and rinse thoroughly followed by 1N HCl and
rinse thoroughly before adding reagents. This step is only needed if the same bottle is continuously
used for the reagent.
14. Clean all platens using a tissue moistened with isopropyl alcohol or methanol.
15. Wipe the pump rollers using a tissue moistened with isopropyl alcohol or methanol. Try to
remove any debris or particulates around the pump rollers and bushings.
16. Pump Startup/Shutdown solution for 5 to 10 minutes until you have obtained good flow through
the system.
The Open Tubular Cadmium Reactor (OTCR) will be cleaned with 1 N HCl followed by a DI rinse
every 2 weeks. Auxiliary pump tubing will be replaced every 6 months.
2.6.2
Maintenance for Turner Trilogy Fluorometer (1/month or more frequently as needed)
At least once a month, or more frequently as needed, the sample chamber and cuvette holder will be
cleaned with a water dampened cotton swab or soft cloth. All components will be dried thoroughly
before reassembling.
2.6.3
Maintenance for Direct Q3
The Direct Q3 is used to produce Milli-Q water (referred to in Section 2.2.1.2.1 and recommended in
section H of the SOP for Ammonia).
At least once a year, or more frequently as needed, the SmartPak filter cartridge and final filter will be
replaced and the system and tank will be sanitized with 30% Hydrogen Peroxide solution.
2.6.4
Maintenance for Plankton Collection and Analysis Equipment
2.6.4.1 Plankton Collection Equipment
The chamber inside the General Oceanographic Environmental flowmeters are filled with fresh water
before every research cruise, so that minimal air is left inside the chamber; they are also checked for
ease of rotation and corrosion. The nets used for zooplankton collection are checked for holes before
each research cruise. Small holes are filled using a glue product called Zap-a-gap®. The cod ends on
the zooplankton collection nets are checked for holes in the mesh before every research cruise. Should a
hole be found, the mesh is replaced; if the mesh starts to separate from the plastic, it is glued back down
using PVC cement.
2.6.4.2 Plankton Analysis Equipment
Fluorettes are checked for holes in the mesh, or separation of the mesh from the plastic, before every
research cruise. If holes are found, the mesh is replaced; if the mesh is separating from the plastic, it is
glued back down using PVC cement.
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The Hensen-Stempel pipettes are cleaned once per year, or more frequently if needed. They are soaked
in warm soapy (dish soap) water, and then soaked in fresh hot water. They are them thoroughly rinsed to
remove all soap, and set aside to dry.
The Folsom plankton splitter is cleaned once per year, or more if necessary. It is scrubbed with dish
soap, and then thoroughly rinsed with hot water until all soap is removed. It is then set aside to dry.
The microscopes are cleaned once per year. The lenses in the eyepieces and eyepiece tubes are wiped
clear of dust using Kimwipes®. The lens over the light condenser is also wiped clean. After every use
the microscope is unplugged and covered with a dust cover.
The multi-channel digital counter used in the enumeration of zooplankton is regularly cleaned using
compressed air. The area under the buttons is flushed of dust and dirt.
2.7
Corrective action contingencies
If results from any analyses of QC checks are unacceptable, corrective actions will be taken as described
for each SOP above. Whenever possible, analyses will be re-run with new QC checks. If results are still
unacceptable, the instrument will be re-calibrated according to manufacturer’s instructions. The Lab
manager is responsible for all corrective actions. The QA Officer must also be consulted. All corrective
actions will be documented in the lab notebook.
2.8
Inspection/Acceptance of Supplies and Consumables
Prior to use, supplies and consumables will be inspected and tested to ensure that they conform to the
required level of quality. Any defective material will be replaced before the sampling event or before
analysis begins. Supplies and consumables consist of: sample containers (whirl paks, polypropylene
sample vial), filters (Whatman GF/F, Nucleopore 0.4 µm), filtration apparatus (syringe and Swinlok
filter holder), preservation solutions (formalin, Utermohls solution), distilled water, laboratory reagents,
and standards (chlorophyll and nutrient).
•
•
Sample containers are either cleaned by the laboratory or purchased new. Containers must be
cleaned according to SOPs prior to use and must be rinsed three times with station water prior to
being filled with sample. Field blanks assess potential contamination of containers and sampling
equipment.
All filtering equipment (the syringe and filter holder) are cleaned prior to use. The equipment
gets a 10% HCl rinse in the followed by a triple rinse of distilled water. Between stations the
equipment gets rinsed with distilled water and a triple rinse with station water.
•
Filters for chlorophyll and dissolved nutrients are used directly from the manufacturer and are
not cleaned or treated.
•
Preservation solutions must be prepared using at least reagent grade chemicals / HPLC grade
solvents. Solutions must be assigned an expiration date of 1 year.
•
Distilled water must be collected into cleaned containers and refreshed prior to each survey.
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•
Laboratory reagents must be at least reagent grade. Dry reagents must be assigned an expiration
date of no more than 5 years; be stored in a clean, dry environment, away from light, and be
traceable to receipt and certificate of analysis. Reagent solutions must be assigned an expiration
date of no more than 1 year and be stored appropriately. The laboratory must maintain a
chemical tracking inventory.
•
Laboratory standards must be certified as at least 96% pure or the lot-specific analysis purity
must be incorporated into calculation of the standard concentration. Standards must be assigned
an expiration date “as received” based on the manufacturer’s expiration date, or a date consistent
with laboratory SOPs and stored as recommended by the manufacturer.
All supplies and consumables are purchased from Fisher Scientific, Sigma Aldrich, or Astoria Pacific.
Chlorophyll standards are purchased once a year from Turner Designs. Nutrient standards are purchased
yearly from Astoria Pacific. Nutrient samples for inter-laboratory comparison and internal quality
control are purchased twice a year (spring and fall) from USGS as part of the Standard Reference
Sample Program (http://bqs.usgs.gov/srs/).
2.9
Data Acquisition Requirements (non-direct measurements)
Data from previous and on-going monitoring conducted by MWRA and CCS will be utilized to assess
the state of CCB and SBNMS. Other possible data from non-direct measurements that may be used
include satellite imagery and mooring data. These secondary data are used “as received” and not
censored.
2.10 Data Management
2.10.1 Hydrographic Data
The hydrographic data generated during the survey consists of rapidly sampled, high-resolution
measurements of conductivity, temperature, depth, DO, fluorescence, and PAR. Data will be logged
internally and downloaded using Seabird SeaTerm software. For surface PAR, data are logged directly
onto a computer using LOGGER-2100 software supplied by Biospherical Instruments, Inc.
SBE Data Processing Software from Seabird will be used to process the raw data from the instrument.
Using this software, data are first converted from the raw data to scientific units (i.e. conductivity,
temperature). Only data from the downcast are used. In order to match the response of the temperature
and conductivity sensors, the data have to be filtered. A low pass filter of 0.5 seconds is used to match
the time constants of temperature and salinity. A second low-pass filter of 2 seconds is applied to
pressure readings to separate data that are collected at a less than minimum descent rate. Temperature
and conductivity are advanced 0.5 seconds to account for misalignment of sensors and a loop edit
function is used to remove data collected during a cast in which the CTD either decelerates or travels
backwards (loops) due to rolling of the vessel. Finally depth, potential temperature, and salinity are
derived from pressure, temperature and conductivity data, respectively, and density (sigma-t) is
calculated based on temperature, salinity and pressure.
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2.10.2 Field Data
All data from field notebooks will be manually entered into the appropriate database format.
2.10.3 Laboratory Data
All laboratory data will either be electronically transferred from the instrument or manually read from
the instrument display (or optical field of a microscope) and entered onto a standard data form. Forms
used for data entry are included in Appendix B. Data in laboratory notebooks will be manually entered
when necessary. All data reduction will be performed electronically either by the instrument software or
in a spreadsheet and will be validated according to procedures described in Section 4.0. Laboratory
replicates will be reported as mean sample values. All field replicates will be reported as individual
sample values. The format for final data submission to MWRA is shown in Table 2-6 . Data will be
submitted electronically as Microsoft Excel spreadsheets.
Table 2-6. Specifications for data sets
a.
Event data (one record for each survey)
Fi Data type & format
Description
Field
Required
Identifier of sampling event (survey)
EVENT_ID
Y
alphanumeric, maximum 10 characters
Name of the event, e.g. "Cape Cod Bay
adjunct to BWQM survey WN141"
EVENT_NAME
Y
alphanumeric, maximum 100
characters
Platform name, e.g. "R/V Shearwater" or
"F/V Columbia")
PLAT_NAME
Y
alphanumeric, maximum 20 characters
Name of chief scientist on board, e.g.
"Amy Costa"
CHIEF_SCIENTIST
Y
alphanumeric, maximum 20 characters
Brief comments on survey regarding
exceptions from standard procedures or
unusual circumstances affecting entire
survey
COMMENTS
alphanumeric, maximum 150
characters
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b.
Station data (three records for each survey – one for each station visit)
F Data type & format
Description
Field
Required
Identifier of sampling event (survey), e.g. "
WN141_PCCS"
EVENT_ID
Y
alphanumeric, maximum 10
characters
Identifier for station, e.g. "F01", "F02",
"F29"
STAT_ID
Y
alphanumeric, maximum 100
characters
Station arrival date and time (local time)
STAT_ARRIV_LOCAL
Y
date
Decimal degrees north latitude when
arriving on station
LATITUDE
Y
alphanumeric, maximum 20
characters
Decimal degrees west longitude (always
negative) latitude when arriving on station
LONGITUDE
Y
alphanumeric, maximum 20
characters
Code describing position accuracy
NAV_QUAL
Y
alphanumeric, maximum 10
characters
Echosounder depth in meters
DEPTH_TO_BOTTOM
Y
number
Brief comments regarding exceptions from
standard procedures or unusual
circumstances for station visit
COMMENTS
alphanumeric, maximum 150
characters
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c.
Samples
Required
F Data type & format
Description
Field
Identifier of sampling event (survey)
EVENT_ID
Y
alphanumeric, maximum 10
characters
Identifier for station.
STAT_ID
Y
alphanumeric, maximum 10
characters
Station arrival date and time (local
time)
STAT_ARRIV _LOCAL
Y
date
Sample identifier
SAMPLE_ID
Y
alphanumeric, maximum 15
characters
Code for type of gear used to collect
sample.
GEAR_CODE
Y
alphanumeric, maximum 12
characters
Depth of sample, from water surface
to bottom of sample, in m.
DEPTH
Y
number (2 decimal places)
Depth of water sample, from water
surface to top of sample, in m.
DEPTH_TOP
number (2 decimal places)
Date and time sample was taken
(local time)
SAMPLE_DATE_
TIME_LOCAL
Date
Sample depth-type code (A=nearsurface, E=near-bottom,
Z=zooplankton net tow)
SAMPLE_DEPTH _CODE
Volume of sample as collected (e.g.
calculated tow volume for
zooplankton tows)
SAMP_VOL
number
Unit of volume measurement.
SAMP_VOL_ UNIT_CODE
alphanumeric, maximum 3
characters
Comments for a given station visit
and sample
COMMENTS
alphanumeric, maximum 150
characters
Y
alphanumeric, maximum 2
characters
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d. Hydrographic measurement data
Description
Field
Identifier of sampling event (survey)
EVENT_ID
Identifier for station.
STAT_ID
Y
Station arrival date and time (Local Time).
STAT_ARRIV
_LOCAL
DEPTH
Y
alphanumeric, maximum 10
characters
alphanumeric, maximum 10
characters
Date
Y
number (2 decimal places)
Depth (in meters= decibars) at which data were
collected.
Date and time when data were collected (Local
Time).
Code for parameter measured.
PROF_DATE_
TIME_LOCAL
PARAM_CODE
Result for parameter.
Value qualifier.
VALUE
VAL_QUAL
Code for the unit of measurement
UNIT_CODE
Code for method.
METH_CODE
Code for instrument used.
INSTR_CODE
Comments for the sensor measurement
COMMENTS
Required
Field
Y
Data type & format
Date
Y
alphanumeric, maximum 20
characters
Number
alphanumeric, maximum 4
characters
alphanumeric, maximum 12
characters
alphanumeric, maximum 13
characters
alphanumeric, maximum 11
characters
alphanumeric, maximum 150
characters
e. Nutrient measurement data
Description
Field
Identifier of sampling event
(survey)
Sample identifier
EVENT_ID
Required
Field
Y
SAMPLE_ID
Y
Identifier for bottle
BOTTLE_ID
Y
Code for parameter measured.
PARAM_CODE
Y
Result for parameter.
Value qualifier.
VALUE
VAL_QUAL
Code for unit of measurement
UNIT_CODE
Code for method.
METH_CODE
Code for instrument used.
INSTR_CODE
Comments on this result
COMMENTS
Data type & format
alphanumeric, maximum 10
characters
alphanumeric, maximum 15
characters
alphanumeric, maximum 15
characters
alphanumeric, maximum 20
characters
number
alphanumeric, maximum 4
characters
alphanumeric, maximum 20
characters
alphanumeric, maximum 13
characters
alphanumeric, maximum 10
characters
alphanumeric, maximum 150
characters
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f. Plankton measurement data
Description
Field
Identifier of sampling event
(survey)
Sample identifier
EVENT_ID
SAMPLE_ID
Y
Subsample (bottle) identifier
BOTTLE_ID
Y
Code for species
SPEC_CODE
Y
Taxonomic name for species
DESCR
Y
Qualifier for species code,
including sex and/or life stage
codes. Default = ‘null’ for when
sex or life stage is unknown or not
relevant.
Count of cells for that species
Value qualifier.
SPEC_QUAL
Code for the unit of measurement
UNIT_CODE
Code for method.
METH_CODE
Number of cells counted
Number of colonies counted
Number assigned by the
laboratory to the sample.
Comments on the record.
RAW_COUNT
COLONY
LAB_SAMPLE_ID
VALUE
VAL_QUAL
COMMENTS
Required
Field
Y
Y
Data type & format
alphanumeric, maximum 10
characters
alphanumeric, maximum 15
characters
alphanumeric, maximum 15
characters
alphanumeric, maximum 17
characters
alphanumeric, maximum 80
characters
alphanumeric, maximum 4
characters
number
alphanumeric, maximum 4
characters
alphanumeric, maximum 12
characters
alphanumeric, maximum 13
characters
floating point
floating point
alphanumeric, maximum 35
characters
alphanumeric, maximum 150
characters
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3.0 ASSESSMENT/OVERSIGHT
3.1
Assessments and Response Actions
3.1.1
Performance Audit
The QA Officer will conduct an initiation audit and, as needed, laboratory and field inspections to
ensure that laboratory analyses and data recording and entry are carried out in accordance with this
QAPP. Deviations from the QAPP will be reported directly to the Program Manager and the
appropriate corrections will be made. All deviations will be noted in the respective field notebook, lab
notebook or electronic file and tracked by the QA Officer.
3.1.2
Corrective Action
All field and laboratory personnel share responsibility for identifying and resolving problems
encountered in the routine performance of their duties. The Program Manager will be responsible for
identifying and resolving any problems that have not been adequately addressed by technical personnel
as well as any problems that require changes in this QAPP and/or require consultation with MWRA.
The Program Manager is also accountable to MWRA for overall performance of this monitoring
program. Issues regarding scheduling (e.g. synoptic sampling with MWRA’s Massachusetts Bay
monitoring consultant) will be reported to MWRA and dealt with under their guidance.
3.2
Reports to Management
A survey report detailing the sampling that was conducted will be submitted to MWRA within a week
after the survey has been completed. Additionally, quarterly reports will be submitted to MWRA which
will include the hydrographic data and laboratory data (nutrients, phytoplankton, zooplankton) collected
during the surveys conducted in the respective quarter.
All data collected during the year will be presented at the MWRA annual technical workshop held each
spring. Following the completion of the workshop, MWRA will be provided with a digital copy of the
Power Point slides, and a two page abstract describing the major results of the year.
4.0 DATA VALIDATION AND USABILITY
4.1
Data Review, Validation and Verification Requirements
No data will be used until they are validated and verified as described in 4.2. The adherence to the data
quality objectives (Table 2-3) and laboratory QC’s, (Table 2-4, Table 2-5), RPD’s of duplicate plankton
counts, notations in field and laboratory notebooks, cross-checks of data entry, and the results of the
audits will be used to objectively and consistently determine whether the data are useable for the
purposes of this project.
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4.2
Validation and Verification Methods
To assess the quality and usability of the data, several methods of data validation and verification are
used throughout the data collection, analysis, and reporting process and overseen by the QA Officer.
Sample containers are pre-labeled to ensure completeness and accuracy of sample collection. Manual
entry of field sampling data is verified for correctness and completeness by comparing the field survey
log book to the post-survey report. Data downloaded directly from instrumentation are date and time
stamped and can be cross-referenced to the field survey log book.
Manual data entry of laboratory data (e.g. zooplankton and phytoplankton) will be verified by 100%
double keypunching and using the computer to check for differences. All other laboratory data is
downloaded directly from the instrument.
Calculations performed on the data (e.g. plankton concentration) will be done in Excel and verified
manually at a frequency to ensure that formulas are correct, appropriate, and consistent, and that
calculations are accurately reported.
Additional data validation is achieved by following the protocols for holding times, instrument
calibration and maintenance, quality control sample results, and other criteria for data quality
requirements as outlined in this QAPP.
4.3
Reconciliation with User Requirements
A key step in the evaluation of the validated data to determine if it meets the user requirements is the
presentation of the results at the MWRA’s annual technical workshop in the spring. This will provide a
means for peer review of the data and interpretation of results. Data collected for this project will also
be compared with data collected during the on-going Cape Cod Bay Monitoring Program and Right
Whale Habitat Program conducted by CCS. A synthesis of the data collected from these programs will
provide a context from which to evaluate nutrient loading and the possible effects on the food chain
from phytoplankton to right whales.
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5.0 REFERENCES
Borkman, D. 1994. Phytoplankton and Nutrients in Buzzards Bay, Massachusetts 1987-1988. M.S.
Thesis. University of Massachusetts Dartmouth, Dartmouth, MA. 203 pp.
Borkman D, Pierce RW, Turner JT. 1993. Dinoflagellate blooms in Buzzards Bay, Massachusetts. Pp.
211-216 in Smayda, T.J., and Y. Shimizu (Eds.), Proceedings of the Fifth International
Conference on Toxic Marine Phytoplankton, Elsevier.
Costa A, Larson E, Stamieszkin K. 2010. Quality Assurance Project Plan (QAPP) for water column
monitoring in Cape Cod Bay 2010-2011. Boston: Massachusetts Water Resources Authority.
Report 2010-20. 82 p.
Guillard RRL. 1973. Division rates. Pages 289-311 In: J.R. Stein, (Ed.) Phycological Methods.
Cambridge Univ. Press.
Leeney RH, Stamieszkin K, Jaquet N, Mayo CA, Osterberg D, Marx MK. 2008. Surveillance,
monitoring and management of North Atlantic right whales in Cape Cod Bay and adjacent
waters. Final Report submitted to the Division of Marine Fisheries, Commonwealth of
Massachusetts.
Libby PS, Fitzpatrick MR, Buhl RL, Lescarbeau GR, Leo WS, Borkman DG, Turner JT, Oviatt CA.
2010. Quality assurance project plan (QAPP) for water column monitoring 2010: Tasks 4-9 and
13. Boston: Massachusetts Water Resources Authority. Report 2010-02. 105 p.
Turner JT, Borkman DG, Pierce RW. 1995. Should Red Tide Dinoflagellates be Sampled Using
Techniques for Microzooplankton Rather than Phytoplankton? Pp. 737-742 in P. Lassus et al.
(Eds.), Harmful Marine Algal Blooms, Lavoisier, Paris, France
32
APPENDIX A: Standard Operating Procedures
SOP-001: General Laboratory Safety
SOP-002: General Labware Cleaning Procedure
SOP-003: Nitrate+Nitrite Analysis
SOP-004: Ortho-Phosphate Analysis
SOP-005: Ammonia Analysis
SOP-006: TN and TP Analysis
SOP-007: Chlorophyll a Analysis
SOP-008: Phytoplankton Analysis
SOP-009: Zooplankton Analysis
SOP-010: Silicate Analysis
33
Standard Operating Procedure 001
General Laboratory Safety
Date: Nov 2007
Revision: 2
Purpose and Description: Provide guidance on appropriate lab safety protocols. Lab complies with most
OSHA requirements for worker safety.
Lab Safety Officer: Amy Costa
Emergency Numbers:
Hazardous Materials Hotline
800-319-2783
Provincetown Fire Department 508-487-7023
Provincetown Health Center
508-487-9395
General Health and Safety Information
1. Eye protection, gloves, and lab coats are available and required when working with chemicals
2. Closed-toed shoes are required
3. Locations of accident and safety equipment:
a. First-aid kit is located on wall to right of sink
b. Eye wash station is located to the right of the sink
c. Shower is located downstairs
d. Spill pads are located under sink
4. Report any accidents immediately to Amy Costa and/or Rich Delaney
5. Nearest Medical Facility is the Provincetown Health Center located at 49 Harry Kemp Way
6. All liquid chemicals must be stored below eye level
7. All chemicals must be properly labeled and stored at all times. Hazardous labels must indicate
what harm the chemical represents.
8. MSDS sheets are located in the file cabinet in folder labeled MSDS
9. Laboratory must comply with local building/fire codes.
10. Wastes must be properly stored in their own container (i.e. acetone waste in container labeled
“Acetone Waste,” acid waste in container labeled “Acid Waste,” etc.)
11. Disposal of hazardous waste will be done through Barnstable County’s Cape Cod Cooperative
Extension. (Hazardous waste generator status: very small quantity generator, limited primarily
to copper sulfate required for nitrate/nitrite analyses and activation of cadmium coil and
acetone.)
Hazardous Wastes Requiring Special Disposal:
Acetone
Copper Sulfate
Acid Wash (10% HCl)
34
Standard Operating Procedure 002
General Labware Cleaning Procedure
Date: Nov 2007
Revision: 2
Purpose and Description: Outline appropriate techniques for cleaning labware and sampling containers.
Only glass sample containers will be reused. Whirl paks and disposable polypropylene tubes will be
discarded. Clean labware and sampling containers are required to ensure that results for the assays
analyzed in the laboratory are accurate.
Method Description: All labware is washed in non-phosphate detergents (i.e. Liqui-Nox), stored under
the sink in a 1 gallon container. Glassware is soaked in a bath of dilute hydrochloric acid (10%) kept in
labeled glass tub.
Glassware Cleaning Procedure
1. Empty non-hazardous contents of bottles down the drain and hazardous contents into appropriate
waste containers
2. Remove labels. This is expedited by soaking bottles in tap water
3. Wash in soapy water (Liqui-Nox)
4. Rinse with tap water at least 3 times
5. Soak glassware for at least 24 hours in tub of 10% HCL. Make sure glassware is completely
submerged
6. Rinse with DI water
7. Fill with DI water and allow glassware to soak for at least 1-2 hours
8. Rinse thoroughly – at least 3 times, inside and out – with DI water
9. Air dry, inverted
10. After drying, store with either parafilm or aluminum foil across the top, or with caps loosely
attached
Plasticware Cleaning Procedures: Do not soak in acid bath.
1. Empty non-hazardous contents of bottles down the drain and hazardous contents into appropriate
waste containers
2. Remove labels. This is expedited by soaking bottles in tap water
3. Wash in soapy water (Liqui-Nox)
4. Rinse with tap water at least 3 times
5. Rinse thoroughly – at least 3 times, inside and out – with DI water
6. Air dry, inverted
35
Standard Operating Procedure 003
Nitrate+Nitrite Analysis
Date: Nov 2007
Revision: 2
Primary Method: EPA 353.4
A. Scope and Application
This method is used for the determination of nitrite or nitrate plus nitrite in seawater and is applicable to
many ranges.
B. Summary of Method
Nitrate is reduced quantitatively to nitrite by cadmium metal in the form of an open tubular cadmium
reactor (OTCR). The nitrite thus formed plus any originally present in the sample is determined as an
azo dye at 540 nm following its diazotization with sulfanilamide and subsequent coupling with
N-1-naphthylethylenediamine.(1) These reactions take place in acidic solution. Nydahl provides a good
discussion of nitrate reduction by cadmium metal, while the specific details of OTCR's are given by
Patton.(2,3) The information concerning mechanisms and kinetics of the color forming reactions can be
found in References 3 and 4.
C. Sample Handling and Preservation
All samples will be filtered using 0.4 µm Nucleopore filters. Samples must be kept frozen until
analysis. Holding time should not exceed 28 days.
D. Raw Materials Required
NOTE:
Chemicals should be of ACS grade or equivalent.
Ammonium Chloride NH4Cl (FW 53.50)
Chloroform CHCl3 (FW 119.38)
Cupric Sulfate, Pentahydrate CuSO4•5H2O (FW 249.69)
Deionized Water (ASTM Type I or II)
Detergent TX-10 (API p/n 90-0760-04)
Hydrochloric Acid, Concentrated HCl (FW 36.46)
Imidazole, C3H4N2 (FW 68.08)
Low Nutrient Seawater (LNSW)*
Magnesium Sulfate MgSO4•7H2O (FW 246.48)*
N-1-naphthylethylenediamine Dihydrochloride C12H14N2•2HCl (FW 259.18)
Potassium Nitrate KNO3 (FW 101.11)
Sodium Bicarbonate NaHCO3 (FW 84.01) *
Sodium Chloride NaCl (FW 58.44) *
Sodium Nitrite NaNO2 (FW 69.0)
Sulfanilamide C6H8N2O2S (FW 172.21)
*See Operating Notes for information on matrix choices.
36
E. Reagent Preparation
1.
Copper Sulfate Solution 2% (1 L)
Cupric Sulfate ........................................................................................ 20 g
CuSO4•5H2O (FW 249.69)
Deionized Water
Dissolve 20 g of cupric sulfate in approximately 900 ml of deionized water contained in a 1 L
volumetric flask. Dilute the solution to the mark with deionized water and mix well. Stable at room
temperature.
2.
Stock Ammonium Chloride-Copper Sulfate (1 L)
Ammonium Chloride ...........................................................................250 g
NH4Cl (FW 53.50)
Copper Sulfate Solution 2%............................................................... 2.5 ml
Deionized Water
Dissolve 250 g of ammonium chloride in 900 ml of deionized water contained in a 1 L beaker. Add 2.5
ml of 2% copper sulfate solution. Transfer the solution to a 1 L volumetric flask and dilute to the mark
with deionized water. Store refrigerated at
2-8ºC.
3.
10% HCl (2 L)
Hydrochloric Acid Concentrated ...................................................... 200 ml
HCl (FW 36.46)
Deionized Water
Carefully add 200 ml of hydrochloric acid to about 1000 ml of deionized water. Cool and dilute to 2000
ml.
4.
Stock Imidazole Buffer (2 L)
Imidazole................................................................................................6.8 g
C3H4N2 (FW 68.08)
Stock Ammonium Chloride-Copper Sulfate........................................ 30 ml
10% HCL ....................................................................................... as needed
Deionized Water
Dissolve 6.8 g imidazole in about 1500 ml deionized water. Add 30 ml of stock ammonium chloridecopper sulfate solution. Adjust the pH to 7.8 - 7.85 with 10% HCl. Dilute to 2000 ml with deionized
water and mix well. Filter to 0.45 µm. Stable at room temperature.
5.
Working Imidazole Buffer (500 ml)
37
Stock Buffer ..................................................................................... 500 ml
Detergent TX-10 ............................................................................ 40 drops
Add 40 drops TX-10 to each 500 ml of Stock Buffer required. Mix well.
6.
NED Reagent (1 L)
N-1-naphthylethylenediamine Dihydrochloride ...................................1.0 g
C12H14N2•2HCl (FW 259.18)
Deionized Water
Dissolve 1.0 g N-1-napthylethylenediamine dihydrochloride in about 800 ml deionized water contained
in a 1 liter volumetric flask. Dilute to the mark with deionized water. Filter to 0.45 µm. Store in a
brown bottle and refrigerate when not in use. Reagent is stable for several months. Discard if colored.
7.
Stock SAN Reagent (1 L)
Sulfanilamide ...................................................................................... 10.0 g
C6H8N2O2S (FW 172.21)
10% HCl
Dissolve 10.0 g of sulfanilamide in about 800 ml of 10% HCl contained in a 1 L volumetric flask.
Dilute to the mark with 10% HCl and mix well. Filter to 0.45 µm. Stable at room temperature, but may
be refrigerated.
8.
Working SAN Reagent
Stock SAN Reagent ........................................................................... 250 ml
Detergent TX-10 .............................................................................. 30 drops
Add 30 drops TX-10 to 250 ml of stock SAN reagent and mix well.
9.
Artificial Seawater (ASW) (4L)
Sodium Chloride ................................................................................128.5 g
NaCl (FW 58.44)
Magnesium Sulfate ..............................................................................28.5 g
MgSO4•7H2O (FW 246.48)
Sodium Bicarbonate ...........................................................................0.672 g
NaHCO3 (FW 84.01)
Deionized Water
Dissolve 128.5 g of sodium chloride, 28.5 g of magnesium sulfate and 0.672 g of sodium bicarbonate in
about 3 liters of deionized water. Dilute to 4 liters with deionized water. These reagents must be high
quality, reagent grade to avoid excessive nutrient or trace metal contamination.
38
10.
Sampler Wash
See Operating Notes.
11.
Startup/Shutdown Solution
Deionized Water .............................................................................. 1000 ml
Detergent TX-10 .............................................................................. 2 – 4 ml
Add 2 to 4 ml of TX-10 to 1000 ml of deionized water. Mix well.
Open Tubular Cadmium Reactor (OTCR)(3)
12.
The Astoria analytical cartridge uses an Open Tubular Cadmium Reactor coil to reduce nitrate to nitrite.
Nitrogen is used to segment the analytical stream to prevent a pH increase due to reaction between
oxygen in ambient air and cadmium. Contact with oxygen will also deactivate the OTCR.
A. OTCR Activation
The OTCR (API p/n 303-0500-24) is a coiled cadmium tube (24") that has been cleaned of
manufacturing oils inside and coated with plastic outside. The outside diameter is 0.090 inches, with
an inside diameter of 0.050 inches, and a wall thickness of 0.020 inches. Short lengths of 0.034" ID
polyethylene are sleeved to the reactor coil to allow installation of the reactor in the manifold. These
sleeves are joined by a N-13 (N-2) nipple.
B. Reagents for OTCR Activation
1. Stock Imidazole Buffer
2. Copper Sulfate Solution
3. 1.0 N Hydrochloric Acid (100 ml)
Hydrochloric Acid, concentrated .................................................................. 8.3 ml
HCl (FW 36.46)
Deionized Water
Add 8.3 ml of concentrated hydrochloric acid to about 70 ml of deionized water contained in a 100
ml volumetric flask. Dilute to the mark with deionized water.
C. Procedure
NOTE:
Do not introduce air into the cadmium tube during this process.
1.
Detach one end of the polyethylene tubing from the N-13 (N-2) nipple.
2.
Using a 10 cc plastic syringe fitted with 0.040” ID PVC tubing and a short 0.034” ID
polyethylene extension, flush the OTCR with the described solutions using the following
procedure:
a)
Deionized Water
39
b)
1.0 N Hydrochloric Acid
CAUTION: The hydrochloric acid may cause pitting of the cadmium reactor interior surface if
left in the OTCR for longer than a few seconds. After the HCl flush, proceed quickly
to Step C.
c) Deionized Water
d) 2% Copper Sulfate
Slowly flush the OTCR with 10 cc of 2% copper sulfate. Repeat. Precipitated copper may be
observed exiting the reactor (black particles).
e) Deionized Water
Flush with deionized water until no more precipitated copper is flushed from the reactor.
This requires a forceful flush. Repeat 2-3 times.
f) Stock Imidazole Buffer
Fill the OTCR with Stock Buffer. The reactor should be stored with stock buffer when not in
use.
D. Installation of the OTCR
The analytical cartridge is provided with a jumper of 0.034"ID polyethylene sleeved at both ends in
the position where the OTCR is to be installed.
1. With the N-13 nipple in place, pump reagents segmented with nitrogen until a stable flow is
established.
NOTE:
The working buffer must be in the cartridge before the OTCR is installed.
2. Turn the pump off and disconnect the N-13 in the jumper connection.
3. Install the OTCR in the jumper, attaching each free end with one N-13 nipple.
4. Resume pumping and wait until a stable bubble pattern is established before
proceeding with the determinations.
E. Removal of the OTCR
1. Before the reagent lines are removed from the reagents, stop the pump, remove the
OTCR and reconnect the N-13 nipple in the jumper connection.
2. Resume pumping. Place the reagent lines in Startup/Shutdown solution and pump
until the cartridge has been thoroughly rinsed.
3. Attach the syringe to the N-13 nipple on the OTCR. Draw 10 to 15 ml of Stock
Buffer through the OTCR. Leaving buffer in the OTCR, remove the syringe and join
the tubing ends with the N-13 nipple.
NOTE:
Do not leave any air in the OTCR. It must be stored filled with Stock Buffer.
F. Reduction Efficiency and Stabilization of the OTCR
In the OTCR, nitrate is reduced to nitrite. However, under some conditions reduction may proceed
further with nitrite being reduced to hydroxylamine and ammonia. These reactions are pH dependent.
40
NO-3 + 2H+ + 2e¯ = NO-2+ H2O
NO-2 + 6H+ + 4e¯ = NO3 NOH+ + H2O
NO-2 + 8H+ + 6e¯ = NH4+ + 2H2O
(1)
(2)
(3)
At the buffered pH of the reactions, equation 1 predominates. However, if the cadmium surface is
overly active, equation 2 will proceed sufficiently to give low results. If the cadmium surface is
insufficiently active, there will be a low recovery of nitrate as nitrite.(3) This latter is defined as poor
reduction efficiency.
To determine the reduction efficiency, run a high level nitrite calibrant followed by a nitrate calibrant of
the same nominal concentration. A reduction efficiency range of 90% - 110% is acceptable. The
reduction efficiency is calculated as follows:
Peak Height (NO-3) x 100 = % efficiency
Peak Height (NO-2)
If the response of the nitrite is as expected but the reactor efficiency is poor, it may be necessary to
repeat the activation procedure. However, if the nitrite
response is much less than expected, it is an indication that the nitrite is being
further reduced and stabilization of the OTCR is necessary.
G. Stabilization
When an OTCR is first activated, it may be necessary to stabilize the activity of the reactor. In order to
stabilize the OTCR, pump a mid or high calibrant
continuously and record the steady state signal. Continue the steady state until a drift is no longer
observed. Return the sampler probe to wash and proceed with determinations when the baseline has
stabilized. An alternate procedure for stabilizing the OTCR is to pump a more concentrated nitrate
solution through the column for 5-10 minutes, but do not attempt to monitor the signal.
F. Calibrants
Specific Stock and Working Calibrant preparation instructions can be found on the back of the flow
diagram. Be sure to use the flow diagram which covers the concentration range you wish to analyze.
Working calibrants may be prepared to cover alternate ranges by adding the appropriate volumes of
stock or intermediate calibrant to 100 ml volumetric flasks that contain approximately 80 ml of sampler
wash solution. Dilute the solution to
100 ml with sampler wash solution and mix well.
The following formula can be used to calculate the amount of stock (or intermediate) calibrant to be
used.
C1V1 = C2V2
Where:
C1 = desired concentration (in mg/L) of working calibrant to be prepared
V1 = final volume (in ml) of working calibrant to be prepared (generally 100 ml)
41
C2 = concentration (in mg/L) of stock (or intermediate) calibrant
V2 = volume (in ml) of stock (or intermediate) calibrant to be used
Rearranging the equation to solve for V2 yields:
V2 = C1V1
C2
For example, to prepare a 1.0 mg/L working calibrant from a 1000 mg/L stock calibrant, use 0.1 ml (100
µl) of the stock calibrant in 100 ml final volume.
V2 = (1.0 mg/L) (100 ml)
1000 mg/L
V2 = 0.1 ml
Add this amount of stock calibrant to the volumetric flask and then dilute to volume with the sampler
wash solution.
G. Operation Procedure
1.
Set up the cartridge as shown in the flow diagram. Check all tubing and connections. Replace if
necessary.
2.
Place reagent lines in startup solution.
3.
Turn on power to all units and latch platens to begin liquid flow.
4.
Verify that the bubble size and spacing is consistent throughout the cartridge. If bubbles are
splitting up as they enter or exit a coil, check and replace fittings if necessary. The bubbles should
flow smoothly without dragging. If dragging occurs, add more TX-10 to the startup solution.
5.
Check all reagent containers on the instrument for particulate matter. Reagents should be filtered.
Be sure all containers are properly labeled and filled before pumping reagents.
6.
After a stable baseline has been verified on the startup solution, place reagent lines in reagent
bottles.
7.
If using data collection software, set up the appropriate sample table.
8.
Allow reagents to run for 5 to 10 minutes and verify a stable baseline.
9.
Once the reagent baseline is satisfactory, add the OTCR into the cartridge flow. Always connect
the inlet first and the outlet second. It is important to avoid the introduction of air into the coil
during this procedure.
10. Once the OTCR is on-line, run for 5-10 minutes and then re-verify the bubble pattern and baseline
stability. Make any necessary adjustments.
11. Load the sampler tray with calibrants, blanks, samples, and QC or monitor samples.
12. Select the appropriate parameters for the detector and sampler. (See Flow Diagram at the end of
42
methodology.)
13. Begin analysis.
14. At the end of analysis remove the OTCR from the cartridge. Disconnect the outlet first, then the
inlet. Flush the OTCR with buffer which contains no surfactant (TX-10).
15. Place all reagent lines in startup solution. Pump for 5 to 10 minutes to flush all of the reagents out
of the cartridge.
16. Turn off the power to all units and release pump platens.
H. Operating Notes
1.
The OTCR may be conditioned by running a mid scale standard through the manifold for 10-15
minutes.
2.
Life expectancy of the OTCR varies and is difficult to predict. It is recommended that a nitrite
standard of the same nominal concentration as the high scale standard be run as a check on column
reduction efficiency.
3.
There are special considerations when running seawater samples on any flow system.
A. Standards
Primary standards should be prepared from the best grade of chemicals available. Certificates
of Analysis are available from the chemical manufacturer. These should be consulted to
identify impurities.
Standard material should be oven dried for two hours at 110°C before weighing.
It is advisable to periodically verify the concentrations of the working standards. This can be
done by running standards against standards from an outside source.
The matrix of the standards should be consistent with that of the samples. If deionized water
standards are used it becomes important to determine the salt effects of each individual test.
(See number 2 under Operating Notes.)
B. Matrix
Optimal system performance can be expected if the sample matrix is carried over into the
sampler wash solution and the standards. Care should be taken when using deionized water
wash solution with seawater standards. Many investigators recommend segregating the samples
by salinity and running as a group to make corrections easier.
There are many options with respect to the matrix of the calibrant and sampler wash solutions.
The relative merits of several types of material are presented here. (1)
Deionized Water
Advantages:
43
1.
2.
3.
4.
The quality of the water is usually well known.
The quality of the water is usually not highly variable.
The prepared standards are relatively stable with time.
Large volumes of water are easily available.
Disadvantages:
1. The chemical factors may be different than in salt solution (salt effect).
Artificial Seawater Solution ( and/or Deionized Water - Sodium Chloride Solution)
Advantages:
1. Salt effects on the chemical factors are minimized.
2. Sodium chloride solution is easy to prepare and is not expensive.
Disadvantages:
1. Ammonium impurity is quite large in sodium chloride.
2. Large quantities of sodium chloride are sometimes required.
Low Nutrient Seawater
Advantages:
1. Salt effects are eliminated.
2. In certain regions of the ocean it is easily obtained.
Disadvantages:
1.
2.
3.
4.
It always contains some nutrients.
If not used immediately it must be filtered to remove any particulate matter.
Often it is difficult to obtain when working in eutrophic waters.
Storage is difficult, so large quantities are not easily obtained.
4.
As stated above, the system is optimal when the sample matrix is carried over into the sampler
wash solution and the standards. If this is not possible (i.e. if deionized water is used for the
sampler wash and/or calibrants), it is advisable to check for refractive index disturbance effects.
This can be accomplished by removing the sodium hypochlorite reagent. After the initial sample
run is finished, place the data collection in pause, replace the hypochlorite reagent with deionized
water, wait for a stable baseline, autozero the detector and run the samples again. The peaks seen
are due to the refractive index disturbance.
5.
As stated above, the system is optimal when the sample matrix is carried over into the sampler
wash solution and the standards. If this is not possible (i.e. if deionized water is used for the
sampler wash and/or calibrants), it is advisable to check for refractive index disturbance effects.
This can be accomplished by running without the NED present. After the initial sample run is
finished, place the data collection in pause, replace the NED reagent with deionized water, wait for
a stable baseline, autozero the detector and run the sample again. The peaks that are seen are due
to the refractive index disturbance.
44
6.
If bubbles are sticking in a debubbler, cleaning the debubbler will allow bubbles to escape
smoothly out the debubble line. Bubbles sticking in the debubbler can cause a loss in the overall
precision of the peak height. To clean, soak the debubbler for 2-3 hours in a mixture of 20-30%
ContradNF (API p/n 80-0007-04) and hot tap water. Rinse thoroughly.
7. If the flowrate of the sample pump tube is ≤ 226 µl/minute (a blk/blk pump tube) a helper line
must be added when the cartridge is run alone. See Section 9 of the Astoria Analyzer Operation
Manual for information on how to add a helper line.
NOTE:
If the sample line is debubbled, a helper line is not necessary.
8. Cover all reagents and other solutions to avoid interference due to dust and other particulates. This
will also help prevent contamination of the solutions from absorbance of analytes in the air.
I. References
1.
2.
3.
4.
5.
6.
Standard Methods for the Examination of Water and Wastewater: Centennial Edition, 21st Ed.,
2005, American Public Health Association, Washington, D.C. (Method referenced: Automated
Cadmium Reduction, 4500 – NO3-F, pp. 4-125 – 4-126).
F. Nydahl, Talanta, 23, Pages 349-357 (1976).
Patton, C.J., Doctoral Dissertation, Michigan State University, 1982, Page 87-121.
Fox, J.B., Anal. Chem., 51, 1493 (1979).
Automated Nutrient Analysis in Seawater, Technical Report, Brookhaven National Laboratory,
Whitledge, Veidt, et. al., May 1986.
Methods and Guidance for Analysis of Water, 1999, Office of Water, Environmental Protection
Agency (USEPA), Cincinnati, OH (Method referenced: 353.3)
ACKNOWLEDGEMENTS
Astoria® and FASPac are trademarks of Astoria-Pacific, Inc., Clackamas, Oregon
Parafilm is a registered trademark of American National Can, Norwalk, CT
Contrad®NF and Neutrad® are registered trademarks of Decon Labs., Inc., Bryn Mawr, Pennsylvania
45
Standard Operating Procedure 004
Ortho-Phosphate Analysis
Date: Nov 2007
Revision: 2
Primary Method: EPA 365.5
A. Scope and Application
This method is used for the determination of ortho-phosphate (dissolved reactive phosphate) in seawater.
The applicable range of this method is 0.02 – 3.0 µM phosphate. However, this method is also
applicable to other ranges.
B. Summary of Method
Ortho-phosphate reacts with molybdenum (VI) and antimony (III) in an acidic medium to form a
phosphoantimonylmolybdenum complex. This complex is subsequently reduced by ascorbic acid to a
heteropolyblue with an absorbance maximum at 880 nm.
C. Sample Handling and Preservation
All samples will be filtered using 0.4 µm Nucleopore filters. Samples must be kept frozen until
analysis. Holding time should not exceed 28 days.
D. Raw Materials Required
NOTE: Chemicals should be of ACS grade or equivalent.
Ammonium Molybdate, (NH4)6Mo7O24·4H2O (FW 1235.86)
Antimony Potassium Tartrate, K2Sb2C8H4O12·3H2O (FW 667.87)
Ascorbic Acid, C6H8O6 (FW 176.13)
Deionized Water (ASTM Type I or Type II)
Low Nutrient Seawater (LNSW)*
Magnesium Sulfate MgSO4·7H2O (FW 246.48)*
Potassium Dihydrogen Phosphate, KH2PO4 (FW 136.09)
Sodium Bicarbonate NaHCO3 (FW 84.01)*
Sodium Chloride NaCl (FW 58.44)*
Sodium Lauryl Sulfate, C12H25O4SNa (FW 288.38)
Sulfuric Acid Concentrated, H2SO4 (FW 98.08)
* See operating notes for information on matrix choices.
46
E. Reagent Preparation
1.
Sodium Lauryl Sulfate (SLS), 15% w/w
Deionized Water .............................................................................................. 85 ml
Sodium Lauryl Sulfate (SLS)..............................................................................15 g
C12H25O4SNa (FW 288.38)
Dissolve 15 g SLS in 85 ml deionized water contained in a 250 ml Erlenmeyer flask. Gentle
warming may be needed for complete dissolution.
2.
Sulfuric Acid, 5 N (1000 ml)
CAUTION: Mixing sulfuric acid with water generates a great amount of heat.
Sulfuric Acid, concentrated. .......................................................................... 140 ml
H2SO4 (FW 98.08)
Deionized Water
Cautiously add 140 ml concentrated sulfuric acid to 600 ml deionized water contained in a 1000
ml Erlenmeyer flask. Cool to room temperature and transfer to a 1000 ml volumetric flask.
Dilute to the mark with deionized water.
3.
Antimony Potassium Tartrate (50 ml)
Antimony Potassium Tartrate ..........................................................................0.15 g
K2Sb2C8H4O12·3H2O (FW 667.87)
Deionized Water
Dissolve 0.15 g antimony potassium tartrate in 40 ml deionized water contained in a 50 ml
volumetric flask. Dilute to the mark with deionized water. Store at 2-8° C in a dark bottle.
4.
Ammonium Molybdate (150 ml)
Ammonium Molybdate .........................................................................................6 g
(NH4)6Mo7O24·4H2O (FW 1235.86)
Deionized Water
Dissolve 6 g ammonium molybdate in 75 ml deionized water. Add deionized water to final
volume of 150 ml and mix well. Store at 2-8 °C in a polyethylene bottle.
5.
Ascorbic Acid (300 ml)
Ascorbic Acid ....................................................................................................5.4 g
47
C6H8O6 (FW176.13)
Deionized Water
Dissolve 5.4 g ascorbic acid in 150 ml of deionized water. Add deionized water to a final
volume of 300 ml and mix well. Stable for 10 days if stored at 2 – 8 °C.
6.
Color Reagent (100 ml)
Sulfuric Acid, 5 N ............................................................................................ 50 ml
Antimony Potassium Tartrate ............................................................................ 5 ml
Ammonium Molybdate .................................................................................... 15 ml
Ascorbic Acid .................................................................................................. 30 ml
Add reagents in the order stated and mix after each addition. Add 3 – 5 ml SLS and filter to 0.45
µm. Prepare reagent daily.
7.
Artificial Seawater (ASW) (4 L) (See Operating Notes)
Sodium Chloride ............................................................................................128.5 g
NaCI (FW 58.44)
Magnesium Sulfate ..........................................................................................28.5 g
MgSO4·7H2O (FW 246.48)
Sodium Bicarbonate .......................................................................................0.672 g
NaHCO3 (FW 84.01)
Deionized Water
Dissolve 128.5 g sodium chloride, 28.5 g magnesium sulfate and 0.672 g sodium bicarbonate in
about 3 liters of deionized water. Dilute to 4 liters with deionized water. These reagents must be
high quality, reagent grade to avoid excessive nutrient or trace metal contamination.
8.
Startup/Shutdown Solution (100 ml)
Deionized Water ............................................................................................ 100 ml
SLS, 15% ........................................................................................................... 2 ml
9.
Sampler Wash Solution
See Operating Note #1.
F. Calibrants
See Nitrate + Nitrite Analysis SOP for information on calibrant preparation.
G. Operation Procedure
48
1.
Set up the cartridge as shown in the flow diagram. Check all tubing and connections.
Replace if necessary.
2.
Place reagent lines in startup solution.
3.
Turn on power to all units including heat bath and latch pump platens to begin liquid flow.
4.
Verify that the bubble size and spacing is consistent throughout the cartridge. If bubbles are
splitting up as they enter or exit a coil or heat bath, check and replace fittings if necessary.
The bubbles should flow smoothly without dragging. If dragging occurs, add more SLS to
the startup solution.
5.
Check all reagent containers on the instrument for particulate matter. Reagents should be
filtered weekly. Be sure all containers are properly labeled and filled before pumping
reagents.
6.
After the heat bath has reached the desired temperature and a stable baseline has been
verified on the startup solution, place reagent lines in reagent bottles.
7.
If using data collection software, set up the appropriate sample table.
8.
Allow reagents to run for 5 to 10 minutes and verify a stable baseline.
9.
Load the sampler tray with calibrants, blanks, samples, and QC or monitor samples.
10. Select the appropriate parameters for the detector and sampler. (See Flow Diagram.)
11. Begin analysis.
12. At the end of analysis place all reagent lines in startup/shutdown solution and turn off the
heat bath. Pump startup/shutdown solution for 20 to 30 minutes to flush all of the reagents
out of the cartridge and to allow the heat bath to cool.
13. Turn off the power to all units and release pump platens.
H. Operating Notes
See Nitrate + Nitrite Analysis SOP for information on sample matrices.
If the flowrate of the sample pump tube is ≤ 226 µl/minute (a blk/blk pump tube) a helper line
must be added when the cartridge is run alone. See Section 9 of the Astoria Analyzer Operation
Manual for information on how to add a helper line.
NOTE:
If the sample line is debubbled, a helper line is not necessary.
49
1. Low sensitivity and noise in the baseline can be caused by debris in the flowcell. Particulate
matter from the reagents and samples can become lodged in the flowcell restricting the
amount of light that is passed through the flowcell. Flushing the flowcell with approximately
10 ml of sampler wash solution with a syringe will dislodge any debris in the flowcell.
Following proper filtration procedures for the reagents and samples will reduce the frequency
of this occurring.
2. To prevent the accumulation of background contamination forming in the color reagent, keep
the reagent bottle covered at all times. Baseline drift may also be reduced by placing the
color reagent in an ice bath during analysis.
3. If increased carryover and drift are experienced, make sure the ascorbic acid and ammonium
molybdate solutions are fresh.
4. If bubbles are sticking in a debubbler, cleaning the debubbler will allow bubbles to escape
smoothly out the debubble line. Bubbles sticking in the debubbler can cause a loss in the
overall precision of the peak height. To clean, soak the debubbler for 2-3 hours in a mixture
of 20-30% ContradNF (API p/n 80-0007-04) and hot tap water. Rinse thoroughly.
5. For chronic carryover and drift problems, the following cleaning solution can be used to
flush the analytical cartridge and flowcell.
Potassium Iodide Cleaning Solution (55 ml)
Potassium Iodide ...................................................................................................1 g
KI (FW 166.00)
5 N Sulfuric Acid (See Reagent Preparation) .................................................. 25 ml
H2SO4 (FW 98.08)
Deionized Water .............................................................................................. 30 ml
Add 1 g KI to about 25 ml 5 N sulfuric acid. Stir vigorously until a strong yellow-orange
color has formed. This may take at least one hour. Add about 30 ml deionized water. The
solution will darken over time, and is usable for one month. Pump the cleaning solution
through all lines in the cartridge for 10 to 15 minutes, followed by startup/shutdown solution.
6. Acid washed glassware should be used for all reagents and calibrants. Commercial
detergents containing phosphorus should never be used to clean glassware used in
phosphorus determination. Wash the glassware with 1:1 hydrochloric acid and rinse it
thoroughly with deionized water. Store the glassware filled with deionized water. If the
glassware is reserved for use only in phosphorus determination, treatment with hydrochloric
acid is necessary only occasionally.
I. References
1.
Standard Methods for the Examination of Water and Wastewater: Centennial Edtion, 21st
Ed., 2005, American Public Health Association, Washington, D.C. (Method referenced:
50
Automated Ascorbic Acid Reduction, 4500-P-F, pp. 4-155 – 4-156).
2. Methods for Chemical Analysis of Water and Wastewater,
March 1984, EPA-600/4-79-020, "Sample Preservation", p. xvii, Environmental Monitoring
and Support Laboratory, Office of Research and Development, U.S. Environmental
Protection Agency Cincinnati, OH 45286.
3. Methods and Guidance for Analysis of Water, 1999, Office of Water, Environmental
Protection Agency (USEPA), Cincinnati, OH (Method referenced: 365.5)
4. Automated Nutrient Analysis in Seawater, Technical Report, Brookhaven National
Laboratory, Whiteledge, Veidt, et. al., May 1986.
ACKNOWLEDGEMENTS
Astoria® and FASPac™ are trademarks of Astoria-Pacific, Inc., Clackamas, Oregon
Dowfax™ 2A1 is a trademark of Dow Chemical, USA
Contrad®NF and Neutrad® are registered trademarks of Decon Labs., Inc., Bryn Mawr,
Pennsylvania
51
Standard Operating Procedure 005
Ammonia: Fluorometric
Date: Nov 2011
Revision: 1
Primary Method: EPA 350.1
A. Scope and Application
This method is used for the determination of ammonia in seawater. The applicable range of this
method is 0.05 to 5 μM.
B. Summary of Method
The sample is mixed with o-phthaldialdehyde and sodium sulfite in a borate-buffered solution at
75°C. After sufficient mixing, the sample concentration is measured by fluorescence
spectroscopy using 360nm excitation and 420-470nm emission wavelengths. The increase in
fluorescence is directly proportional to the ammonia concentration.
C. Interferences
Inorganic salts can have a depressive effect1; Copper 6% (>300μg/L), Iron 0.5% (1- 3mg/L),
Mercury 5% (10mg/L), Sulfide 2.3% (>10μM). Particulate matter should be removed or
centrifuged to prevent clogging in the system.
D. Sample Handling and Preservation
Samples should be analyzed directly. Samples should be frozen as soon as possible if not
analyzing immediately.
E. Raw Materials Required
Ammonium Sulfate (NH4)2SO4 (FW 132.15) Brij®-35, 30% w/v (API p/n 90-0710-04) Deionized Water (ASTM Type I or II) Ethanol, 200 Proof (FW 46.07) Optional, See Operating Notes
Low Nutrient Seawater (LNSW)*
Magnesium Sulfate MgSO4·7H2O (FW 246.48) Nitrogen gas source o-phthaldialdehyde (OPA) C8H6O2 (FW 134.1), CAS [643-79-8]
Sodium Bicarbonate NaHCO3 (FW 84.01)*
Sodium Chloride NaCl (FW 58.44)* Sodium Sulfite Na2SO3 (FW 126.0) Sodium Tetraborate Na2B4O7⋅10H2O (FW 381.37)
*See operating notes for information on matrix choices.
NOTE: Chemicals should be of ACS grade or equivalent.
52
F. Reagent Preparation
1. Stock Sodium Sulfite Solution, (250
Sodium Sulfite ........................................................................................ 2 g
Na2SO3 (FW 126.0)
Deionized Water
Dissolve 2 g sodium sulfite in approximately 200 ml of deionized water contained in a 250-ml
volumetric flask, mix well, and dilute to the mark. Store in a sealed container. This solution is
stable for 1 month.
2. Borate Buffer Solution (1 L)
Sodium Tetraborate.............................................................................. 30 g
Na2B4O7⋅10H2O (FW 381.37)
Deionized Water
Dissolve 30 g of sodium tetraborate in about 900 ml of deionized water contained in a 1- L
volumetric flask. Dilute to the mark with deionized water and mix well. Keep tightly closed.
Stable for several months.
3. OPA Working Reagent (250 ml)
o-phthaldialdehyde ...............................................................................0.2 g
C8H6O2 (FW 134.1)
Borate buffer solution ........................................................................250 ml
Sodium sulfite solution........................................................................0.5 ml
Add 0.2 g o-phthaldialdehyde to about 125 ml of borate buffer solution contained in a 250-ml
Erlenmeyer flask and add a magnetic stir bar. Stir solution using a stir plate until OPA is
dissolved. Transfer solution to a 250-ml graduated cylinder. Add 0.5 ml of sodium sulfite
solution. Dilute to the 250 ml mark with buffer solution and mix well. Add 1 ml of Brij®-35.
Prepare as needed. See Operating Note #9 for alternate preparation instructions.
4. Sampler Wash Solution
Artificial Seawater, See Operating Note #13
CAUTION: o-phthaldialdehyde is light sensitive. Avoid prolonged exposure to light.
5. Artificial Seawater (ASW) (4 L) (See Operating Notes)
Sodium Chloride...................................................................128.5 g
NaCl (FW 58.44)
Magnesium Sulfate ................................................................ 28.5 g
MgSO4•7H2O (FW 246.48)
Sodium Bicarbonate...............................................................0.672 g
NaHCO3 (FW 84.01)
53
Deionized Water
Dissolve 128.5 g of sodium chloride, 28.5 g of magnesium sulfate, and 0.672 g of sodium
bicarbonate in approximately 3 L of deionized water. Dilute to 4 L with deionized water. These
reagents must be high quality reagent grade to avoid excessive nutrient or trace metal
contamination.
6. Startup/Shutdown Solution
Brij®-35, 30% .........................................................................1-2 ml
Deionized water
Add 1-2 ml Brij®-35 to 1 L of deionized water. Mix well.
G. Calibrants
See Nitrate + Nitrite Analysis SOP for information on calibrant preparation.
H. Operation Procedure
1. Set up the cartridge as shown in the flow diagram. Check all tubing and connections. Replace
if necessary.
2. Place reagent lines in startup solution.
3. Turn on power to all units including heat bath. Latch platens to begin liquid flow. Open
nitrogen pillow.
4. Verify that the bubble size and spacing is consistent throughout the cartridge. If the bubbles
are splitting up as they enter or exit a coil or heat bath, check and replace any suspect
fittings. The bubbles should flow smoothly without dragging. If dragging is observed,
add more Brij®-35 to the startup solution.
5. Check all reagent containers on the instrument for particulate matter. Be sure all containers are
properly labeled and filled before pumping reagents. Filter to 0.45 μm if necessary.
6. After heat bath has reached desired temperature and a stable baseline has been verified using
startup solution, place reagent lines in appropriate reagent containers.
7. Open data collection software and set up the appropriate sample table.
8. Allow reagents to run for 10 to 15 minutes and verify a stable baseline.
9. Load the sampler tray with calibrants, blanks, samples, and QC or monitor samples.
10. Select the appropriate parameters for the detector and sampler. (See Flow Diagram.)
11. Begin analysis.
12. At the end of analysis place all reagent lines in startup solution and turn off heat bath. Pump
startup solution for 20 to 30 minutes to flush all of the reagents out of the cartridge and to
allow the heat bath to cool.
13. Release pump platens and turn off the power to all units.
14. Close the nitrogen pillow.
54
I. Operating Notes
See Nitrate + Nitrite Analysis SOP for information on sample matrices.
1. Prepare ammonia free water by passing distilled water through a mixture of strongly acidic
cation and strongly basic anion exchange resins.5
2. To prevent ammonia contamination from the air, segment the analytical stream with nitrogen
or draw air through a 5 N sulfuric acid solution.
3. If bubbles are sticking in a debubbler, cleaning the debubbler will allow bubbles to escape
smoothly out the debubble line. Bubbles sticking in the debubbler can cause a loss in the
overall precision of the peak height. To clean, soak the debubbler for 2-3 hours in a
mixture of 20-30% Contrad
NF (APIand
-0007-04)
p/nhot
80 tap water. Rinse
thoroughly.
4. If the flowrate of the sample pump tube is ≤ 226 μl/minute (a blk/blk pump tube) a helper line
must be added when the cartridge is run alone. See Section 8 of the Astoria Analyzer
Operation Manual for information on how to add a helper line.
5. Cover all reagents and other solutions to avoid interference due to dust and other particulates.
This will also help prevent contamination of the solutions from absorbance of analytes in
the air.
6. The OPA reagent can be made by an alternate procedure. The instructions are as follows:
• OPA stock solution: Dissolve 2 g of OPA in 50 ml of pure ethanol (200 proof). The stock
solution needs to be stored refrigerated in an amber glass bottle. The stock is stable for 2 months.
• To 250 ml of borate buffer add 5 ml of OPA solution, 0.5 ml sodium sulfite solution. Add 10
drops of Brij-35 and mix thoroughly. Transfer to an amber glass bottle and let stand
approximately 24 hours. Working solution can be stored in the dark for 1 month.
7. If there is a significant drop in peak height even after making fresh OPA reagent, remake the
sodium sulfite solution then add it to the prepared OPA reagent.
8. Due the ease of contamination it is necessary to fill wash cups directly from the sampler wash
container. The transfer should be performed in one step rather than multiple steps (i.e.
large pipette).
9. It is highly recommended that 16x100mm plastic test tubes are used for sampling. They help
reduce outside contamination.
10. Artificial seawater does not need to be used. Salted water (NaCl) is sufficient as sampler wash
solution. The refractive index is small regardless of what salinity the sampler wash is, but
a salinity of 35 provides the best results.
55
J. References
1. Kérouel, R. et al, Marine Chemistry, 57, 265-275(1997).
2. Holmes, R.M. et al, Canadian Journal of Fisheries and Aquatic Sciences, 56, 1801-1808
(1999).
3. Jones, R. et al, Journal of Limnology and Oceanography, 36, 814-819(1991).
4. Automated Nutrient Analysis in Seawater, Technical Report, Brookhaven
Laboratory, Whiteledge, Veidt, et. al., May 1986.
National
5. Methods for Chemical Analysis of Water and Wastes, March 1984, EPA-600/4-79-020,
"Nitrogen, Ammonia", Method 350.1 (Colorimetric, Automated Phenate) STORET NO.
Total 00610, Dissolved 00608.
ACKNOWLEDGMENTS
Astoria® and FASPac™ are trademarks of Astoria-Pacific, Inc., Clackamas, Oregon
Brij®-35 is a registered trademark of ICI Americas, Wilmington, Delaware
Contrad®NF and Neutrad® are registered trademarks of Decon Labs., Inc., Bryn Mawr,
Pennsylvania
56
Standard Operating Procedure 006
TN and TP Analysis
Date: Feb 2012
Primary Method: Oviatt and Hindle (1994)
A. Scope and Application
This method is used for the determination of Total Nitrogen and Total Phosphorous in seawater and is
applicable to many ranges.
B. Summary of Method
Alkaline persulfate digestion oxidizes all forms of inorganic and organic nitrogen to nitrate and
hydrolyzes all forms of inorganic and organic phosphorous to ortho-phosphate. After digestion,
samples are analyzed as described in SOPs for nitrate/nitrite and for ortho-phosphate with the
exception of substituting the imidazole buffer with an ammonium chloride buffer described
below.
C. Sample Handling and Preservation
Samples must be kept frozen until analysis. Holding time should not exceed 28 days.
D. Raw Materials Required
NOTE: Chemicals should be of ACS grade or equivalent.
Sodium hydroxide (NaOH, FW=40.0)
Potassium persulfate (K2S2O8, FW=270.33)
Boric acid (H3BO3, FW=61.83)
Glycine (C2H5NO2•HCl, FW=111.5)
Glycerophophate (C3H7O6PNa2•5H2O, FW=306.1)
Glucose (C6H12O6, FW=180.2)
Ammonium chloride (NH4Cl, FW=53.49)
Ammonium hydroxide (NH4OH, FW=35.05)
Detergent TX-10 (API p/n 90-0760-04)
E. Reagent Preparation
1.
Sodium Hydroxide, 1.0 N
Sodium Hydroxide ...........................................................................................10.0 g
NaOH (FW 40.00)
Ultrapure Water
Dissolve 10.0 g of sodium hydroxide in about 180 mL of DI water in a 200 mL volumetric flask
[Caution: When NaOH dissolves in water, heat is released.] After dissolution is complete, allow
57
the resulting solution to cool and dilute it to the mark with DI water. Make day of sample
digestion.
2.
Alkaline Persulfate Digestion Reagent
Potassium Persulfate ........................................................................................25.0 g
K2S2O8 (FW=270.33)
Sodium Hydroxide, 1.0 N ............................................................................. 175 mL
Boric Acid ........................................................................................................15.0 g
H3BO3, FW=61.83
Ultrapure Water
Add 25.0 g of potassium persulfate, 15.0 g of boric acid, and 175 mL of 1.0 N sodium hydroxide
solution to about 250 mL of DI water in 1000-mL Pyrex media bottle. Cap the bottle, swirl its
contents, and place it in an magnetic stirrer dissolution is complete (about 10 minutes). Remove
the bottle from the magnetic stirrer and pour solution into 500 ml volumetric flask. Add
ultrapure water to the 500 ml mark. Prepare this reagent day of sample digestion.
3.
Glycine Digest-Check Stock Solution
Glycine .............................................................................................................3.98 g
C2H5NO2•HCl (FW=111.5)
Ultrapure Water
Dissolve 3.98 g glycine in about 400 mL of DI water in a 500 mL volumetric flask. Dilute this
solution to the mark with DI water and mix it thoroughly by manual inversion and shaking.
Transfer the stock digest-check solution to a 500 mL Pyrex media bottle in which it is stable for
6 months at 4°C.
4.
Glycerophosphate Digest-Check Stock Solution
Glycerophosphate ..........................................................................................1.976 g
C3H7O6PNa2•5H2O (FW=306.1)
Ultrapure Water
Dissolve 1.976 g glycerophosphate in about 400 mL of DI water in a 500 mL volumetric flask.
Dilute this solution to the mark with DI water and mix it thoroughly by manual inversion and
shaking. Transfer the stock digest-check solution to a 500 mL Pyrex media bottle in which it is
stable for 6 months at 4°C.
5.
Glucose Digest-Check Stock Solution
Glucose ..........................................................................................................1.564 g
C6H12O6 (FW=180.2)
Ultrapure Water
58
Dissolve 1.564 g glucose in about 400 mL of DI water in a 500 mL volumetric flask. Dilute this
solution to the mark with DI water and mix it thoroughly by manual inversion and shaking.
Transfer the stock digest-check solution to a 500 mL Pyrex media bottle in which it is stable for
6 months at 4°C.
6.
Mixed Digest-Check Solution
Glycerine digest-check stock solution .............................................................. 1 mL
Glycerophosphate digest-check stock solution ................................................. 1 mL
Glucose digest-check stock solution ............................................................... 10 mL
Ultrapure Water
Dispense 1 mL each of glycine and glycerophosphate stock digest-check solutions, 10 mL of the
glucose digest-check stock solution into a 250 mL volumetric flask that contains about 200 mL
of DI water. Dilute the contents of the flask to the mark with DI water and mix it thoroughly by
manual inversion and shaking. Transfer the mixed digest-check solution to a 250 mL Pyrex
media bottle in which it is stable for 1 month at 4°C.
7.
Ammonium Chloride Solution
Ammonium chloride ...........................................................................................30 g
NH4Cl (FW=53.49)
Ultrapure water
Dissolve 30.0 g of ammonium chloride in approximately 800 ml of ultrapure water in a 1000 ml
volumetric flask. Dilute to mark and store at room temperature for up to 1 month.
8.
Ammonium Chloride Buffer
Ammonium chloride solution ......................................................................... 50 mL
Ultrapure water ............................................................................................. 100 mL
Ammonium hydroxide ................................................................................... 250 µL
NH4OH (FW=35.05)
TX-10 ............................................................................................................ 1.5 mL
Mix ammonium chloride solution, ultrapure water, ammonium hydroxide and TX-10 in a 250 ml
flask. Prepare day of sample analysis.
F. Sample Preparation
Alkaline persulfate digests are prepared by dispensing samples and digestion reagent into 45-mL,
screw-cap, borosilicate glass vials (Type 1, class B) in the volume ratio of 4 + 1 (i.e. 20.000 mL
59
sample, 5.000 mL digestion reagent). All tubes are capped tightly and mixed thoroughly by
manual inversion. The capped tubes are placed in a room temperature DI water bath. The level
of the bath should reach approximately ¼ inch below the level of the liquid in the sample vials.
With the lid closed, the water bath is heated to at least 100°C and sample vials are boiled gently
for 15 minutes. After 15 minutes, the vials are cooled to room temperature (overnight) in the
water bath with the cover remaining on.
Note that the tightly capped digests can be stored at room temperature for several days before
their nitrogen and phosphorous concentrations are determined as described in SOPs for
nitrate/nitrite and ortho-phosphate.
G. Operation Procedure
Follow procedure described in SOPs for nitrate/nitrite and ortho-phosphate with one exception:
the imidazole buffer used in the nitrate/nitrite analysis should be replaced with an ammonium
chloride buffer.
H. References
1. Ameel, J.J., R.P. Axler, and C.J. Owen. 1993. Persulfate Digestion for Determination of
Total Nitrogen and Total Phosphorous in Low-Nutrient Waters.
American
Environmental Laboratories 10:8-10.
2. Green, L. 2006. Standard Operating Procedure 016: Total Phosphorous and Nitrogen
Analysis. University of Rhode Island Watershed Watch.
3. Oviatt, C.A. and K.M. Hindle. 1994. Manual of Biological and Geochemical Techniques
in Coastal Areas. MERL Series, Report#1, Third Edition. Section 1.6, pp. 88-91, “Total
Dissolved and Total Particulate Nitrogen and Phosphorous.” The University of Rhode
Island, Kingston, Rhode Island.
60
Standard Operating Procedure 007
Chlorophyll a Analysis
Date: Nov 2007
Revision: 2
EPA Method: 445.0
Field Sample Filtration: Generally two replicate samples are filtered from each water sample
within 2 hours of sample collection. If filtration cannot take place immediately after sample
collection, water samples must be refrigerated. In subdued light water samples are filtered
through 0.7 um glass fiber filters (Whatman GF/F). Individual filters are folded in half, wrapped
first in blotting filters (for example a piece of white paper towel or white coffee filter), and then
in squares of aluminum foil. These foil packs are then placed in a Whirlpak labeled with the
following information: location, date, amount of water filtered. The filters are kept frozen until
just prior to extraction and analysis.
Lights must be kept off during analysis and preparations. Lighting should be the
minimum that is necessary to read instructions and operate fluorometer.
Extraction Procedure: A glass fiber filter, through which has been filtered a known aliquot of
water, is placed in a glass 16 x 150 mm test tube containing 10 ml of 90% acetone. The vial is
capped, shaken vigorously and allowed to steep 18 - 24 hours at 4 deg. C. Prior to analysis the
rack of vials is brought to room temperature.
Instrumentation: Turner Trilogy Laboratory Fluorometer
Calibration: The fluorometer is calibrated at the beginning of each monitoring season with 2
liquid pure chlorophyll a standards and reagent blanks following the manufacturer’s instructions.
At the time of calibration a solid secondary standard is also analyzed and the formula for
calculating chlorophyll a in samples is determined. That solid standard is analyzed with each
batch of samples. When sample concentration is calculated an allowance for instrument drift is
made using the daily batch readings of the solid standard. Blanks of 90% acetone, and an unused
filter extracted with 90% acetone are set up with each rack of samples.
Cuvettes: 12x35 mm glass test tubes
Fluorometer Operation:
1.
Make sure Trilogy is off.
2.
Insert the Chlorophyll (acidification) Optical Application Module
3.
Close lid and turn power on. Use the touch screen to identify the type of Optical
Application Module installed.
Procedure:
1.
The fluorometer should be on with Home screen displayed.
2.
Select Chl A (chlorophyll, acidification)
3.
In raw fluorescence mode, measure a blank (90% acetone) and the secondary standard.
Record on data sheet.
61
4.
On the screen, select Mode to switch to the calibration mode.
5.
Use stored calibration – select the most recent stored calibration.
6.
Samples: After the 24 hr extraction is complete, be sure cap is tightly sealed, and invert
test tube several times to mix contents. Work in dim light; wear surgical gloves to keep
acetone from your hands.
7.
Remove cap, poor 5 ml into sample cuvette.
8.
Check cuvette to be sure there are no finger prints on glass; polish with a Kimwipe if
necessary.
9.
Open the lid and insert the test tube. Close the lid.
10. Touch Measure Fluorescence to commence measurement. The sample will be measured
for 6 seconds and report the average reading for the sample.
11. Remove sample, add 0.15 mL of 0.1 N HCl, cover and mix well. Wipe cuvette again
with Kimwipe. Wait 90 sec and read a second time.
12. Record chlorophyll a and pheophytin a values on data sheet.
13. When finished measuring samples, switch to raw fluorescence mode and measure bland
and secondary standard again. Record on data sheet.
14. Dispose of all acetone in Acetone Waste container. Rinse with tap water
15. Soak all test tubes and stoppers in soapy water (Liqui Nox) for 24 hours.
16. Rinse well with tap water and soak in DI water for 24 hours.
17. Rinse 3 times with DI water and dry in test tube rack.
Calculations: When in direct concentration mode the following calculations will be use to
calculate corrected chlorophyll a and pheophytin values.
Variables stored during calibration phase of fluorometer
Cstand[1] = Concentration of standard 1
Fblank = Fluorescence of Blank value
Fstand[1],B = Fluorescence of standard 1 before acidification
Fstand[1],A = Fluorescence of standard 1 after acidification
Fm = Acidification Ratio = (Fstand[1],B – Fblank) / (Fstand[1],A – Fblank)
Variables required from the sample analysis phase
Fsamp,B = Fluorescence of sample before acidification
Fsamp,A = Fluorescence of sample after acidification
Vsolvent = Volume of solvent used to extract sample
Vwater = Volume of water filtered
Interpolation equation used in end calculation of chlorophyll a and pheophytin concentrations
62
Interp,B = Cstand[1] * (Fsamp,B - Fblank) / (Fstand[1],B - Fblank)
Interp,A = Cstand[1] * (Fsamp,A - Fblank) / (Fstand[1],B - Fblank)
End calculation for corrected chlorophyll and pheophytin a
Chlorophyll a concentration = [Fm/(Fm-1)] * (Interp,B - Interp,A) * (Vsolvent/ Vwater)
Pheophytin a concentration = [Fm/(Fm-1)] * [(Fm * Interp,A) - Interp,B] * (Vsolvent/ Vwater)
References:
EPA Method 445.0
Standard Methods for the Examination of Water and Wastewater: Centennial Edition, 21st Ed.,
Method 10200H, pp. 10-19 – 10-26 (2005).
Turner Designs Trilogy Laboratory Fluorometer User’s Manual
[www.turnerdesigns.com/t2/doc/manuals/TrilogyUsersManual.pdf]
63
Standard Operating Procedure 008
Phytoplankton Collection and Analysis
The following methods for the collection, identification, and enumeration of phytoplankton
species are similar to those described in Libby et al. (2002, 2005, 2006, and 2010) and have been
used in Massachusetts Water Resources Authority Harbor Outfall Monitoring Projects HOM3
through HOM6. Thus data from current efforts will be comparable to data sets of previous years.
Collection
A Niskin bottle will be used to collect surface water at a depth of 1-2 meters at each station. A
portion of the water from the Niskin sampling bottle (800 mls) will be measured with into a
clean graduated cylinder that has been thrice-rinsed with the sampling bottle water. The sample
will be mixed with Utermöhl’s solution (1 ml Utermöhl’s per 100 ml water sampled) in a 1-L
HDPE bottle and stored in a cooler until it can be further processed in the lab. Utermöhl's
solution is prepared as described in Guillard (1973): 100 g potassium iodide, 50 g iodine, and 50
g sodium acetate each are dissolved incrementally in distilled water to a final volume of 1 L.
Sample Preparation
At the laboratory, the Utermöhl's-preserved whole seawater samples will be prepared for analysis
by concentrating the sample by gravitational settling as described by Borkman (1994), Borkman
et al. (1993), and Turner et al. (1995). Samples will be settled in glass graduated cylinders with
no more than a 5-to-1 height-to-width ratio. The preserved samples are stored at ambient
temperature and in the dark until analysis. Prior to analysis, the 800 ml of seawater will be
settled to 50 ml and decanted by low vacuum aspiration.
Counting and Abundance Estimates
One ml of the concentrate will be examined and phytoplankton counted in a gridded SedgwickRafter chamber using a Olympus BH-2 research microscope with phase contrast optics.
Phytoplankton abundance is calculated by dividing the number of cells counted by the volume
examined in a gridded Sedgwick-Rafter chamber. The theoretical maximum possible volume that
would be examined would be an entire Sedgwick-Rafter chamber (1 ml). The grid subdivides the
chamber into µl divisions so that if an entire chamber is not counted, an exact volume can still be
determined. Typical volumes counted are one row of the chamber (50 1-µl cells or 1/20 of 1
ml). The volume of sample examined is dependent on number of cells encountered and how long
it takes to reach cut-offs of 75 entities (unicellular forms, colonies, or chains) of each of the top 3
taxa, and 400 entities total. Calculation of abundance also accounts for the concentration factor
used in the settling process. Normally, the volume processed is 800 ml of whole-water sample,
settled to 50 ml of concentrate, for a 16:1 ratio.
The following equation results in the abundance estimate for cells counted:
C * [VS / VC] [ 1000 / VTOT] = cells/ L.
64
where C = cells counted
VS = Volume of concentrated sample
VC = Volume of sample examined
VTOT = Original volume
Final abundance estimates will be reported in units of 106 cells per liter.
References
Borkman, D. 1994. Phytoplankton and Nutrients in Buzzards Bay, Massachusetts 1987-1988.
M.S. Thesis. University of Massachusetts Dartmouth, Dartmouth, MA. 203 pp.
Borkman D, RW Pierce, JT Turner. 1993. Dinoflagellate blooms in Buzzards Bay,
Massachusetts. Pp. 211-216 in Smayda, T.J., and Y. Shimizu (Eds.), Proceedings of the Fifth
International Conference on Toxic Marine Phytoplankton, Elsevier.
Guillard RRL. 1973. Division rates. Pages 289-311 In: J.R. Stein, (Ed.) Phycological Methods.
Cambridge Univ. Press.
Libby PS, Gagnon C, Albro C, Mickelson M, Keller AA, Borkman D, Turner JT, Oviatt CA.
2002. Combined work/quality assurance plan for baseline water quality monitoring: 2002-2005.
Boston: Massachusetts Water Resources Authority. Report ENQUAD ms-074. 79 p.
Libby PS, Gagnon C, Albro C, Mickelson M, Keller A, Borkman D, Turner J, Oviatt CA. 2005.
Combined work/quality assurance plan for baseline water quality monitoring: 2004-2005.
Boston: Massachusetts Water Resources Authority. Report ENQUAD ms-074 Version 1. 76 pp
+ apps.
Libby PS, Mansfield A, Buhl R, Lescarbeau G, Leo W, Keller AA, Borkman DG, Turner JT and
Oviatt CA. 2006. Combined work/quality assurance project plan (QAPP) for water column
monitoring 2006 - 2007, tasks 4, 5, 6, 7, 8, 11. Boston: Massachusetts Water Resources
Authority. Report 2006-03. 119 p.
Libby S, Fitzpatrick M, Buhl, R, Lescarbeau G, Leo W, Borkman D, Turner J, Oviatt CA. 2009b.
Quality assurance project plan (QAPP) for water quality monitoring: 2008-2009, Revision 1.
Boston: Massachusetts Water Resources Authority. Report 2008-02. 92 p.
Libby PS, Fitzpatrick MR, Buhl RL, Lescarbeau GR, Leo WS, Borkman DG, Turner JT, Oviatt
CA. 2010. Quality Assurance Project Plan (QAPP) for water column monitoring 2010: Tasks 4-9
and 13. Boston: Massachusetts Water Resources Authority. Report 2010-2. 105 p.
Turner JT, DG Borkman, RW Pierce. 1995. Should Red Tide Dinoflagellates be Sampled Using
Techniques for Microzooplankton Rather than Phytoplankton? Pp. 737-742 in P. Lassus et al.
(Eds.), Harmful Marine Algal Blooms, Lavoisier, Paris, France.
65
Standard Operating Procedure 009
Zooplankton Collection and Analysis
Collection
At each station, zooplankton is collected from the upper 19 meters of the water column. Samples
are collected using standard 333-micrometer (µm) mesh conical nets fitted with General
Oceanics helical flow meters. The flow meters are calibrated at the beginning of each year to
attain the most accurate correction constant possible. The 333 µm mesh has been experimentally
determined to represent the filtering ability of right whale baleen; however, any mesh size can be
used to sample with these protocols.
Water column collections are initiated by vertically dropping a 60 cm-diameter net on-station.
When the net has dropped the full 19 meters, the net is pulled obliquely through the water
column by the boat until a mark on the rope reaches the surface, indicating that the net is now
horizontal at the surface of the water column. At this point, the net is retrieved.
Once on board, the samples are washed from the nets carefully with a sea-water hose. The
sample is concentrated into the bottom of the net to the collection bucket. From there it is
concentrated further into a 333 µm mesh fluorette (PVC piping with 333 micron mesh at one
end, 11.5 cm inside diameter, 9.5 cm length). This is rinsed into a sample jar and preserved with
10% buffered formalin. Samples are then placed into a cooler for the remainder of the cruise.
Subsampling the zooplankton net samples
If the sample is sparse enough, the entire sample is enumerated. Usually, the sample will have to
be subsampled. To enumerate and identify the zooplankton in a net sample that is to be
subsampled, all zooplankton are poured into a small fluorette (PVC piping with 333 micron
mesh at one end, 5 cm inside diameter, 12cm length ) and rinsed with fresh water to remove
excess formalin. Once well rinsed, the entire sample is rinsed with fresh water in a squirt bottle
into a glass beaker. The goal is to subsample 200 organisms in 5 ml of sample, so an
appropriately sized beaker is approximated depending on the density and size of the organisms in
the sample. Water is added to increase the volume of the entire sample to a known volume, read
off the beaker. Again, this volume is approximated to get at least 200 organisms in 5 ml of
sample. The lowest total volume used is 125 ml, to prevent poor subsampling. A HensenStempel® Pipette is used to first stir the contents of the beaker, suspending and equally
distributing the sample throughout the beaker, and then to subsample 5 ml of the sample from the
middle of the beaker. The 5 ml is transferred directly into a glass watch-glass and the pipette’s
sample chamber is rinsed with fresh water from a squirt bottle, into the watch-glass, to ensure
that no organisms from the subsample are lost. The sample is now ready to be enumerated and
identified.
66
Sample Splitting
If the zooplankton net sample is too large to be sub-sampled in such a way that there will be
approximately 200 organisms per 5 ml of sample, the sample must be split; for this a Folsom
plankton splitter is used. First, the splitter is leveled by adjusting its legs. The sample is poured
from a glass beaker (see steps above for transferring the sample into the glass beaker) into the
opening in the main part of the splitter, oriented so that when it is rotated downward, the sample
is split and exits the holes in the splitter, into the holding chambers. As the splitter is rotated, a
squirt bottle is used to be sure that all organisms are included. Half of the split sample can now
be put back into a beaker. If the sample is still too dense, the process is repeated.
Sample Enumeration and Identification
Once the desired subsample is in the watch-glass, it is placed under a Leica L2® dissecting
microscope. Light source and zoom adjustments are dependent on the personal preferences of
the individual counting and identifying the organisms. An Interface Systems® 20 channel digital
counter is used to enumerate the organisms as they are identified. A number of keys and guides
to Gulf of Maine zooplankton are used (see “zooplankton identification references”). 35 types of
organisms are identified to a particular taxonomic level and some are staged (see zooplankton lab
datasheet for details). As the individual identifying and enumerating counts an organism, he or
she moves it aside so that no organism is counted more than once. If organisms are floating, they
can be removed and then counted after the rest of the sample has been finished, for ease of
accurate enumeration and identification. Once all organisms in the subsample have been
identified, enumerated, and recorded with the digital counter (and paper should there be more
than 20 taxa identified), the data on the digital counter are recorded onto the zooplankton lab
datasheet. If less than 200 organisms have been found to make up the subsample, another 5 ml
subsample is taken, processed, and added to the previous subsample counts; this may be done as
many times as necessary, unless the volume of the sample being subsampled is no longer enough
in which to completely submerge the pipette chamber. If this occurs, the individual enumerating
must begin again. When taking additional subsamples, no water or organisms can be added to
the total sample being subsampled. Upon counting 200 organisms or more, the recorded
subsample volume counted is adjusted to reflect the number of ml counted. After the count has
been completed and recorded, the entire sample is transferred back into a fluorette and then back
into the sample jar. Formalin is added in a volume dependent on the sample far size (Table 1),
and water is added to fill the sample jar to the top. The point of contact between the sample jar
and sample lid is wrapped in Parafilm® and the sample is archived.
Zooplankton Density Calculations
The following formula is used for calculating the org/m3 from the oblique net tow count data:
Org/m3 = O x (VT/VC) x [1/(mE - mS) x CN]
67
Where O is the counted organisms, VT is the total sample volume, VC is the counted sample
volume, mE is the flow meter end reading, mS is the flow meter start reading, and CN is the net
constant.
Table 1. Sample jar volume and volume of formalin added for sample archiving
Jar size (ml) Formalin added (ml)
40
3
75-80
6
175
12
310-325
22
Zooplankton Identification References
Gerber RP. An Identification Manual to the Coastal and Estuarine Zooplankton of the Gulf of
Maine Region. Freeport: Freeport Village Press, 2000.
Johnson WS and Allen DM. Zooplankton of the Atlantic and Gulf Coasts. Baltimore: Johns
Hopkins University Press, 2005.
Smith DL and Johnson KB. A Guide to Marine Coastal Plankton and Marine Invertebrate
Larvae. Dubuque: Kendall/Hunt Publishing Company, 1996.
Todd CD, Laverack MS and Boxshall GA. Coastal Marine Zooplankton. Cambridge: Cambridge
University Press, 1996.
68
Standard Operating Procedure 010
Silicate Analysis
Date: Sep 2002
Primary Method: EPA 366
A. Scope and Application
This method is used for the determination of silicate in seawater and is applicable to many
ranges.
B. Summary of Method
Silicomolybdic acid is formed by the reaction of silicate with molybdic acid. The silicomolybdic
acid is reduced by stannous chloride to form molybdenum blue with an absorbance maximum at
820 nm.(1-4)
C. Interferences
Interference from orthophosphate and tannin is eliminated by the use of tartaric acid. Filter turbid
samples before determination. Color absorbing at the analytical wavelength will interfere.
D. Sample Handling and Preservation
Collect samples in plastic containers. Analyze samples as soon as possible. Refrigerate samples
at 2-8°C if immediate analysis is not possible.
E. Raw Materials Required
Ammonium Molybdate (NH4)6Mo7O24•4H2O (FW 1235.95)
Chloroform CHCl3 (FW 119.38) Deionized Water (ASTM Type I or II) Hydrochloric Acid, concentrated, HCl, (FW 36.46)
Low Nutrient Seawater (LNSW)* Magnesium Sulfate MgSO4•7H2O (FW 246.48)* Sodium Bicarbonate NaHCO3 (FW 84.01)* Sodium Chloride NaCI (FW 58.44)* Sodium Hexafluorosilicate Na2SiF6 (FW 188.06) Sodium Lauryl Sulfate (SLS) CH3(CH2)10CH2OSO3Na (FW 288.38)
Stannous Chloride SnCl2•2H2O (FW 225.65) Sulfuric Acid, concentrated H2SO4 (FW 98.08) Tartaric Acid H2C4H4O6 (FW 150.09)
*See Operating Notes for information on matrix choices.
NOTE: Chemicals should be of ACS grade or equivalent.
69
F. Reagent Preparation
1. Sodium
(SLS)
15%Lw/w
auryl Sulfate
Sodium Lauryl Sulfate .......................................................................... 15 g
CH3(CH2)10CH2OSO3Na (FW 288.38)
Deionized Water ................................................................................. 85 ml
Dissolve 15 g of sodium lauryl sulfate in 85 ml of deionized water contained in a 250 ml
Erlenmeyer flask. It may be necessary to warm the mixture in a water bath to dissolve.
2. Stock Molybdic Acid (1 L)
Ammonium Molybdate....................................................................... 10.8 g
(NH4)6Mo7O24•4H2O (FW 1235.95)
Sulfuric Acid....................................................................................... 2.8 ml
H2SO4, concentrated (FW 98.08)
Deionized Water
While stirring, cautiously add 2.8 ml of sulfuric acid to approximately 900 ml of deionized water
contained in a 1 L volumetric flask. Dissolve 10.8 g of ammonium molybdate in the acidic
solution. Dilute the solution to the mark with deionized water and mix it well. Filter to 0.45 μm.
Store the reagent in a plastic container. Do not refrigerate this reagent. Discard the solution if it
becomes blue.
3. Working Molybdic Acid Reagent (100 ml)
Stock Molybdic Acid ......................................................................... 100 ml
SLS, 15 % w/w ................................................................................ 4 drops
Mix together 100 ml of ammonium molybdate and 4 drops of SLS. Prepare daily the quantity
sufficient for the day’s run.
4. Tartaric Acid, 20% w/v (1 L)
Tartaric Acid ....................................................................................... 200 g
H2C4H4O6 (FW 150.09)
Deionized Water
Chloroform....................................................................................... 2 drops
CHCl3
Dissolve 200 g of tartaric acid in approximately 700 ml of deionized water contained in a 1 L
volumetric flask. Dilute the solution to the mark with deionized water and mix it well. Filter to
0.45 μm. Add 2 drops of chloroform. Store the reagent in a plastic container and refrigerate it at
2-8°C. Filter every 10 days.
70
5. Stock Stannous Chloride (100 ml)
Stannous Chloride ............................................................................. 50.0 g
SnCl2•2H2O (FW 225.65)
Hydrochloric Acid................................................................................ 50 ml
HCl, concentrated, (FW 36.46)
Deionized Water
While stirring, cautiously add 50 ml of hydrochloric acid to 30 ml of deionized water contained
in a plastic volumetric flask. Dissolve 50 g of stannous chloride in the acidic solution. Heating
may be necessary to obtain complete dissolution. Dilute to 100 ml with deionized water and mix
well. Store the stock solution in a tightly closed plastic container and freeze it at less than -10°C.
6. Hydrochloric Acid 1.2 N (1 L)
Hydrochloric Acid.............................................................................. 100 ml
HCl, concentrated, (FW 36.46)
Deionized Water
Cautiously, while stirring, add 100 ml of hydrochloric acid to approximately 800 ml of deionized
water contained in a 1 L volumetric flask. Dilute the solution to the mark with deionized water
and mix it well. Filter to 0.45 μm. Store the solution in a plastic container.
7. Working Stannous Chloride Reagent
Stock Stannous Chloride ................................................................... 2.0 ml
Hydrochloric Acid, 1.2 N ................................................................... 100 ml
Mix together 2.0 ml of stock stannous chloride and 100 ml of 1.2 N hydrochloric acid in a plastic
container. Prepare the reagent fresh daily.
8. Startup/Shutdown Solution
Add 2 ml of 15% SLS per 100 ml of deionized water.
9. Artificial Seawater (ASW) (4 L)
Sodium Chloride...............................................................................128.5 g NaCI (FW 58.44)
Magnesium Sulfate............................................................................ 28.5 g MgSO4•7H2O (FW
246.48)
Sodium Bicarbonate ........................................................................ 0.672 g NaHCO3 (FW 84.01)
71
Deionized Water
Dissolve 128.5 g of sodium chloride, 28.5 g of magnesium sulfate and 0.672 g of sodium
bicarbonate in about 3 liters of deionized water. Dilute to 4 liters with deionized water. These
reagents must be high quality, reagent grade to avoid excessive nutrient or trace metal
contamination.
10. Sampler Wash
See Operating Notes.
G. Calibrants
Specific Stock and Working Calibrant preparation instructions can be found on the back of the
flow diagram. Be sure to use the flow diagram which covers the concentration range you wish to
analyze.
Working calibrants may be prepared to cover alternate ranges by adding the appropriate volumes
of stock or intermediate calibrant to 100 ml volumetric flasks that contain approximately 80 ml
of sampler wash solution. Dilute the solution to
100 ml with sampl
well.
The following formula can be used to calculate the amount of stock (or intermediate) calibrant to
be used.
C1V1 = C2V2
Where:
C1 =desired
ntration
(in mg/L)
conceof working calibrant to be prepared
V 1 =final
volume (in ml) of working calibrant to be prepared (generally 100 ml) C2 = concentration (in
mg/L) of stock (or intermediate) calibrant
V 2 =volume (in ml)
calibrant to be used
Rearranging the equation to solve for V2 yields:
V2 = C1V1 C2
For example, to prepare a 1.0 mg/L working calibrant from a 1000 mg/L stock calibrant, use 0.1
ml (100 μl) of the stock calibrant in 100 ml final volume.
V2 = (1.0 mg/L) (100 ml) 1000 mg/L
V2 = 0.1 ml
Add this amount of stock calibrant to the volumetric flask and then dilute to volume with the
sampler wash solution.
H. Operation Procedure
Set up the cartridge as shown in the flow diagram. Check all tubing and connections. Replace if
72
necessary.
Place reagent lines in startup solution. Turn on power to all units and latch pump platens to begin
liquid flow.
Verify that the bubble size and spacing is consistent throughout the cartridge. If bubbles are
splitting up as they enter or exit a coil, check and replace fittings if necessary. The bubbles
should flow smoothly without dragging. If dragging occurs, add more SLS to the startup
solution.
Check all reagent containers on the instrument for particulate matter. Reagents should be filtered.
Be sure all containers are properly labeled and filled before pumping reagents.
After a stable baseline has been verified on the startup solution, place reagent lines in reagent
bottles. Due to the lower amount of SLS in the reagents, the flow and bubble pattern may drag
slightly.
NOTE: Leave the stannous chloride reagent line in the startup solution for 5 minutes after
adding the other reagents. This will allow the tartaric acid to reach the cartridge first.
If using data collection software, set up the appropriate sample table. Allow reagents to run for 5
to 10 minutes and verify a stable baseline.
Load the sampler tray with calibrants, blanks, samples, and QC or monitor samples.
15.
Select the appropriate parameters for the detector and sampler. (See Flow Diagram.)
16.
Begin analysis.
17.
At the end of analysis place all reagent lines in startup solution. Pump startup solution for
10 to 15 minutes to flush all of the reagents out of the cartridge.
18.
Turn off the power to all units and release pump platens.
I. Operating Notes
1. The use of glass containers should be avoided. Prepare all reagents and calibrants in plastic
containers, or transfer all reagents and calibrants to plastic containers immediately
following preparation.
2. Thepowderedstannouschloride,SnCl2•2H2O,shouldbestoredfrozenatlessthan- 10°C. Allow to
come to room temperature before opening.
3. Preparefreshworkingstannouschlorideifunstablebaselines,poorpeakshapesor reduced sensitivity
are experienced.
4. PolyFlow tubing(APIp/n303-2674-01) isused for transmission tubing on this cartridge to help
73
achieve smooth flow and reduce carryover.
5. High quality SLS is important. Fisher catalog numbers 02674-25, BP166-100 or BP166-500
are acceptable.
6. There are special considerations when running seawater samples on anyflow system.
A .
Standards
Primary standards sh
available. Certificates of Analysis are available from the chemical manufacturer. These
should be consulted to identify impurities.
n
Standard
dried formaterial should be
two hours at 110°C before weighing.
I t is advisable to periodica
concentrations of the working standards. This can be done by running standards against
standards from an outside source.
The matrix
istent
with of the standard
that of the samples. If deionized water standards are used it becomes important to
determine the salt effects of each individual test. (See number 2 under Operating Notes.)
NOTE: First move the stannous chloride reagent line to startup solution for 5 minutes
before moving the other reagent lines. This will allow the stannous chloride line to rinse out
before the tartaric acid is removed.
J. References
11.
Truesdale,V.W.,C.J.Smith,“TheFormationofMolybdosilicicAcidsfromMixed Solutions of
Molybdosilicic Acids from Mixed Solutions of Molybdate and Silicate”, Analyst, March
1975, vol. 100, Pg. 203-212.
12.
Armstrong, F.A.J., C.R. Stearns, and J.D. Stickland. 1967. The measurement of
upwelling and subsequent biological processes by means of the Technicon 
AutoAnalyzer
 and
-Sea
Res.
associated
14(3): 381-389.
equipment. Deep
13.
Atlas, E. L.W. Hager, L.I. Gordon and P.K. Park. 1971. A practical manual for use of the
Technicon
 AutoAn
alyzer
 in seawater nutr
215. Oregon State University, Dept of Oceanography, Ref. No. 71-22. 48 pp.
14.
Gordon, L.I., J.M. Krest and A.A. Ross. in preparation. Continuous Flow Analysis of
silicic acid in seawater: Reducing sensitivity to laboratory temperature fluctuations.
15.
AutomatedNutrientAnalysisinSeawater,TechnicalReport,BrookhavenNational
Laboratory, Whitledge, Veidt, et. al., May 1986.
74
APPENDIX B: Data Forms
Field Data Sheet: Survey Data
Field Data Sheet: Station Data
Lab Data Sheet: Chlorophyll
Lab Data Sheet: Phytoplankton
Lab Data Sheet: Zooplankton
75
Survey Data
VESSEL
Alert
Seaway
Shearwater
Date
High Tide
Low Tide
Weather
Sea State
Wind
Time
Begin
End
Comments
76
Station Data
Trip
Station
Time
Depth
CTD
Secchi
Depth
Turb
Sample
Nutrients
Surface
Depth
Chl a
77
Lab Data Sheet: Chlorophyll
Lab Name__________________________________
Date Extracted_____________
Time Extracted_____________
Lab Analyst________________________________
Secondary Standard___________/______________
Acetone Blank_______________/______________
Calibration Used _____________
Sample
Location
Sample
Date
Volume
Filtered
Date Measured_____________
Time Measured_____________
Volume
Solvent
Chl a
Pheo
Notes
78
Lab Data Sheet: Phytoplankton
C
D
c
D
c
Bacteriastrum spp.
Centric diatom sp. group 1 diam <10
microns
D
c
Cerataulina pelagica
D
c
Chaetoceros atlanticus
D
c
Chaetoceros borealis
D
c
Chaetoceros compressus
D
c
Chaetoceros debilis
D
c
Chaetoceros decipiens
D
c
Chaetoceros didymus
D
c
Chaetoceros laciniosus
D
c
Chaetoceros laciniosus
D
c
Chaetoceros lauderi
D
c
Chaetoceros lorenzianus
D
c
D
c
D
c
Chaetoceros socialis
Chaetoceros sp. group 1 diam <10
microns
Chaetoceros sp. group 2 diam 10-30
microns
D
c
Chaetoceros subtilis
D
c
Corethron criophilum
D
c
Coscinodiscus oculus-iridis
D
c
D
c
D
c
Coscinodiscus radiatus
Coscinodiscus sp. group 2 diam 40-100
microns
Coscinodiscus sp. group 3 diam >100
microns
D
c
Dactyliosolen blavyanus
D
c
Dactyliosolen fragilissimus
D
c
Detonula confervacea
D
c
Ditylum brightwellii
D
c
Eucampia cornuta
D
c
Guinardia delicatula
D
c
Guinardia flaccida
D
c
Guinardia striata
Cf
79
D
c
Lauderia annulata
D
c
Leptocylindrus danicus
D
c
Leptocylindrus minimus
D
c
Lithodesmium undulatum
D
c
Melosira nummuloides
D
c
Paralia sulcata
D
c
Porosira glacialis
D
c
Pseudosolenia calcar-avis
D
c
Proboscia alata
D
c
Rhizosolenia hebetata
D
c
Rhizosolenia setigera
D
c
Skeletonema costatum
D
c
Stephanodiscus spp.
D
c
Stephanopyxis turris
D
c
Thalassiosira anguste-lineata
D
c
Thalassiosira nordenskioeldii
D
c
D
c
D
c
Thalassiosira rotula
Thalassiosira sp. group 1 diam <20
microns
Thalassiosira sp. group 3 10-20 microns
length
D
p
Amphora spp.
D
p
Asterionella formosa
D
p
Asterionellopsis glacialis
D
p
Bellerochea malleus
D
p
Cocconeis scutellum
D
p
Cocconeis spp.
D
p
Cylindrotheca closterium
D
p
Grammatophora marina
D
p
Gyrosigma spp.
D
p
Isthmia nervosa
D
p
Licmophora spp.
D
p
Odontella aurita
D
p
Odontella sinensis
D
p
Odontella spp.
D
p
Pennate diatom sp. group 1 <10 microns
80
length
D
p
D
p
D
p
D
p
Pennate diatom sp. group 2 10-30
microns length
Pennate diatom sp. group 3 31-60
microns length
Pennate diatom sp. group 4 61-100
microns length
Pennate diatom sp. group 5 >100 microns
length
D
p
Pleurosigma spp.
D
p
Pseudonitzschia delicatissma complex
D
p
Pseudonitzschia pungens
D
p
Rhabdonema minutum
D
p
Striatella unipunctata
D
p
Synedra spp.
D
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
p
Thalassionema nitzschioides
Dino
Dinophysis acuminata
Dino
Dinophysis fortii
Dino
Dinophysis norvegica
Dino
Dinophysis tripos
Dino
Phalacroma rotundatum
Gony
Alexandrium fundyense
Gony
Alexandrium spp.
Gony
Amylax triacantha
Gony
Ceratium furca
Gony
Ceratium fusus
Gony
Ceratium lineatum
Gony
Ceratium longipes
Gony
Ceratium macroceros
Gony
Ceratium tripos
81
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Din
o
Gony
Gonyaulax spp.
Gony
Gymn
o
Gymn
o
Gymn
o
Gymn
o
Gymn
o
Gymn
o
Gymn
o
Gymn
o
Scrippsiella trochoidea
Per
Heterocapsa rotundata
Per
Heterocapsa triquetra
Per
Protoperidinium bipes
Per
Protoperidinium claudicans
Per
Protoperidinium depressum
Per
Protoperidinium pallidum
Per
Protoperidinium pentagonum
Per
Protoperidinium quinquecorne
Per
Protoperidinium sp. group 1 10-30
microns width, 10-40 microns length
Per
Protoperidinium sp. group 2 31-75
microns width, 41-80 microns length
Per
Protoperidinium sp. group 3 76-150
microns width, 81-150 microns length
Proro
Prorocentrum micans
Proro
Prorocentrum minimum
Akashiwo sanguinea
Amphidinium crassum
Amphidinium spp.
Gymnodinium sp. group 1 5-20 microns
width, 10-20 microns length
Gymnodinium sp. group 2 21-40 microns
width, 21-50 microns length
Gyrodinium sp. group 1 5-20 microns
width, 10-20 microns length
Gyrodinium sp. group 2 21-40 microns
width, 21-50 microns length
Gyrodinium spirale
82
Din
o
Din
o
Din
o
Proro
Prorocentrum scutellum
Proro
Prorocentrum triestinum
Thecate dinoflagellate
F
Calycomonas wulffii
F
F
Coccolithophorida
Cryptomonas sp. group 1 length <10
microns
Cryptomonas sp. group 2 length >10
microns
F
Dictyocha formosa
F
Dictyocha speculum
F
Dinobryon spp.
F
Ebria tripartita
F
Eutreptia/Eutreptiella spp.
F
Paulinella ovalis
F
F
Phaeocystis pouchetii
Pyramimonas sp. group 1 10-20 microns
length
Unid. micro-phytoflag sp. group 1 length
<10 microns
Unid. micro-phytoflag sp. group 2 length
>10 microns
C
Ciliatea (aloricate)
C
Mesodinium rubrum
C
Tintinnidae (hyaline)
C
Tintinnids (aglomerate)
Chl
Pediastrum spp.
Chl
Scenedesmus spp.
F
F
F
83
Zooplankton Count Sheet
Cruise I.D.
Date of Cruise
Date Counted
Counter Initials
Station
Net (sfc/obl)
Settled Volume
Container
Volume
Counted Volume
Sample Volume
Total Zooplankton
Calanus finmarchicus
C1
C2
C3
C4
C5
C6 F
C6 M
Early (C1 to C4)
Late (C5 & C6)
Centropages spp.
Early (C1 to C4)
C5
C6 F
C6 M
Late (C5 & C6)
Σ Early
typ
ham
typ
ham
typ
ham
typ
ham
typ
ham
typ
ham
84
Σ Late
Pseudocalanus spp.
Para/Clausocalanus
Pseudo Early (C1 to
C4)
Pseudo Late (C5 &
C6)
COPEPODS
Temora longicornis
Tortanus discuadatus
Acartia spp.
Eurytemora spp.
Oithona spp.
Metridia spp.
Paraeuchaeta spp.
harpacticoids
other/unidentified copepods
OTHER ZOOPLANKTON
Cyprids
Nauplii
Chaetognaths
Cladocera
Polychaetes
Larvaceans
Molluscs
Cyphonautes
Gammerid Amphipods
Hyperiid Amphipods
Fish Eggs
Fish Larvae
Euphausiids
Zoea
85
Late larval crustaceans
Mysids
Urchin larvae
Veligers
Medusae
Salps
Ctenophores
Pteropods
Cliones
Ostracods
Other
COMMENTS
86
APPENDIX C: Guidance Protocol on the Interaction with Whales
87
Guidance Protocol on the Interaction with Whales Specifically Northern Right Whales for Vessels
Operated/Contracted by the Commonwealth of Massachusetts
Introduction
The northern right whale is the most endangered large whale in the world. In the western north Atlantic
the population is estimated to be about 300 animals. Massachusetts coastal waters are part of the range of
the northern right whale and Cape Cod Bay has been designated a critical habitat for the whale under the
federal Endangered Species Act because of its high use by the species in the late winter and early spring
for feeding. The Great South Channel, east of Cape Cod, has also been designated critical habitat because
of its importance to the right whale as a feeding area. It has been determined that the most significant
human induced causes of mortality are ship strike and entanglements in fishing gear.
Purpose
The purpose of this protocol is to give guidance to the vessels owned by the Commonwealth and those
operating under contract to the Commonwealth as to proper operational procedures if the vessels should
encounter whales - i.e., sighting and reporting procedures, and entanglement and carcass reporting
protocol.
Applicability
This protocol will apply to all vessels owned by the Commonwealth of Massachusetts and/or contracted
out by the Commonwealth of Massachusetts.
Geographic Scope/Operational Scope
This protocol applies to all applicable vessels operating in or adjacent to Commonwealth waters. When
vessels are operating in the designated critical habitat areas (Cape Cod Bay or the Great South Channel)
heightened operation is applicable, especially during the late winter and spring when the right whales are
expected to be located in these areas.
Sightings of Right Whales
The Executive Office of Environmental Affairs and the National Marine Fisheries Service is interested in
receiving reports from individuals who observe right whales during vessel operations. Reports should be
made to the National Marine Fisheries Service Clearinghouse. Patricia Gerrior, NMFS Right Whale Early
Warning System Coordinator, who manages the Clearinghouse and her numbers are 508-495-2264
(work), 508-495-2393 (fax) and pager 508-585-8473. Please report your name, agency and phone
numbers at which you can be contacted. The vessel's name, the date, time and location of the sighting, the
numbers of whales sighted and any other comments that may be of importance. If a camera or video
camera is available please take some photographs. These photographs should be provided to Pat Gerrior
or Dan McKiernan, Massachusetts Division of Marine Fisheries. They will in turn send copies to the New
England Aquarium for comparison to the Right Whale Photo Identification Catalog. Please remember
that Massachusetts has Right Whale Conservation Regulations (322 CMR 12:00) which establishes
a 500 yard buffer zone surrounding a right whale. Vessels shall depart immediately from any
buffer zone created by the surfacing of a right whale.
Physical Contact with a Whale
If a vessel owned by the Commonwealth of Massachusetts or under contract with the Commonwealth of
88
Massachusetts comes into physical contact with any whale it should be noted in the vessel's logbook. The
vessel's logbook should include the time and location of the incident; weather and sea conditions; vessel
speed; the species of whale struck if known; the nature of any injures to crew, and/or the whale, and/or
damage to the vessel. Also record the name of any other vessels in the area that may have witnessed the
incident or can provide information about circumstances. A copy of the vessel's log for the entire trip
should be submitted to the Director of the Division of Marine Fisheries, the Director of the Division of
Law Enforcement, the Secretary of Environmental Affairs and the National Marine Fisheries Service,
Northeast Region in Gloucester.
If after hitting the whale, the animal is incapacitated or appears to have life threatening injuries and the
vessel is safe and secure, immediately call the Center for Coastal Studies, entanglement hotline at 800900-3622 or via their pager at 508-803-0204 and the Massachusetts Environmental Police
Communications Center at 800-632-8075 or 617-727-6398. Stay with the whale until the Coast Guard or
Center for Coastal Studies arrives on scene.
Entanglements
If the vessel come upon or entangles a right whale immediately notify the Center for Coastal Studies'
entanglement hotline at 800-900-3622 or via their pager at 508-803-0204 and the Massachusetts
Environmental Police Communications Center at 800-632-8075 or 617-727-6398. Do not attempt to
remove any debris from the whale, stay on station with the whale or follow at a safe distance. As
relocating an entangled whale can be extremely difficult, staying on station or following the animal is
very important. However, if following the whale is not possible contact, the Coast Guard and/or the
Center for Coastal Studies and note the last direction the animal was heading and any other pertinent
information that would assist in relocating the whale.
Stranded Whales
For a stranded right whale please notify the Stranding Network immediately call Connie Merigo or
Howard Krum, New England Aquarium, Central Wharf, Boston, MA 02110. The standing Network's
hotline is 617-973-5247 (pager) or as a second resort call 617-973-5246/6551.
QUICK REFERENCE
Sightings & Photographs
Patricia Gerrior, NMFS Right Whale Early Warning System Coordinator, manages the Clearinghouse and
her numbers are 508-495-2264 (work), 508-495-2393 (fax) and pager 508-585-8473
Photographs
Dan McKiernan, Massachusetts Division of Marine Fisheries, 19th Floor, 100 Cambridge Street, Boston,
MA 02202. 617-727-3193 ext. 369.
Entanglements or Injured whales
Center for Coastal Studies, entanglement hotline at 800-900-3622 or pager at 508-803-0204
Massachusetts Environmental Police Communications Center at 800-632-8075 or 617-727-6398.
Stranded Animals
The standing Network's hotline is 617-973-5247 (pager) or as a second resort call 617-973-5246/6551.
Massachusetts Water Resources Authority
Charlestown Navy Yard
100 First Avenue
Boston, MA 02129
(617) 242-6000
www.mwra.com