National Coastal Condition Assessment Laboratory Methods Manual


National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
      Page 1
               United States Environmental Protection Agency
               Office of Water
               Office of Environmental Information
               Washington, DC
               EPA No. 841-R-09-002
   National Coastal Condition Assessment
       Laboratory Methods
                 Manual
    November 2010

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 2
NOTICE
The goal of the National Coastal Condition Assessment (NCCA) is to provide a comprehensive
assessment of the Nation's freshwater, marine shoreline and estuarine waters. The complete
documentation of overall project management, design, methods, and standards is contained in
four companion documents, including:

National Coastal Condition Assessment: Quality Assurance Project Plan (EPA 841-R-09-004)
National Coastal Condition Assessment: Field Operations Manual (EPA 841-R-09-003)
National Coastal Condition Assessment: Laboratory Methods Manual (EPA 841-R-09-002)
National Coastal Condition Assessment: Site Evaluation Guidelines (EPA-841-R-09-OOX)

The suggested citation for this document is:

USEPA. 2009. National Coastal Condition Assessment: Laboratory Methods Manual.
EPA 841-R-09-002. U.S. Environmental Protection Agency, Office of Water and
Office of Research and Development, Washington, DC.

This document (Laboratory Methods Manual) contains information on the methods for analyses
of the samples to be collected during the survey, quality assurance objectives, sample handling,
and data reporting. These methods are based on established methods and/or guidelines
developed and followed in the Agency's Environmental Monitoring and Assessment program.
Methods described in this document are to be used specifically in work relating to the NCCA.
The method outlined in Section 3.0 of this Manual entitled, Enterococci in Water by TaqMan®
Quantitative Polymerase Chain Reaction (qPCR) /Assay, is unpublished and provided as
DRAFT. Copies of this draft method are available upon request. All published references are
available from the National Technical Information Service, 5285 Port Royal Road, Springfield,
VA 22161. Mention of trade names or commercial products in this document does not constitute
endorsement or recommendation for use by EPA. Details on specific methods for sampling and
sample processing and handling prior to sending to the laboratory can be found in the
companion document Field Operations Manual listed above.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                         Date: November 2010
	Page 3

                              TABLE OF CONTENTS

 LIST OF FIGURES	8
 1.0 INTRODUCTION AND GENERAL INSTRUCTIONS	9
   1.1     INTRODUCTION	9
   1.2     LIST OF INDICATORS	9
   1.3     TRAINING AND QUALIFICATIONS	11
   1.4     SAFETY	11
   1.5     PROTOCOLS	11
   1.6     QUALITY CONTROL AND LABORATORY AUDITS	12
 2.0 WATER QUALITY	13
   2.1     PERFORMANCE-BASED METHODOLOGIES	13
   2.2     DISSOLVED INORGANIC NITROGEN-AMMONIA	14
    2.2.1   Saltwater	14
    2.2.2   FRESHWATER	22
   2.3     DISSOLVED INORGANIC NITROGEN NITRATE-NITRITE	28
    2.3.1   Saltwater	28
    2.3.2   FRESHWATER	39
   2.4     TOTAL NITROGEN AND PHOSPHORUS	45
   2.5     TOTAL PHOSPHORUS AND FRESHWATER ORTHOPHOSPHATE	63
    2.5.1   Scope and Application	63
    2.5.2   Summary of Method	63
    2.5.3   Interferences	63
    2.5.4   Safety	63
    2.5.5   Equipment and Supplies	63
    2.5.6   Reagents and Standards	64
    2.5.7   Sample Collection, Preservation and Storage	65
    2.5.8   Quality Control	65
    2.5.9   Calibration and Standardization	67
    2.5.10 Procedure	68
    2.5.11  Data Analysis and Calculations	69
   2.6     ORTHOPHOSPHATE (Saltwater Only)	70
    2.6.1   Scope and Application	70
    2.6.2   Method Summary	70
    2.6.3   Interferences	70
    2.6.4   Equipment and Supplies	70
    2.6.5   Reagent and Standards	71
    2.6.6   Sample Storage	72
    2.6.7   Quality Control	72
    2.6.8   Procedure	74
    2.6.9   Data Analysis and Calculations	75
   2.7     CHLOROPHYLL a	76
    2.7.1   Scope and Application	76
    2.7.2   Method Summary	76
    2.7.3   Interferences	76
    2.7.4   Safety	76
    2.7.5   Equipment and Supplies	76
    2.7.6   Reagents and Standards	77
    2.7.7   Sample Storage	77
    2.7.8   Quality Control	77

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                        Date: November 2010
	Page 4

    2.7.9   Calibration and Standardization	78
    2.7.10  Procedure	79
    2.7.11  Data Anlaysis and Calculations	79
    2.7.12  References	80
 3.0 FECAL INDICATOR	81
   3.1     SCOPE AND APPLICATION	81
   3.2     SUMMARY OF METHOD	81
   3.3     DEFINITIONS OF METHOD	81
   3.4     INTERFERENCES	82
   3.5     HEALTH AND SAFETY WARN INGS	83
   3.6     PERSONNEL QUALIFICATIONS	83
   3.7     EQUIPMENT AND SUPPLIES	83
   3.8     REAGENTS AND STANDARDS	83
   3.9     PREPARATIONS PRIOR TO DNA EXTRACTION AND ANALYSIS	84
   3.10   PROCEDURES FOR PROCESSING AND QPCR ANALYSIS OF SAMPLE
         CONCENTRATES	85
    3.10.1  Sample Processing (DNA Extraction)	85
    3.10.2  Sample Analysis by Enterococcus qPCR	86
   3.11   STORAGE AND TIMING OF PROCESSING / ANALYSIS OF FILTER
         CONCENTRATES	89
   3.12   CHAIN OF CUSTODY	89
   3.13   QUALITY CONTROL/QUALITY ASSURANCE	89
   3.14   METHOD PERFORMANCE	90
   3.15   RECORD KEEPING AND DATA MANAGEMENT	90
   3.16   WASTE MANAGEMENT AND POLLUTION PREVENTION	90
   3.17   REFERENCES	91
   3.18   TABLES, DIAGRAMS, FLOWCHARTS, CHECKLISTS, AND VALIDATION DATA.91
    3.18.1  SOP for "Modified" MagNA Pure LC  DNA Purification Kit III Protocol	96
 4.0 CONTAMINANTS	98
   4.1     SAMPLE PREPARATION FOR METALS ANALYSIS	100
    4.1.1    Microwave Assisted Acid Digestion	100
    4.1.2   Summary of Method	100
    4.1.4   Apparatus and Supplies	101
    4.1.5   Reagents	103
    4.1.6   Procedure	103
    4.1.7   Calculations	107
    4.1.8   Calibration of Microwave Equipment	107
    4.1.9   Quality Control	109
   4.2     METALS IN FISH TISSUE AND SEDIMENT	109
    4.2.1   Inductively Coupled Plasma-Mass  Spectrometry	109
    4.2.2   Inductively Coupled Plasma-Atomic Emission Spectrometry	124
   4.3     MERCURY IN FISH  TISSUE AND SEDIMENTS	142
    4.3.1   Scope of Application	142
    4.3.2   Summary of Method	142
    4.3.3   Sample Handling and Preservation	142
    4.3.4   Interferences	142
    4.3.5   Apparatus	142
    4.3.6   Reagents	143
    4.3.7   Calibration	143
    4.3.8   Procedure	144

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                           Date: November 2010
	Page 5

     4.3.9   Calculation	144
   4.4     SAMPLE PREPARATION FOR ORGANIC COMPOUNDS IN FISH TISSUE AND145
   SEDIMENTS	145
     4.4.1   Ultrasonic Extraction	145
     4.4.2   Apparatus and Materials	145
     4.4.3   Reagents	146
     4.4.4   Procedure	146
     4.4.5   Extract Cleanup	149
     4.4.6   Sample Handling	149
   4.5     ORGANOCHLORINE PESTICIDES IN FISH TISSUE AND SEDIMENTS	150
     4.5.1   Scope and Application	150
     4.5.2   Summary of Method	150
     4.5.3   Interferences	151
     4.5.4   Equipment and Supplies	152
     4.5.5   Reagents and Standards	153
     4.5.6   Gas Chromotography Specifications	155
     4.5.7   Quality Control and Assurance	156
     4.5.8   Calibration and Standardization	158
     4.5.9   Analytical Procedure and Analysis	160
     4.5.10  Quantitation of Multi-Component Analytes	163
     4.5.11   GC/MS Confirmation	165
   4.6     POLYCHLORINATED BIPHENOLS (PCBs) IN FISH TISSUE AND SEDIMENTS 166
     4.6.1   Scope and Application	166
     4.6.2   Summary of Method	166
     4.6.3   Interferences	167
     4.6.4   Equipment and Supplies	168
     4.6.5   Reagents and Standards	169
     4.6.6   GC Specifications	170
     4.6.7   Quality Control and Assurance	171
     4.6.8   Calibration and Standardization	173
     4.6.9   Gas Chromatography Analasis of Sample Extracts	174
     4.6.10  Qualitative Identification	176
     4.6.11   Quantitative Identification	177
     4.6.12  Confirmation	177
   4.7     POLYNUCLEAR AROMATIC HYDROCARBONS (PAHs) IN SEDIMENTS ONLY178
     4.7.1   Scope and Application	178
     4.7.2   Summary of Method	179
     4.7.3   Interferences	179
     4.7.4   Equipment and Supplies	179
     4.7.5   Reagents and Standards	180
     4.7.6   Quality Control	182
     4.7.7   Calibration and Standardization	184
     4.7.8   Procedures	189
     4.7.9   Quantitation	191
 5.0 SEDIMENTS	192
   5.1     SEDIMENT GRAIN SIZE AND CHARACTERIZATION	192
     5.1.1   Scope of Application	192
     5.1.2   Sample Storage and Equipment	192
     5.1.3   Procedures for Silt-Clay Content Determination	192
     5.1.4   Procedures for Percent Water Content	194

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                         Date: November 2010
	Page 6

     5.1.5   Procedures for Sediment Grain Size Distribution	195
     5.1.6   Calculations for Sediment Grain Size Distributions	198
     5.1.7   Determination of Statistical Parameters Of Grain Size	198
   5.2     ASSESSING SEDIMENT TOXICITY USING ESTUARINE AND MARINE
          AMPHIPODS	199
     5.2.1   Scope of Application	199
     5.2.2   Summary of Method	199
     5.2.3   Interferences	199
     5.2.4   Equipment and Supplies	200
     5.2.5   Reagents and Water	201
     5.2.6   Sample Manipulation	202
     5.2.7   Quality Control	202
     5.2.8   Culturing and Maintaining Test Organisms	203
     5.2.9   Procedure	204
   5.3     SEDIMENT TOXICITY USING FRESHWATER AMPHIPODS	208
     5.3.1   Scope of Application	208
     5.3.2   Summary of Method	208
     5.3.3   Interferences	208
     5.3.4   Equipment and Supplies	209
     5.3.5   Reagents and Water	210
     5.3.6   Sample Manipulation	211
     5.3.8   Culturing and Maintaining Test Organisms	212
     5.3.9   Procedure	214
 6.0 INFAUNAL BENTHIC MACROINVERTEBRATE COMMUNITIES	217
   6.1     SCOPE AND APPLICATION	217
   6.2     SUMMARY OF METHOD	217
   6.3     SAMPLE STORAGE AND TREAMENT	217
   6.4     SORTING	217
   6.5     PROCEDURE	217
     6.5.1   Identification and Enumeration - General	217
     6.5.2   Subsampling	218
   6.6     QUALITY ASSURANCE AND QUALITY CONTROL	218
     6.6.1   Sorting QC	218
     6.6.2   TaxonomicQC	219
   6.8     REFERENCES	220
 APPENDIX A	221
 APPENDIX B	222
 APPENDIX C	231

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 7

LIST OF TABLES

Table 1.1. National Coastal Condition Assessment Indicators 10
Table 2.1. Laboratory method performance requirements for water chemistry and chlorophyll a
sample analysis 13
Table 2.2. Concentration Ranges for Working Buffer Solution .... Error! Bookmark not defined.
Table 3.1. PCR Assay Mix Composition (according to Draft EPA Enterococcus TaqMan qPCR
Method) 91
Table 3.2. Batch Calibrator & Enterococcus Standards PCR Run - 7 Samples 91
Table 3.3. Sub-Batch Test Sample PCR Run - 26 Samples & 1 Method Blank 92
Table 3.4. Laboratory Methods: Fecal Indicator (Enterococci) 92
Table 3.5. Parameter Measurement Data Quality Objectives 93
Table 3.6. Laboratory QC Procedures: Enterococci DMA Sequences 94
Table 4.1. Laboratory method performance requirements for contaminants in sediment and fish
tissue 98
Table 4.3. Interference Check Solution Preparation Procedures 114
Table 4.4. Recommended Interference Check Sample Components and Concentrations 115
Table 4.5. Typical Stock Solution Preparation Procedures 130
Table 4.6. Mixed Standard Solutions 131
Table 4.8. Indicator List of Organchlorine Pesticides 150
Table 4.9. Indicator List of Polychlorinated Biphenyls (PCBs) 166
Table 4.10. Indicator List of Polynuclear Aromatic Hydrocarbons (PAHs) 178
Table 5.1. Laboratory method performance requirements for sediment grain size 192
Table 5.2. Sampling Time Intervals 196
Table 5.3. Laboratory method performance requirements for sediment toxicity 199
Table 5.4. Equipment and Supplies for Culturing and Testing Estuarine and Marine Amphipods.
201
Table 5.5. Recommended Test Conditions for Conducting Reference-Toxicity Tests 203
Table 5.6. Test Conditions for Conducting a10-d Sediment Toxicity Test 205
Table 5.7. General Activity Schedule for Conducting 10-d Sediment Toxicity Test 207
Table 5.8. Equipment and Supplies for Culturing and Testing the Freshwater Amphipod H.
azteca 210
Table 5.9. Recommended Test Conditions for Conducting Reference-Toxicity Tests 212
Table 5.10. Recommended Test Conditions for Conducting 10-d Sediment Toxicity Tests....214
Table 5.11. General Activity Schedule for Conducting 10-d Sediment Toxicity Test 216

-------
National Coastal Condition Assessment
Laboratory Methods Manual                                             Date: November 2010
                                                                               Page 8
                                  LIST OF FIGURES

Figure 2.1. Manifold Configuration for Ammonia Analysis	19
Figure 2.2. Manifold Configuration for the Nitrate + Nitrite Analysis using an Open Tubular
Cadmium Reactor	36
Figure 2.3. Manifold Configuration for Nitrate + Nitrite Analysis using a Laboratory Packed
Copper-coated Cadmium Reduction	36
Figure 2.4. Manifold Configuration for Nitrite Analysis	37
Figure 2.5. Ammonia Manifold forTKN Analysis	Error! Bookmark not defined.
Figure 2.6. Phosporous Manifold	68
Figure 2.7. Analytical Scheme	69
Figure 2.8. Manifold Configuration for Orthophosphate	74
Figure 3.1. Batch Sample Analysis Bench Sheet for Draft EPA Enterococcus TaqMan qPCR
Method	95
Figure 3.2. Enterococcus qPCR Analysis Decision Tree (ADT)	96
Figure 4.2. Inductively Coupled Plasma-Atomic Emission Spectrometry	125

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 9

                  1.0 INTRODUCTION AND GENERAL INSTRUCTIONS

 1.1    INTRODUCTION

 This manual describes methods for analyses of the samples collected during the 2010 National
 Coastal Condition Assessment (NCCA), including quality assurance objectives, sample
 handling, and data reporting. The NCCA is a statistical survey of the condition of our nation's
 coastal waters, estuaries, and shorelines.  Probability-based surveys are used to determine the
 state of populations or resources of interest using a representative sample of relatively few
 members or sites.  This random selection design allows data from the subset of sampled sites
 to be applies to the larger target populations (i.e., our coastal waters) and assessments with
 known confidence boundaries to be made.

 Along with  EPA, states, tribes, and other partners will participate in the survey every five years
 as part of the National Aquatic Resource Surveys (NARS) Program. The goals of the NARS are
 threefold:

    •  Address key questions about the quality of the nation's coasts

        o  What percentage of US coastlines is in  good condition with respect to ecological
           integrity,  recreational safety, and other key parameters?
        o  What is the relative importance of identified stressors such as nutrients, metals,
           etc.7

    •   Promote collaboration and build strong state/tribal capacity for monitoring programs.

    •   Provide a nationally consistent data set to examine water quality and develop baseline
        and trend information to evaluate the effectiveness of water protection/remediation
        programs effectiveness.

 With input from the states and other partners, EPA used an unequal probability design to select
 682 marine sites along the coasts  of the continental United States and 225 freshwater sites from
 the nearshore regions of the Great Lakes. Field crews will collect a variety of measurements
 and samples from these predetermined sampling areas which have been assigned longitude
 and latitiude coordinates. Additional sites were also identified for Puerto Rico,  Hawaii, Alaska
 and the Pacific Territory islands to provide an equivalent design for these coastal areas if these
 states and  territories  choose to sample them.

 1.2    LIST OF INDICATORS

 Indicators for the 2010 survey are  presented in Table 1.1. They will remain the same as those
 used previously for the National Coastal Condition Report with a few modifications.  The most
 prominent change in  the 2010 survey is the inclusion of coasts along the Great Lakes; therefore
 both sample collection methods and laboratory methods will reflect freshwater and saltwater
 matrices.

 Based on recommendations from a state workshop held in 2008, the NCCA workgroup decided
 on a few improvements to the original indicators. The changes include:  1) measuring
 Enterococcus levels as a human health indicator; 2) requiring the measurement of
 photosynthetically active radiation (PAR) using instrumentation to help standardize the water
 clarity indicator; 3) for sediment toxicity testing, labs will  use Leptochirus instead of Ampelisca

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 10
sp. for marine sites, and will use Hyalella for freshwater sites; 4) tissue studies will be conducted
using whole fish, and 5) fish community structure, Total Suspended Solids (TSS), and PAHs in
fish tissue will no longer be included.

Table 1.1.  National Coastal Condition Assessment Indicators
Measure/Indicator
Water
Quality
Sediment
Quality
Biological
Quality
Dissolved oxygen
PH
Temperature
Depth
Conductivity
(freshwater)
Salinity (marine)
Seech i/light
measurements
PAR
Nutrients
Chlorophyll
Grain size
Total organic
carbon
Sediment
chemistry
Sediment toxicity
Tissue
Contaminants
Benthic
community
structure
Specific data type
Observable on-site
Observable on-site
Observable on-site
Filtered surface sample for dissolved
inorganic NO2 NO3 NH4 ,PO4;
Unfiltered surface sample for Total N
and P
chlorophyll a
Silt/Clay content
Sediment total organic carbon
15 metals
25 PAHs
20 PCBs
14 pesticides
6 DDT metabolites
1 0-day static bioassay with
Leptochirus or Hyalella
13 metals (no SB orMN)
20 PCBs
14 pesticides
6 DDT metabolites
One sediment grab target for benthic
abundance enumeration and species
identification
Assessment
outcome
Hypoxia/anoxia
Water column
characterization
Societal value and
ecosystem production
Nutrient enrichment
Influencing factor for
extent and severity
for contamination
Influencing factor for
extent and severity
for contamination
Potential biological
response to sediment
contamination
Biological response
to sediment exposure
Environmentally
available contaminant
exposure
Biological response
to site conditions

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 11

1.3 TRAINING AND QUALIFICATIONS

These methods should be used only by trained, qualified laboratory technicians experienced in
the theory and application of aquatic resource methodology. A minimum of 2 years experience
in the particular laboratory technique and associated analytical equipment is required.

1.4 SAFETY

This manual describes procedures which may involve hazardous materials, operations, and
equipment, and it does not purport to address the associated safety issues. While some safety
considerations are included, it is beyond the scope of the manual to encompass all safety
measures necessary to conduct each test.

Development and maintenance of an effective health and safety program in the laboratory
requires an ongoing commitment by laboratory management. It is the laboratory's responsibility
to maintain a safe work environment and a current awareness file of OSHA and other applicable
regulations regarding the safe handling of the samples, chemicals and machinery. A reference
file of material safety data sheets (MSDSs) should be available to all personnel involved with
these analyses.

The collection and handling of sediment samples could subject personal to health and safety
risks. Contaminants in field-collected sediments may include carcinogens, mutagens, and other
potentially toxic compounds. Inasmuch as sediment testing is often begun before chemical
analysis can be completed, worker contact should be kept minimal. Personnel collecting
sediment samples and conducting tests should take all safety precautions necessary for the
prevention of bodily injury and illness which might result from ingestion or invasion of infectious
agents, inhalation or absorption of corrosive or toxic substances through the skin, and
asphyxiation due to lack of oxygen or the presence of noxious gases.

1.5 PROTOCOLS

Participating laboratories must be prepared to receive all or a portion of 1200 samples. Prior to
receiving samples, the laboratory must contact Marlys Cappaert at the Information Management
Center by phone (541-754-4467) or e-mail (cappaert.marlys@epa.gov) to arrange access to
EPA's sample tracking system. All samples must be logged into EPA's tracking system by the
contractor upon receipt. Samples will be tracked according to each unique site_id and sample
number. The laboratory must adhere to strict sample tracking procedures throughout the
laboratory analysis phase. If expected samples do not arrive, the laboratory must immediately
contact Marlys Cappaert.

All cooperating laboratories must work with the Information Management group (Marlys
Cappaert, Cappaert.Marlys@epamail.epa.gov, 541-754-4467,) to ensure their bench sheets
and/or data reporting spreadsheets are compatible with the electronic deliverables the lab will
need to submit. For taxonomic analyses, the laboratory must use a standard, agreed upon key
for identification and all organisms are to be identified to the lowest correct taxonomic level,
usually genus or species. Any questions regarding the standard operating procedure must be
directed to the appropriate Project Officer for the particular contract laboratory.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 12

Weekly tallies of samples received and samples processed are required via e-mail. More
detailed progress reports summarizing work performed and financial expenditures are required
monthly.

1.6 QUALITY CONTROL AND LABORATORY AUDITS

Laboratories participating in the NCCA must adhere to and document the quality control (QC)
elements prescribed for each analytical method. QC requirements routinely associated with
analytical chemistry measurements include: calibration standards, reagent blanks, duplicates,
check samples (spike/recovery), Standard Reference Materials (SRMs), and continuing
calibration curve check samples. Other types of laboratory procedures or tests (e.g., grain size
determination and toxicity testing) will also be conducted for the NCCA; the QC elements
appropriate for each of these methods are specified in this manual.

To ensure that a laboratory is technically competent to perform a particular analysis or
procedure, the NCCA will require that laboratory to conduct an initial demonstration of capability
prior to the laboratory being authorized to analyze NCCA field samples. For most analytical
processes, the demonstration will include documentation of the laboratory's calculated Method
Detection Limits (MDLs) for each analyte of interest and a "blind" analysis of an appropriate
performance evaluation (PE) sample (e.g., an SRM). For other types of laboratory
determinations, appropriate PE exercises will be described.

QC results provide the analyst with an immediate indication to the overall validity of the
analytical process, affording the opportunity to make necessary adjustments to bring the
analysis into control. Post-analysis, documented QC results enable data users to define the
level of quality and reliability of that data.

Quality assurance procedures and practices will include an independent laboratory audit. Each
laboratory is required to maintain at its facility performance records, raw data, and preserved
samples for a minimum of two years from the date the final results are submitted to EPA.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
             Page 13
                                     2.0  WATER QUALITY

2.1     PERFORMANCE-BASED METHODOLOGIES

Suggested analytical methods for water quality indicators are described in section 2.2 - 2.7 of
this manual.  However, some  laboratories participating in the survey may choose to employ
other analytical methods.  Laboratories engaged by EPA or the State may use a different
analytical method as long as the lab is able to achieve the same performance requirements as
the standard methods. Performance data must be submitted to EPA prior to initiating any
analyses.  Methods performance requirements for this program identify detection limit, precision
and accuracy objectives for each indicator.  Method performance requirements for water
chemistry and chlorophyll a sample analysis are shown in Table 2.1

Table 2.1.  Laboratory method performance requirements for water chemistry and chlorophyll a sample
analysis
Analyte
Ammonia (NH3)
Nitrate-Nitrite
(NO3-NO2)
Total Nitrogen
(TN)
Total
Phosphorous (TP)
and
ortho-Phosphate
Nitrate (NO3)
Chlorophyll-a
Units
mgN/L
mg N/L
mg/L
|jgP/L
mg N/L
u.g/L in
extract
Potential
Range
of Samples1
Oto17
0 to 360
(as nitrate)
0.1 to 90
0 to 22,000
(as TP)
0. to 360
0.7 to 11, 000
Method
Detection
Limit Objective2
0.01 marine
(0.7 ueq/L)
0.02 freshwater
0.01 marine
0.02 freshwater
0.01
2.0
0.01 marine
(10.1 ueq/L)
0.03 freshwater
1.5
Transition
Value3
0.10
0.10
0.10
20.0
0.1
15
Precision
Objective4
±0.01 or
±10%
±0.01 or
±10%
±0.01 or
±10%
±2 or
±10%
±0.01 or
±5%
± 1.5 or
±10%
Accuracy
Objective5
±0.01 or
±10%
±0.01 or
±10%
±0.01 or
±10%
±2 or
±10%
±0.01 or
±5%
± 1.5 or
±10%
  Estimated from samples analyzed at the WED-Corvallis laboratory between 1999 and 2005
  The method detection limit is determined as a one-sided 99% confidence interval from repeated measurements of a low-level
  standard across several calibration curves.
  Value for which absolute (lower concentrations) vs. relative (higher concentrations) objectives for precision and accuracy are
  used.
  For duplicate samples, precision is estimated as the pooled standard deviation (calculated as the root-mean square) of all
  samples at the lower concentration range, and as the pooled percent relative standard deviation of all samples at the higher
  concentration range. For standard samples, precision is estimated as the standard deviation of repeated measurements across
  batches at the lower concentration range, and as percent relative standard deviation of repeated measurements across batches
  at the higher concentration range.
  Accuracy is estimated as the difference between the measured (across batches) and target values of performance evaluation
  and/or internal reference samples at the lower concentration range, and as the percent difference at the higher concentration
  range.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 14

2.2 DISSOLVED INORGANIC NITROGEN - AMMONIA

2.2.1 Saltwater

2.2.1.1 Scope and Application

This method may be used for estuarine and coastal waters. The method is based upon the
indophenol reaction adapted to automated gas-segmented continuous flow analysis. A
statistically determined method detection limit of 0.3 ug N/L has been determined from seawater
of four different salinities. The method is linear to 4.0 mg N/L using a Flow Solution System.

2.2.1.2 Method Summary

The automated gas segmented continuous flow colorimetric method is used for the analysis of
ammonia concentration. Ammonia in solution reacts with alkaline phenol and NaDTT at 60°C to
form indophenol blue in the presence of sodium nitroferricyanide as a catalyst. The absorbance
of indophenol blue at 640 nm is linearly proportional to the concentration of ammonia in the
sample. A small systematic negative error caused by differences in the refractive index of
seawater and reagent water, and a positive error caused by the matrix effect (the change in the
colorimetric response of ammonia due to the change in the composition of the solution) on the
color formation, may be corrected for during data processing.

2.2.1.3 Interferences

1. Hydrogen sulfide at concentrations greater than 2 mg S/L can negatively interfere with
ammonia analysis. Hydrogen sulfide in samples should be removed by acidification with
sulfuric acid to a pH of about 3, then stripping with gaseous nitrogen.

2. The addition of sodium citrate and EDTA complexing reagent eliminates the precipitation of
calcium and magnesium when calcium and magnesium in seawater samples mix with high
pH (about 13) reagent solution.

3. Sample turbidity is eliminated by filtration or centrifugation.

4. As noted, refractive index and salt error interferences occur when sampler wash solution
and calibration standards are not matched with samples in salinity. For low concentration
samples (<20 ug N/L), low nutrient seawater with salinity matched to samples, sampler
wash solutions and calibration standards is recommended to eliminate matrix interferences.

2.2.1.4 Safety
Chloroform should be used it in a properly ventilated area, such as in a fume hood.

2.2.1.5 Equipment and Supplies
1. Gas Segmented Continuous Flow Autoanalyzer
• Automatic sampler • Spectrophotometer equipped with a
• Analytical cartridge with tungsten lamp (380-800 nm) or
reaction coils and heater photometer with a 640 nm interference
• Proportioning pump filter (max- 2 nm bandwidth)
• Nitrogen gas (high-purity ' Strip chart recorder or computer based
grade, 99.99%) data acquisition system

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 15
2. Glassware
• Pipettes
60-ml glass or high density polyethylene sample bottles,
glass volumetric flasks and glass sample tubes.
Ammonia is known for its high surface reactivity. When working at low levels (<20 ug N/L),
additional cleansing of labware is mandatory. Plastic bottles and glass volumetric flasks should
be cleaned in an ultrasonic bath with reagent water for 60 minutes. Bottles and sample tubes
made of glass should be cleaned by boiling in reagent water.

3. Supplies
• Analytical balance with
accuracy to 0.1 mg
• Drying oven
• Desiccator
Membrane filters (0.45 urn nominal pore size)
Syringes with syringe filters
Centrifuge
Ultrasonic water bath cleaner
2.2.1.6 Reagents and Standards

2.2.1 .6.1 Stock Reagent Solutions
5 g
1. Complexing Reagent. Dissolve 140 g of sodium citrate dehydrate
of sodium hydroxide (NaOH) and 10 g of disodium EDTA (IS^CioHuOsl^h^O) in
approximately 800 ml reagent water, mix and dilute to 1 L with reagent water. The pH of
this solution is 13. This solution is stable for 2 months.

2. Stock Ammonium sulfate Solution (100 mg N/L). Transfer 0.4721 g of pre-dried (105°C for
2 hours) ammonium sulfate (NH4)2SO4 to a 1000 ml_ volumetric flask containing
approximately 800 ml of reagent water and dissolve. Add a few drops of chloroform as a
preservative. Dilute the solution to 1 L with reagent water. Store the solution in a glass
bottle at 4°C. This solution is stable for 2 months.

3. Low Nutrient Sea Water. Obtain natural or commercially available low nutrient seawater
from surface water (salinity 36%o, < 7 ug N/L) and filter it through 0.3 micron pore size glass
fiber filters. Do not use artificial seawater.

2.2.1.6.2 Working Reagents
1. Brij-35 Start-up solution. Brij-35 is a trade name for polyoxyehtylene(23) lauryl ether
(Ci2H25(OCH2CH2)23OH) and is commercially available. Add 2 mL of Brij-35 surfactant to
1000 mL reagent water and mix gently.

2. Working Complexing Reagent. Add 1 mL Brij-35 to 200 mL of stock complexing reagent.
Prepare daily. This volume of solution is sufficient for an 8-hour run.

3. Sodium Nitroferricyanide Solution. Dissolve 0.25 g of sodium nitroferricyanide
(Na2Fe(CN)5NO'2H2O) in 400 mL reagent water, dilute to 500mL Store in an amber bottle
at room temperature.

4. Phenol Solution. Dissolve 1 .8 g solid phenol (C6H5OH) and 1 .5 g of sodium hydroxide in
100 mL of reagent water. Prepare fresh daily.

5. NaDTT Solution. Dissolve 0.5 g of sodium hydroxide and 0.2 g dichloroisocyanuric acid
sodium salt (NaDTT, NaCsC^NsOs) in 100 mL reagent water. Prepare fresh daily.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 16

6. Colored SYNC Peak Solution. Add 50 uL of blue food coloring to 1000 ml_ reagent water
and mix. Further dilute to obtain a peak of between 25 to 100 percent full scale according to
the AUFS setting used for refractive index measurement.

7. Primary Dilution Standard Solution (5 mg N/L). Prepare by diluting 5.0 ml_ stock standard
solution to 100 mL with reagent water. Prepare fresh daily.

Note. When working with samples of a narrow range of salinities (± 2%o) or samples
containing low ammonia concentrations (< 20 ug N/L), it is recommended that the calibration
solutions be prepared in low nutrient seawater diluted to the salinity of samples, and the
sampler wash solution also be low nutrient seawater diluted to the same salinity. If this
procedure is employed, it is not necessary to perform the matrix effect and refractive index
corrections outlined in sections 2.1.10.1 and 2.1.10.2. When analyzing samples of
moderate or high ammonia concentrations (> 20 pg N/L) and of varying salinities, the
calibration standard solutions and sampler wash solution can be prepared with reagent
water. The corrections for matrix effect and refractive index should be subsequently applied
(sections 2.1.10.1 and 2.1.10.2).
9. Saline Ammonia Standards. If the calibration standards are not prepared to match sample
salinity, then saline ammonia standards must be prepared in a series of salinities in order to
quantify the matrix effect. The following dilutions in 100 mL reagent water are
recommended:
Salinity (%o)
0
9
18
27
35
Low nutrient
seawater (mL)
0
25
50
75
98
Cone, primary
dilution standard (mL)
2
2
2
2
2
ma N/L
0.10
0.10
0.10
0.10
0.10
2.2.1.7 Quality Control

Each laboratory using this method is required to implement a formal quality control (QC)
program. The minimum requirements consist of an initial demonstration of performance,
continued analysis of Laboratory Reagent Blanks (LRB), laboratory duplicates and Laboratory
Fortified Blanks (LFB) with each set of samples.

2.2.1.7.1 Initial Demonstration of Performance

1. The method detection limit (MDL) must be established for the method analyte using a low
level seawater sample containing, or fortified at, approximately 5 times the estimated
detection limit. To determine MDL values, analyze at least seven replicate aliquots of water
which have been processed through the entire analytical method. Calculate the MDL as:

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 17

MDL=(t)(S)
where,
S = the standard deviation of the replicate analysis
t = t value for n-1 degrees of freedom at the 99% confidence limit; t = 3.143 for
six degrees of freedom.

2. The linear dynamic range (LDR) must be determined by analyzing a minimum of eight
calibration standards ranging from 0.002 to 2.00 mg N/L across all sensitivity settings
(absorbance units full scale output range setting) of the detector. Standards and sampler
wash solutions should be prepared in low nutrient seawaterwith salinities similar to the
samples to avoid the necessity to correct for salt error or refractive index. Normalize
responses by multiplying the response by the absorbance units full scale output range
setting. Perform the linear regression of normalized response vs. concentration, and obtain
the constants m and b, where m is the slope and b is the y-intercept. Incrementally analyze
standards of higher concentration until the measured absorbance response (R) of a
standard no longer yields a calculated concentration (Cc) that is within 100 ± 10% of known
concentration (C), where
Cc = (R-b)/m
This concentration defines the upper limit of the LDR. If samples have a concentration that
is >90% of the upper limit of the LDR, they must be diluted and reanalyzed.

2.2.1.7.2 Assessing Laboratory Performance

2. Laboratory Fortified Blank (LFB). The lab should analyze at lease one LFB with each set of
samples. The LFB must be at a concentration within the daily calibration range. The LFB
data are used to calculate percent recovery. If the recovery of the analyte falls outside the
required control limits of 90-110%, the source of the problem should be identified and
resolved before continuing the analysis.

3. The laboratory must use LFB data to assess lab performance against the required control
limits of 90-110%. When sufficient internal performance data become available (usually a
minimum of 20 to 30 analyses), optional control limits can be developed from the percent
mean recovery (x) and standard deviation (S) of the mean recovery. These data can be
used to establish the upper and lower control limits as follows:

Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than the required control limits of 90-
11-%. After each 5-10 new recovery measurements, new control limits can be calculated
using only the most recent 20 to 30 data points. Also the standard deviation (S) data
should be used to establish an ongoing precision statement for the level of concentration
included in the LFB. These data must be kept on file and available for review.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
_ Page 18

2.2.1.7.3 Assessing Analyte Recovery-Laboratory Fortified Sample Matrix (LFM)

2. Calculate percent recovery of anlayte, corrected for background concentration measured in
a separate unfortified sample. These values should be compared with the values obtained
from the LFBs. Percent recoveries may be calculated using the following equation:

R= f Cs - C) x100
S
where,

R = percent recovery
Cs = measured fortified sample addition in mg N/L

C = sample background concentration in mg N/L

S = concentration in mg N/L added to the environmental sample
3. If the recovery of the analyte falls outside the required control limits of 90-1 10%, but the
laboratory performance for that analyte is within the control limits, the fortified sample
should be prepared again and analyzed. If the result is the same after reanalysis, the
recovery problem encountered with the fortified sample is judged to be the matrix related
and the sample data should be flagged.

2.2.1.8 Calibration and Standardization

1 . At least five calibration standards should be prepared fresh daily for system calibration.

2. A calibration curve should be constructed for each sample set by analyzing a series of
calibration standard solutions. A sample set should contain no more than 60 samples. For
a large number of samples make several sample sets with individual calibration curves.

3. Analyze the calibration standards in duplicate before the actual samples.
4. The calibration curve containing five data points or more that bracketed the concentration
of samples should have a correlation coefficient (r) of 0.995 or better and the range should
not be greater than two orders of magnitude.

5. Use a high calibration solution followed by two black cups to quantify system carryover.
The difference in peak heights between two blank cups is due to the carryover from the
high calibration solution. The carryover coefficient (k) is calculated as follows:
Phigh
where,
Phigh = the peak height of the high ammonia standard
Pb1 = the peak height of the first blank sample
Pb2 = the peak height of the second blank sample

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 19
       The carryover coefficient (k) should be measured in seven replicates to obtain a
       statistically significant number.  The carryover coefficient should be remeasured with any
       change in manifold plumbing or upon replacement of pump tubes.

       The carryover correction (CO) of a given peak (i) is proportional to the peak height of the
       preceding sample PM.

                           CO = k x PM

       To correct a given peak height reading, (Pi), subtract the carryover correction.

                           Pi,c=Pi-CO
       where PiiC is the corrected peak height.

       The correction for carryover should be applied to all the peak heights throughout a run.
       The carryover coefficient should be less than 5%.

6.   Place a high standard solution at the end of each sample run to check for sensitivity drift.
    Apply sensitivity drift correction to all the samples.  The sensitivity drift during a run should
    be less than 5%.

Note. Sensitivity drift correction is available in most data acquisition software supplied with
    subanalyzers. It is assumed that sensitivity drift is linear with time. An interpolated drift
    correction factor is calculated for each sample according to the sample position during  the
    run.  Multiply the sample peak height by the corresponding sensitivity drift correction factor
    to obtain the corrected peak height for each sample.

2.2.1.9 Procedure

1.  If samples are stored in a refrigerator,  equilibrate to room temperature prior to analysis.

2.  Turn on the continuous flow analyzer and data acquisition components and warm up for at
   least 30 minutes.

3.  Set up cartridge and pump tubes as shown in  Figure 2.1.
(j
**,
OW
Dobubbler
r-i ,
, m
J Detector
' 8+Onm
S
Coal o)
f \ 	
c
asle

-
,_0__9_-
8 _o_
J .
L> 	
e
	 O-
.-o— a._
4
— on
0 3
2 01
1
Manifold
Wash To Sair

_^.
Heater
&
f
B
V
	 /• 	
©
"N /
)
/
. s


0.41
0.41
0.10
0.10
0.10
1.01
0.25
0.32
1.57

Pump
mUmtn
_J




£
Nhrofenicyanide
NaDTT
Phenol
-| Sample
N.lrogen
Comptoxing Roagent
Reagent Water
or Low Nutrient Seawater
Sample:Wash = 30":30"
Figure 2.1.  Manifold Configuration for Ammonia Analysis

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 20

4. Set the spectrophotometer wavelength to 640 nm and turn on lamp.

5. Set the absorbance unit full scale (AUFS) range on the spectrophotometer at the
appropriate setting according to the highest concentration of ammonia in the samples. The
highest setting appropriate for this method is 0.2 AUFS for 6 mg N/L.

6. Choose an appropriate wash solution for sampler wash. For analysis of samples with a
narrow range of salinities (±2%o) or for samples containing low ammonia concentrations (<20
ug N/L), it is recommended that the calibration solutions be prepared in low nutrient
seawater diluted to the salinity of samples, and that the sampler wash solution also be low
nutrient seawater diluted to the same salinity. For samples with varying salinities and higher
ammonia concentrations (>20 ug N/L), it is suggested that the reagent water used for the
sampler wash solution and for preparing calibration standards and procedures in sections
2.1.10.1 and 2.1.10.2 be employed.

7. Begin pumping the Brij-35 start-up solution through the system and obtain a steady
baseline. Place the reagents on-line. The reagent baseline will be higher than the start-up
solution baseline. After the reagent baseline has stabilized, reset the baseline.

Note. To minimize the noise in the reagent baseline, clean the flow system by sequentially
pumping the sample line with reagent water, 1 N HCI solution, reagent water, 1 N A/a OH
solution for a few minutes each at the end of the daily analysis. Make sure to rinse the
system well to prevent precipitation ofMg(OH)2 when seawater is introduced into the
system. Keep the reagents and samples free of particulates and filter if necessary.

If the baseline drifts upward, pinch the waste line fora few seconds to increase back
pressure. If absorbance drops down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach a syringe at the waste outlet of the
flowcell. Air bubbles in the flow cell can often be eliminated by simply attaching a syringe for
a few minutes or, if not, dislodged by pumping the syringe piston. Alternatively, flushing the
flowcell with alcohol was found to be effective in removing trapped air.
8. The sampling rate is approximately 60 samples per hour with 30 second of sample time and
30 seconds of wash time. Place a blank after every ten samples.

2.2.1.10 Data Analysis and Calibration

Concentrations of ammonia in samples are calculated from the linear regression, obtained from
the standard curve in which the concentration of the calibration standards are entered as the
independent variable and their corresponding peak heights are the dependent variable.

2.2.1.10.1 Refractive Index Correction

1. If reagent water is used as the wash solution, the analyst has to quantify the refractive
index correction due to the difference in salinity between sample and wash solution. The
following procedures are used to measure the relationship between the sample salinity and
the refractive index on a particular detector.

2. Analyze a set of ammonia standards in reagent water with color reagent using reagent
water as the wash and obtain a linear regression of peak height versus concentration.
Then replace reagent water wash solution with low nutrient seawater wash solution.

Note. In ammonia analysis absorbance of the reagent water is higher than that of the low
nutrient seawater. When using reagent water as a wash solution, the change in refractive

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 21

index causes the absorbance ofseawaterto become negative. To measure the
absorbance due to refractive index change in different salinity samples, low nutrient
seawater must be used as the wash solution to bring the baseline down.

3. Replace the phenol solution and NaDTT solution with reagent water. All other reagents
remain the same. Replace the synchronization sample with the colored SYNC peak
solution.

4. Prepare a series of different salinity samples by diluting the low nutrient seawater.
Commence analysis and obtain peak heights for different salinity samples. The peak
heights for the refractive index correction must be obtained at the same AUFS range
setting and on the same spectrophotometer as the corresponding standards.

5. Using low nutrient seawater as the water wash, a maximum absorbance will be observed
for reagent water. No change in refractive index will be observed in the seawater sample.
Assuming the absolute absorbance for reagent water (relative to the seawater baseline) is
equal to the absorbance for seawater (relative to reagent water baseline), subtract the
absorbance of samples of various salinities from that of reagent water. The results are the
apparent absorbance due to the change in refractive index between samples of various
salinities relative to the reagent water baseline.

6. For each sample of varying salinity, calculate the apparent ammonia concentration due to
refractive index from its peak height corrected to the reagent water baseline and the
regression equation of ammonia standards obtained with color reagent being pumped
through the system. Salinity is entered as the dependent variable. The resulting
regression allows the analyst to calculate apparent ammonia concentration due to
refractive index when sample salinity is known. Thus, the analyst would not be required to
obtain refractive index peak heights for all samples.

7. The magnitude of refractive index correction can be minimized by using a low refractive
index flowcell. It is important that the refractive index correction must be calculated for the
particular detector. The refractive index must be redetermined whenever a significant
change in the design of the flowcell or new matrix is encounter.

A typical equation is:
Apparent ammonia (urn N/L) = 0.0134 + 0.0457(5)

where S is the sample salinity in parts per thousand.

The apparent ammonia concentration due to refractive index so obtained should then be
added to samples of corresponding salinity when reagent water was used as the wash
solution for sample analysis.

2.2.1.10.2 Matrix Effect Correction

1. When calculating concentrations of samples of varying salinities from standards and wash
solution prepared in reagent water, it is necessary to first correct for refractive index errors,
then correct for the change in color development due to the differences in composition
between samples and standards (matrix effect). Even where refractive index correction may
be small, the correction for matrix effect can be appreciable.

2. Plot the salinity of the saline standards as the independent variable, and the apparent
concentration of ammonia (mg N/L) from the peak height (corrected for refractive index)
calculated from the regression of standards in reagent water, as the dependent variable for

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 22

all saline standards. The resulting regression equation allows the analyst to correct the
concentrations of samples of known salinity for the color enhancement due to matrix effect.
3. The matrix effect becomes a single factor independent of sample salinity. A typical equation
to correct for matrix effect is:

Corrected concentration (mg N/L) = Uncorrected concentration /1.17 (mg N/L)

4. Results of sample analysis should be reported in mg N/L (ppm) or in ug N/L (ppb).

2.2.2 FRESHWATER

2.2.2.1 Scope and Application

1. This method covers the determination of ammonia in freshwater.

2. The applicable range is 0.01-2.0 mg/L NH3as N. Higher concentrations can be
determined by sample dilution. Approximately 60 samples per hour can be analyzed.

3. This method is described for macro glassware; however, micro distillation equipment
may also be used.

2.2.2.2 Summary of Method

1. The sample is buffered at a pH of 9.5 with a borate buffer in order to decrease hydrolysis of
cyanates and organic nitrogen compounds, and is distilled into a solution of boric acid.
Alkaline phenol and hypochlorite react with ammonia to form indophenol blue that is
proportional to the ammonia concentration. The blue color formed is intensified with sodium
nitroprusside and measured colorimetrically.

2. Reduced volume versions of this method that use the same reagents and molar ratios are
acceptable provided they meet the quality control and performance requirements stated in
the method.

3. Limited performance-based method modifications may be acceptable provided they are fully
documented and meet or exceed requirements expressed in Section 2.2.2.8 Quality Control.

2.2.2.3 Interferences

1. Cyanate, which may be encountered in certain industrial effluents, will hydrolyze to some
extent even at the pH of 9.5 at which distillation is carried out.

2. Residual chorine must be removed by pretreatment of the sample with sodium thiosulfate or
other reagents before distillation.
3. Method interferences may be caused by contaminants in the reagent water, reagents,
glassware, and other sample processing apparatus that bias analyte response.

2.2.2.4 Safety

1. The toxicity or carcinogenicity of each reagent used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and exposure
should be as low as reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 23

2. Each laboratory is responsible for maintaining a current awareness file of OSHA regulations
regarding the safe handling of the chemicals specified in this method. A reference file of
Material Safety Data Sheets (MSDS) should be made available to all personnel involved in
the chemical analysis. The preparation of a formal safety plan is also advisable.

3. The following chemicals have the potential to be highly toxic or hazardous, consult MSDS.

- Sulfuric acid - Sodium nitroprusside
- Phenol

2.2.2.5 Equipment and Supplies

1. Balance - Analytical, capable of accurately weighing to the nearest 0.0001 g.

2. Glassware - Class A volumetric flasks and pipets as required.

3. An all-glass distilling apparatus with an 800-1000 ml_ flask.
4. Automated continuous flow analysis equipment designed to deliver sample and reagents
in the required order and ratios.

- Sampling device (sampler) - Colorimetric detector
- Multichannel pump - Data recording device
- Reaction unit or manifold

2.2.2.6 Reagents and Standards

1. Reagent water - Ammonia free: Such water is best prepared by passage through an ion
exchange column containing a strongly acidic cation exchange resin mixed with a strongly
basic anion exchange resin. Regeneration of the column should be carried out according to
the manufacturer's instructions.

Note: All solutions must be made with ammonia-free water.
2. Boric acid solution (20 g/L): Dissolve 20 g H3BO3 in reagent water and dilute to 1 L.

3. Borate buffer: Add 88 ml_ of 0.1 N NaOH solution to 500 ml_ of 0.025 M sodium tetraborate
solution (5.0 g anhydrous Na2B4O7 or 9.5 g Na2B4O7CioH2O per L) and dilute to 1 L with
reagent water.

4. Sodium hydroxide, 1 N: Dissolve 40 g NaOH in reagent water and dilute to 1L.

5. Dechlorinating reagents: A couple of dechlorinating reagents may be used to remove
residual chlorine prior to distillation. These include:
- Sodium thiosulfate: Dissolve 3.5 g Na2S2O3C5H2O in reagent water and dilute to 1 L.
One ml_ of this solution will remove 1 mg/L of residual chlorine in 500 ml_ of sample.
- Sodium sulfite: Dissolve 0.9 g Na2SO3 in reagent water and dilute to 1 L. One ml_
removes 1 mg/L Cl per 500 ml_ of sample.

6. Sulfuric acid 5 N: Air scrubber solution. Carefully add 139 ml_ of conc.sulfuric acid to
approximately 500 ml_ of reagent water. Cool to room temperature and dilute to 1 L with
reagent water.
7. Sodium phenolate: Using a 1-L Erlenmeyer flask, dissolve 83 g phenol in 500 ml_ of
distilled water. In small increments, cautiously add with agitation, 32 g of NaOH.
Periodically cool flask under water faucet. When cool, dilute to 1 L with reagent water.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 24

8. Sodium hypochlorite solution: Dilute 250 ml_ of a bleach solution containing 5.25% NaOCI
(such as "Clorox") to 500 ml_ with reagent water. Available chlorine level should
approximate 2%-3%. Since "Clorox" is a proprietary product, its formulation is subject to
change. The analyst must remain alert to detecting any variation in this product significant
to its use in this procedure. Due to the instability of this product, storage over an extended
period should be avoided.

9. Disodium ethylenediamine-tetraacetate (EDTA) (5%): Dissolve 50 g of EDTA (disodium
salt) and approximately six pellets of NaOH in 1L of reagent water.
10. Sodium nitroprusside (0.05%) Dissolve 0.5 g sodium nitroprusside in 1 L of reagent water.

11. Stock solution: Dissolve 3.819 g of anhydrous ammonium chloride, NH4CI, dried at 105°C,
in reagent water, and dilute to 1 L. 1.0 ml_ = 1.0 mg NH3-N.

12. Standard Solution A: Dilute 10.0 ml_ of stock solution to 1 L with reagent water. 1.0 ml_ =
0.01 mg NH3-N.
13. Standard Solution B: Dilute 10.0 ml_ of standard solution A to 100.0 ml_ with reagent water.
1.0 ml_ = 0.001 mg NH3-N.

2.2.2.7 Sample Collection, Preservation and Storage

1. Samples should be collected in plastic or glass bottles. All bottles must be thoroughly
cleaned and rinsed with reagent water. Volume collected should be sufficient to insure a
representative sample, allow for replicate analysis.

2. Samples must be preserved with H2SO4 to a pH <2 and cooled to 4°C at time of collection.

3. Samples should be analyzed as soon as possible after collection. If storage is required,
preserved samples are maintained at 4°C and may be held for up to 28 days.

2.2.2.8 Quality Control

Each laboratory using this method is required to operate a formal quality control (QC) program.
The minimum requirements of this program consist of an initial demonstration of laboratory
capability, and the periodic analysis of laboratory reagent blanks, fortified blanks and other
laboratory solutions as a continuing check on performance. The laboratory is required to
maintain performance records that define the quality of the data that are generated.

2.2.2.8.1 Initial Demonstration of Performance

2. Linear Calibration Range (LCR) - The LCR must be determined initially and verified every
six months or whenever a significant change in instrument response is observed or
expected. The initial demonstration of linearity must use sufficient standards to insure that
the resulting curve is linear. The verification of linearity must use a minimum of a blank and
three standards. If any verification data exceeds the initial values by ± 10%, linearity must
be reestablished. If any portion of the range is shown to be nonlinear, sufficient standards
must be used to clearly define the nonlinear portion.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 25

3. Quality Control Sample (QCS) -- When beginning the use of this method (on a quarterly
basis or as required to meet data-quality needs) verify the calibration standards and
acceptable instrument performance with the preparation and analyses of a QCS. If the
determined concentrations are not within ±10% of the stated values, performance of the
determinative step of the method is unacceptable. The source of the problem must be
identified and corrected before either proceeding with the initial determination of MDLs or
continuing with on-going analyses.

4. Method Detection Limit (MDL) ~ MDLs must be established for all analytes, using reagent
water (blank) fortified at a concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven replicate aliquots of the fortified
reagent water and process through the entire analytical method. Perform all calculations
defined in the method and report the concentration values in the appropriate units.
Calculate the MDL as follows:

MDL=(t)x(S)
where,

t = value for a 99% confidence level and a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates]

S = standard deviation of the replicate analyses

MDLs should be determined every six months, when a new operator begins work or
whenever there is a significant change in the background or instrument response.

2.2.2.8.2 Assessing Laboratory Performance

1. Laboratory Reagent Blank (LRB) - The laboratory must analyze at least one LRB with each
batch of samples. Data produced are used to assess contamination from the laboratory
environment. Values that exceed the MDL indicate laboratory or reagent contamination
should be suspected and corrective actions must be taken before continuing the analysis.

a. Upper Control Limit = x + 3S

b. Lower Control Limit = x - 3S

4. The optional control limits must be equal to or better than the required control limits of 90-
110%. After each 5-10 new recovery measurements, new control limits can be calculated
using only the most recent 20-30 data points. Also, the standard deviation (S) data should
be used to establish an on-going precision statement for the level of concentrations included
in the LFB. These data must be kept on file and be available for review.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 26

5. Instrument Performance Check Solution (IPC) - For all determinations the laboratory must
analyze the IPC (a mid-range check standard) and a calibration blank immediately following
daily calibration, after every 10th sample (or more frequently, if required) and at the end of
the sample run. Analysis of the IPC solution and calibration blank immediately following
calibration must verify that the instrument is within ±10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is still within ±10%. If the calibration
cannot be verified within the specified limits, reanalyze the IPC solution. If the second
analysis of the IPC solution confirms calibration to be outside the limits, sample analysis
must be discontinued, the cause determined and/or in the case of drift, the instrument
recalibrated. All samples following the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must be kept on file with the sample
analyses data.

2.2.2.8.3 Assessing Analyte Recovery and Data Quality

2. Calculate the percent recovery for each analyte, corrected for concentrations measured in
the unfortified sample, and compare these values to the designated LFM recovery range 90-
110%. Percent recovery may be calculated using the following equation:

R = Cs - C x 100
s
where,

R = percent recovery

Cs = fortified sample concentration
C = sample background concentration

s = concentration equivalent of analyte added to sample

3. If the recovery of any analyte falls outside the designated LFM recovery range and the lab
performance for that analyte is shown to be in control, the recovery problem encountered
with the LFM is judged to be either matrix or solution related, not system related.

4. Where reference materials are available, they should be analyzed to provide additional
performance data. The analysis of reference samples is a valuable tool for demonstrating
the ability to perform the method acceptably.

2.2.2.9 Calibration and Standardization

1. Prepare a series of at least three standards, covering the desired range, and a blank by
diluting suitable volumes of standard solutions to 100 mL with reagent water.

2. Process standards and blanks as described in Section 2.2.2.10, Procedure.
3. Place appropriate standards in the sampler in order of decreasing concentration and
perform analysis.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 27

4. Prepare standard curve by plotting instrument response against concentration values. A
calibration curve may be fitted to the calibration solutions concentration/response data
using computer or calculator based regression curve fitting techniques. Acceptance or
control limits should be established using the difference between the measured value of the
calibration solution and the "true value" concentration.
5. After the calibration has been established, it must be verified by the analysis of a suitable
QCS. If measurements exceed ±10% of the established QCS value, the analysis should be
terminated and the instrument recalibrated. The new calibration must be verified before
continuing analysis.

6. Periodic reanalysis of the QCS is recommended as a continuing calibration check.

2.2.2.10 Procedure

1. Preparation of equipment: Add 500 mL of reagent water to an 800 ml_ Kjeldahl flask. The
addition of boiling chips that have been previously treated with dilute NaOH will prevent
bumping. Steam out the distillation apparatus until the distillate shows no trace of ammonia.

2. Sample preparation: Remove the residual chorine in the sample by adding the
dechlorinating agent equivalent to the chlorine residual. To 400 mL of sample add 1 N
NaOH, until the pH is 9.5.

3. Distillation: Transfer the sample, the pH of which has been adjusted to 9.5, to an 800 mL
Kjeldahl flask and add 25 mL of the borate buffer. Distill 300 mL at the rate of 6-10 mL/min.
into 50 mL of 2% boric acid contained in a 500 mL Erlenmeyer flask.

Note: The condenser tip or an extension of the condenser tip must extend below the level of the
boric acid solution.

4. Since the intensity of the color used to quantify the concentration is pH dependent, the acid
concentration of the wash water and the standard ammonia solutions should approximate
that of the samples.

5. Allow analysis system to warm up as required. Feed wash water through sample line.

6. Arrange ammonia standards in sampler in order of decreasing concentration of nitrogen.
Complete loading of sampler tray with unknown samples.

7. Switch sample line from reagent water to sampler and begin analysis.

2.2.2.11 Data Analysis and Calculations

1. Prepare a calibration curve by plotting instrument response against standard concentration.
Compute sample concentration by comparing sample response with the standard curve.
Multiply answer by appropriate dilution factor.

2. Report only those values that fall between the lowest and the highest calibration standards.
Samples exceeding the highest standard should be diluted and reanalyzed.

3. Report results in mg NH3-N/L.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 28

2.3 DISSOLVED INORGANIC NITROGEN - NITRATE-NITRITE

2.3.1 Saltwater

2.3.1.1 Scope and Application

1. This method provides a procedure for determining nitrate and nitrite concentrations in
estuarine and saltwater. Nitrate is reduced to nitrite by cadmium and the resulting nitrite
determined by formation of an azo dye.

2. A statistically determined method detection limit (MDL) of 0.075 ug N/L has been
determined in seawaters of five different salinities. The method is linear to 5.0 mg N/L using
a Flow Solution System.
3. Approximately 40 samples per hour can be analyzed.

2.3.1.2 Method Summary

An automated gas segmented continuous flow colorimetric method for the analysis of nitrate
concentration is used. Samples are passed through a copper-coated cadmium reduction
column. Nitrate in the sample is reduced to nitrite in a buffer solution. The nitrite is then
determined by diazotizing with sulfanilamide and coupling with n-1-naphthylethylenediamine
dihydrochloride to form a color azo dye. The absorbance measured at 540 nm is linearly
proportional to the concentration of nitrite + nitrate in the sample. Nitrate concentrations are
obtained by subtracting nitrite values, which have been separately determined without the
cadmium reduction procedure, from nitrite + nitrate values.

There is no salt error in this method. The small negative error caused by differences in the
refractive index of seawater and reagent water is readily corrected during the data processing.

2.3.1.3 Interferences

1. Hydrogen sulfide at concentrations greater than 0.1 mg S/L can interfere with nitrite analysis
by precipitating on the cadmium column. Hydrogen sulfide in samples must be removed by
precipitation with cadmium or copper salt.

2. Iron, copper, and other heavy metals at concentrations greater than 1 mg/L alter reduction
efficiency of the cadmium column. The addition of EDTA will complex these metal ions.
3. Phosphate at a concentration greater than 0.1 mg/L decreases the reduction efficiency of
cadmium. Dilute samples if possible or remove phosphate with ferric hydroxide prior to
analysis.

4. Particulates inducing turbidity should be removed by filtration.

2.3.1.4 Equipment and Supplies

1. Gas Segmented Continuous Flow Autoanalyzer

o Automatic sampler
o Analytical cartridge with reaction coils and heater
o Open tubular Cadmium Reactor or laboratory prepared packed copper-coated
cadmium reduction column (prepared according to sec. 2.2.5.3 and 2.2.5.4)

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 29

o Proportioning pump
o Spectrophotometer equipped with a tungsten lamp (380-800 nm) or photometer
with a 540 nm interference filter (max. 2 nm bandwidth)
o Strip chart recorder or computer based data acquisition system
o Nitrogen gas (high-purity grade, 99.99%)

2. Glassware and Supplies

o Pipettes
o 60-ml high density polyethylene sample bottles, glass volumetric flasks and glass
sample tubes.
o Analytical balance with accuracy to 0.1 mg
o Drying oven
o Desiccator
o Membrane filters (0.45 urn nominal pore size)
o Syringes with syringe filters
o pH meter with a glass electrode and reference electrode.

2.3.1.5 Reagents and Standards

2.3.1.5.1 Stock Reagent Solutions

1. Stock Sulfanilamide Solution. Dissolve 10 g sulfanilamide (CeHs^C^S) in 1 L of 10% HCI.

2. Stock Nitrate Solution (100 mg N/L). Transfer 0.4928 g of pre-dried (105°C for 1 hour)
sodium nitrite (NaNO2) to a 1000 ml_ volumetric flask containing approximately 800 ml of
reagent water and dissolve. Dilute the solution to 1 L with reagent water. Store the
solution in a polyethylene bottle at 4°C. This solution is stable for 6 months.

3. Stock Nitrite Solution (100 mg N/L). Transfer 0.7217 g of pre-dried (105°C for 1 hour)
potassium nitrate (KNO3) to a 1000 ml_ volumetric flask containing approximately 800 ml of
reagent water and dissolve. Dilute the solution to 1 L with reagent water. Store the
solution in a polyethylene bottle at 4°C. This solution is stable for 3 months.

Note. High purity nitrite salts may not be available. /Assays given be reagent manufacturers are
usually in the range of 95-97%. The impurity must be taken into account for the weight
taken.

4. For marine samples- Low Nutrient Sea Water. Obtain natural or commercially available
low nutrient seawater from surface water (salinity 35-36%o, < 7 ug N/L) and filter it through
0.3 micron pore size glass fiber filters. Do not use artificial seawater.

2.3.1.5.2 Working Reagents

1. Brij-35 Start-up solution. Brij-35 is a trade name for polyoxyehtylene(23) lauryl ether
(Ci2H25(OCH2CH2)23OH) and is commercially available. Add 2 mL of Brij-35 surfactant to
1000 mL reagent water and mix gently.

2. Working Sulfanilamide Solution. Add 1 mL Brij-35 to 200 mL of stock sulfanilamide solution.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 30

Note. Adding surfactant Brij-35 to sulfanilamide solution instead of to the buffer solution is to
prevent the Brij-35 from being adsorbed on the cadmium surface, which may result in
decreasing surface reactivity of the cadmium and reduce the lifetime of the column.

3. NED Solution. Dissolve 1 g of NED (N-1-naphthylehtylenediamine dihydrochloride,
Ci2H14N2*HCI) in 1 L of reagent water.

4. Imidazole Buffer Solution. Dissolve 13.6 g of imidazole (C3H4N2) IN 4L reagent water. Add
2ml concentrated HCI. Adjust the pH to 7.8 with diluted HCI. Store in a refrigerator.

5. Copper Sulfate Solution (2%). Dissolve 20g of copper sulfate (CuSO4«5H2O) in 1L reagent
water.

6. Colored SYNC Peak Solution. Add 50 uL of red food coloring to 1000 ml_ reagent water
and mix. Further dilute to obtain a peak of between 25 to 100 percent full scale according
to the AUFS setting used for refractive index measurement.

7. Primary Dilution Standard Solution (5 mg N/L). Prepare by diluting 5.0 ml_ of stock
standard solution to 100 ml_ with reagent water. Prepare fresh daily.

Note. This solution should be prepared as an intermediate concentration appropriate for further
dilution in preparing calibration solutions. Therefore the concentration must be adjusted
according to the desired calibration concentration range.
8. Calibration Standards. Prepare a series of calibration standards by diluting suitable
volumes of a primary dilution standard to 100 ml_ with reagent water or low nutrient
seawater. The concentration range should bracket the expected concentrations of samples
and not span more than two orders of magnitude. At least five calibration standards with
equal concentration increments should be used to construct the calibration curve. Prepare
daily.
If nitrate + nitrite and nitrite are analyzed simultaneously by splitting a sample into two
analytical systems, a nitrate and nitrite mixed standard should be prepared. The total
concentration (nitrate+nitrite) must be assigned to the concentrations of calibration
standards in the nitrate+nitrite system.

When analyzing samples of varying salinities, it is recommended that the calibration
standard solutions and sampler wash solution be prepared in reagent water and corrections
for refractive index be made to the sample concentrations determined.

9. Saline Nitrate and Nitrite Standards. If the calibration standard solutions are not prepared to
match sample salinity, then saline nitrate and nitrite standards must be prepared in a series
of salinities in order to quantify the salt error, the change in the colorimetric response of
nitrate due to the change in the composition of the solution. The following dilutions in 100
ml_ reagent water are recommended:
Salinity (%o)
0
9
18
27
35
Low nutrient
seawater (ml_)
0
25
50
75
98
Cone, primary
dilution standard (ml_)
2
2
2
2
2
ma N/L
0.10
0.10
0.10
0.10
0.10

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 31

 2.3.1.5.3      Open Tubular Cadmium Reactor

 For this method, the lab may use either a commercial Open Tubular Cadmium Reactor (OTCR)
 or a laboratory-prepared packed copper-coated cadmium reduction column.  If an OTCR is
 used, it should be activated as follows.

 Prepare reagent water, 0.5N HCI solution and 2% CuSO4 solution in three 50 ml_ beakers.  Fit
 three 10-mL plastic syringes with unions.  First flush the OTCR with 10 ml_ reagent water.  Then
 flush it with 10 mL 0.5N HCI solution for 3 seconds, immediately followed by flushing with a
 couple of syringe volumes of reagent water. Slowly flush with CuSO4 solution until a large
 amount of black precipitated copper comes out.  Finally flush with reagent water with imidazole
 buffer for short term storage.

 2.3.1.5.4      Packed Cadmium Reduction Column

 If a laboratory-prepared packed copper-coated cadmium reduction column is to be used, the
 following procedures should be used.

 1.   File a cadmium stick to obtain freshly prepared cadmium filings.  Sieve the filings and retain
     the fraction between 25 and 60 mesh size (0.25-0.71 mm).  Wash the filings two times with
     10% HCI followed with reagent water.

 2.   Decant the reagent water and add 50 mL of 2%  CuSO4 solution. While swirling, brown
     flakes of colloidal  copper will appear and the blue color of the solution will fade.  Decant the
     faded solution and add fresh CuSO4 solution and swirl. Repeat until the blue color no
     longer fades.
 3.   Wash the filings with reagent water until all the blue color is gone and the supernatant is
     free of fine particles. Keep filings submersed in  reagent water to avoid exposure to air.

 4.   The column can be prepared in a plastic or a glass tube 2mm ID. Plug one end with glass
     wool. Fill the column with water and transfer Cd filings in suspension using a 10 mL pipette
     tip connected to one end of the column.  While gently tapping the tube and pipette tip  let Cd
     filings pack tightly and uniformly in the column without trapping air bubbles.

 5.   Insert another glass wool plug at the top of the column.  If a U-shape tube is used, the
     pipette tip is connected to the other end and the  procedure repeated.  Connect both ends of
     the column  using  a plastic tube filled with buffer solution to form a closed loop.

 6.   If a packed  cadmium column has not been used for several days, it should be reactivated
     prior to sample  analysis.

 2.3.1.5.5      Stabilization of OTCR or Packed Cadmium Reduction Column

 1.   Pump the buffer and other reagent solutions through the manifold and obtain a stable
     baseline.

 2.   Pump 0.7 mg-N/L nitrite standard solution continuously through the sample line and record
     the steady state signal.
 3.   Stop the pump and install the column on the manifold.  Ensure no air bubbles have been
     introduced into the manifold during the installation. Resume the pumping and confirm a
     stable baseline.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 32

4. Pump 0.7 mg-N/L nitrate solution continuously through the sample line and record the
signal. The signal will increase slowly and reach steady state in about 10-15 minutes. This
steady state signal should be close to the signal obtained from the same concentration of a
nitrite solution without the column on line.

5. The reduction efficiency of the column can be determined by measuring the absorbance of
a nitrate standard solution followed by a nitrite standard solution of the same concentration.
Reduction efficiency is calculated as follows:

Reduction efficiency = Absorbance of Nitrate
Absorbance of Nitrite

2.3.1.6 Quality Control

Each laboratory using this method is required to implement a formal quality control (QC)
program. The minimum requirements consist of an initial demonstration of performance,
continued analysis of Laboratory Reagent Blanks (LRB), laboratory duplicates and
Laboratory Fortified Blanks (LFB) with each set of samples.

2.3.1.6.1 Initial Demonstration of Performance

1. The method detection limit (MDL) must be established for the method analyte using a low
level seawater sample containing, or fortified at, approximately 5 times the estimated
detection limit. To determine MDL values, analyze at least seven replicate aliquots or
water which have been processed through the entire analytical method. Calculate the MDL
as follows:

MDL=(t)(S)
where,
S = the standard deviation of the replicate analysis

t = t value for n-1 degrees of freedom at the 99% confidence limit; t = 3.143 for
six degrees of freedom.
2. The linear dynamic range (LDR) must be determined by analyzing a minimum of eight
calibration standards ranging from 0.002 to 2.00 mg N/L across all sensitivity settings
(absorbance units full scale output range setting) of the detector. Standards and sampler
wash solutions should be prepared in low nutrient seawater with salinities similar to the
samples to avoid the necessity to correct for salt error or refractive index. Normalize
responses by multiplying the response by the absorbance units full scale output range
setting. Perform the linear regression of normalized response vs. concentration, and obtain
the constants m and b, where m is the slope and b is the y-intercept. Incrementally analyze
standards of higher concentration until the measured absorbance response (R) of a
standard no longer yields a calculated concentration (Cc) that is within 100 ± 10% of known
concentration (C), where

Cc = (R-b)/m
This concentration defines the upper limit of the LDR. If samples are found to have a
concentration that is > 90% of the upper limit of the LDR, they must be diluted and
reanalyzed.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 33

2.3.1.6.2 Assessing Laboratory Performance

1. Laboratory Reagent Blank (LRB). The lab should analyze at least one LRB with each set
of samples. Should an analyte value in the LRB exceed the MDL, then laboratory or
reagent contamination should be suspected. When the LRB value constitutes 10% or more
of the analyte concentration determined for a sample, duplicates of the sample must be
reprepared and analyzed after the source of contamination has been corrected and
acceptable LRB values have been obtained.
2. Laboratory Fortified Blank (LFB). The lab should analyze at lease one LFB with each set of
samples. The LFB must be at a concentration within the daily calibration range. The LFB
data are used to calculate percent recovery. If the recovery of the analyte falls outside the
required control limits of 90-110%, the source of the problem should be identified and
resolved before continuing the analysis.

Lower Control Limit = x - 3S

Optional control limits must be equal to or better than required control limits of 90-110%.
After each 5 to 10 new recovery measurements, new control limits can be calculated using
only the most recent 20 to 30 data points. Also the standard deviation (S) data should be
used to establish an ongoing precision statement for the level of concentration included in
the LFB. These data must be kept on file and available for review.

2.3.1.6.3 Assessing Analyte Recovery-Laboratory Fortified Sample Matrix (LFM)

1. The laboratory should add a known amount of analyte to a minimum of 5% of the total
number of samples or one LFM per sample set, whichever is greater. The analyte added
should be 2-4 times the ambient concentration and should be at least four times greater
thean the MDL.
2. Calculate percent recovery of anlayte, corrected for background concentration measured in
a separate unfortified sample. These values should be compared with the values obtained
from the LFBs. Percent recoveries may be calculated using the following equation:

R= f Cs - O x100
S
where,

R = percent recovery
Cs = measured fortified sample addition in mg N/L
C = sample background concentration in mg N/L
S = concentration in mg N/L added to the environmental sample

3. If the recovery of the analyte falls outside the required control limits of 90-110%, but the
laboratory performance for that analyte is within the control limits, the fortified sample
should be prepared again and analyzed. If the result is the same after reanalysis, the

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 34

recovery problem encountered with the fortified sample is judged to be the matrix related
and the sample data should be flagged.

2.3.1.7 Calibration and Standardization

1. At least five calibration standards should be prepared fresh daily for system calibration.

3. Analyze the calibration standards in duplicate before the actual samples.

4. The calibration curve containing five data points or more that bracketed the concentration
of samples should have a correlation coefficient (r) of 0.995 or better and the range should
not be greater than two orders of magnitude.
5. Use a high calibration solution followed by two black cups to quantify system carryover.
The difference in peak heights between two blank cups is due to the carryover from the
high calibration solution. The carryover coefficient (k) is calculated as follows:

k = Phi_ • Ph?
Phigh
where,

Phigh = the peak height of the high ammonia standard
Pb1 = the peak height of the first blank sample
Pb2 = the peak height of the second blank sample

The carryover coefficient (k) should be measured in seven replicates to obtain a
statistically significant number. The carryover coefficient should be remeasured with any
change in manifold plumbing or upon replacement of pump tubes.

The carryover correction (CO) of a given peak (i) is proportional to the peak height of the
preceding sample PM.

CO = k x PM

To correct a given peak height reading, (Pi), subtract the carryover correction.

Pi,c=Pi-CO
where PiiC is the corrected peak height. The correction for carryover should be applied to
all the peak heights throughout a run. The carryover coefficient should be less than 5%.

6. Place a high standard nitrate solution followed by a nitrite standard solution of the same
concentration at the beginning and end of each sample run to check for change in reduction
efficiency of the column. The decline of reduction efficiency during a run should be < 5%.

7. Place a high standard solution end of each sample run to check for sensitivity drift. Apply
sensitivity drift correction to all the samples. Sensitivity drift during a run should be < 5%.

Note. Sensitivity drift correction is available in most data acquisition software supplied
with subanalyzers. It is assumed that sensitivity drift is linear with time. An interpolated
drift correction factor is calculated for each sample according to the sample position
during the run. Multiply the sample peak height by the corresponding sensitivity drift
correction factor to obtain the corrected peak height for each sample.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 35

2.3.1.8 Procedure

1. If samples are stored in a refrigerator, equilibrate to room temperature prior to analysis.

2. Turn on the continuous flow analyzer and data acquisition components and warm up for at
least 30 minutes.
3. Set up cartridge according to the type of cadmium reducer used - OTCR Figure 2.2 or
packed cadmium column Figure 2.3. Configuration for analysis of nitrite alone is shown in
Figure 2.4.

Note. When a gas segmented flow stream passes through the OTCR, particles derived from
the column were found to increase baseline noise and to cause interference at low level
analysis. Packed cadmium columns are therefore preferred for nitrite analysis at low
concentrations.
4. Set the spectrophotometer wavelength to 540 nm and turn on lamp.

5. Set the absorbance unit full scale (AUFS) range on the spectrophotometer at the
appropriate setting according to the highest concentration of nitrate in the samples. The
highest setting appropriate for this method is 0.2 AUFS for 0.7 mg N/L.

6. Begin pumping the Brij-35 start-up solution through the system and obtain a steady
baseline. Place the reagents on-line. The reagent baseline will be higher than the start-up
solution baseline. After the reagent baseline has stabilized, reset the baseline.

Note. To minimize the noise in the reagent baseline, clean the flow system by sequentially
pumping the sample line with reagent water, 1 N HCI solution, reagent water, 1 N A/a OH
solution for a few minutes each at the end of the daily analysis. Make sure to rinse the
system well to prevent precipitation ofMg(OH)2 when seawateris introduced into the
system. Keep the reagents and samples free of particulates and filter if necessary.

If the baseline drifts upward, pinch the waste line fora few seconds to increase back
pressure. If absorbance drops down rapidly when back pressure increases, this indicates
that there are air bubbles trapped in the flow cell. Attach a syringe at the waste outlet of
the flowcell. Air bubbles in the flow cell can often be eliminated by simply attaching a
syringe fora few minutes or, if not, dislodged by pumping the syringe piston. Alternatively,
flushing the flowcell with alcohol was found to be effective in removing trapped air.

Note. To minimize noise in the reagent baseline, clean the flow system by sequentially
pumping the sample line with reagent water, 1 N HCI solution, reagent water, 1 N A/a OH
solution for a few minutes each at the end of the daily analysis. Make sure to rinse the
system well to prevent precipitation ofMg(OH)2 when seawateris introduced into the
system. Keep the reagents and samples free of particulates and filter if necessary.

If the baseline drifts upward, pinch the waste line fora few seconds to increase back
pressure. If absorbance drops down rapidly when back pressure increases, it indicates that
there are air bubbles trapped in the flow cell. Attach a syringe at the waste outlet of the
flowcell. Air bubbles in the flow cell can often be eliminated by attaching a syringe for a few
minutes or, if not, dislodged by pumping the syringe piston. Alternatively, flushing the
flowcell with alcohol was found to be effective in removing trapped air.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
            Page 36
r-F
[_
elector!
To Waste
OTCR
Debubbler
-4 	 £. 	 	 v . ' , . . , y
J V
\
\
1

I V


(
3
i o wastes
-(-
~o I
x A—
10 Lr
Q_ ®
8
	 	 pte^
o 7
6
0
_o ,s 	
4
	 7-TZ
-n?
? 0
\j-
1

Manifold
Wash To S
8
/
)
X
)
\
/
\
)

A
0.41
0.41
0.10
0.10
0.32
0.25
1.12
1.57

Pump
mL/min
)

NED
Sulfanilamide
B Sample
	 	
Nitrogen
Buffer
Reagent Water
or Low Nutrient Seawat
Sample:Wasrt = 60":
Figure 2.2. Manifold Configuration for the Nitrate + Nitrite Analysis
using an Open Tubular Cadmium Reactor



Debubbler
n y

l_
elector!
To Waste
Cd
Column
J X
>

'(
(

|
o— -
\J
S



C

r
6 o
o. 5
4
r
2 f

r
y-
Manifold
Wash To S

TO Wasted"
-£.
) ,

) f
\
} ^
/ \


0.41
0.41
0.10
0.10
025
0.32
1.12
1.57

Pump
mL/min
J




r
NED
Sulfanilamide
Nitrogen
~| Sample
i — i
Buffer
Reagent Water
or Low Nutrient Seawa
Sample:Wash = 60":
Figure 2.3. Manifold Configuration for Nitrate + Nitrite Analysis using a Laboratory Packed Copper-
coated Cadmium Reduction

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 37
Debubbler
stectorL
540nnQ g
f E
To Waste /"

T.
8
0 "*
6
0
0 s
4
-/•s —
0 >
2 0=
1
Manifold
Wash To S

t /

) ;

*"***
"N
0.41
0.10
0.10
0.25
1,01
1,57

Pump
mL/min
)
NED
Sulfanilamide
Air
Sample
Reagent Water
or Low Nutnent Seawat
Sample: Wash = 60":
Figure 2.4. Manifold Configuration for Nitrite Analysis
8. Check the reduction efficiency of the OTCR or packed cadmium column. If the reduction
efficiency is less than 90%, reactivate and restabilize. Ensure reduction efficiencies reach at
least 90% before analyzing samples.

9. The sampling rate is approximately 60 samples per hour with 30 second of sample time and
30 seconds of wash time. Place a blank after every ten samples.

2.3.1.9 Data Analysis and Calibration

Concentrations of nitrate in samples are calculated from the linear regression, obtained from
the standard curve in which the concentration of the calibration standards are entered as the
independent variable and their corresponding peak heights are the dependent variable.
2.3.1.9.1
Refractive Index Correction
1. If reagent water is used as the wash solution, the analyst has to quantify the refractive index
correction due to the difference in salinity between sample and wash solution. The following
procedures are used to measure the relationship between the sample salinity and the
refractive index on a particular detector.

2. Analyze a set of nitrate or nitrite standards in reagent water with color reagent using reagent
water as the wash and obtain a linear regression of peak height vs concentration. Then
replace reagent water wash solution with low nutrient seawater wash solution.

Note. The change in absorbance due to refractive index is small therefore low concentration
standards should be used to bracket the expected absorbances due to refractive index.

Note. In nitrate and nitrite analysis absorbance of the reagent water is higher than that of the
low nutrient seawater. When using reagent water as a wash solution, the change in
refractive index causes the absorbance of seawater to become negative. To measure the
absorbance due to refractive index change in different salinity samples, low nutrient
seawater must be used as the wash solution to bring the baseline down.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 38

3. Replace the NED solution with reagent water. All other reagents remain the same.
Replace the synchronization sample with the colored SYNC peak solution.
4. Prepare a series of different salinity samples by diluting the low nutrient seawater.
Commence analysis and obtain peak heights for different salinity samples. The peak
heights for the refractive index correction must be obtained at the same AUFS range
setting and on the same spectrophotometer as the corresponding standards.

6. For each sample of varying salinity, calculate the apparent nitrate or nitrite concentration
due to refractive index from its peak height corrected to the reagent water baseline and the
regression equation of ammonia standards obtained with color reagent being pumped
through the system. Salinity is entered as the dependent variable and the apparent nitrate
or nitrite concentration due to refractive index is entered a the dependent variable. The
resulting regression allows the analyst to calculate apparent nitrate or nitrite concentration
due to refractive index when sample salinity is known. Thus, the analyst would not be
required to obtain refractive index peak heights for all samples.

Apparent nitrate (urn N/L) = 0.01047(5)
Apparent nitrite (urn N/L) = 0.00513 (S)
where S is the sample salinity in parts per thousand.
The apparent nitrate and nitrite concentration due to refractive index so obtained should
then be added to samples of corresponding salinity when reagent water was used as the
wash solution for sample analysis.

If nitrate and nitrite concentrations are greater than 100 and 50 urn N/L, respectively, the
correction for refractive index is negligible and this procedure can be optional.

2.3.1.9.2 Salt Error Correction

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 39

all saline standards. The resulting regression equation allows the analyst to correct the
concentrations of samples of known salinity for color enhancement due to salt error.
3. Results of sample analysis should be reported in mg N/L (ppm) or in ug N/L (ppb).

2.3.2 FRESHWATER

2.3.2.1 Scope and Application

1. This method covers the determination of nitrite singly, or nitrite and nitrate combined in
freshwater samples.

2. The applicable range is 0.05-10.0 mg/L nitrate-nitrite nitrogen. The range may be extended
with sample dilution.

2.3.2.2 Summary of Method

1. A filtered sample is passed through a column containing granulated copper cadmium to
reduce nitrate to nitrite. The nitrite (that was originally present plus reduced nitrate) is
determined by diazotizing with sulfanilamide and coupling with N-(l-naphthyl)-
ethylenediamine dihydrochloride to form a highly colored azo dye which is measured
colorimetrically. Separate (not combined nitrate-nitrite) values are readily obtained by
carrying out the procedure first with, and then without, the Cu-Cd reduction step.

2. Reduced volume versions of this method that use the same reagents and molar ratios are
acceptable provided they meet the quality control and performance requirements stated in
the method.

3. Limited performance-based method modifications may be acceptable provided they are
fully documented and meet or exceed requirements expressed in the NCCA QAPP.

2.3.2.3 Interferences

1. Build up of suspended matter in the reduction column will restrict sample flow. Since nitrate
and nitrite are found in a soluble state, samples may be pre-filtered.

2. Low results might be obtained for samples that contain high concentrations of iron, copper
or other metals. EDTA is added to the samples to eliminate this interference.
3. Residual chlorine can produce a negative interference by limiting reduction efficiency.
Before analysis, samples should be checked and if required, dechlorinated with sodium
thiosulfate.
4. Samples that contain large concentrations of oil and grease will coat the surface of the
cadmium. This interference is eliminated by pre-extracting the sample with an organic
solvent.

5. Method interferences may be caused by contaminants in the reagent water, reagents,
glassware, and other sample processing apparatus that bias analyte response.

2.3.2.4 Safety

1. The toxicity or carcinogenicity of each reagent used in this method has not been fully
established. Each chemical should be regarded as a potential health hazard and exposure

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 40

should be as low as reasonably achievable. Cautions are included for known extremely
hazardous materials or procedures.
2. Each laboratory is responsible for maintaining a current awareness file of OSHA
regulations regarding the safe handling of the chemicals specified in this method. A
reference file of Material Safety Data Sheets (MSDS) should be made available to all
personnel involved in the chemical analysis. The preparation of a formal safety plan is also
advisable.

3. The following chemicals have the potential to be highly toxic or hazardous, consult MSDS.
- Cadmium - Sulfuric acid
- Phosphoric acid - Chloroform
- Hydrochloric acid

2.3.2.5 Equipment and Supplies

1. Balance -- Analytical, capable of accurately weighing to the nearest 0.0001 g.
2. Glassware -- Class A volumetric flasks and pipets as required.
3. Automated continuous flow analysis equipment designed to deliver sample and reagents
in the required order and ratios.

- Sampling device (sampler) - Colorimetric detector
- Multichannel pump - Data recording device
- Reaction unit or manifold

2.3.2.6 Reagents and Standards

1. Granulated cadmium: 40-60 mesh. Other mesh sizes may be used.
2. Copper-cadmium: The cadmium granules (new or used) are cleaned with dilute HCI and
copperized with 2% solution of copper sulfate in the following manner:

a. Wash the cadmium with HCI (Section 7.6) and rinse with distilled water. The color of
the cadmium so treated should be silver.
b. Swirl 10 g cadmium in 100 ml_ portions of 2% solution of copper sulfate for five
minutes or until blue color partially fades, decant and repeat with fresh copper sulfate
until a brown colloidal precipitate forms.
c. Wash the copper-cadmium with reagent water (at least 10 times) to remove all the
precipitated copper. The color of the cadmium so treated should be black.

3. Preparation of reduction column. The reduction column is a U-shaped, 35 cm length, 2 mm
I.D. glass tube (see Note). Fill the reduction column with distilled water to prevent
entrapment of air bubbles during the filling operations. Transfer the copper-cadmium
granules to the reduction column and place a glass wool plug in each end. To prevent
entrapment of air bubbles in the reduction column, be sure that all pump tubes are filled with
reagents before putting the column into the analytical system.

Note: Other reduction tube configurations, including a 0.081 I.D. pump tube, can be used in
place of the 2 mm glass tube, if checked as in.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 41

4. Reagent water: Because of possible contamination, this should be prepared by passage
through an ion exchange column comprised of a mixture of both strongly acidic-cation and
strongly basic-anion exchange resins. The regeneration of the ion exchange column
should be carried out according to the manufacturer's instructions.

5. Color reagent: To approximately 800 ml_ of reagent water, add, while stirring, 100 mL
cone, phosphoric acid, 40 g sulfanilamide and 2 g N-1-naphthylethylenediamine
dihydrochloride. Stir until dissolved and dilute to 1 L. Store in brown bottle and keep in the
dark when not in use. This solution is stable for several months.
6. Dilute hydrochloric acid, 6N: Add 50 ml_ of cone. HCI to reagent water, cool, and dilute to
100ml_.
7. Copper sulfate solution, 2%: Dissolve 20 g of CuSO4C5H2O in 500 ml_ of reagent water
and dilute to 1 L.

8. Wash solution: Use reagent water for unpreserved samples. For samples preserved with
H2SO4, use 2 ml_ H2SO4 per liter of washwater.

9. Ammonium chloride-EDTA solution: Dissolve 85 g of reagent grade ammonium chloride
and 0.1 g of disodium ethylenediamine tetracetate in 900 mL of reagent water. Adjust the
pH to 9.1 for preserved or 8.5 for non-preserved samples with cone, ammonium hydroxide
and dilute to 1 L. Add 0.5 mL Brij-35 (CASRN 9002-92-0).

10. Stock nitrate solution: Dissolve 7.218 g KNO3 and dilute to 1 L in a volumetric flask with
reagent water. Preserve with 2 mL of chloroform per liter. Solution is stable for six months.
1 mL= 1.0 mg NO3-N.
11. Stock nitrite solution: Dissolve 6.072 g KNO2 in 500 mL of reagent water and dilute to 1 L
in a volumetric flask. Preserve with 2 mL of chloroform and keep under refrigeration. 1.0
mL= 1.0 mg NO2-N.

12. Standard nitrate solution: Dilute 1.0 mL of stock nitrate solution to 100 mL. 1.0 mL = 0.01
mg NO3-N. Preserve with .2 mL of chloroform. Solution is stable for six months.

13. Standard nitrite solution: Dilute 10.0 mL of stock nitrite) solution to 1000 mL. 1.0 mL = 0.01
mg NO2-N. Solution is unstable; prepare as required.

2.3.2.7 Sample Collection, Preservation and Storage

1. Samples are collected in plastic or glass bottles. All bottles are thoroughly cleaned and
rinsed with reagent water. Volume collected must be sufficient to insure a representative
sample, allow for replicate analysis.

2. Samples should be analyzed as soon as possible after collection. If storage is required,
preserved samples are maintained at 4°C and may be held for up to 28 days.

2.3.2.8 Quality Control

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 42

2.3.2.8.1 Initial Demonstration of Performance

1. The initial demonstration of performance is used to characterize instrument performance
(determination of LCRs and analysis of QCS) and laboratory performance (determination of
MDLs) prior to performing analyses by this method.
2. Linear Calibration Range (LCR) - The LCR must be determined initially and verified every
six months or whenever a significant change in instrument response is observed or
expected. The initial demonstration of linearity must use sufficient standards to insure that
the resulting curve is linear. The verification of linearity must use a minimum of a blank and
three standards. If any verification data exceeds the initial values by ± 10%, linearity must
be reestablished. If any portion of the range is shown to be nonlinear, sufficient standards
must be used to clearly define the nonlinear portion.
3. Quality Control Sample (QCS) -- When beginning the use of this method (on a quarterly
basis or as required to meet data-quality needs) verify the calibration standards and
acceptable instrument performance with the preparation and analyses of a QCS. If the
determined concentrations are not within ±10% of the stated values, performance of the
determinative step of the method is unacceptable. The source of the problem must be
identified and corrected before either proceeding with the initial determination of MDLs or
continuing with on-going analyses.

MDL=(t)x(S)
where,

t = value for a 99% confidence level and a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates]
S = standard deviation of the replicate analyses

MDLs should be determined every six months, when a new operator begins work or
whenever there is a significant change in the background or instrument response.

2.3.2.8.2 Assessing Laboratory Performance

1. Laboratory Reagent Blank (LRB) ~ The laboratory must analyze at least one LRB with each
batch of samples. Data produced are used to assess contamination from the laboratory
environment. Values that exceed the MDL indicate laboratory or reagent contamination
should be suspected and corrective actions must be taken before continuing the analysis.

2. Laboratory Fortified Blank (LFB) - The laboratory must analyze at least one LFB with each
batch of samples. Calculate accuracy as percent recovery. If the recovery of any analyte
falls outside the required control limits of 90-110%, that analyte is judged out of control, and
the source of the problem should be identified and resolved before continuing analyses.
3. The laboratory must use LFB analyses data to assess laboratory performance against the
required control limits of 90-110%. When sufficient internal performance data become

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 43

available (usually a minimum of 20-30 analyses), optional control limits can be developed
from the percent mean recovery (x) and the standard deviation (S) of the mean recovery.
These data can be used to establish the upper and lower control limits as follows:

Upper Control Limit = x + 3S
Lower Control Limit = x - 3S

The optional control limits must be equal to or better than the required control limits of 90-
110%. After each 5-10 new recovery measurements, new control limits can be calculated
using only the most recent 20-30 data points. Also, the standard deviation (S) data should
be used to establish an on-going precision statement for the level of concentrations included
in the LFB. These data must be kept on file and be available for review.

4. Instrument Performance Check Solution (IPC) - For all determinations the laboratory must
analyze the IPC (a mid-range check standard) and a calibration blank immediately following
daily calibration, after every 10th sample (or more frequently, if required) and at the end of
the sample run. Analysis of the IPC solution and calibration blank immediately following
calibration must verify that the instrument is within ±10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is still within ±10%. If the calibration
cannot be verified within the specified limits, reanalyze the IPC solution. If the second
analysis of the IPC solution confirms calibration to be outside the limits, sample analysis
must be discontinued, the cause determined and/or in the case of drift, the instrument
recalibrated. All samples following the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must be kept on file with the sample
analyses data.

2.3.2.8.3 Assessing Analyte Recovery and Data Quality

1. Laboratory Fortified Sample Matrix (LFM) ~ The laboratory must add a known amount of
analyte to a minimum of 10% of the routine samples. In each case the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The analyte concentration must be high
enough to be detected above the original sample and should not be less than four times the
MDL. The added analyte concentration should be the same as that used in the laboratory
fortified blank.

R = Cs - C x 100
s
where,
R = percent recovery
Cs = fortified sample concentration
C = sample background concentration
s = concentration equivalent of analyte added to sample

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 44

2.3.2.9 Calibration and Standardization

1. Prepare a series of at least three standards, covering the desired range, and a blank by
diluting suitable volumes of the standard nitrate solution. At least one nitrite standard should
be compared to a nitrate standard at the same concentration to verify the efficiency of the
reduction column.

2. Set up manifold. Care should be taken not to introduce air into the reduction column.

3. Place appropriate standards in the sampler in order of decreasing concentration and
perform analysis.

4. Prepare standard curve by plotting instrument response against concentration values. A
calibration curve may be fitted to the calibration solutions concentration/response data using
computer or calculator based regression curve fitting techniques. Acceptance or control
limits should be established using the difference between the measured value of the
calibration solution and the "true value" concentration.
5. After the calibration has been established, it must be verified by the analysis of a suitable
quality control sample (QCS). If measurements exceed ±10% of the established QCS value,
the analysis should be terminated and the instrument recalibrated. The new calibration must
be verified before continuing analysis. Periodic reanalysis of the QCS is recommended as a
continuing calibration check.

Note: Condition column by running 1 mg/L standard for 10 minutes if a new reduction column is
being used. Subsequently wash the column with reagents for 20 minutes.

2.3.2.10 Procedure

1. If the pH of the sample is below 5 or above 9, adjust to between 5 and 9 with either cone.
HCIorconc. NH4OH.

2. Allow system to equilibrate as required. Obtain a stable baseline with all reagents, feeding
reagent water through the sample line.

3. Place appropriate nitrate and/or nitrite standards in sampler in order of decreasing
concentration and complete loading of sampler tray.

4. Switch sample line to sampler and start analysis.

2.3.2.11 Data Analysis and Calculations

2. Report only those values that fall between the lowest and the highest calibration standards.
Samples exceeding the highest standard should be diluted and reanalyzed.
3. Report results in mg/L as nitrogen.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
_ Page 45

 2.4    TOTAL NITROGEN AND PHOSPHORUS

 2.4.1  Scope and Application

 These methods are intended for determination of total nitrogen (organic nitrogen + ammonium +
 nitrate + nitrite) and phosphorus (all forms) in filtered and whole-water samples by alkaline
 persulfate digestion. They were validated for determination of total nitrogen and total
 phosphorus in drinking water, wastewater, and water-suspended sediment. Their applicability to
 bottom materials was not investigated. Analytical ranges are 0.03 to 5.00 mg-N/L for dissolved
 and total nitrogen and 0.01 to 2.00 mg-P/L for dissolved and total phosphorus.

 2.4.2  Method Summary and Analytical Considerations
 Filtered and whole-water samples are dispensed into glass culture tubes, dosed with alkaline
 persulfate reagent, capped tightly, and digested  in an autoclave at250°F (121°C) and 17 Ib/in2
 (1 17.2 kPa) for 1 hour. The alkaline persulfate digestion procedure oxidizes all forms of
 inorganic and organic nitrogen to nitrate and hydrolyzes all forms of inorganic and organic
 phosphorus to orthophosphate. Nitrate and orthophosphate in alkaline persulfate digests are
 determined in parallel with a 2-channel photometric, air-segmented continuous flow analyzer.

 Digest preparation protocols and reagent formulations were adapted from previously published
 procedures (Valderrama, 1981; Hosomi and Sudo, 1986;  Ameel and others, 1993; D'Elia and
 others, 1997; American Public Health Association, 1998b). Two other reports (Nydahl, 1978;
 Cabrera and Beare, 1993) provided insight into the potential for low nitrogen recovery in
 samples containing high concentrations of dissolved and  particulate organic carbon.

 Quantitative recovery of nitrogen and phosphorus by alkaline persulfate digestion depends
 critically on a progressive  decrease in pH (initial  pH  >12, final pH < 2.2)  during the 1-hour
 course of the digestion (Hosomi and Sudo, 1986). These  dynamic reaction  conditions are
 achieved by formulating the digestion reagent with approximately equimolar concentrations of
 persulfate and hydroxide ions — 0.05 M, initial pH >12 after 1+2 dilution by samples in this
 method. Under these initially alkaline conditions, dissolved and suspended  nitrogen in samples
 oxidize to nitrate. As the digestion proceeds, bisulfate ions resulting from thermal decomposition
 of persulfate first neutralize and then acidify the reaction mixture by the  following chemical
 reaction:
 After all of the persulfate has decomposed, the digest mixture pH approaches 2, and under
 these acidic conditions, dissolved and suspended phosphorus hydrolyze to orthophosphate.
 The foregoing discussion indicates that analysis of samples with variable and unknown acidity
 or alkalinity by alkaline persulfate digestion methods will be problematic. Users of this method
 are cautioned that amending FCA and WCA samples with concentrations of sulfuric acid other
 than those specified in USGS field manual protocols (Wilde and others, 1998) likely will result in
 undetected method failure and possible reporting of erroneous results.
 As is the case for Kjeldahl digestion, alkaline persulfate digestion converts all forms of
 phosphorus to orthophosphate. Thus alkaline persulfate digestion dissolved and total
 phosphorus (DPAIkP and TPAIkP) concentrations can be compared directly with Kjeldahl
 digestion dissolved and total phosphorus (KDP and KTP) concentrations by graphical and
 statistical analysis. This  is not the case, however, for Kjeldahl dissolved and total nitrogen (KDN
 and KTN) concentrations and alkaline persulfate digestion dissolved and total nitrogen (DNAIkP

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 46

and TNAIkP) concentrations. In principle, organic nitrogen, but not nitrate or nitrite, is reduced to
ammonium during Kjeldahl digestion. Determining ammonium in Kjeldahl digests, therefore,
measures organic nitrogen + ammonium. Alkaline persulfate digestion oxidizes all forms of
nitrogen to nitrate. Determining nitrate + nitrite in alkaline persulfate digests, therefore,
measures total nitrogen (organic nitrogen + ammonium + nitrite + nitrate). To reconcile this
difference between the two methods, nitrate + nitrite concentrations were subtracted from
DNAIkP and TNAIkP concentrations prior to graphical and statistical comparisons with KDN and
KTN concentrations throughout this report. For this purpose and as a quality-control (QC)
check, all filtered and whole-water samples selected for alkaline persulfate digestion also were
analyzed for dissolved nitrate + nitrite, ammonium, and orthophosphate on the same day that
digests were prepared. Particulates were removed from acidified, whole-water samples (WCA
bottle type) by 0.45-um filtration prior to dissolved nutrient determinations.
A 2-channel, air-segmented continuous flow analyzer was configured for simultaneous
photometric determination of nitrate + nitrite and orthophosphate in alkaline persulfate digests.
Nitrate + nitrite was determined by a cadmium-reduction, Griess-reaction method (Wood and
others, 1967) equivalent to U.S. Environmental Protection Agency (USEPA) method 353.2 (U.S.
Environmental Protection Agency, 1993) and U.S. Geological Survey (USGS) method I-2545-90
(Fishman, 1993, p. 157) except that sulfanilamide and N-(1-naphthy)ethylenediamine reagents
were separate rather than combined. The analytical cartridge diagram is shown in figure 1.
Orthophosphate was determined by a phosphoantimonylmolybdenum blue method (Murphy and
Riley, 1962; Pai and others, 1990), which is equivalent to the 2-reagent variants (separate
molybdate and ascorbic acid reagents) of USEPA method 365.1 (U.S. Environmental Protection
Agency, 1993) and USGS method 1-2601-90 (Fishman, 1993). The analytical cartridge diagram
is shown in Figure 2.

2.4.3 Interferences
2.4.3.1 Alkaline Persulfate Digestion

1. Chloride concentrations up to 1,000 mg/L (the highest tested for this report) do not interfere.
Furthermore, because good results are obtained for seawater in 2 + 1 mixture with digestion
reagent (D'Elia and others, 1997), chloride concentrations of about 10,000 mg/L apparently
are tolerated provided that calibrants are matrix matched. Higher chloride concentrations,
however, are likely to interfere because of reaction with persulfate to form oxychlorides or
chlorine that might deplete persulfate required to oxidize inorganic and organic nitrogen
species to nitrate. Resulting active chlorine species also can interfere in colorimetric
reactions used to determine nitrate and orthophosphate in digests.
2. Sulfate concentrations up to 1,000 mg/L (the highest tested for this report) do not interfere.

3. Organic carbon concentrations greater than 150 mg/L interfere because of reaction with
persulfate to form carbon dioxide, thus depleting persulfate required to oxidize inorganic and
organic nitrogen species to nitrate.
4. Overacidification of FCA and WCA samples at collection sites can result in low recovery of
inorganic and organic nitrogen at the NWQL. The possibility of Overacidification can be
avoided by exclusive use of the sulfuric acid field-amendment solution—one vial containing
1 mL of 4.5 N H2SO4 (One Stop Shopping number FLD-438) per 120 mL of sample—which
is specified in the USGS National Field Manual (Wilde and others, 1998).

5. Nitrate and nitrite do not contribute to KDN and KTN concentrations in principle, but in
practice, positive and negative interferences by these ions are well known—see, for
example, American Public Health Association, 1998c; Patton and Truitt, 2000. This

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 47
   interference can confound comparison of KN and NAIkP concentrations when dissolved
   nitrate concentrations are greater than about 0.1 mg NO3-N/L.
6.  Suspended particles remaining in digests must be removed by sedimentation and
   decantation or filtration prior to colorimetric analyses.

2.4.3.2Colorimetric Nitrate + Nitrite Determination
1.  Typically, concentrations of substances with potential to interfere in cadmium-reduction,
   Griess-reaction nitrate + nitrite methods are negligible in ambient surface- and ground-water
   samples. For specific details of inorganic and organic compounds that might interfere in the
   color reaction, see Norwitz and Keliher (1985, 1986), as well as more general information by
   the American Public Health Association (1998a).

2.  Sulfides, which are often present in anoxic water and well known to deactivate cadmium
   reduction reactors, are oxidized during the alkaline persulfate digestion and are  unlikely to
   interfere.

2.4.3.3 Colorimetric Orthophosphate Determination
1.  Barium,  lead, and silver can interfere by forming insoluble phosphates, but their
   concentrations in natural-water samples usually are  less than the interference threshold
   (Fishman, 1993).

2.  Interference from silicate, which also can form reduced heteropoly  acids with molybdenum
   (Zhang and others, 1999), is negligible under reaction conditions used for this report.

3.  Arsenate, AsO43, but not arsenite, AsO33, can interfere by forming reduced heteropoly
   acids analogous to those formed by Orthophosphate (Johnson, 1971). Because  of the
   possibility that arsenite might be oxidized to arsenate by persulfate, both  species at
   concentrations up to 20 mg-As/L in deionized water were digested  and analyzed. With
   reference to Table 2, it is apparent that a major fraction of arsenite is oxidized to arsenate
   during alkaline persulfate digestion and that interference by either species up to 1  mg-As/L
   is negligible.

Table 2. Data from a study of arsenate and  arsenite interference in alkaline persulfate total phosphorus
determinations [mg-As/L, milligrams of arsenic per liter; mg-P/L, milligrams of phosphorus per liter; nd, not
detected; =, nearly equal to; ±, plus or minus]
AsO43" added
mg-As/L
0.5
1.0
2.0
5.0
10.0
20.0
PO43" found
mg-P/L
nd
nd
= 0.05
0.32 ±0.01
1.14±0.13
Off scale
AsO33" added
mg-As/L
0.5
1.0
2.0
5.0
10.0
20.0
PO43" found
mg-P/L
nd
nd
nd
0.29 ±0.04
0.91 ±0.06
Off scale

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 48

2.4.4 Instrumentation and Auxiliary Analyses

1. RFA-300™, third-generation, air-segmented continuous flow analyzers (Alpkem) were used
to automate photometric determination of nitrate + nitrite and orthophosphate in alkaline
persulfate digests and dissolved ammonium, nitrate + nitrite, and orthophosphate in filtered-
and whole-water samples prior to digestion. Modules in these systems include 301
samplers, 302 peristaltic pumps, 313 analytical cartridge bases, 314 power modules, 305A
photometers, and a personal computer (PC)-based data acquisition and processing system.
Alternative instrumentation—flow injection analyzers, sequential injection analyzers, other
second- or third-generation continuous flow analyzers, or automated batch analyzers—also
could be used to automate photometric finishes.

2. Photometric data were acquired and processed automatically using FASPac™ version 1.34
software (Astoria-Pacific, Clackamas, Ore.). This software operates under Microsoft
Windows on a PC platform and includes a model 350 interface box that controls the sampler
and digitizes analog photometer outputs with 16-bit resolution. Other data acquisition
systems could be used provided that the A/D converter has 16-bit resolution and is capable
of acquiring data at frequencies ranging from 0.5 to 2 Hz, that is, from 30 points/min to 120
points/min. As a general rule, data acquisition frequencies for air-segmented continuous
flow analyzers should match the roller lift-off frequency of the peristaltic pump (Patton and
Wade, 1997), that is, 0.5 Hz forTechnicon AutoAnalyzer II™ and 1.5 Hz for Alpkem RFA-
300 equipment. Data acquisition frequencies in the range of 2 to 5 Hz are suitable for
photometric flow-injection analyzers.

3. Operating characteristics for this equipment are listed in Table 3.
4. Dissolved ammonium, nitrate + nitrite, and orthophosphate in undigested samples were
determined photometrically by USGS automated continuous flow methods I-2522-90, I-
2545-90 (2-reagent variant), and 1-2601-90 (2-reagent variant), respectively. These methods
are described in Fishman (1993).

5. The pH of WCA samples was estimated with narrow range (0-2.5) colorimetric pH-indicating
test strips to detect improperly acidified samples that had pH values outside the expected
range of 1.6 to 1.9.
6. WCA samples were processed through 5-mL capacity UniPrep™ syringeless filters
equipped with 0.45-um nylon membranes (Whatman, Clifton, N.J.) to remove suspended
solids prior to determination of dissolved ammonium, nitrate + nitrite, and orthophosphate.
These syringeless filters also were used to remove suspended solids from WCA-sample
digests prior to photometric analysis when simple sedimentation and decantation into
analyzer cups failed to do so.

2.4.5 Apparatus

1. Samples were digested in an autoclave (model number STME, Market Forge Industries,
Inc., Everett, Mass.) operated at250°F (121°C) and 17 Ib/in2 (117.2 kPa) for 1 hour.
2. Filtered and chilled sample (FCC bottle type) digests were prepared robotically using a
large-scale, syringe-pump-based x-y-z sample dispenser/diluter module (model number ML-
4200, Hamilton Company, Reno, Nev.). This system is equipped with four probes and four
10-mL syringe pumps that operate in tandem under control of DOS-based Eclipse™
software (Hamilton Company, Reno, Nev.). Custom modifications to the ML-4200 system,
including a pneumatically actuated probe expander, fixtures, and a variety of bottle and test-
tube racks, were obtained from another vendor (Robotics Plus, Houston, Tex.).

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 49
3.  Whole-water (WCA bottle type) sample digests were prepared manually using EDP Plus™
   electronic, digital pipets (Rainin Instruments) equipped with a 10-mL liquid end.
4.  Digestion vessels were 20 x 150  mm Pyrex®, screw-cap culture tubes (VWR 53283-810;
   Fisher 14-957-76E or 14-959-37C; or equivalent), and 18-415 linerless polypropylene caps
   (Comair Glass, Inc., Vineland, N.J.—Part number 14-0441-004).
Table 3. Settings and operational details of Alpkem RFA-300 continuous flow analyzers used for this
study [nm, nanometer; mm, millimeter; mg-N/L, milligrams nitrogen per liter; mg-P/L, milligrams
phosphorus per liter; =, nearly equal to; min, minute; ml, milliliter; -, not applicable; °C, degrees Celsius;
s, second; h, hour]
Instrumental conditions
Analytical wavelength
Flow cell path length
Calibration range
Standard calibration control setting
Segmentation rate (bubbles min-1)
Heated reaction coil volume
Heated reaction coil temperature
Dwell time (seconds)
Sample time (volume)
Wash time (volume)
Analysis rate, sample-to-wash ratio
Nitrate + nitrite
540 nm
10 mm
0.05 to 5.0 mg-N/L
=1.1
90
None used
-
140
25 s (95 uL)
10s(38uL)
=103/h,5:2
Orthophosphate
880 nm
15 mm
0.01 to 2.0 mg-P/L
=1.5
90
2mL
37°C
260
25 s (31 uL)
10s (12 ML)
=103/h, 5:2
2.4.6   Reagents
This section provides detailed instructions for preparing digestion and colorimetric reagents. All
references to deionized water (Dl) refer to NWQL in-house Dl water, which is equivalent to
ASTM type I Dl water (American Society for Testing and Materials, 2001, p. 107-109) for
nutrient analysis. All volumetric glassware and reagent and calibrant storage containers should
be triple rinsed with dilute (~5 percent v/v) hydrochloric acid and Dl water just prior to use.
Additionally storage containers for reagents and calibrants should be triple rinsed with small
portions of the solutions before they are filled.

2.4.6.1 Digestion Reagents

NOTE: The alkaline persulfate digestion reagent for FCA and WCA samples contains an
additional amount of sodium hydroxide that is calculated to neutralize the sulfuric acid added to
these samples at collection sites.

1.   Sodium hydroxide, 1.5M (for FCC samples): Dissolve 60 g of sodium hydroxide (NaOH,
    FW=40.0) in about 800 mL of Dl water in a 1-L volumetric flask. [Caution: When NaOH
    dissolves in water, heat is released.] After dissolution is complete, allow the resulting
    solution to cool and dilute it to the mark with Dl water. Transfer this reagent to a plastic
    bottle in which it is stable at room temperature for 6 months.

2.   Sodium hydroxide, 2.3 M (for FCA and WCA samples): Dissolve 92 g of sodium hydroxide
    (NaOH, FW=40.0) in about 800 mL of Dl water in a 1-L volumetric flask. After dissolution is

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 50

complete, allow the resulting solution to cool and dilute it to the mark with Dl water. Transfer
this reagent to a plastic bottle in which it is stable at room temperature for 6 months.
3. Alkaline persulfate digestion reagent (for FCC samples): Add 18.0 g of potassium persulfate
(K2S2O8, FW=270.33) and 45 ml_ of 1.5 M sodium hydroxide solution to about 350 ml_ of Dl
water in a graduated 500-mL Pyrex™ media bottle (Corning number 1395-500 or
equivalent). Cap the bottle, swirl its contents, and place it in an ultrasonic bath until
potassium persulfate dissolution is complete (about 10 minutes). Remove the bottle from the
ultrasonic bath, dry its outer surfaces, and then add enough Dl water to bring the volume to
450 ml_. (Make a line on the side of the bottle that indicates this volume to within ±5 ml_.)
Swirl the bottle to mix its contents and then divide the resulting solution among four, 125-mL
clear plastic bottles used with the robotic digest preparation system. Prepare this reagent
daily.
4. Alkaline persulfate digestion reagent (forFCA and WCA samples): Add 18.0 g of potassium
persulfate (K2S2O8, FW=270.33) and 45 ml_ of 2.3 M sodium hydroxide solution to about
350 ml_ of Dl water in a graduated 500-mL Pyrex™ media bottle (Corning number 1395-500
or equivalent). Then complete preparation of this reagent exactly as described. Prepare this
reagent daily.

NOTE: Reagent volumes in (450 ml_) are sufficient to prepare 80 digests plus a 15-percent
excess for rinsing and providing a liquid level in the 125-mL bottles necessary to prevent air
aspiration during robotic dispensing operations. For manual digest preparation, a 400-mL
volume of digestion reagent should be sufficient.

2.4.6.2 Colorimetric Reagents
Sampler wash reservoir solution (0.05 M sodium bisulfate): Dissolve 6.9 g of sodium bisulfate
(NaHSO4»H2O, FW=138.08) in about 800 mL of Dl water in a graduated 1-L Pyrex™ media
bottle. Dilute this solution to the mark with Dl water, mix it well, and store it tightly capped at
room temperature.

NOTE: This solution matches the matrix of sample digests. Use it as the matrix for continuing
calibration verification (CCV) solutions and any other undigested check samples.

2.4.6.3 Orthophosphate Determination

1. Stock potassium antimony tartrate reagent: Dissolve 3.0 g of antimony potassium tartrate
[K(SbO)C4H4O7«1/2 H2O, FW=333.93] in about 800 mL of Dl water in a 1-L volumetric flask.
Dilute this solution to the mark with Dl water and mix it well. Transfer this reagent to a plastic
bottle in which it is stable for 6 months at room temperature.

2. Stock ascorbic acid reagent: Dissolve 4.5 g of ascorbic acid (C6H8O6, FW=176.1) in about
200 mL of Dl water in a 250-mL volumetric flask. Dilute this solution to the mark with Dl
water, mix it well, and transfer to a 250-mL glass bottle that has been previously rinsed with
5 percent (v/v) hydrochloric acid solution and Dl water. This reagent is stable for 2 weeks at
4°C.
3. Stock sodium lauryl sulfate reagent (15 percent w/w): Add 340 mL of Dl water to 60 g of
sodium lauryl sulfate [SLS, CH3(CH2)11OSO3Na, FW=288.38] in a 500-mL Pyrex™ media
bottle. Cap the bottle and place it in an ultrasonic bath until the SLS dissolves completely
(about 30 minutes). Manual inversion of the bottle at 5-minute intervals speeds dissolution.
Transfer this solution to a plastic bottle in which it is stable indefinitely at room temperature.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 51

4. Acidic molybdate-antimony reagent Using a graduated cylinder, cautiously add 72 ml_ of
concentrated sulfuric acid (H2SO4, sp. gr. 1.84) to about 700 ml_ of Dl water in a 1-L
volumetric flask. Work in a hood and manually swirl or magnetically stir the flask during each
addition of sulfuric acid. Next add 7.7 g of ammonium molybdate [(NH4)6Mo7O24»4H2O,
FW=1235.86] to the hot sulfuric acid solution. Manually swirl or magnetically stir the
contents of the flask until the ammonium molybdate dissolves. Then add 50 ml_ of stock
antimony potassium tartrate solution and again mix the contents of the flask thoroughly.
After the resulting solution has cooled, dilute it to the mark with Dl water, mix it well, and
transfer it to a clean 1-L plastic bottle in which it is stable for 1 year at room temperature.

5. Sodium lauryl sulfate diluent reagent: Use a 100-mL graduated cylinder to dispense 10 mL
of stock SLS and 90 mL of Dl water into a small plastic bottle. Manually swirl the bottle to
mix its contents. Prepare this reagent daily.
6. Ascorbic acid reagent Use a 50-mL graduated cylinder to dispense 5 mL of the stock
ascorbic acid reagent and 25 mL of Dl water into an amber glass reagent bottle. Manually
swirl the bottle to mix its contents. Prepare this solution daily.
7. Startup/shutdown solution: Add 1 mL of stock SLS reagent to 100 mL of Dl water in a small
plastic bottle. Thoroughly rinse the bottle and prepare a fresh solution every few days or as
needed.

2.4.6.4 Nitrate Determination
1. Copper (II) sulfate reagent (2 percent w/v): Dissolve 20 g of copper sulfate pentahydrate
(CuSO4»5H2O, FW=249.7) in about 800 mL of Dl water in a 1-L volumetric flask. Dilute this
solution to the mark with Dl water, mix it well, and transfer it to a 1-L plastic bottle. This
reagent is stable for several years at room temperature.
2. Imidazole buffer, 0.1 M, (pH 7.5): In a hood, cautiously add 5.0 mL of concentrated

hydrochloric acid (HCI, ~12 M) and 1.0 mL of 2 percent copper sulfate solution to 1,600 mL
of Dl water in a 2-L volumetric flask. Mix the contents of the flask thoroughly and then add
13.6 g of imidazole (C3H4N2, FW=68.08). Again swirl or shake the flask until the imidazole
dissolves. Dilute the resulting solution to the mark with Dl water, mix it well, and transfer it
into two 1-L plastic bottles. This reagent is stable for 6 months at room temperature.
NOTE: Add 250 uL of Brij-35 surfactant to 250 mL of imidazole buffer each time its container is
refilled on the continuous flow analyzer. Do not add Brij-35 to the bulk buffer solution.
3. Packed bed cadmium reactor. Cadmium reactors are prepared by slurry packing 40- to 60-
mesh, copperized cadmium granules into 6-cm lengths of PTFE Teflon™ tubing (1.6 mm i.d.
x 3.2 mm o.d.). Cadmium granules are retained in the column with hydrophilic plastic frits
(40-um nominal pore size). Detailed instructions for preparing copperized cadmium granules
and packing them into columns can be found in NWQL standard operating procedure (SOP)
IM0384.0 (or subsequent revisions; available on request).
4. Sulfanilamide reagent ('SAN"): Use a graduated cylinder to dispense 100 mL of
concentrated hydrochloric acid (HCI, 36.5-38.0 percent, «12 M) into about 700 mL of Dl
water in a 1-L volumetric flask. Work in a hood and manually swirl or magnetically stir the
flask during each addition of HCI. Add 10.0 g of SAN (C6H8N2O2S, FW=172.20) to the
warm hydrochloric acid solution. Manually shake, sonicate, or magnetically stir the contents

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 52

of the flask until the SAN dissolves. After the resulting solution has cooled, dilute it to the
mark with Dl water, mix it well, and transfer it to a clean 1-L plastic bottle in which it is stable
for 1 year at room temperature.

5. N-(1-Naphthyl)ethylenediamine dihydrochloride reagent ('NED"): Dissolve 1.0 g NED
(C12H14N2-2HCI, FW=259.2) in about 800 ml_ of Dl water in a 1-L volumetric flask. Dilute
the resulting solution to the mark with Dl water and mix well by manually shaking the flask.
Transfer this reagent to a 1-L amber glass bottle in which it is stable for 6 months at room
temperature.
6. Startup/shutdown solution: Add 250 uL of Brij-35 surfactant to 250 mL of Dl water in a
plastic bottle. Thoroughly rinse the bottle and prepare a fresh solution every few days or as
needed.

2.4.7 Calibrants and Quality-Control Solutions
This section provides detailed instructions for preparing calibrants, matrix spike solution, quality-
control check solutions, and digestion check solution.

1. Potassium nitrate stock calibrant solution, 1 mL =2.5 mg-N: Dissolve 1.805 g of potassium
nitrate (KNO3, FW=101.1) in about 80 mLof Dl water in a 100-mL volumetric flask. Dilute
this solution to the mark with Dl water and mix it thoroughly by manual inversion and
shaking. Transfer the stock calibrant to a 100-mL Pyrex™ media bottle in which it is stable
for 6 months at 4°C.

2. Potassium di-hydrogen phosphate stock calibrant solution, 1 mL = 1.0 mg-P: Dissolve
0.4394 g potassium di-hydrogen phosphate (KH2PO4, FW=136.09) in about 80 mL of Dl
water in a 100-mL volumetric flask. Dilute this solution to the mark with Dl water and mix it
thoroughly by manual inversion and shaking. Transfer the stock calibrant to a 100-mL
Pyrex™ media bottle in which it is stable for 6 months at 4°C.

3. Sulfuric acid «1.8 M: Use a 25-mL graduated cylinder to dispense 10 mL of concentrated
sulfuric acid (H2SO4, sp. gr. 1.84) into about 75 mL of Dl water in a 100-mL volumetric flask.
After the solution cools, dilute it to the mark with Dl water, mix well, and transfer it to a 125-
mL plastic bottle. Make a new batch of this acid each time acidified working calibrants and
blanks are prepared and use the remainder to prepare acidified blank solution as needed.

4. Mixed stock calibrant solution, 1 mL= 1.25 mg-N and 0.5 mg-P. Dispense equal volumes
(minimum of 2 mL each) of nitrate and phosphate stock calibrants into a small beaker and
mix them thoroughly. Prepare this solution each time working calibrants are prepared.
5. Working calibrant solutions (for FCC samples): Use two adjustable, digital pipets (ranges 10
to 100 uL and 100 to 1,000 uL) to dispense the volumes of mixed stock calibrant (7.4) listed
in table 4 into 250-mL volumetric flasks that each contains about 200 mL of Dl water. Dilute
the working calibrants to the mark with Dl water and mix them thoroughly by manual
inversion and shaking. Transfer the working calibrants to 250-mL Pyrex™ media bottles in
which they are stable for 4 weeks at 4°C.

6. Acidified working calibrant solutions (forFCA and WCA samples): Prepare these calibrants
identically to those described in section 7.5, except add 2.5 mL of 1.8 M H2SO4 to each
flask before diluting it to the mark with Dl water.

7. Check standards (for FCC samples): Check standards in three concentration ranges, which
were designated Low, High, and Very high, were prepared from a concentrated commercial
nutrient QC mixture (Demand™, Environmental Resource Associates, Arvada, Colo.), as

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 53

listed in table 5. Transfer check standards to 1-L Pyrex™ media bottles in which they are
stable for 2 months at 4°C. Each of these check standards was dispensed, digested, and
analyzed along with every batch of filtered and whole-water samples analyzed for this study.

8. Acidified check standards (forFCA and WCA samples): Prepare these check standards
identically to those described in section 7.7, except add 10.0 ml_ of 1.8 M H2SO4 to the
flasks before diluting them to the mark with Dl water.

9. Spike Solutions

a. Nitrogen stock spike solution (1 mL = 0.50 mg-N)\ Dissolve 0.955 g ammonium chloride
(NH4CI, FW=53.49) in about 400 mL of Dl water in a 500-mL volumetric flask. Dilute this
solution to the mark with Dl water and mix it thoroughly by manual inversion and
shaking. Transfer the stock spike solution to a 500-mL Pyrex™ media bottle in which it is
stable for 6 months at 4°C.

b. Phosphorus stock spike solution (1 mL = 0.20 mg-P): Dissolve 0.439 g potassium
dihydrogen phosphate (KH2PO4, FW=136.1) in about 400 mL of Dl water in a 500-mL
volumetric flask. Dilute this solution to the mark with Dl water and mix it thoroughly by
manual inversion and shaking. Transfer the stock spike solution to a 500-mL Pyrex™
media bottle in which it is stable for 6 months at 4°C.

c. Mixed spike solution (100 uL = 0.005 mg-N and 0.002 mg-P): Dispense 1 mL each of
ammonium chloride and orthophosphate stock spike solutions into a 10-mL volumetric
flask and dilute to the mark with Dl water. Transfer the mixed spike solution to a 15-mL,
screw-cap polyethylene centrifuge tube in which it is stable for 2 weeks at 4°C.

NOTE: An equivalent mixed spike solution can be prepared more conveniently from stock
calibrants by diluting 500 uL of each to 25 mL in a volumetric flask.
10. Digest-Check Stock Solutions

a. Glycine digest-check stock solution (1 mL = 1.0 mg-N): Dissolve 3.98 g glycine
(C2H5NO2-HCI, FW=111.5) in about 400 mL of Dl water in a 500-mL volumetric flask.
Dilute this solution to the mark with Dl 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.
b. Glycerophosphate digest-check stock solution (1 mL = 0.4 mg-P): Dissolve 1.976 g
glycerophosphate (C3H7O6PNa2»5H2O, FW=306.1) in about 400 mL of Dl water in a
500-mL volumetric flask. Dilute this solution to the mark with Dl 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.

c. Glucose digest-check stock solution (1 mL = 1.25 mg-C): Dissolve 1.564 g glucose
(C6H12O6, FW=180.2) in about 400 mL of Dl water in a 500-mL volumetric flask. Dilute
this solution to the mark with Dl 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.

d. Mixed digest-check solution (for FCC samples—nominal concentration 4 mg-N/L, 1.6
mg-P/L, and 50 mg-C/L): Dispense 1 mL each of glycine and glycerophosphate stock
digest-check solutions and 10 mL of the glucose digest-check stock solution into a 250-
mL volumetric flask that contains about 200 mL of Dl water. Dilute the contents of the
flask to the mark with Dl water and mix it thoroughly by manual inversion and shaking.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
            Page 54
       Transfer the stock digest-check solution to a 250-mL Pyrex™ media bottle in which it is
       stable for 1 month at 4°C.
    e.  Acidified mixed digest-check solution (for FCA and WCA samples): Prepare this digest-
       check solution identically to the one described ealier, except add 2.5 ml_ of 1.8 M
       H2SO4 to the flask before diluting its contents to the mark with Dl water. Transfer the
       acidified mixed digest-check solution to a 250-mL Pyrex™ media bottle in which it is
       stable at 4°C for 1 month.
Table 4. Volumes of mixed calibrant and amendment solution required to prepare working calibrants and
blanks for determination of total nitrogen and phosphorus by the alkaline persulfate digestion method.
Final volumes are 250 ml [uL, microliter; ml, milliliter; mg-N/L, milligrams nitrogen per liter; mg-P/L,
milligrams phosphorus per liter; M, molarity (moles per liter); FCA, filtered, chilled, acidified (bottle type);
WCA, whole water, chilled, acidified (bottle type)]
Calibrant
identity
C1
C2
C3
C4
C5
C6
C7
Mixed calibrant
volume (pL)
1,000
750
500
250
100
62
0
Volume 1.8 M
H2SO4 1 (mL)
2.5
2.5
2.5
2.5
2.5
2.52
2.5
Nominal
concentration
(mg-N/L)
5.00
3.75
2.50
1.25
0.50
0.03
0
Nominal
concentration
(mg-P/L)
2.00
1.50
1.00
0.50
0.20
0.012
0
 Add H2SO4 only to acidified calibrants.z Prepare 1 L of C6 (24 uL of mixed calibrant and 10 mL of 1.8
M H2SO4, if appropriate, diluted to 1 L with Dl water) to minimize dispensing error.
Table 5. Volumes of Environmental Resource Associates (ERA) Demand™ nutrient concentrate used to
prepare 1-liter volumes of check standards used in this study
Check standard
identity
Low
High
Very high
ERA Demand™
volume (pL)
100
500
1,000
Volume 1. 8 M
H2SO4 1 (mL)1
10.0
10.0
10.0
Nominal
concentration
(mg-N/L)
0.22
1.09
2.20
Nominal
concentration
(mg-P/L)
0.11
0.54
1.08
 Add H2SO4 only to acidified check standards as described in section 7.8.
2.4.8  Sample Preparation
Alkaline persulfate digests are prepared by dispensing samples and digestion reagent into 30-
mL, screw-cap, Pyrex™ culture tubes in the volume ratio of 2 + 1. For filtered samples (FCC
bottle types) that were prepared robotically, 9.5-mL volumes of samples, blanks, calibrants, and
reference materials were dosed with 4.75-mL volumes of alkaline persulfate digestion reagent.
This is the maximum sample volume that could be delivered by the robotic dispenser/diluter
system's 10.000-mL syringes because 0.500 mL of their capacity is expended in the creation of
air gaps that minimize interaction between samples and the Dl water carrier fluid. Whole-water

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 55

samples (WCA bottle types) that require vigorous shaking (and in a few cases, continuous
magnetic stirring) just prior to dispensing operations were prepared manually with conventional,
high-precision, hand-held electronic pipets (Rainin EDP Plus™). Here dispensed volumes of
sample and digestion reagent were 10.000 and 5.000 ml_, respectively. After robotic or manual
sample and reagent-dispensing operations are complete, 100 uL of mixed spike solutio is added
manually to the designated tube. Then all tubes are capped tightly and mixed thoroughly either
by manual inversion (three times) or with a vortex mixer (3, 5-second cycles). The capped tubes
positioned in a purpose-built, 80-position stainless-steel rack then are placed in an autoclave
where they are digested at 121°C and 117.2 kPa for 1 hour. Table 6 lists the rack protocol
suggested for a batch of 80 tubes consisting of up to 64 samples plus six calibrants, four blanks,
three quality-control (QC) check solutions, one digest-check solution, one duplicate sample, and
one spiked sample. A step-by-step procedure for alkaline persulfate digest preparation is
provided in NWQL SOP IM0384.0.

NOTE: When samples contain large quantities of suspended solids, continuous stirring during
sample aspiration might provide the only means of obtaining representative aliquots.
When the digestion cycle is complete and pressure and temperature gages on the autoclave
indicate 0 kPa and less than 80°C, remove the alkaline persulfate digests from the autoclave
and allow them to cool sufficiently to be handled comfortably. Then mix the contents of each
capped digestion tube by manual inversion (three times) or with a vortex mixer (three, 5-second
cycles). FCC and FCA digests can be poured into analyzer cups immediately after mixing. Wait
about 1 hour after mixing WCA digests to allow suspended solids to settle. If it is not possible to
decant or pipet a clear supernatant solution from digest tubes into analyzer cups, then
suspended solids must be removed by 0.45-um filtration prior to colorimetric analysis. Note that
tightly capped digests can be stored at room temperature for several days (4 days was the
maximum delay tested) before their nitrogen and phosphorus concentrations are determined by
automated colorimetry.

2.4.9 Instrument Performance

An 80-tube batch of samples, calibrants, and reference materials can be prepared robotically
and made ready for digestion in about 1 hour. Digestion time—including warm up, cool down,
and postdigestion mixing—is about 2 hours. The NWQL Nutrients Unit has two autoclaves, each
of which can hold two, 80-tube racks of alkaline persulfate digests. Nitrate and orthophosphate
in alkaline persulfate digests can be determined simultaneously with the 2-channel air-
segmented continuous flow analyzer at a rate of about 100 samples per hour with less than 1
percent interaction. Thus, using a combination of robotic and manual sample preparation, up to
six racks (384 actual samples out of 480 total tubes) of alkaline persulfate digests can be
prepared in an 8-hour day. This estimate assumes the use of both NWQL autoclaves and a
combination of robotic (FCC samples) and manual (WCA samples) sample preparation.
Likewise, up to six racks of previously digested samples can be analyzed for nitrate and
orthophosphate in an 8-hour day. This production rate assumes that digest analysis can lag
sample digestion by 1 to 3 days.

2.4.10 Calibration
With a second-order polynomial least-squares curve-fitting function (y = a+bx+cx2, where y is
the baseline and blank-corrected peak height and x is the nominal concentration), calibration
plots with correlation coefficients (r2) greater than 0.999 are achieved routinely. Typical
calibration plots for nitrate and orthophosphate in alkaline persulfate digests are shown in
Figures 3 and 4.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                               Date: November 2010
	Page 56

 NOTE: In addition to baseline drift correction, a digestion blank correction must be applied to
 calibrants, check standards, and samples prior to calculation of final results.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 57
2.4.11  Procedure and Data Evaluation

Set up the continuous flow analyzer analytical cartridges as shown in figures 1 and 2. Turn on
electrical power to all system modules and put fresh sampler wash reservoir solution and
reagents on-line. After about 10 minutes, verify that the sample and reference outputs of both
photometers are set at about 5 volts. A suggested sampler tray protocol for automated
determination of nitrate and orthophosphate in alkaline persulfate digests is listed in table 7.

NOTE: To minimize errors that result from contaminated analyzer cups, rinse them several
times with the solution they are to contain before placing them on the analyzer sampler tray.
NOTE: The full-scale absorbance range control (STD CAL)  of photometers should not require
daily adjustment. Between-analysis/between-day variations in baseline-absorbance level and
calibration curve slope of about ±5 percent are acceptable. Adjustment of the STD CAL control
to compensate for larger variations  in sensitivity or baseline (reagent blank) levels will only mask
underlying problems, such as incipient light source failure, partially clogged flow cells, or
contaminated or improperly prepared reagents, any of which could compromise analytical
results.

Table 6. Suggested rack protocol for alkaline persulfate digest preparation [ID, identification; QC, quality
control; yyyy, year; ddd, Julian day]
Tube
number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
ID
C1
C2
C3
C4
C5
C6
C7 (blank)
blank
blank
blank
QC low
Digest check
QC high
QC very high
yyyydddOOOl
yyyyddd0002
yyyydddOOOS
yyyyddd0004
yyyydddOOOS
yyyyddd0006
Tube
number
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
ID
yyyyddd007
yyyydddOOS
yyyyddd009
yyyyddddO
yyyydddd 1
yyyyddd012
yyyydddd 3
yyyydddd 4
yyyydddd 5
yyyydddd 6
yyyydddd 7
yyyydddd 8
yyyydddd 9
yyyyddd020
yyyyddd021
yyyyddd022
yyyyddd023
yyyyddd024
yyyyddd025
yyyyddd026
Tube
number
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ID
yyyyddd027
yyyyddd028
yyyyddd029
yyyydddOSO
yyyyddd031
yyyyddd032
yyyydddOSS
yyyyddd034
yyyyddd035
yyyyddd036
yyyyddd037
yyyydddOSS
yyyyddd039
yyyyddd040
yyyyddd041
yyyyddd042
yyyyddd043
yyyyddd044
yyyyddd045
yyyyddd046
Tube
number
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
ID
yyyyddd047
yyyyddd048
yyyyddd049
yyyyddd050
yyyyddd051
yyyyddd052
yyyyddd053
yyyyddd054
yyyyddd055
yyyyddd056
yyyyddd057
yyyyddd058
yyyyddd059
yyyyddd060
yyyyddd061
yyyyddd062
yyyyddd063
yyyyddd064
Duplicate
Spike

-------
National Coastal Condition Assessment
Laboratory Methods Manual
                                                                       Date: November 2010
                                                                                   Page 58
    to
    t—
    _i
    o
    ui
    O
    03
    Cc
    O
    03
    m
       4.00
 3.00
 2.00
 1.00
       0.00
y = a +• bm + ex.1
Parameter Value Enror r*
a 0..010 0.010 0.99994
b 0,,S&6 0.013
c -0.005 0.003 /
JO
/
•

/
- ^
x".
X
mg-N.'L
5.00
3.75
2.50
1.25
OJ50
0.10
0.00
1
^
Volts
4.322
3.272
2.175
1 .'22
0.469
0.081
0.000
-
-
-

-
-

r
           0.00          1.2G          2.50          3.75          5.00

              NITROGEN CONCENTRATION. IN MILLIGRAMS PER LITER


Figure 3. Typical calibration graph for total nitrogen determined as nitrate in alkaline persulfate digests.
   cfl
   O
   >
   LU
   O
   03
   CC
   o
   cfl
   03
      4.00
      3/0-Q
2.00
1.00
                 y = a i- bx -»• c*"

      PBramgSsr  Value   _Error

           a      o.oos   o.ooe

           b      2.132   0.018

           c      0.024   0.009
                                        0.99998
           0.0           0.5           1.0           1.5            2.0
              PHOSPHORUS CONCENTRATION, IN MiLL'GBAttS PER LITER


Figure 4. Typical calibration graph for total phosphorus determined as orthophosphate in alkaline
persulfate digests.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 59
Table 7. Suggested analyzer sample tray protocol for automated determination of nitrate and
orthophosphate in alkaline persulfate digests [#, number; ID, identification; SYNC, synchronization peak;
CO, carry-over peak; W, wash; UB, undigested blank; DB, digested blank; CCV, continuing calibration
verification; QC, quality control; yyyy, year; ddd, Julian day]
Cup#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
ID
SYNC
CO (C6)
(C6)
W
C1
C2
C3
C4
C5
C6
C7
W
CCV
UB1
QC low2
Digest check3
QC high2
QC very high2
yyyydddOOOl
yyyyddd0002
yyyydddOOOS
yyyyddd0004
yyyydddOOOS
Cup
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
1 Undigested blank (sampler wash
sections
# ID
yyyyddd006
yyyyddd007
yyyydddOOS
yyyyddd009
yyyyddddO
yyyydddd 1
yyyyddd012
yyyydddd 3
yyyydddd 4
yyyydddd 5
yyyydddd 6
yyyydddd 7
yyyydddd 8
yyyydddd 9
yyyyddd020
yyyyddd021
yyyyddd022
yyyyddd023
yyyyddd024
yyyyddd025
yyyyddd026
yyyyddd027
yyyyddd028
reservoir solution,
Cup#
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
see section
7.7 and 7.8. SDigest-check sample; see sections 7.10.4
ID
yyyyddd029
yyyydddOSO
yyyyddd031
yyyyddd032
UB
W(DB)
yyyydddOSS
yyyyddd034
yyyyddd035
yyyyddd036
yyyyddd037
yyyydddOSS
yyyyddd039
yyyyddd040
yyyyddd041
yyyyddd042
yyyyddd043
yyyyddd044
yyyyddd045
yyyyddd046
yyyyddd047
yyyyddd048
yyyyddd049
6.2.1).2NWQL
and 7. 10. 5.
Cup
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90


Check

# ID
yyyyddd050
yyyyddd051
yyyyddd052
yyyyddd053
yyyyddd054
yyyyddd055
yyyyddd056
yyyyddd057
yyyyddd058
yyyyddd059
yyyyddd060
yyyyddd061
yyyyddd062
yyyyddd063
yyyyddd064
Duplicate
Spike
UB
CCV
UB
W(DB)


Standard, see

2.4.12 Calculations
Instrument calibration requires preparing a set of solutions (calibrants) in which the analyte
concentration is known. These calibrants are digested along with samples and used to establish
a calibration function that is estimated from a least-squares fit of nominal calibrant
concentrations (x) in relation to peak absorbance (y). A second-order polynomial function (y =
a+bx+cx2) usually provides improved concentration estimates at the upper end of the calibration
range than a more conventional linear function (y = a+bx). Accuracy is not lost when a second-
order fit is used, even if the calibration function is strictly linear, because, in this case, the value
estimated for the quadratic parameter c will approach zero.
Before the calibration function can be estimated, the baseline absorbance component of
measured peak heights, including drift (continuous increase or decrease in the baseline
absorbance  during the course of an analysis), if present, needs to be removed. Baseline
absorbance  in continuous flow analysis is analogous to the reagent blank absorbance in batch
analysis. Correction for baseline absorbance is an automatic function of most data acquisition
and processing software sold by vendors of continuous flow analyzers.

NOTE: These correction algorithms are based on linear interpolation between initial and
intermediate or final baseline measurements, and so they do not accurately correct for abrupt,

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 60

step-changes in baseline absorbance that usually indicate partial flow-cell blockage. It is
prudent, therefore, to reestablish baseline absorbance at intervals of 20 samples or so.
After peaks are baseline corrected, they need to be digestion-blank corrected. This correction
can be applied in several ways:

1. Subtract the baseline-corrected absorbance of the digestion blank—compute an average
concentration if multiple digested blanks are included in each block—from the baseline-
corrected absorbance of all calibrants, check standard, and samples in the block. Then
estimate regression parameters (a, b, and c terms) for the calibration function by using a
second-order polynomial least-squares algorithm. For second and higher order calibration
functions, use the Newton-Raphson successive approximations algorithm (Draper and
Smith, 1966; Swartz, 1976, 1977, 1979) to convert corrected peak heights into
concentrations.

2. Designate digestion blanks as a calibrant with a nominal concentration of zero. In this case
the resulting calibration function will have a positive y-intercept that approximates the
baseline-corrected absorbance of the digestion blank. If this method is used, be sure that
the curve-fitting algorithm does not force a zero y-intercept by including one or more
"dummy" (0,0) points in the data set used for calibration.

3. Designate digested blanks as baseline correction samples—that is, "W" in the FasPac™
software used to acquire and process data at the NWQL. In this case initial, intermediate (if
included), and final baselines are interpolated between digested blank peak maxima. Thus,
baseline and digestion blanks are corrected in a single operation.

NOTE: Digestion blanks were corrected for data in this report by using method 3. However,
analytical results calculated by the other two methods should be equivalent. Regardless of the
blank correction algorithm chosen, make sure that it is documented in the SOP and that
analysts understand it. The SOP for these methods must be updated whenever any changes in
data acquisition and processing software or in calculation algorithms are implemented.

Most software packages provide a data base for entering appropriate dilution factors. Usually
these factors can be entered before or after samples are analyzed. If dilution factors are
entered, reported concentrations will be compensated automatically for the extent of dilution.
The dilution factor is the number by which a measured concentration must be multiplied to
obtain the analyte concentration in the sample prior to dilution. For example, dilution factors of
2, 5, and 10 indicate that sample and diluent were combined in proportions of 1+1, 1+4, and
1+9, respectively.

2.4.13 Reporting Results
Total nitrogen (lab codes 2754, 2755, 2756)
• 2 decimal places for concentrations up to 5.00 mg-N/L
• 2 significant figures for concentrations greater than 5.00 mg-N/L
Total phosphorus (lab codes 2757, 2758, 2759)
• 2 decimal places for concentrations up to 2.00 mg-P/L
• 2 significant figures for concentrations greater than 2.00 mg-P/L

2.4.14 Detection Levels, Bias, and Precision

1. Method detection limits (MDL) for composited, low-concentration FCC and WCA samples
(five of each) were estimated using the U.S Environmental Protection Agency (1997)

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 61

     protocol—see Table 8. Target concentrations for nitrogen and phosphorus in FCC and
     WCA composite samples were 0.05 mg-N/L and 0.02 mg-P/L, respectively. The MDL for
     nitrogen was 0.015 mg-N/L and for phosphorus was 0.007 mg-P/L. Laboratory reporting
     levels (LRL) will be about twice the MDL concentrations.

 2.   Table 9 lists the average and standard deviation of 9987L, 9987H, and 9987VH QC check
     solutions that were included in every rack of alkaline persulfate digests. Most probable
     values (MPVs) and standard deviations in table 9 were published by the USGS Branch of
     Quality Systems for the 2002 water year (12-month period ending September 30 each year
     is called the "water year"). In all cases, total nitrogen and total phosphorus concentrations
     determined for these reference materials by the alkaline persulfate digestion method were
     tightly centered around published MPVs and well within published control  limits.

 3.   Spike Recoveries: Median, 90th and 10th percentiles of percent spike recoveries measured
     in samples collected during high-flow and low-flow conditions are listed in  table 10. Median
     spike recoveries for nitrogen (0.5 mg-N/L as glycine) ranged from about 92 tolOO percent
     and for phosphorus (0.2 mg-P/L as glycerophosphate) from about 86 to 108 percent.
 4.   Duplication of Results: Median,  10% percentiles, and 90% percentiles for  concentration
     differences for duplicate samples collected during the nominally high- and low-flow
     conditions are listed  in table 11. Median concentration differences between duplicate
     analyses are about the same as the MDLs. Larger tenth-percentile differences for whole-
     water samples that were collected during nominally high-flow conditions in relation to those
     of filtered water samples likely reflect the difficulty of obtaining reproducible aliquots from
     samples that contain large amounts of suspended solids. Such samples were purposely
     chosen as duplicates to assess "worst-case" digest-preparation sampling  precision.

 Table 8. Data and calculations used to estimate method detection limits (MDL) for nitrogen and
 phosphorus in unacidified (FCC) and acidified (WCA) samples following alkaline persulfate digestion.
 Low-concentration FCC and WCA samples (five of each) were composited for these determinations [mg-
 N (-P)/L, milligrams nitrogen (or phosphorus) per liter; %, percent; MDL, method detection limit]
Concentration found (mg-N/L or mg-P/L)
Target concentration
[mg-N <-P)/L]

0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
0.05 (0.02)
Average
Standard deviation
Number of values
Degrees of freedom
f-value (1 -sided, 99%)
MDL
Dissolved
nitrogen
(unacidified)
0.064
.078
.072
.066
.067
.066
.071
.063
.068
.005
8
7
2.998
0.15
Total nitrogen
(acidified)

0.041
.042
.035
.035
.032
.039
.026
.035
.035
.005
8
7
2.998
0.15
Dissolved
phosphorus
(unacidified)
0.026
.024
.026
.029
.026
.023
.022
.026
.025
.002
8
7
2.998
.007
Total phosphorus
(acidified)

0.033
.029
.029
.027
.029
.027
.026
.026
.028
.002
8
7
2.998
.007

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                                 Date: November 2010
	Page 62

 Table 9. Most probable values and standard deviations for reference samples 9987L, 9987H, and
 9987VH along with averages and standard deviations of these reference materials that were included in
 every rack of alkaline persulfate digests [ID, identification of reference sample; MPV, most probable
 value; FCC, filtered, chilled (bottle type); WCA, whole water, chilled, acidified (bottle type); mg-N/L,
 milligrams nitrogen per liter; mg-P/L, milligrams phosphorus per liter; ±, plus or minus]
       ID             MPV
                               	High-flow samples  	Low-flow samples
	      FCC'           WCA*           FCC3           WCA4
                 Alkaline persulfate dissolved and total nitrogen concentration (mg-N/L)
 9987L           0.22 ±0.08      0.21 ± 0.03     0.21 ± 0.03       0.19 ±0.03      0.20 ±0.02
 9987H           1.09 ±0.15      1.09 ±0.03     1.09 ±0.03       1.06 ±0.08      1.04 ±0.04
 9987VH         2.20 ±0.24      2.27 ± 0.05     2.18 ±0.06       2.16 ±0.07      2.13 ±0.06
                 Alkaline persulfate dissolved and total phosphorus concentration (mg-P/L)
 9987L           0.108 ±0.008    0.105 ±0.004   0.104 ±0.004    0.107 ±0.006    0.105 ±0.004
 9987H           0.54 ±0.02      0.54 ± 0.01      0.5580.02       0.57 ± 0.02      0.54 ±0.01
 9987VH	1.08 ±0.05      1.13±0.02     1.10±0.03       1.13±0.03      1.09 ±0.02

 1 Number of points: n = 19; 2n = 21; 3n = 21; 4n = 18.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 63

2.5 TOTAL PHOSPHORUS AND FRESHWATER ORTHOPHOSPHATE

2.5.1 Scope and Application
1. This method covers the determination of specified forms of phosphorus in marine or
freshwater, and the determination of orthophosphate in freshwater. To determine
orthophosphate is saltwater, use the method outlined in section 2.6.

2. The methods are based on reactions specific for the orthophosphate ion. The most
commonly measured forms are total and dissolved phosphorus, total and dissolved
orthophosphate. Hydrolyzable phosphorus is normally found only in sewage-type samples.
Insoluble forms of phosphorus are determined by calculation.

3. The applicable range is 0.01-1.0 mg P/L. 20 - 30 samples per hour can be analyzed.

2.5.2 Summary of Method

1. Ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute
solutions of phosphorus to form an antimony-phosphomolybdate complex. This complex is
reduced to an intensely blue-colored complex by ascorbic acid. The color is proportional to
the phosphorus concentration.

2. Only orthophosphate forms a blue color in this test. Polyphosphates (and some organic
phosphorus compounds) may be converted to the orthophosphate form by manual sulfuric
acid hydrolysis. Organic phosphorus compounds may be converted to the orthophosphate
form by manual persulfate digestion. The developed color is measured automatically.

3. Reduced volume versions of this method that use the same reagents and molar ratios are
acceptable provided they meet the quality control and performance requirements stated in
the method.

2.5.3 Interferences

1. No interference is caused by copper, iron, or silicate at concentrations many times greater
than their reported concentration in seawater. However, high iron concentrations can cause
precipitation of, and subsequent loss, of phosphorus.

2. The salt error for marine samples ranging from 5-20% salt content was found to be <1 %.
3. Arsenate is determined similarly to phosphorus and should be considered when present in
concentrations higher than phosphorus. However, at concentrations found in sea water, it
does not interfere.

4. Sample turbidity must be removed by filtration prior to analysis for orthophosphate. Samples
for total or total hydrolyzable phosphorus should be filtered only after digestion. Sample
color that absorbs in the photometric range used for analysis will also interfere.

2.5.4 Safety
1. Sulfuric acid (Sections 2.5.6.2 and 2.5.6.7) has the potential to be highly toxic or hazardous,
consult Material Safety Data Sheet.

2.5.5 Equipment and Supplies

1. Balance ~ Analytical, capable of accurately weighing to the nearest O.OOOI g.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 64

2. Glassware -- Class A volumetric flasks and pipets as required. All glassware used in the
determination must be washed with hot 1:1 HCI and rinsed with distilled water. The acid-
washed glassware should be filled with distilled water and treated with all the reagents to
remove the last traces of phosphorus that might be adsorbed on the glassware.

3. Hot plate or autoclave.

4. Automated continuous flow analysis equipment designed to deliver and react sample and
reagents in the required order and ratios.

5. Sampling device (sampler)

6. Multichannel pump
7. Reaction unit or manifold

8. Colorimetric detector

9. Data recording device

2.5.6 Reagents and Standards

1. Reagent water: Distilled or deionized water, free of the analyte of interest. ASTM type II or
equivalent.

2. Sulfuric acid solution, 5N: Slowly add 70 ml_ of cone. H2SO4 (CASRN 7664-93-9) to
approximately 400 ml_ of reagent water. Cool to room temperature and dilute to 500 ml_ with
reagent water.

3. Antimony potassium tartrate solution: Weigh 0.3 g K(SbO)C4H4O6 •1/4H2O (CASRN 28300-
74-5) and dissolve in 50 ml_ reagent water in 100 ml_ volumetric flask, dilute to volume.
Store at 4°C in a dark, glass-stoppered bottle.

4. Ammonium molybdate solution: Dissolve 4 g (NH4)6Mo7O24 »4H2O (CASRN 12027-67-7) in
100 ml_ reagent water. Store in a plastic bottle at 4°C.
5. Ascorbic acid, 0.1M: Dissolve 1.8 g of ascorbic acid (CASRN 50-81-7) in 100 ml_ of reagent
water. The solution is stable for about a week if prepared with water containing no more
than trace amounts of heavy metals and stored at 4°C.

6. Combined reagent: Mix the above reagents in the following proportions for 100 ml_ of the
mixed reagent: 50 ml_ of 5N H2SO4, 5 ml_ of antimony potassium tartrate solution, 15 mL of
ammonium molybdate solution, and 30 mL of ascorbic acid solution. Mix after addition of
each reagent. All reagents must reach room temperature before they are mixed and must be
mixed in the order given. If turbidity forms in the combined reagent, shake and let stand for a
few minutes until the turbidity disappears before processing. This volume is sufficient for a
four hour operation. Since the stability of this solution is limited, it must be freshly prepared
for each run.

Note: A stable solution can be prepared by not including the ascorbic acid in the combined
reagent. If this is done, the mixed reagent (molybdate, tartrate, and acid) is pumped through
the distilled water line and the ascorbic acid solution (30 mL of 7.5 diluted to 100 mL with
reagent water) through the original mixed reagent line.
7. Sulfuric acid solution, 11 N: Slowly add 155 mL cone. H2SO4 to 600 mL reagent water.
When cool, dilute to 500 mL.

8. Ammonium persulfate (CASRN 7727-54-0).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 65

9. Acid wash water: Add 40 ml_ of sulfuric acid solution to 1 L of reagent water and dilute to 2
L. (Not to be used when only orthophosphate is being determined).
10. Phenolphthalein indicator solution (5 g/L): Dissolve 0.5 g of phenolphthalein (CASRN 77-
09-8) in a solution of 50 ml_ of isopropyl alcohol (CASRN 67-63-0) and 50 ml_ of reagent
water.

11. Stock phosphorus solution: Dissolve 0.4393 g of predried (105°C for one hour)
12. Potassium phosphate monobasic KH2PO4 (CASRN 7778-77-0) in reagent water and dilute
to 1000 mL 1.0ml_ = 0.1 mg P.
13. Standard phosphorus solution: Dilute 10.0 mL of stock solution to 100 mL with reagent
water. 1.0mL = 0.01 mg P.

14. Standard phosphorus solution: Dilute 10.0 mL of standard solution to 100 mL with reagent
water. 1.0 mL = 0.001 mg P.

2.5.7 Sample Collection, Preservation and Storage

1. Samples must be preserved with H2SO4 to a pH <2 and cooled to 4°C at time of collection.

2. Samples should be analyzed as soon as possible after collection. If storage is required,
preserved samples are maintained at 4°C and may be held for up to 28 days.

2.5.8 Quality Control

Note. Each laboratory using this method is required to operate a formal quality control (QC)
program. The minimum requirements of this program consist of an initial demonstration of
laboratory capability, and the periodic analysis of laboratory reagent blanks, fortified blanks and
other laboratory solutions as a continuing check on performance. The laboratory is required to
maintain performance records that define the quality of the data that are generated.

2.5.8.1 Initial Demonstration of Performance

1. The initial demonstration of performance is used to characterize instrument performance
(determination of LCRs and analysis of QCS) and laboratory performance (determination of
MDLs) prior to performing analyses by this method.
2. Linear Calibration Range (LCR) ~ The LCR must be determined initially and verified every six
months or whenever a significant change in instrument response is observed or expected.
The initial demonstration of linearity must use sufficient standards to insure that the resulting
curve is linear. The verification of linearity must use a minimum of a blank and three
standards. If any verification data exceeds the initial values by ±10%, linearity must be
reestablished. If any portion of the range is shown to be nonlinear, sufficient standards must
be used to clearly define the nonlinear portion.
3. Quality Control Sample (QCS) - When beginning the use of this method, on a quarterly basis
or as required to meet data-quality needs, verify the calibration standards and acceptable
instrument performance with the preparation and analyses of a QCS. If the determined
concentrations are not within ±10% of the stated values, performance of the determinative
step of the method is unacceptable. The source of the problem must be identified and
corrected before either proceeding with the initial determination of MDLs or continuing with
on-going analyses.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 66

4. Method Detection Limit (MDL) - MDLs must be established for all analytes, using reagent
water (blank) fortified at a concentration of two to three times the estimated instrument
detection limit. To determine MDL values, take seven replicate aliquots of the fortified reagent
water and process through the entire analytical method. Perform all calculations defined in
the method and report the concentration values in the appropriate units. Calculate the MDL
as follows:
MDL=(t)x(S)
where,
t = value for a 99% confidence level and a standard deviation estimate with n-1 degrees
of freedom [t = 3.14 for seven replicates]

S = standard deviation of the replicate analyses

MDLs should be determined every six months, when a new operator begins work, or
whenever there is a significant change in the background or instrument response.

2.5.8.2 Assessing Laboratory Performance

1. Laboratory Reagent Blank (LRB) -- The laboratory must analyze at least one LRB with each
batch of samples. Data produced are used to assess contamination from the laboratory
environment. Values that exceed the MDL indicate laboratory or reagent contamination
should be suspected and corrective actions must be taken before continuing the analysis.

3. The laboratory must use LFB analyses data to assess laboratory performance against the
required control limits of 90-110%. When sufficient internal performance data become
available (usually a minimum of 20-30 analyses), optional control limits can be developed
from the percent mean recovery (x) and the standard deviation (S) of the mean recovery.
These data can be used to establish the upper and lower control limits as follows:
Upper Control Limit = x + 3S
Lower Control Limit = x - 3S
The optional control limits must be equal to or better than the required control limits of 90-
110%. After each 5 to 10 new recovery measurements, new control limits can be calculated
using only the most recent 20-30 data points. Also, the standard deviation (S) data should
be used to establish an on-going precision statement for the level of concentrations included
in the LFB. These data must be kept on file and be available for review.

4. Instrument Performance Check Solution (IPC) - For all determinations the laboratory must
analyze the IPC (a mid-range check standard) and a calibration blank immediately following
daily calibration, after every tenth sample (or more frequently, if required) and at the end of
the sample run. Analysis of the IPC solution and calibration blank immediately following
calibration must verify that the instrument is within ±10% of calibration. Subsequent
analyses of the IPC solution must verify the calibration is still within ±10%. If the calibration
cannot be verified within the specified limits, reanalyze the IPC solution. If the second
analysis of the IPC solution confirms calibration to be outside the limits, sample analysis
must be discontinued, the cause determined and/or in the case of drift the instrument

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 67

recalibrated. All samples following the last acceptable IPC solution must be reanalyzed. The
analysis data of the calibration blank and IPC solution must be kept on file with the sample
analyses data.

2.5.8.3 Assessing Analyte Recovery and Data Quality

1. Laboratory Fortified Sample Matrix (LFM) -- The laboratory must add a known amount of
analyte to a minimum of 10% of the routine samples. In each case the LFM aliquot must be
a duplicate of the aliquot used for sample analysis. The analyte concentration must be high
enough to be detected above the original sample and should not be less than four times the
MDL. The added analyte concentration should be the same as that used in the laboratory
fortified blank.
2. Calculate the percent recovery for each analyte, corrected for concentrations measured in
the unfortified sample, and compare these values to the designated LFM recovery range 90-
110%. Percent recovery may be calculated using the following equation:

R = Cs - C x100
s

where,
R = percent recovery
Cs = fortified sample concentration
C = sample background concentration
s = concentration equivalent of analyte added to sample

3. If the recovery of any analyte falls outside the designated LFM recovery range and the
laboratory performance for that analyte is shown to be in control, the recovery problem
encountered with the LFM is judged to be either matrix or solution related, not system
related.
4. Where reference materials are available, they should be analyzed to provide additional
performance data. The analysis of reference samples is a valuable tool for demonstrating
the ability to perform the method acceptably.

2.5.9 Calibration and Standardization

1. Prepare a series of at least three standards, covering the desired range, and a blank by
pipetting and diluting suitable volumes of working standard solutions into 100 mL volumetric
flasks. Suggested ranges include 0.00-0.10 mg/L and 0.20-1.00 mg/L.

2. Process standards and blanks as described in section 2.5.10.
3. Set up manifold as shown in Figure 2.6.

4. Prepare flow system as described in section 2.5.10.

5. Place appropriate standards in the sampler in order of decreasing concentration and
perform analysis.
6. Prepare standard curve by plotting instrument response against concentration values. A
calibration curve may be fitted to the calibration solutions concentration/response data using
computer or calculator based regression curve fitting techniques. Acceptance or control

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 68
   limits should be established using the difference between the measured value of the
   calibration solution and the "true value" concentration.
7.  After the calibration has been established, it must be verified by the analysis of a suitable
   quality control sample (QCS). If measurements exceed ±10% of the established QCS value,
   the analysis should be terminated and the instrument recalibrated. The new calibration must
   be verified before continuing analysis. Periodic reanalysis of the QCS is recommended as a
   continuing calibration check.


w.










"~---^


,STF,





~~~-






\^






^~~~~-^
TOF/CTOMP



HEATING BATH
37"C

nmo

TUBE


5 TURNS
ryrn


COLORIMETER
650 -660 urn or 880 urn
SOmmW/C









5 TURNS
nnm
















WASTE




mUmm
8.42


032


032
0.23

OM

PUMP




















SAMPLE


AIR


DISTILIJED WATER
MKTID REAGENT

FROM F/C


30 PEE HOUR
SAMPLE W SIC.
WASH 40 SIC.
Figure 2.6. Phosporous Manifold

2.5.10  Procedure

2.5.10.1 Phosphorus

1.   Add 1 ml_ of sulfuric acid solution to a 50 ml_ sample and/or standard in a 125 mL
    Erlenmeyer flask.

2.   Add 0.4 g of ammonium persulfate.

3.   Boil gently on a pre-heated hot plate for approximately 30-40 minutes or until a final volume
    of about 10 mL is reached. Do not  allow sample to go to dryness. Alternately, heat for 30
    minutes in an autoclave at 21 °C  (15-20 psi).

4.   Cool and dilute the sample to 50 mL. If sample is not clear at this point, filter.

5.   Determine phosphorus as outlined in Figure 2.7 with acid wash water in wash tubes.

2.5.10.2 Hydrolyzable Phosphorus

1.   Add 1 mL of sulfuric acid solution to a 50 mL sample and/or standard in a 125 mL
    Erlenmeyer flask.

2.   Boil gently on a pre-heated hot plate for 30-40 minutes until a final volume of about 10 mL
    is reached. Do not allow sample to go to dryness. Alternatively, heat for 30 minutes in an
    autoclave at 121 °C (15-20 psi).

3.   Determine phosphorus as outlined in Figure 2.7 with acid wash water in wash tubes.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
                                                                    Date: November 2010
                                                                               Page 69
CO
C73
tn
O3



\
/
Res






SAM

s
\
/
X
due




\
J
Orthc

/
/

HJj






\
/
Filter (through
\


Direct
Colorimetry
ssolved
phosphate




\
\
\
<'

Total Sample (No Filtration)

Direct
/ Colorimetry
\
Orthophosphate
0.45
\
/
Fill
\
\
/
H2SO
/Hydr
/ Color

\
\
/
n membrane filter)
/
\
ate
/
/

\
4
rfysis &
imetr>;
Jiss, Hydrolyzable
& Orthophosphate

\
{
Ph


\
/
H2SO
/Hydn
/ Color

/
\
rfysis &
imetry

Sydroiyzable &
Orthophosphate








\
\
\
'




Persulfate
/Digestion &
/ Colorimetry
\
Phosphorous

Persulfate
/Digestion &
/ Colorimetry
hssolved
osphorous




Figure 2.7. Analytical Scheme
2.5.1 1  Data Analysis and Calculations
1.
2.
   Prepare a calibration curve by plotting instrument response against standard concentration.
   Compute sample concentration by comparing sample response with the standard curve.
   Multiply answer by appropriate dilution factor.
   Report only those values that fall between the lowest and the highest calibration standards.
   Samples exceeding the highest standard should be diluted and reanalyzed. Any sample with
   a computed value is less than 5% of its immediate predecessor must be rerun.

3.  Report results in mg P/L.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 70

2.6 ORTHOPHOSPHATE (Saltwater Only)

2.6.1 Scope and Application

This method is for saltwater only. For determination of orthophosphate in freshwater, see
method in section 2.5 above.

This method provides a procedure for the determination of low-level orthophosphate
concentrations normally found in marine waters by way of automated colorimetric analysis. In
this method, the two reagents are added separately for greater reagent stability and facility of
sample separation.

2.6.2 Method Summary

Ammonium molybdate and antimony potassium tartrate react in an acidic medium with dilute
solutions of phosphate to form an antimony-phospho-molybdate complex. This complex is
reduced to an intensely blue-colored complex by ascorbic acid. The color produced is
proportional to the phosphate concentration present in the sample. Positive bias caused by
differences in the refractive index of seawater and reagent water is corrected for prior to data
reporting.

2.6.3 Interferences

1. Interferences caused by copper, arsenate and silicate are minimal relative to the
orhtophosphate determination because of the extremely low concentrations normally
found in estuarine and coastal waters. High iron concentrations can cause precipitation
of and subsequent loss of phosphate from the dissolved phase. Hydrogen sulfide
effects, such as occur in samples collected from deep anoxic basins, can be treated by
simple dilution of the sample since high sulfide concentrations are most often associated
with high phosphate values.

2. Sample turbidity is removed by filtration prior to analysis.

3. Refractive index interferences are corrected for in Section 2.1.9.

2.6.4 Equipment and Supplies

1. Continuous Flow Automated Analytical System:

o Sampler
o Manifold of analytical Cartridge equipped with 37°C heating bath
o Proportioning pump
o Colorimeter equipped with 1.5 x 50 mm tubular flow cell and 880 nm filter
o Phototube that can be used for 600-900 nm range
o Strip chart recorder or computer based data system

2. Phosphate-free glassware and polyethylene bottles

3. Membrane or glass fiber filters, 0.45 urn nominal pore size

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 71

2.6.5 Reagent and Standards

2.6.5.1 Stock Reagent Solutions

1. Ammonium Molybdate solution (40 g/L). dissolve 20.0 g of ammonium molybdate
tetrahydrate ((NH4)6Mo7O24'4H2O) in approximately 400 ml_ of reagent water and dilute
to 500 ml_. Store in a plastic bottle out of direct sunlight. This reagent is stable for
approximately three months.
2. Antimony Potassium Tartrate solution (3.0 g/L). Dissolve 0.3 g of antimony potassium
tartrate (K(SbO)C4H4O6*1/4H2O) in approximately 90 ml_ of reagent water and dilute to
100ml_. This reagent is stable for approximately three months.

3. Ascorbic Acid solution (18.0 g/L). Dissolve 18.0 g ascorbic acid (C6H6O6) in
approximately SOOmL of reagent water and dilute to 1L. Dispense approximately 75mL
into clean polyethylene bottles and freeze. The stability of frozen ascorbic acid is three
month. Thaw overnight in the refrigerator before use. The stability of thawed reagent is
less than 10 days.

4. Sodium Lauryl Sulfate solution (30.0 g/L). Sodium dodecyl sulfate
(CH3(CH2)iiOSO3Na). Dissolve 3.0 g of sodium lauryl sulfate in approximately 80 mL of
reagent water and dilute to 100 mL. This solution is the wetting agent and its stability is
approximately three weeks.

5. Sulfuric Acid solution (4.9 N). Slowly add 136 mL of concentrated sulfuric acid (H2SO4)
to approximately 800 mL reagent water. After the solution is cooled, dilute to 1 L with
reagent water.

6. Stock Phosphorus solution. Dissolve 0.439 g of pre-dried (105°C for 1 hour) monobasic
potassium phosphate (KH2PO4) in reagent water and dilute to 1000mL (1.0 mL = 0.100
mg P). The stability of this stock standard is approximately 3 months when kept
refrigerated.

7. Low Nutrient Seawater. Obtain low nutrient seawater (36%o; <0.0003 mg P/L) or
dissolve 31 g analytical reagent grade sodium chloride, 10g analytical grade magnesium
sulfate, and 0.05g analytical grade sodium bicarbonate in 1L of reagent water.

2.6.5.2 Working Reagents

1. Reagent A. Mix 100 mL of 4.9N H2SO4, 30 mL of ammonium molybdate solution, 10 mL
of antimony potassium tartrate solution and 2.0 ml of sodium lauryl sulfate for a total
volume of 142 mL. Prepare fresh daily.

2. Reagent B. Mix 0.5 mL of the sodium lauryl sulfate solution to 75 mL of ascorbic acid
solution. Stability is about 10 days when refrigerated.

3. Refractive Reagent A. Add 50 mL of 4.9N H2SO4 to 20 mL of reagent water. Add 1 mL
of sodium lauryl sulfate. Prepare fresh daily.

4. Secondary Phosphorous solution. Take 1.0 mL of Stock Phosphorous solution and dilute
to 100 mL with reagent water (1.0 mL = 0.0010 ng P). Refrigerate and prepare fresh
every 10 days.

5. Prepare a series of standards by diluting suitable volumes of standard solutions to 100
mL with reagent water. Prepare daily. When working with samples of known salinity, it

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 72

is recommended that the standard curve concentrations be prepared in low-nutrient
seawater diluted to match the salinity of the samples. Doing so obviates the need to
perform the refractive index correction outlined in section 2.4.10.1. When analyzing
samples of varying salinities, it is recommended that the standard curve be prepared in
reagent water and refractive index corrections be made to the sample concentrations.
The following dilutions are suggested.
ml Secondary
Phosphorous sol'n
0.1
0.2
0.5
1.0
2.0
4.0
5.0
Cone.
ma P/L
0.0010
0.0020
0.0050
0.0100
0.0200
0.0400
0.0500
2.6.6 Sample Storage

Sample should be refrigerated and stored at 4°C for up to 24 hours. If samples cannot be
analyzed within 24 hours, then freezing at -20°C for a maximum period of two months is
acceptable.

2.6.7 Quality Control

2.6.7.1 Initial Demonstration of Performance

1. The method detection limit (MDL) must be established for the method analyte using a
low level seawater sample containing, or fortified at, approximately 5 times the estimated
detection limit. To determine MDL values, analyze at least seven replicate aliquots or
water which have been processed through the entire analytical method. Calculate the
MDL as follows:

MDL=(t)(S)
where,

S = the standard deviation of the replicate analysis
t = t value for n-1 degrees of freedom at the 99% confidence limit; t = 3.143 for six
degrees of freedom.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 73

concentration, and obtain the constants m and b, where m is the slope and b is the y-
intercept. Incrementally analyze standards of higher concentration until the measured
absorbance response (R) of a standard no longer yields a calculated concentration (Cc)
that is within 100 ± 10% of known concentration (C), where

Cc = (R-b)/m
This concentration defines the upper limit of the LDR. If samples are found to have a
concentration that is > 90% of the upper limit of the LDR, they must be diluted and
reanalyzed.

2.6.7.2 Assessing Laboratory Performance

2. Laboratory Fortified Blank (LFB). The lab should analyze at lease one LFB with each
set of samples. The LFB must be at a concentration within the daily calibration range.
The LFB data are used to calculate percent recovery. If the recovery of the analyte falls
outside the required control limits of 90-110%, the source of the problem should be
identified and resolved before continuing the analysis.

3. The laboratory must use LFB data to assess lab performance against the required
control limits of 90-110%. When sufficient internal performance data become available
(usually a minimum of 20 to 30 analyses), optional control limits can be developed from
the percent mean recovery (x) and standard deviation (S) of the mean recovery. These
data can be used to establish the upper and lower control limits as follows:

Upper Control Limit = x + 3S

Lower Control Limit = x - 3S
The optional control limits must be equal to or better than the required control limits of
90-110%. After each 5 to 10 new recovery measurements, new control limits can be
calculated using only the most recent 20 to 30 data points. Also the standard deviation
(S) data should be used to establish an ongoing precision statement for the level of
concentration included in the LFB. These data must be kept on file and available for
review.

2.6.7.3 Assessing Analyte Recovery-Laboratory Fortified Sample Matrix (LFM)

2. Calculate percent recovery of analyte, corrected for background concentration measured
in a separate unfortified sample. These values should be compared with the values
obtained from the LFBs. Percent recoveries may be calculated using the equation:

R= f Cs - C) x100

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 74
             where,
             R = percent recovery
             Cs = measured fortified sample addition in mg N/L
             C = sample background concentration in mg N/L
             S = concentration in mg N/L added to the environmental sample

   3.  If the recovery of the analyte falls outside the required control limits of 90-110%, but the
       laboratory performance for that analyte is within the control limits, the fortified sample
       should be prepared again and analyzed.  If the result is the same after reanalysis, the
       recovery problem encountered with the fortified sample is judged to be the matrix related
       and the sample data should be flagged.

2.6.8   Procedure

   1.  Bring samples to room temperature.

   2.  Assemble manifold as shown in Figure 2.8 below. The  tubing, flow rates, sample wash
       ratio, sample rate,  etc. are based on a Technicon AutoAnalyzer II system.  Specifications
       for similar segmented flow analyzers vary, so slight adjustments may be necessary.
TeSwf
3
. 	
«•!
1 Bal
ipteW
re 	
•tog
h
Oebu
Colo
880 t
ash Receptacle
STUme
J
bt*er
SC.|-W

mnnr;

CtowaMB












2.0
1
0.32
1,8
0.23
0,16
0,42
Pump
Walar (GRN/GRNJ
Air (BJkjBlk)
' Samjte JYEU^EL)
"ZL.
Sismpior
9:1
Beaaool A (CWNWHf)
^^a^rtBpW"***!
Wtoats from FfC 

SO X 1.8 nun ID BC
1S9-B021-O4PhciU>lube
   Figure 2.8.  Manifold Configuration for Orthophosphate
   3.  Allow both colorimeter and recorder to warm up for 30 minutes. Obtain a steady
       baseline with reagent water pumping through the system, add reagents to the sample
       stream and after the reagent water baseline has equilibrated, note the rise (reagent
       water baseline) and adjust baseline.

       For analysis of samples with a narrow salinity range, it is advisable to use low nutrient
       seawater matched to sample salinity as wash water in the sampler in place of reagent

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 75

water. For samples with a large salinity range, it is suggested that reagent wash water
and procedure be employed.
4. The sampling rate should be about 40 samples per hour with a 9:1 sample:wash ratio.

5. Place standards in sampler in order of decreasing concentration. Complete filling the
sampler tray with samples, LRBs, LFBs, and LFMs.

2.6.9 Data Analysis and Calculations

Concentrations of orthophosphate are calculated from the linear regression obtained from the
standard curve in which the concentrations of the calibration standards are entered as the
independent variable and the corresponding peak height is the dependent variable.

2.6.9.1 Refractive Index Correction

1. Obtain a second set of peak heights for all samples and standards with Refractive
Reagent A being pumped through the system in place of Reagent A. This "apparent"
concentration due to coloration of the water should be subtracted from the
concentrations obtained with Reagent A pumping through. Reagent B remains the same
and is also pumped through the system. Peak heights for the refractive index correction
must be obtained at the same standard calibration setting and on the same colorimeter
as the corresponding samples and standards.

2. Subtract the refractive index peak heights from the heights obtained for the
orthophosphate determination. Calculate the regression equation using the corrected
sample peak heights.

3. When a large data set has been amassed in which each sample's salinity in which each
sample's salinity is known, a regression for the refractive index correction on a particular
colorimeter can be calculated. For each sample, the apparent orthophosphate
concentration due to refractive index is calculated from its peak height obtained with
Refractive Reagent A and Reagent B, and the regression of orthophosphate standards
obtained with orthophosphate Reagent A and Reagent B. Its salinity is entered as the
independent variable and its apparent orthophosphate concentration due to its refractive
index in that colorimeter is entered as the dependent variable. The resulting regression
equation allows the analyst to subtract an apparent orthophosphate concentration when
the salinity is known, as long as other matrix effects are not present. Thus, the analyst
would not be required to obtain the refractive index peak heights for all samples after a
large data set has been found to yield consistent apparent orthophosphate
concentrations due to salinity.

4. A typical equation is:

mg P/L apparent PO43 = 0.000087 x Salinity (%o)

where
0.000087 is the slope of the line.
5. Results should be reported in mg PO43 - P/L (ppm) or urn PO43 - P/L (ppb).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 76

2.7 CHLOROPHYLL a

2.7.1 Scope and Application
This method is for the low level determination of chlorophyll a (chl a) in marine and freshwater
phytoplankton using fluorescence detection. This method may be modified to determine levels
of chlorophyll a only by using a set of very narrow bandpass excitation and emission filters that
nearly eliminate the spectral interference caused by the presence of pheophytin a and
chlorophyll b. Separate equations are used for this modified method.

2.7.2 Method Summary
Chlorophyll-containing phytoplankton are concentrated by filtering at low vacuum through a
glass fiber filter. The pigments are extracted from the phytoplankton in 90% acetone with the
aid of a mechanical tissue grinder and allowed to steep. The filter slurry in centrifuged to clarify
the solution. Fluorescence is measured before and after acidification. Sensitivity calibration
factors are used to calculate the concentration in the sample extract. The concentration in the
water sample is reported in ug/L.

2.7.3 Interferences
1. Any substance extracted from the filter or acquired from laboratory contamination that
fluoresces in the red region of the spectrum may interfere with the accurate measurement of
both chlorophylls a and b.
2. The relative amounts of chlorophylls a, b and c vary with the taxonomic composition.
Chlorophylls b and c may significantly interfere with chlorophyll a measurements depending
on the amount present. Due to the spectral overlap of chlorophyll a with pheophytin a and
chlorophyll a, underestimation of chlorophyll a occurs. The degree of interference depends
on the ration of a:b.
3. Quenching effects are observed in highly concentrated solutions or in the presence of high
concentrations of other chlorophylls or carotenoids. Minimum sensitivity settings on the
fluorometer should be avoided; samples should be diluted instead.
4. Fluorescence is temperature dependent with higher sensitivity occurring at lower
temperatures. Samples, standards, blanks and quality control standards must be at the
same temperature to prevent errors and/or low precision. Samples should be analyzed at
ambient temperature. Ambient temperature should not fluctuate more than ± 3°C between
calibrations or recalibration of the fluorometer will be necessary.
5. Samples must be clarified by centrifugation prior to analysis.
6. All photosynthetic pigments are light and temperature sensitive. Work must be performed in
subdued light and all standards, QC materials and filter samples must be stored in the dark
to prevent degradation.

2.7.4 Safety

The grinding of filters during the extraction process should be conducted in a fume hood due to
the volatilization of acetone by the tissue grinder.

2.7.5 Equipment and Supplies

1. Fluorometer. Equipped with a high intensity F4T.5 blue lamp, red sensitive photomultiplier,
and filters for excitation (CS-5-60) and emission (CS-2-64).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 77

Note. The modified method requires excitation filter (436FS10) and emission filter (680FS10).
2. Centrifuge capable of 675 g.
3. Tissue grinder, Teflon pestle (50 mm X 20 mm) with grooves in the tip with %" stainless
steel rod long enough to chuck onto a suitable drive motor and 30-mL capacity glass
grinding tube.
4. Filters, glass fiber, 47-mm or 25-mm, nominal pore size of 0.7 urn unless otherwise justified
by data quality objectives.
5. Aluminum foil
6. Assorted labware.

2.7.6 Reagents and Standards
1. Acetone, HPLC grade.
2. Hydrochloric acid (HCI), concentrated.
3. Chlorophyll a free of chlorophyll b. May be commercially obtained.
4. Water. ASTM Type I water is required. Suitable water may be obtained by passing distilled
water through a mixed bed of anion and cation exchange resins.
5. HCI Solution (0.1 N). Add 8.5 ml_ of concentrated HCI to aout 500 ml_ and dilute to 1 L.
6. Aqueous Acetone Solution (90% acetone/10% water). Carefully measure 100 Ml of water
into a 1-L graduated cylinder. Transfer to a 1-L flask or storage bottle. Measure 900 ml_ of
acetone into the graduated cylinder and transfer to the flask or bottle containing the water.
Mix, label and store appropriately.
7. Chlorophyll Stock Standard Solution (SSS). The dry standard received from supplier
should be stored at -20°C or -70°C. Prepare SSS just prior to use. Tap the ampoule of dry
standard until all the dried chlorophyll is in the bottom of the ampoule. In subdued light,
carefully break the tip off the ampoule and transfer the entire contents into a 50-mL
volumetric flask. Dilute to volume with 90% acetone, label the flask, and wrap in aluminum
foil to protect from light. The concentration of the solution must be determined
spectrophotometrically using a multiwavelength spectrophotometer. The concentration of
all dilutions must be determined spectrophotometrically each time they are made.
8. Chlorophyll a Primary Dilution Standard solution (PDS). Add 1 ml_ of the SSS to a clean
100-mL flask and dilute to volume with the aqueous acetone solution. If exactly 1 mg of
pure chlorophyll a was used to prepare the SSS, the concentration of the PDS is 200 ug/L.
Prepare fresh just prior to use.

2.7.7 Sample Storage
Sampled filters should be stored frozen (-20°C or -70°C) in the dark until extraction. Filters can
be stored frozen at these temperatures for up to 24 days without significant loss of chlorophyll a.

2.7.8 Quality Control

Each laboratory is required to operate a formal quality control (QC) program. The minimum
requirements of this program consist of an initial demonstration of laboratory capability and the
continued analysis of laboratory reagent blanks, field duplicates and quality control samples as
a continuing check on performance. The laboratory is required to maintain performance records
that define the quality of the data generated.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 78

2.7.8.1 Initial Demonstration of Performance

1. The laboratory must demonstrate performance prior to sample analysis.
2. Linear Dynamic Range (LDR). The LDR is determined by analyzing a minimum of 5
calibration standards ranging in concentration from 0.2 ug chl a /L to 200 ug chl a/L across
all sensitivity settings of the fluorometer. If using an analog fluorometer or a digital
fluorometer requiring manual changes in sensitivity settings, normalize responses by
dividing the response by the sensitivity multiplier. Perform the linear regression of
normalized response v. concentration and obtain the constants m and b, where m is the
slope and b is the y-intercept. Incrementally analyze standards of higher concentration until
the measured fluorescence response, R, of a standard no longer yields a calculated
concentration, Cc, that is ± 10% of the known concentration, C, where Cc = (R-b)/m. That
concentration defines the upper limit of the LDR for your instrument. If samples have a
concentration that is 90% of the upper limit of the LDR, dilute the samples and reanalyze.
3. Instrumental Detection Limit (IDL). Zero the fluorometer with a solution of 90% acetone on
the maximum sensitivity setting. Pure chlorophyll a in 90% acetone should be serially
diluted until it is no longer detected by the fluorometer on a maximum sensitivity setting.
4. Estimated Detection Limit (EDL). Several blank filters are extracted according to the
procedure outlined in section 2.7.10. A solution of pure chlorophyll a in 90% acetone is
serially diluted until it yields a response that is 3X the average response of blank filters.
5. Quality Control Sample (QCS). Verify the calibration standards and acceptable instrument
performance with the analysis of a QCS. If the determined value is not within the
confidence limits established by project data quality objectives, then the determinative step
of this method is unacceptable. The source of the problem must be identified and corrected
before performing this method.

2.7.8.2 Assessing Laboratory Performance

Laboratory Reagent Blank (LRB). The laboratory must analyze at least 1 blank filter with each
sample batch. The LRB should be the last filter extracted. LRB values that exceed the IDL
indicate contamination from the lab environment. When LRB values constitute 10% or more of
the analyte level determined for a sample, fresh samples of filed duplicates must be analyzed
after the contamination has been corrected and acceptable LRB values have been obtained.

2.7.9 Calibration and Standardization

Prepare 0.2, 2, 5, 20 and 200 ug chl a/L calibration standards from the PDS (section 2.5.6.9).
Allow the instrument to warm up for at least 15 minutes. Measure the fluorescence of each
standard at sensitivity settings that provide midscale readings. Obtain response factors for
chlorophyll a for each sensitivity setting as follows:

Fs = Ca/Rs
where,
Fs = response factor for sensitivity setting, S.
Rs = fluorometer reading for sensitivity setting, S.
Ca = concentration of chlorophyll a.

Note. If you are using special narrow bandpass filters for chlorophyll a determination, DO NOT
acidify. Use the "unconnected" chl a calculation described in section 2.5.11.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 79

2.7.10 Procedure

2.7.10.1 Extraction of Filter Samples
1. If samples are frozen, remove them from the freezer but keep them in the dark. Set up the
tissue grinder and have on hand tissues and squirt bottles containing water and acetone.
Workspace lighting should be the lowest possible while still being able to read instructions
and operate machinery.
2. Remove filter from container and place in glass grinding tube. The filter may be torn into
smaller pieces to facilitate extraction. Push filter to the bottom of the tube with a glass rod.
With a volumetric volumetric pipette, add 4 ml_ of aqueous acetone solution to the grinding
tube. Grind the filter until it is converted to a slurry. Do not overheat sample by overgrinding.
3. Pour the slurry into a 15-mL screw-cap centrifuge tube and, using a 6-mL volumetric pipette,
rinse the pestle and the grinding tube with 90% acetone. Add the rinse to the centrifuge
tube with the filter slurry. Cap the tube and shake it vigorously. Place in the dark before
proceeding to the next filter extraction. Thoroughly rinse the pestle, grinding tube and glass
rod alternatively with water and acetone ending with acetone.
4. Shake each tube vigorously before placing all of to steep in the dark at 4°C. Samples
should be allowed to steep for a minimum of 2 hrs but not to exceed 24 hrs. The tubes
should be shaken at least once during the steeping process.
5. After steeping is complete, shake the tubes vigorously and centrifuge samples for 15 min. at
675 g or for 5 min at 1000 g. Samples should be allowed to come to ambient temperature
before analysis. Recalibrate the fluorometer if the room temperature has fluctuated ± 3°C
from last calibration.

2.7.10.2 Sample Analysis

1. After the fluorometer has warmed up for at least 15 minutes, use 90% acetone solution to
zero the instrument on the sensitivity setting that will be used for sample analysis.
2. Pour or pipette the supernatant of the extracted sample into a sample cuvette. For a cuvette
that holds 5 ml_ of extraction solution, 0.15 ml_ of the 0.1 N HCI solution should be used.
3. Choose a sensitivity setting that yields a midscale reading. If the concentration of
chlorophyll a in a sample is > 90% of the upper limit of the LDR, then dilute the sample with
90% acetone solution and reanalyze.
4. Record the fluorescence measurement and sensitivity setting.
5. Remove the cuvette from the fluorometer and acidify the extract to a final concentration of
0.003 NHCI using the 0.1 N NCI solution. Use a Pasteur pipette to thoroughly mix the
sample be aspirating and dispensing the sample into the cuvette, keeping the pipette tip
below the surface of the liquid to avoid aerating the sample.
6. Wait 90 seconds and measure fluorescence again.
Note. If you are using special narrow bandpass filters, DO NOT acidify samples. Use the
uncorrected chl a calculations described in the following section (section 2.5.11).

2.7A1 Data Anlaysis and Calculations

2.7.11.1 Uncorrected Chlorophyll a
1. Calculate the chlorophyll a concentration in the extract as follows:

CE,u= RbxFs

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 80

              where,
              CE,u    = uncorrected chlorophyll a concentration (|jg/L) in the extract solution
              Rb     = fluorescence response of sample extract before acidification
              Fs     = fluorescence response factor for sensitivity setting S.
 2.  Calculate the "uncorrected" concentration of chlorophyll a in whole water sample as follows:
              Cs,u =  CF_M x Extract volume (L) x DF
                           Sample volume (L)
              where,
              Cs,u    = uncorrected chl-a concentration (ug/L)  in the whole water sample
              Extract volume = volume (L) of extraction prepared before any dilutions
              DF     = dilution factor
              Sample volume = volume (L) of whole water sample.
 3.  LRB and QCS  data should be reported with each sample data set.

 2.7.11.2 Corrected Chlorophyll a

 1.  Calculate the chlorophyll a concentration in the extract as follows:
              CE,c=  Fs(r/r-1)(Rb-Ra)
       where,
       CE,C   = corrected chlorophyll a concentration (ug/L) in the extract solution analyzed
       Fs  =  response factor for the sensitivity setting S
       r      = the before-to-after acidification ratio of a pure chlorophyll a solution
       Rb     = fluorescence of sample extract before acidification
       Ra     = fluorescence of sample extract after acidification

 2.  Calculate the "corrected" concentration of chlorophyll a in the whole water sample as
    follows:
              Cs,c=  CF_M x Extract volume (L) x DF
                           Sample volume (L)
              where,
              Cs,c   =  corrected chlorophyll a concentration  (ug/L) in the whole water sample
              Extract volume = volume (L) of extraction prepared before any dilutions
              DF     = dilution factor
              Sample volume = volume (L) of whole water sample.
    3.  LRB and QCS data should be reported with each sample data set.

 2.7.12 References
 USEPA. 1997.  Methods for the Determination of Chemical Substances in Marine and
      Estuarine Environmental Matrices-2nd Edition.  EPA No. 600-R-97-072. U.S.
      Environmental Protection Agency, Office of  Research and Development, Washington, DC.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 81

                                3.0 FECAL INDICATOR

 3.1    SCOPE AND APPLICATION

 This document describes the application of Draft EPA Enterococcus TaqMan qPCR Method for
 the processing and qPCR analysis of water sample concentrates from coastal marine waters
 and the freshwaters of the Great Lakes (NCCA 2010) for the purpose of determining water
 quality by Real-Time Quantitative Polymerase Chain Reaction (qPCR) assays that determine
 the concentration of the fecal indicator Enterococcus bacteria, by measuring the concentration
 of their DMA in the water sample.

 This method facilitates the microbiological determination of water quality of water bodies at
 remote locations from which collected water samples cannot feasibly be analyzed for the
 enumeration of viable (culturable) indicator bacteria because they cannot be transported to an
 analytical laboratory within 6 hours of collection time for analysis by membrane filtration and / or
 selective media inoculation and incubation (e.g.  MPN  broth analysis) methods (EPA Method
 1600). Instead, water samples to be analyzed by Enterococci levels by qPCR are concentrated
 by "field" filtration within 6 hours after collection of the samples.  The filter concentrates, inserted
 into sterile sample extraction tubes containing sterile glass beads or ceramic beads are quickly
 frozen on dry ice until transport to the analytical  laboratory by air courier where they are
 analyzed up to one year after being received.  This method extends the window of time
 available for analysis due to the stability of the Enterococcus cell DMA whose concentration
 directly correlates with the number of Enterococcus cells.

 3.2    SUMMARY OF METHOD

 Aliquots of each water sample have previously been filtered aseptically, the filters have been
 folded inwardly in half four times to form an umbrella or folded in half and rolled up into a
 cylinder and then inserted into sterile sample extraction tubes containing sterile glass beads or
 Roche MagNA Lyser Green Beads™ (actually siliconized white ceramic beads in a green
 capped tube).  Extraction tubes containing filter concentrates (retentates) have been stored on
 dry ice until transport to the  analytical laboratory by air courier. The filter retentate vials can also
 be preserved in -20° to -80°  C freezers after being frozen on  dry ice.  Filter concentrates in vials
 will be shipped by air courier on dry ice to the analytical team at EPA New England Regional
 Laboratory. Filter concentrates received by NERL staff will be subjected to DMA extraction
 procedures and subsequently analyzed by the Draft EPA Enterococcus TaqMan qPCR Method
 along with modifications to the QA/QC procedures described below.  The laboratory methods
 are summarized in Table 3.4 of Section 3.18.  Each filter is subjected to bead-beating in
 extraction buffer to lyse cells and produce a clarified extract.  A sub-sample of the diluted extract
 is subjected to Enterococcus qPCR analysis. This processing can be completed up to  1 year
 after cell concentration if the sample filter retentates are maintained frozen at -20 to -80°C.

 3.3    DEFINITIONS OF METHOD

 Batch Size: The number of samples that will be processed by filter extraction with the  same
 batch (volume) of SAE buffer and analyzed by the same qPCR assay(s) using the same batch
 of qPCR master mix. A batch is covered for quantitation purposes by the same "batch" calibrator
 samples, a minimum of three, analyzed during the same week.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 82

Bottle Blank: Analyte-free water is collected into a sample container, of the same lot number as
the containers used for collection of the environmental samples. Analysis of this sample is
performed to evaluate the level of contamination, if any, introduced into the environmental and
control samples from the sample container(s) from a common vendor's lot.

DNA: Deoxyribo-Nucleic Acid, double-stranded genetic molecules containing sequences of the
four nucleotide bases, adenine, thymine, guanidine, and cytosine that encode rRNA, mRNA,
and tRNA involved in protein synthesis.

Field Filter Blank: A volume of sterile PBS, free of target organisms (i.e. Enterococcus) filtered
through a sterile filter and processed in parallel with all other samples to serve as a sentinel for
detection of reagent contamination or contamination transferred between samples by
processing and analysis.

Field Replicates: Samples collected from coastal marine waters and the Great Lakes that are
collected at the same sampling site one right after the other with only slight temporal variation.
They are not "splits" of the same sample volume.

Filtrate: Sample liquid or buffer rinsate passing through the filter into the vacuum flask.
Laboratory Quality Samples: Mock samples created in the lab such as lab blanks, lab-fortified
blanks (LFBs), and Lab-Fortified Matrices (LFMs) used to assure lack of sample contamination
and to measure analytical recovery during performance of sample processing and analysis
methods.

Performance Testing (PT) / Performance Evaluation Sample (PES): Calibrator samples
(filters spiked with E. faecalis grown in Brain Heart Infusion Broth) and Laboratory Fortified
Blanks (Phosphate Buffered Saline; PBS) spiked with Enterococcus faecalis cells from BHI
Broth suspension will be assayed by EPA Method 1600 and Draft EPA Enterococcus TaqMan
qPCR Method to ascertain method performance. Ball-T Bioballs® which contain a specified
number of E. faecalis cells may also be acquired to determine the performance of the Relative
Quantitation Method. Purified E. faecalis DNA acquired from American Type Culture Collection
and TIB Mol Biol Inc. is used to test the performance of the Absolute Quantitation Method.

Retentate: The sample residue retained by the filter after the sample is vacuum-filtered. The
retentate contains particulates, microbiota, and macrobiota from which the DNA is extracted into
buffer by bead-beating for subsequent qPCR analysis.

Rinsate: The volume of phosphate buffered saline (PBS) applied to a sample's filter retentate in
order to "wash" any residual fine particles, smaller than the filter's nominal pore size, through
the retentate and the filter.

Sample Processing Control (SPC): A surrogate homologue analyte (e.g. Salmon DNA) spiked
into each sample to determine the recovery of target analyte and/or detect assay inhibition
caused by matrix effects.

Standards: Known amounts or numbers of copies of Enterococcus genomic DNA analyzed by
the Enterococcus qPCR assay to generate a Standard Curve (Log Copy Number vs. Crossing
Point Value) in order to determine Enterococcus genomic copy numbers in "Unkown" test
sample extracts by Absolute Quantitation Method.

3.4 INTERFERENCES
a. Low pH (acidic) water
b. Humic and fulvic acid content

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 83
c. Suspended solids (e.g. fecal matter) and participates (sand, dirt)
d. Excessive algal growth

3.5 HEALTH AND SAFETY WARNINGS

All proper personal protection clothing and equipment (e.g. lab coat, protective eyeware /
goggles) must be worn or applied. When working with potential hazardous chemicals (e.g. 95%
ethanol) or biological agents (fecally-contaminated water) avoid inhalation, skin contact, eye
contact, or ingestion. If skin contact occurs remove clothing immediately and wash / rinse
thoroughly. Wash the affected skin areas thoroughly with large amounts of soap and water. If
available consult the MSDS for prompt action, and in all cases seek medical attention
immediately. If inhalation, eye contact or ingestion occurs, consult the MSDS for prompt action,
and in all cases seek medical attention immediately.

3.6 PERSONNEL QUALIFICATIONS

All laboratory personnel shall be trained in advance in the use of equipment and procedures
used during the sample extraction and qPCR analysis steps of this SOP. All personnel shall be
responsible for complying with all of the quality assurance / quality control requirements that
pertain to their organizational / technical function. All personnel shall be responsible for being
aware of proper health and safety precautions and emergency procedures.
3.7 EQUIPMENT AND SUPPLIES

Clean powderless latex or vinyl gloves
Goggles or Face Shield
Micropipettors
Cepheid SmartCycler PCR Thermocycler
Roche MagNA Lyser
Roche MagNA Pure LC (automated nucleic
acid isolation and purification platform)
High Speed Microfuge

Roche MagNA Lyser Rotor Cooling Block
2-mL tube racks
Cepheid Smart tubes
MagNA Pure LC sample processing
cartridges, reagent trays, and pipette tips

3.8 REAGENTS AND STANDARDS
Semi-conical, screw cap microcentrifuge
tubes (PGC, #506-636 or equivalent) pre-
filled with 0.3 + 0.02 g Acid-washed glass
beads (Sigma, # G-1277 or equivalent).
Filled tubes are autoclaved 15-min. Liquid
Cycle (Slow Exhaust).
Or
Roche MagNA Lyser Green Bead tubes
(Roche Applied Science, #03-358-941-001)
sterile, siliconized 3-mm diameter ceramic
beads in a siliconized 2-mL microfuge tube.

Permanent marking pens (fine point and
regular point) for labeling tubes
Bench Sheets & Printouts of Computer
Software Sampling Loading Screen
a. Qiagen AE Buffer (Qiagen 19077)
b. Salmon DNA (Sigma D1626)
c. Frozen tubes of Enterococcus faecalis (ATCC #29212) calibrator cell stock
d. Purified Enterococcus faecalis (ATCC #292120) genomic DNA, 10-ug
e. TaqMan® Environmental PCR Master Mix (ABI #4396838)
f. Enterococcus PCR primers and TaqMan® probe (TIB Mol Biol Inc.)

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 84

g. Sketa PCR primers and TaqMan® probe (TIB Mol Biol Inc.)
h. Bovine Serum Albumen (BSA; Sigma Cat. #6-4287)
i. MagNA Pure LC DMA Isolation Kit III for Fungi & Bacteria (Roche Biochemical Division)

3.9 PREPARATIONS PRIOR TO DNA EXTRACTION AND ANALYSIS

Determine / Estimate the sample batch size (number of samples) for one-week of sample
processing and qPCR analysis. The batch size is the number of sample filter concentrates that
will be extracted by bead-beating with the same batch (volume) of SAE buffer and analyzed by
the same qPCR assay(s) using the same batch of qPCR master mix. A batch is covered for
quantitation purposes by the batch calibrator samples, (a minimum of three) whose 5-fold and
25-fold diluted extracts are analyzed at the outset of the week along with a reagent blank. Fill
out a batch sample analysis bench sheet. (See Section 3.18.)

1. Micropipettors are calibrated annually and tested for accuracy on a weekly basis. Follow
manufacturer instructions for calibration check. Measure three replicate volumes per pipettor
and keep log book of their weights on a calibrated balance scale.

2. Preparation of stock Salmon Sperm (SS) DNA: Dissolve Salmon DNA in PCR grade water
to a concentration equal to approximately 10 ug/mL Determine concentration of Salmon
testes DNA stock by measuring the OD26o in a UV spectrophotometer or by PicoGreen DNA
Concentration Determination Protocol utilizing a NanoDrop UV fluorometer. A DNA solution
with an OD26oOf 1.0 has a concentration equal to approximately 50 ug/mL depending on the
GC content of the DNA's sequence(s).
3. Dilute Salmon testes DNA stock with AE buffer to make 0.2 ug/mL Salmon DNA Extraction
Buffer (SAE). Extraction buffer may be prepared in advance and stored at 4 °C for a
maximum of 1 week.

Note: Determine the total volume of Salmon DNA Extraction Buffer required for each day or
week by multiplying the extraction volume (i.e. 600-uL) times the total number of samples to
be analyzed including controls, water samples, and calibrator samples. For example, for 18
samples, prepare enough Salmon/DNA extraction buffer for 24 extraction tubes (18) /6 = 3,
therefore, 3 extra tubes for water sample filtration blanks (method blanks) and 3 extra tubes
for calibrator samples). Note that the number of samples is divided by 6 because you should
conduct one method blank for every 6 samples analyzed. Additionally, prepare excess
volume to allow for accurate dispensing of 600 uL per tube, generally 1 extra tube. Thus, in
this example, prepare sufficient Salmon DNA Extraction Buffer for 24 tubes plus one extra.
The total volume SAE needed per sample is 600 uL. Hence for the SAE volume for 25
sample tubes is equal to 15,000 uL. Dilute the Salmon DNA working stock 1:50, for a total
volume needed (15,000 jjL) 50 = 300 jjL of 10 jjg/mL Salmon DNA working stock. The AE
buffer needed is the difference between the total volume and the Salmon testes DNA
working stock. For this example, 15,000 uL - 300 uL = 14,700 uL AE buffer needed.
4. Make stocks of ATCC Enterococcus faecalis purified genomic DNA to be diluted to specific
concentrations for use as internal standards in individual qPCR runs whose results are used
to generate the weekly Enterococcus qPCR Standard Curve for quantitation purposes.
5. Use Enterococcus faecalis genomic DNA (10-ug) to make a Frozen E. faecalis DNA
Reference Stock (20-uL) at a concentration of 2.89 x 106 GEQs per uL

6. Dilute 10-uL of the Frozen E. faecalis Reference DNA stock 363-fold to a final volume of
3,630 uL AE buffer. Aliquot 20-uL volumes into many 200-uL microfuge tubes and store

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 85

frozen at -20 °C. The net concentration of Enterococcus GEQs is 8,000 / |jl_. Each week
perform a series of 10-fold and 4-fold dilutions from one thawed tube of the 8,000 GEQ/uL
standard solution to create 800 GEQ/uL, 80 GQ/uL and 20 GEQ/uL standard solutions. The
analyst performs Enterococcus qPCR upon duplicate 5-uL volumes of each of the four
standards yielding a Standard Curve of Log GEQs ENT versus Ct value from which the
assays "efficiency" is subsequently calculated in the Relative Quantitation EXCEL
Spreadsheet.

7. Make Enterococcus faecalis calibrator filter samples:
i. Assemble calibrator positive control samples by thawing tubes of E. faecalis cell
stocks, diluting their contents (10-uL) up to 1-mL AE buffer and spotting 10-uL on
sterile PC filter previously folded and inserted into a pre-chilled Green Bead tube.
ii. Spot a sufficient number of calibrator filter samples for the entire study to insure
uniform, consistent relative quantitation of study samples. Store the calibrator filter
samples in -20°C freezer and thaw individual calibrators (three per week) for extraction
with each week's batch of samples.
iii. The calibrator sample filters are spotted with 104 or 10s Enterococcus faecalis cells
and this number is incorporated into the Relative Quantitation EXCEL spreadsheet.

8. Prior to and after conducting work with cells and / or genomic DMA standards, disinfect and
inactivate (render non-amplifiable) DMA in the Sample Extraction Hood, the qPCR Cabinet,
and the qPCR Sample Loading Hood with 10% bleach and >_15-min. exposure to high
intensity germicidal (254 nm) ultraviolet light.

3.10 PROCEDURES FOR PROCESSING AND QPCR ANALYSIS OF SAMPLE
CONCENTRATES

3.10.1 Sample Processing (DNA Extraction)

Typically, 100-mL volumes of surface water are filtered according to Draft EPA Enterococcus
TaqMan qPCR Method for processing and analysis by PCR assays. Due to the limitations of
field crew sampling time and the performance limitations of the manually-operated vacuum
pumps used in the field sampling operations, two 100-mL or four 50-mL (optional) surface water
samples will be filtered depending on difficulty of filtration. Lower volumes (< 50-mL) were
acceptable if suspended particulates hinder the filtering of the standard 50-mL volume during
the National Lakes & Ponds but should not be necessary in the NCCA if the prescribed 0.4
micron pore size Polycarbonate filters (Whatman Nucleopore Cat. #1111007) are correctly
employed. If the filtration of 100-mL volumes is not possible after diligent efforts by field crews,
lesser volumes may be acceptable but only if equal replicate volumes are filtered. Field crews
should make every effort to filter 100-mL of water sample per filter, filtering 50-mL sub-samples
at a time. If filtration has slowed to a drip by the end of finishing the first 50-mL sub-sample the
field crew should make the decision to filter four filters of 50-mL volumes instead of two filers of
100-mL each. If a sample is highly turbid with extreme amounts of Total Suspended Solids field
crew members should filter 25-mL sub-sample aliquots at a time in their attempt to reach the
100-mL or 50-mL target volume. Although every attempt should be made to filter a minimum of
50-mL of sample per filter, if only volumes less than 50-mL can be filtered then at least make
sure all four of the replicate filters contain an equivalent volume of sample filtered through them
(e.g. 4x30-mL).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 86

Filtration of lower sample volumes has necessitated modifications to Draft EPA Enterococcus
TaqMan qPCR Method which are directed by the Analysis Decision Tree (ADT; Section 3.18.8).
In accordance with the ADT, if < 50-mL of a water sample is filtered per filter replicate, then the
laboratory analyst extracts two replicate filters in parallel and combines equivalent volumes of
the filter extracts to form one composite filter extract. Each individual filter is extracted with only
300-ul_ of SAE Extraction Buffer instead of the usual prescribed 600-ul_ volume of SAE buffer.
Halving the SAE buffer volume enables the analyst to maintain an equivalent Method Detection
Limit and maintain a similar Sample Equivalence Volume (SEQ; i.e. water sample volume per
extract volume) in the extract volumes (e.g. 5-uL) of each sample filter concentrate added to the
PCR reactions.

1. Pre-chill MagNA Lyser Rotor Cooling Block in -20°C freezer. Label 1.7-mL sterile microfuge
tubes with sample ID number to match them with Green Bead Tubes. Two supernatant
recovery tubes and one "5-fold" dilution tube is needed per sample and should be labeled
accordingly. The dilution tube shall be filled with 80-uL AE buffer using a micropipettor.
2. To extract sample filters, uncap green bead tube (cold) and add 0.6-mL (600-uL) SAE
Buffer (Qiagen AE Buffer spiked with Salmon DMA). Re-cap tubes tightly.

3. Insert Green Bead tubes of samples into MagNA Lyser and bead-beat for 60-sec (1-min) at
5,000 rpm at Room Temperature. Transfer sample tubes to microfuge. Spin tubes at
12,000 rpm for 2-min. Being careful to move filter aside, recover and transfer up to 400-uL
of supernatant (sans debris) to new tube with a P-200 or P-1000 micropipettor.

4. Spin the supernatant tubes for 5-min at 14,000 rpm at Room Temperature. Recover >350-
uL supernatant and transfer to new 1.7-mL tube. When all samples in a batch have been
extracted transfer dilute 20-uL of DMA extract (2nd supernatant) five-fold (5X) in 80-uL AE
buffer (sans SS-DNA) and store at 4°C for qPCR assays. (If supernatant, 5X and even 25X
sample dilutions possess dark pigment and exhibit severe qPCR inhibition in Sketa assays,
consider extracting replicate filters of samples using the Modified MagNA Pure LC DMA
Isolation Protocol (see Section 3.18.9).

3.10.2 Sample Analysis by Enterococcus qPCR

3.10.2.1 Preparation ofqPCR Assay Mix

1. To minimize environmental DMA contamination, routinely treat all work surfaces with a 10%
bleach solution, allowing the bleach or reagent to contact the work surface for a minimum of
15 minutes prior to rinsing with sterile water. If available, turn on UV light for 15 minutes.
Alternatively use commercial products (e.g. DMA Away or DMA ZAP) according to vendor
instructions.

2. Using a micropipettor with aerosol barrier tips, add PCR grade water to the lyophilized
primers and probe to create stock solutions of 500 uM primer and 100 uM probe and dissolve
by extensive vortexing. Pulse centrifuge to coalesce droplets. Store stock solutions at -20°C.

3. Prepare working stocks of Enterococcus, and Salmon DMA primer/probe mixes by adding 10
uL of each Enterococcus or Salmon DMA primer stock and 4 uL of respective probe stock to
676 uL of PCR grade water, and vortex. Pulse centrifuge to create pellet. Use a micropipettor
with aerosol barrier tips for all liquid transfers. Transfer aliquots of working stocks for single
day use to separate tubes and store at 4°C.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 87

4. Using a micropipettor, prepare assay mix of the Enterococcus, and Salmon DMA reactions in
separate, sterile, labeled 1.7 ml_ microcentrifuge tubes as described in
a. Table 3.1.

5. Finger vortex the assay mix working stocks; then pulse microcentrifuge to coalesce droplets.
Return the primer/probe working stocks and other reagents to the refrigerator.

6. Thaw and finger vortex sample extract (dilution) tubes that will be assayed in PCR run.
Microfuge a few seconds to coalesce droplets. Finger mix and spin the standards and
calibrator samples (dilutions). Temporarily store all samples in 4°C refrigerators until use in
assay or return to long term storage at -20°C. Discard disposable gloves and put on a new
pair.

7. Set 32 Smart tubes in Cepheid Racks in PCR cabinet along with micro-pippetors and
expose to germicidal UV lamp for 15-min.

8. Pipette 20-ul_ of respective Master Mix into each labeled Smart tube. Transfer Smart tubes
(racks) from PCR cabinet to disinfected Sample Loading Fume Hood.

9. Using P-10 or P-20 micro-pipettor load each Smart tube with 5- uL volume of respectively
designated sample extract (dilution), standard, or buffer blank (SAE). Cap each sample's
Smart tube after loading.

10. Check to make sure each Smart tube is properly labeled and identifiable by sample number
or l-core position (e.g. A4). Insert loaded Smart tubes into Smart Tube microfuge. Close lid
and spin 5-sec. Pop lid to stop. Remove Smart Tubes from microfuge and insert into proper
position in SmartCycler.

Enterococcus (Enterol) and Salmon (Sketa) qPCR assays (Draft EPA Enterococcus TaqMan
qPCR Method) will be performed upon 5-uL aliquots of un-diluted & 5X diluted extracts of
sample unknowns, calibrator, field blank, and lab blank. A "No Template Controls" (NTC) shall
be analyzed on an ongoing basis to ensure that the Master Mix PCR reagents are not
contaminated. To minimize the number of Enterococcus qPCR reactions needed to be
performed upon samples, Sketa qPCR assays will be performed upon the 5-fold diluted DMA
extracts of samples before any Enterococcus qPCR assays are run in order to screen samples
for the presence and dilution of PCR inhibitors by comparison with the undiluted and 5-fold
dilution DMA extract of the calibrator samples and unused portions of SAE buffer. Each
sample's lowest dilution DMA extract not exhibiting PCR inhibition in the Sketa qPCR assay will
be re-assayed by the Enterococcus qPCR assay and it's results will be used for quantitation of
Enterococcus DMA sequences and CCEs.

Detection of reduced levels of Salmon DMA (higher instrument Ct values) is indicative of
technical error during extract dilution or excessive levels of PCR inhibitors or nuclease activity
which could impact detection of the Enterococcus DMA target sequences in the Enterococcus
PCR assay. Alternatively, the high Sketa Ct value may be indicative of the occurrence of a
technical error during extract dilution. If a test sample's Ct value is less than 3 cycles different
than the blank negative control and calibrator samples, indicating only negligible or marginal
inhibition (the Sketa Assay is more sensitive to inhibitors than the ENT Assay), an aliquot of its
five-fold diluted extract is analyzed in the Enterococcus Assay. If an abundance of PCR
inhibitors or DMA nucleases are present in a sample extract which are causing a greater
increase in an extract's Ct value (> 3 cycles increase), then the extract is diluted an additional
five-fold (net 25-fold dilution) and re-assayed by both the Sketa and ENT assays. If the
inhibition is not ameliorated by the additional dilution, which should restore the Sketa Ct value to

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                            Date: November 2010
	Page 88

 that of the 25-fold diluted calibrator samples' extracts, the following actions are taken by the
 analyst.  First, the analyst re-dilutes the sample's undiluted DMA extract five-fold and re-
 analyzes the dilution with the Sketa PCR assay to confirm that Ct variance is not due to a
 dilution error.  If the Ct difference is not attributed to a dilution error, replicate sample filters of
 the "inhibited" samples are subjected to DMA extraction and purification by the MagNA Pure LC
 automated platform loaded with the Roche DMA Isolation Kit III (Bacteria;  Fungi) reagents (see
 Section 3.18.9).

 The EPA Modified MagNA Pure LC extraction process which includes the spiking of the Lysis
 Binding Buffer with the Salmon (I PC) DMA is more effective, but more costly, than Draft EPA
 Enterococcus TaqMan qPCR Method in neutralizing severe levels of  PCR inhibitors and DMA
 nucleases present in some environmental samples, especially those containing high levels of
 algae or phytoplankton. The purified DMA extract yielded by MagNA  Pure extraction of the few
 (<5%) "severely inhibited" samples is subsequently analyzed by the Sketa and Enterococcus
 qPCR assays and the number of Enterococcus CCEs per 100-mL determined by the Delta CT
 and Delta Delta CT Relative Quantitation Methods. While the MagNA Pure LC extraction
 method is not 100% conservative (no partitioning or recovery issues)  like Draft EPA
 Enterococcus TaqMan qPCR Method, it typically exhibits DNA recoveries in the range of 25-
 50%.  DNA recoveries and Enterococcus CCE concentrations are calculated using only the
 Delta-Delta Ct Relative Quantitation Method.  The relative DNA recoveries are determined by
 comparison of the Sketa results from purified DNA eluates of each test sample with those of the
 extracted lab blank and calibrator samples. The absolute DNA recovery is calculated by
 comparison of the former Sketa results with those of elution buffer spiked  with an amount of
 Salmon DNA equivalent to the amount in the Salmon-spiked Lysis Binding Buffer added to each
 sample filter lysate during the MagNA Pure LC DNA extraction process.

 The "Unknown" and "Control" sample extracts whether processed using the SAE buffer or
 MagNA Pure LC Kit III reagents are analyzed according to the Cepheid SmartCycler
 Enterococcus and Sketa qPCR protocols described in Appendix A of  the Draft EPA
 Enterococcus TaqMan qPCR Method with Ct determination made by  the software using Manual
 Determination (equivalent of Fit Points Method of Roche LightCycler)  with the fluorescence
 threshold set at 8.0 units which enables uniform analysis and comparability of all samples'
 qPCR results.

 Sample analysis sequence for SmartCycler:

 Example: For analyses on a single 16-position SmartCycler, calibrator samples and water
 samples are analyzed  in separate runs and a maximum of 6 water samples (or 2 replicates of 3
 samples) are analyzed per run, as described in Table 2 and Table 3 of Section 3.18 (below).

 Enterococcus and Sketa (Salmon DNA = SPC) qPCR results are exported to an EXCEL
 spreadsheet in which relative quantitation calculations are performed  by analysts. The Draft
 EPA Enterococcus TaqMan qPCR Method results are reported in terms (units of measure) of
 Number of Enterococcus Sequences and Number of Enterococcus Calibrator Cell Equivalents
 (CCEs) per 100-mL sample volume. The qPCR results are converted to this standardized unit of
 measure based on the volume of water sample actually filtered (e.g.,  10-mL, 25-mL, or 50-mL).

 Note: Samples with Enterococcus qPCR results below the Reporting  Limits (qPCR results with
 Ct values below 35.5 cycles) are qualified as "Estimates". The RL varies proportionally to the
 volume of sample filtered by each sample crew at a specific site. Reporting limits (RL) and

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                            Date: November 2010
	Page 89

 Method Detection Limits (MDLs) will be higher among samples for which a volume of water <50-
 mL is filtered.

 Enterococcus qPCR results are flagged if some part of the sample collection, hold-time,
 processing, shipment, storage, sample extraction, or qPCR analysis are compromised and did
 not meet the requirements of the Sampling and Analysis SOPs.

 3.11   STORAGE AND TIMING OF PROCESSING / ANALYSIS OF FILTER
       CONCENTRATES

 When a sufficient number of water sample filter concentrates (filters and retentates) have been
 received by NERL and qPCR analytical reagents have been obtained the samples will be
 logged into LIMS. Sample processing and qPCR will commence and results will be entered into
 the LIMS upon completion of analysis.

 3.12   CHAIN OF CUSTODY

 Follow the Sample Control Procedures, Field Sampling Form / Enterococci Filtration / Sample
 Processing Standard Operating Procedures.

 Field Sampling forms and NCAA 2010 Sample Tracking EXCEL Spreadsheet shall be consulted
 to determine if a sample has been properly preserved during collection and transport prior to
 analysis and that it has  passed all criteria permitting its analysis. The qPCR results of samples
 exceeding established criteria or whose associated field / lab blanks had positive Enterococcus
 qPCR detections of DMA shall be flagged.

 3.13   QUALITY CONTROL / QUALITY ASSURANCE

 The Data Quality Objectives and the Laboratory QC Procedures are listed and summarized in
 Tables 5 and 6 of section 3.18 below.

 The number of field blanks (dilution buffer only) shipped by field crews during their first visit to
 subsequently "re-visited" marine and freshwater sampling sites represents a frequency of 5-10%
 of the total number of samples extracted  and analyzed by qPCR. All field  blanks (negative
 controls) will be extracted and analyzed by qPCR for the detection of Enterococcus. The blanks
 will be analyzed in these cases to insure  that positive detections in field samples are not due  to
 contamination by sampling crews.

 One Lab / Method Blank (LB; sterile filters) will be run  per extraction sub-batch in order to insure
 the sterility (lack of DMA contamination) in the SAE buffer and pipette tips used to process all of
 the samples. The LB sample will be processed and diluted like all other "Unknown" samples

 Up to four replicate filter concentrates (retentates) derived from the field filtration of 100-mL or
 50-mL sample volumes of every sample will be received by NERL and stored at -20 to -80°C.
 One filter retentate of each sample  (and duplicates for 10% of samples) will be extracted to
 obtain DMA lysates for Enterococcus qPCR analysis. The remaining filter concentrates will be
 archived for possible extraction and analysis at a later time if needed.

 Enterococcus and Sketa qPCR analysis will be performed upon 5-uL volumes of the non-diluted
 and 5-fold diluted (in AE buffer) extracts which will be added to 20-uL qPCR Master Mix

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 90

volumes and analyzed in the Cepheid SmartCycler qPCR instrument in accordance with Draft
EPA Enterococcus TaqMan qPCR Method.

Duplicate Enterococcus and Sketa qPCR assays will be performed upon 10% of the sample
extracts (diluted and un-diluted) each week (batch) to determine qPCR assay variance.

3.14 METHOD PERFORMANCE

Method Performance will be determined by the use of Performance Testing (PT) / Performance
Evaluation Samples (PES). Calibrator samples (filters spiked with frozen stocks of E. faecalis
grown in Brain Heart Infusion Broth) and Lab-Fortified Matrices (LFMs; duplicate sample filters
spiked with frozen stocks of E. faecalis grown in Brain Heart Infusion Broth) will be extracted
and assayed by Draft EPA Enterococcus TaqMan qPCR Method Enterococcus and Sketa
qPCR assays in order to ascertain method performance. The LFMs are performed upon
several samples (approx. 5% frequency) per batch, typically samples exhibiting non-detection of
Enterococcus, in order to determine method performance and also to insure that non-detects
are not due to poor DMA recovery caused by matrix effects.

3.15 RECORD KEEPING AND DATA MANAGEMENT

Laboratory analysts shall follow the EPA OEME Laboratory Data Management SOP.

Each lab analyst shall record all details pertaining to sample processing and analysis in a
designated, bound laboratory notebook.

Pertinent sample collection and analysis data shall be entered into the Laboratory Information
Management System (LIMS) and SeaGate Crystal Reports shall be generated as required by
the EPA (TOPO).

An EXCEL spreadsheet of sample analysis data and associated calculations used to derive a
field sample's or control sample's Enterococcus genomic DMA (GEQ) and Cell Equivalent
(CEQ) concentration shall be uploaded to the NCCA 2010 database stored on a computer
server in Corvallis, Oregon.

3.16 WASTE MANAGEMENT AND POLLUTION PREVENTION

During the sample processing procedures there may be hazardous waste produced. The waste
must be handled and disposed of in accordance with federal, state, and municipal regulations.

All recyclable and non-recyclable materials for disposal will be properly sorted for their
respective waste streams and placed into proper containers for janitorial staff to collect and
process according to EPA guidelines.

All ethanol used shall be consumed by ignition or evaporation. Volumes of ethanol remaining at
the end of the project can be stored for later use in a flammable cabinet or disposed of through
appropriate hazardous waste disposal vendors.

Reagent ethanol shall be contained in screw cap tubes along with the filter forceps to sterilize
the latter and to prevent ethanol spillage during transport between sampling sites.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 91
After the DMA extract is recovered from the sample filter after bead-beating in buffer and
centrifugation, the filter and bead-tube will be discarded in autoclave bags and sterilized for 30-
min at 12VC/30 psi to inactivate any potential pathogens that may be associated with the
samples.

3.17  REFERENCES

USEPA Region 1 (New England) OEME NERL Standard Operating Procedure for the Collection
of Chemical & Biological Ambient Water Samples (ECASOP-Ambient Water Sampling 2;
January 31, 2007)

Draft EPA Enterococcus TaqMan qPCR Method for Quantitation of Enterococci in Water and
Wastewater by TaqMan® Quantitative Polymerase Chain Reaction (qPCR) Assay.
December 2006 (12/15/06 a)

USEPA NERL OEME Draft Bench SOP for Real-Time PCR Method Quantifying Enterococci in
Recreational Water Samples (August 2006)
3.18   TABLES, DIAGRAMS, FLOWCHARTS, CHECKLISTS, AND VALIDATION DATA

Table 3.1. PCR Assay Mix Composition (according to Draft EPA Enterococcus TaqMan qPCR Method)
Reagent
Sterile H O
Bovine Serum Albumen (20 mg/mL)
TaqMan® 2X Environmental Master
Primer/probe working stock solution
Volume/Sample (multiply by # samples to be analyzed
day)
per
1.5 ML
2.5uL
12.5 uL
3.5 uL*
     Note: This will give a final concentration of 1 fjM of each primer and 80 nM of probe in the reactions.
     Prepare sufficient quantity of assay mix for the number of samples to be analyzed per day including
     calibrators and negative controls plus at least two extra samples. It is strongly recommended that
     preparation of assay mixes be performed each day before handling ofDNA samples.

Table 3.2. Batch Calibrator & Enterococcus Standards PCR Run - 7 Samples
Sample Description*
3 Calibrators (5- and/or
25-fold dilution)
3 Calibrators (5- and/or
25-fold dilution)
4 Enterococcus faecalis
DMA Standards
No template control
(reagent blank)
Quantity Samples
3
3
4
1
PCR Assay
Master Mix
Salmon DNA (Sketa)
Enterococcus
Enterococcus
Enterococcus
Quantity PCR Reactions
6
6
8
1
     Diluted equivalently to the water samples

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
            Page 92
Table 3.3. Sub-Batch Test Sample PCR Run - 26 Samples & 1 Method Blank
Sample Description*
Water samples, (5-fold dilution)
Method blank or Sample PCR Reaction
Duplicate, (1- or 5-fold dilution)
Non-diluted SAE Buffer
Water samples, (1- or 5-fold dilution)
Method blank or Sample PCR Reaction
Duplicate, (1- & 5-fold dilution)
Quantity
Samples
26
1
1
26
1
PCR Assay
Master Mix
Enterococcus
Enterococcus
Enterococcus
Salmon DMA
Salmon DMA
Quantity PCR
Reactions
26
1
1
26
1
* Use of 5-fold diluted samples for analysis is currently recommended if only one dilution can be analyzed.
 Analyses of undiluted water sample extracts have been observed to cause a significantly higher
 incidence of PCR inhibition while 25-fold dilutions analyses may unnecessarily sacrifice sensitivity.
Table 3.4. Laboratory Methods: Fecal Indicator (Enterococci)
Variable or
Measurement

Sample Collection


Sub-sampling


Sub-sample
(& Buffer Blank)




Preservation &
Shipment


DMA Extraction
(Recovery)


EPA Draft
Enterococcus
TaqMan qPCR
Method
(Enterococcus &
Sketa SPC qPCR)
QA
Class

C


N


N




c



c



c
Expected
Range and/or
Units

NA


NA


NA




-40Cto+40 C



10-141%



<60 (RL) to
>1 00,000 ENT
CCEs/100-mL
Summary of Method
Sterile sample bottle submerged
to collect 250-mL sample 6-12"
below surface at 1-m from shore
2 x 1 00-mL or 4 x 50-mL sub-
samples poured in sterile 50-mL
tube after mixing by inversion 25
times.
Up to 1 00-mL sub-samples
filtered through sterile
polycarbonate filter. Funnel rinsed
with 2 x 1 0-mL buffer. Filter

folded or rolled, then inserted in
tube then frozen on dry ice.
Batches of sample tubes shipped
on dry ice to lab for analysis.


Bead-beating of filter in buffer
containing Extraction Control
(SPC) DMA. DMA recovery
measured
5-uL aliquots of sample extract
are analyzed by ENT & Sketa
qPCR assays along with blanks,
calibrator samples & standards.
Field and lab duplicates are
analyzed at 5% frequency. Field
blanks analyzed along with test
samples.
References
NCCA Field
Operations
Manual 2010
NCCA
Laboratory
Methods Manual
2008

NCCA
Laboratory
Methods Manual

2008

NCCA
Laboratory
Methods Manual
2008
EPA Draft
Enterococcus
TaqMan qPCR
Method
EPA Draft
Enterococcus
TaqMan qPCR
Method;
NERL NCCA
2010qPCR
Analytical SOP
       C = critical, N = non-critical quality assurance classification.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
            Page 93
Table 3.5. Parameter Measurement Data Quality Objectives
Variable or
Measurement
DMA Extraction
(Recovery)
Enterococcus & SPC
qPCR
SPC & ENT DMA
sequence numbers of
Calibrators &
Standards by AQM
ENT CCEs by dCf
ROM
ENT CCEs by ddCf
ROM
QA
Class
C
C
RSD =
40%
RSD =
55%
RSD =
55%
Expected
Range and/or
Units
10-141%
<60 to >1 0,000
ENT CEQs
/100-mL
80%
40%
50%
Summary of Method
Bead-beating of filter in buffer
containing Extraction Control
(SPC) DNA. DNA recovery
measured
5-uL aliquots of sample
extract are analyzed by ENT
& Sketa qPCR assays along
with blanks, calibrator
samples & standards.
Field and lab duplicates are
analyzed at 5% frequency.
Field blanks analyzed at end
of testing only if significant
detections observed.
95%
95%
95%
References
EPA Draft
Enterococcus
TaqMan qPCR
Method
EPA Draft
Enterococcus
TaqMan qPCR
Method
NERL NCCA 2008
2009 qPCR
Analytical SOP
(QAPP)



  C = critical, N = non-critical quality assurance classification.
  *AQM = Absolute Quantitation Method; ROM = Relative Quantitation Method;
  SPC = Sample Processing Control (Salmon DNA/ Sketa); CCEs = Calibrator Cell Equivalents

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 94
Table 3.6.  Laboratory QC Procedures: Enterococci DMA Sequences
Check or
Sample
Description
Frequency
Acceptance Criteria
Corrective Action
SAMPLE PROCESSING
Re-process sub-
samples
(duplicates)
10% of all
samples
completed per
laboratory
Percent Similarity >70%
If <70%, re-process additional
sub-samples
qPCR ANALYSIS
Duplicate
analysis by
different
biologist within
lab
Independent
analysis by
external
laboratory
Use single stock
of E. faecalis
calibrator
10% of all
samples
completed per
laboratory
None
ForallqPCR
calibrator
samples for
quantitation
Percent Congruence <40%
RSD
Independent analysis TBD
All calibrator sample Cp (Cf)
must have an RSD <_40%.
If >30%, determine reason and if
cause is systemic, re-analyze all
samples in question.
Determine if independent
analysis can be funded and
conducted.
If calibrator Cp (CO values
exceed an RSD value of 30% a
batch's calibrator samples shall
be re-analyzed and replaced with
new calibrators to be processed
and analyzed if RSD not back
within range.
DATA PROCESSING & REVIEW
100%
verification and
review of qPCR
data
All qPCR
amplification
traces, raw and
processed data
sheets
All final data will be checked
against raw data, exported
data, and calculated data
printouts before entry into
LIMS and upload to
Corvallis, OR database.
Second tier review by contractor
and third tier review by EPA.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 95
Purified DNA Extracts
NCCA Batch #

Batch
Sample #















Sample
ID#














QA/QC
Qual
Code














Sample Vol
(mL)
Filtered














Dates
Vol. SAE
Buffer
Added (uL)














Color
of
Filter
















25X
Dilution
Needed?














Comments














Figure 3.1. Batch Sample Analysis Bench Sheet for Draft EPA Enterococcus TaqMan qPCR Method

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 96
2010 NCCA Enterococcus qPCR Analysis Decision Tree
Confirm water volume filtered for NRSA sample
filter to be processed and analyzed by qPCR
Process sample filter retentates by adding
600-|jL SAE buffer and bead beating
Perform Sketa qPCR assay upon 5-pL
of 5 fold dilution of DMA extracts
Add 300-|jL SAE Buffer to
each of 2 filter replicates,
bead-beat as per protocol
Perform Sketa qPCR
upon 5-|jL aliquot of
non-diluted &3to5-
fold diluted DMA eluate
Purify DMA free of
nucleases and inhibitors
using DNAEZ Kit or MagNA
Pure DMA Isolation Kit III
Sketa CT
> 3 Cycles

> Cal CT
I '
Enter Sketa and ENT
qPCRCTs with Volume
& Dilution intoCalc
Template. Determine
CCEs/100-mL
Created 10/25/07
Updated 1/2/08
Revised 02/09/10
Dilute 3-5
fold more
and re-assay
by Sketa
qPCR.
Figure 3.2. Enterococcus qPCR Analysis Decision Tree (ADT)
3.18.1 SOP for "Modified" MagNA Pure LC DNA Purification Kit III Protocol

1. Pre-warm the MagNA Pure LC DNA Isolation Kit III Lysis Buffer to 65 °C in waterbath.
Quickly pipette 260-uL of warm Lysis Buffer (un-amended) into each "Green Bead" tube with
filter (preserved after filtration temporarily on ice or during long-term storage in freezer).
Shake tube 5-10 sec to mix buffer with beads and filter. Let stand at RT until batch of 16
samples (including positive control LFB or LFM and negative control LB samples) have all
had Lysis Buffer and had their caps sealed tight. Leave water bath on to use during 30-
minute Proteinase K treatment period.
2. Load the 16 samples into MagNA Lyser Rotor Plate and insert into MagNA Lyser. Tighten
the three handscrews of the locking mechanism. Close the lid tightly. Set controls to shake
for 60-sec at 5,000 rpm. Press the start button.
3. When the shake cycle has ended press the Open Lid Button. Open the lid and unlock the
locking mechanism screws. Remove tube plate and set on bench top MagNA Lyser tube
ring hub. Remove tubes, insert into tube styrofoam water bath float and cool tubes in ice
water for 2-min. or place directly into 24-place microfuge rotor, pre-chilled in freezer.
4. Insert tubes into centrifuge rotor symmetrically in order to balance rotor. Close lid of
centrifuge. Set spin parameters for 3,000 rpm for 1-min at 4°C. Press Start button.
Centrifuge to collect drops and foam off of cap down into tube.
5. When centrifuge stops, open lid and remove tubes from rotor. Uncap tubes in order and
add 40-uL of Proteinase K (dissolved in Lysis Buffer Elution Buffer). Re-cap tubes and mix
lysate by inversion. Do not vortex. Knock beads and filter down from cap into bottom of tube
by tapping tubes on bench countertop.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 97

6. Insert tubes into styrofoam floating rack. Incubate tubes 30-min at 65°C in water bath. Set
timer for 15-min. At end of 15-min remove rack from water bath and inverts several times to
mix samples and tap beads and filter back down into tube. Re-place rack in 65°C waterbath
for 15-min. for total of 30-min.
7. Repeat steps 3 to 8 to process 16 more samples in parallel for loading MagNA Pure LC
sample cartridge with 32 DMA extracts for downstream processing in the robotic platform.
8. After 30-min in 65 °C waterbath remove tubes from water bath and place in MagNA Lyser
Bead Beater for 15 seconds at 5,000 rpm. After 15 seconds of bead-beating, place in ice
bath for 5-min to cool.
9. Insert tubes in centrifuge rotor and spin 3-min at 12,000 rpm and 4 °C to pellet sediment and
cell debris. When spinning is complete, open lid of centrifuge and rotor and mark side of
outer side of cap where pellet should have formed.
10. Carefully remove rotor from centrifuge and set on bench. Remove tubes one at a time from
rotor and use 200-uL pipettor and sterile aerosol-proof tips to transfer approximately 150uL
lysate supernatant from tube to wells in MagNA Pure LC Sample Cartridge in pre-
designated order.
11. When all 16 sample supernatants transferred to sample cartridge put adhesive film over
cartridge to prevent contamination and evaporation. Put sample cartridge in ice water bath
or fridge to maintain 4 °C.
12. Repeat steps 9 to 13 for second batch of 16 samples (lysates). Re-cover sample cartridge
with adhesive film for storage. Centrifuge sample cartridge opposite a balance cartridge for
75-sec (1-min, 15-sec) at 2800 rpm in IEC centrifuge (or equivalent) with rotor adaptors for
microtiter plates in place. Insert the film-covered sample cartridge in MagNA Pure LC
platform.
13. Load the MagNA Pure LC platform with volumes of extraction kit reagents prescribed by
MagNA Pure LC computer software for the number of samples being extracted. Before
closing the platform lid and starting the extraction process add 1.5-uL of 0.27 mg/mL
Salmon DNA Stock per 1mL Lysis Binding Buffer (blue soapy solution) as the Sample
Processing Control (SPC). If the amount of Salmon DNA stock to be added is less than 10-
uL, dilute the Salmon DNA stock so that a volume > 10-uL can be pipetted into the Lysis
Binding Buffer. Rinse pipette tip up and down three times in Lysis Binding Buffer.
14. Remove film from top of sample cartridge and re-insert in Roche MagNA Pure LC platform
set up with DNA Purification Kit III (Fungi; Bacteria) reagents in tubs, tips, tip holders, and
processing / elution cartridges. Close platform lid and after checking off checklist of loaded
items (e.g. reagents, tips) lock the lid and start the automated DNA III Extraction Protocol
which purifies each sample's DNA and elutes it into 100-uL Elution Buffer.
15. When extraction process is complete, unlock the MagNA Pure LC platform lid and remove
the sample eluate cartridge. Cover the cartridge with adhesive film and store at 4 C until
qPCR analysis. Store cartridge at < -20 °C for long term preservation.
16. Prepare Elution Buffer Control from 9.3ug/mL Salmon DNA Stock by diluting a small
volume to 37.2 pg/1000uL (1-mL). This control sample is only analyzed by the Sketa qPCR
assay. The Ct value obtained represents that value expected in Sketa qPCR assays of
each MagNA Pure LC purified sample if 100% of the Salmon DNA was recovered and
detected. Vortex to mix on low speed briefly prior qPCR analysis. Centrifuge for 1.5-min to
coalesce droplets. Remove film to aliquot sub-samples and re-place with new film cover to
restore at cool temperatures.

-------
  National Coastal Condition Assessment
  Laboratory Methods Manual
Date: November 2010
            Page 98
                                    4.0 CONTAMINANTS
  PERFORMANCE-BASED METHODOLOGIES

  Suggested analytical methods for contaminants in sediment and fish tissue are described in
  section 4.0 of this manual.  However, some laboratories participating in the survey may choose
  to employ other analytical methods. Laboratories engaged by EPA or the State may use a
  different analytical method as long as the lab is able to achieve the same performance
  requirements as the standard methods. Performance data must be submitted to EPA prior to
  initiating any analyses. Methods performance requirements for this program identify detection
  limit, precision and accuracy objectives for each indicator. Method performance requirements
  for contaminants in sediment and fish tissue are shown in Table 4.1

  Table 4.1.  Laboratory method performance requirements for contaminants in sediment and fish tissue
Inorganic
Analytes
Aluminum
Antimony
Arsenic
Cadmium
Chromium
Copper
Iron
Lead
Manganese
Mercury
Nickel
Selenium
Tin
Zinc
Organic
Analytes
PAHs
PCS congeners
Chlorinated
pesticides/DDTs
TOC
MDL Objective
- Fish Tissue
(wet weight,
ug/g (ppm))
10.0
Not measured
2.0
0.2
0.1
5.0
50.0
0.1
Not measured
0.01
0.5
1.0
0.05
50.0
MDL Objective
- Fish Tissue
(wet weight,
ng/g (ppb))
NA
2.0
2.0
Not measured
MDL Objective
- Sediments
(dry weight,
ng/g (ppm»
1500
0.2
1.5
0.05
5.0
5.0
500
1.0
1.0
0.01
1.0
0.1
0.1
2.0
MDL Objective
- Sediments
(dry weight,
ng/g (ppb))
10
1.0
1.0
100
Maximum Allowable
Accuracy1
Tissue
35%
35%
35%
35%
35%
35%
35%
35%
35%
35%
35%
35%
35%
35%
Sediment
20%
20%
20%
20%
20%
20%
20%
20%
20%
20%
20%
20%
20%
20%
Maximum Allowable
Accuracy1
Tissue
20%
20%
20%
20%
Sediment
35%
35%
35%
35%
Maximum Allowable
Precision2
Tissue
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
Sediment
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
30%
Maximum Allowable
Precision2
Tissue
30%
30%
30%
30%
Sediment
30%
30%
30%
30%
Completeness
Objective3
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
95%
Completeness
Objective3
95%
95%
95%
95%
Accuracy (bias) goals are expressed either as absolute difference (± value) or percent deviation from "true" value.  Precision goals
  are expressed as relative percent difference (RPD) or relative standard deviation (RSD) between two or more replicate
  measurements.3 Completeness goal is the percentage of expected results that are obtained successfully.

-------
National Coastal Condition Assessment
Laboratory Methods Manual                                               Date: November 2010
                                                                                 Page 99

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 100

4.1 SAMPLE PREPARATION FOR METALS ANALYSIS

4.1.1 Microwave Assisted Acid Digestion

1. This method is applicable to the microwave assisted acid digestion of siliceous matrices,
and organic matrices including biological tissues. This method is applicable for the following
elements:

Aluminum Beryllium Copper Mercury Sodium
Antimony Cadmium Iron Molybdenum Strontium
Arsenic Calcium Lead Nickel Thallium
Boron Chromium Magnesium Potassium Vanadium
Barium Cobalt Manganese Selenium Zinc
Other elements and matrices may be analyzed by this method if performance is
demonstrated for the analyte of interest, in the matrices of interest, at the concentration
levels of interest.
2. This method is a rapid multi-element microwave assisted acid digestion prior to analysis
protocol so that decisions can be made about the material. Digests and alternative
procedures produced by the method are suitable for analysis by flame atomic absorption
spectrometry (FLAA), cold vapor atomic absorption spectrometry (CVAA), graphite furnace
atomic absorption spectrometry (GFAA), inductively coupled plasma atomic emission
spectrometry (ICPAES), inductively coupled plasma mass spectrometry (ICP-MS) and
other analytical elemental analysis techniques where applicable. Due to the rapid advances
in microwave technology, consult your manufacturer's recommended instructions for
guidance on their microwave digestion system and refer to this manual's "Disclaimer" when
conducting analyses using this method.
3. The goal of this method is total sample decomposition and with judicious choice of acid
combinations this is achievable for most matrices. Selection of reagents which give the
highest recoveries for the target analytes is considered the optimum method condition.

4.1.2 Summary of Method

A representative sample is digested in concentrated nitric acid and usually hydrofluoric acid
using microwave heating with a suitable laboratory microwave system. The method has several
additional alternative acid and reagent combinations including hydrochloric acid and hydrogen
peroxide. The method has provisions for scaling up the sample size to a maximum of 1.0 g. The
sample and acid are placed in suitably inert polymeric microwave vessels. The vessel is sealed
and heated in the microwave system. The temperature profile is specified to permit specific
reactions and incorporates reaching 180 ± 5. After cooling, the vessel contents may be filtered,
centrifuged, or allowed to settle and then decanted, diluted to volume, and analyzed by the
method found in section 4.3 of this manual.

4.1.3 Interferences

1. Gaseous digestion reaction products, very reactive, or volatile materials that may create
high pressures when heated and may cause venting of the vessels with potential loss of
sample and analytes. The complete decomposition of either carbonates, or carbon based
samples, may cause enough pressure to vent the vessel if the sample size is greater than
0.25 g. Variations of the method due to very reactive materials are specifically addressed in
section 4.1.6.2.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 101

2. Most samples will be totally dissolved by this method with judicious choice of the acid
combinations. A few refractory sample matrix compounds, (e.g., TiO2, alumina, other oxides)
may not be totally dissolved and in some cases may sequester target analyte elements.

3. The use of several digestion reagents that are necessary to either completely decompose
the matrix or to stabilize specific elements may limit the use of specific analytical
instrumentation methods. Hydrochloric acid is known to interfere with some instrumental
analysis methods such as flame atomic absorption (FLAA) and inductively coupled plasma
atomic emission spectrometry (ICP-AES). The presence of hydrochloric acid may be
problematic for graphite furnace atomic absorption (GFAA) and inductively coupled plasma
mass spectrometry (ICP-MS). Hydrofluoric acid, which is capable of dissolving silicates, may
require the removal of excess hydrofluoric acid or the use of specialized non-glass
components during instrumental analysis. This method enables the analyst to select other
decomposition reagents that may also cause problems with instrumental analyses requiring
matrix matching of standards to account for viscosity and chemical differences.

4.1.4 Apparatus and Supplies

4.1.4.1 Microwave

1. The temperature performance requirements necessitate the microwave decomposition
system sense the temperature to within ± 2.5°C and automatically adjust the microwave field
output power within 2 seconds of sensing. Temperature sensors should be accurate to ±
2°C (including the final reaction temperature of 180°C). Temperature feedback control
provides the primary control performance mechanism for the method. Due to the flexibility in
the reagents used to achieve total analysis, temperature feedback control is necessary for
reproducible microwave heating.

Alternatively, for a specific set of reagent(s) combination(s), quantity, and specific vessel
type, a calibration control mechanism can be developed similar to previous microwave
methods. Through calibration of the microwave power, vessel load and heat loss, the
reaction temperature profile described in section 4.6.2 can be reproduced. The calibration
settings are specific for the number and type of vessel used and for the microwave system
in addition to the variation in reagent combinations. Therefore no specific calibration settings
are provided in this method. These settings may be developed by using temperature
monitoring equipment for each specific set of equipment and reagent combination. They
may only be used if not altered as previously described in other methods. In this
circumstance, the microwave system provides programmable power which can be
programmed to within ± 12 W of the required power.

Typical systems provide a nominal 600 W to 1200 W of power. Calibration control provides
backward compatibility with older laboratory microwave systems without temperature
monitoring or feedback control and with lower cost microwave systems for some repetitive
analyses. Older lower pressure vessels may not be compatible.

2. The temperature measurement system should be periodically calibrated at an elevated
temperature. Pour silicon oil (a high temperature oil into a beaker and adequately stirred to
ensure a homogeneous temperature. Place the microwave temperature sensor and a
calibrated external temperature measurement sensor into the beaker. Heat the beaker to a
constant temperature of 180 ± 5°C. Measure the temperature with both sensors. If the
measured temperatures vary by more than 1 - 2°C, the microwave temperature

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 102

measurement system needs to be calibrated. Consult the microwave manufacturer's
instructions about the specific temperature sensor calibration procedure.
CAUTION: The use of microwave equipment with temperature feedback control is required to
control the unfamiliar reactions of unique or untested reagent combinations of unknown
samples. These tests may require additional vessel requirements such as increased
pressure capabilities.
3. The microwave unit cavity is corrosion resistant and well ventilated. All electronics are
protected against corrosion for safe operation.

CAUTION: There are many safety and operational recommendations specific to the model and
manufacturer of the microwave equipment used in individual laboratories. A listing of these
specific suggestions is beyond the scope of this method, and requires the analyst to consult
the specific equipment manual, manufacturer, and literature for proper and safe operation of
the microwave equipment and vessels.
4. The method requires essentially microwave transparent and reagent resistant suitably inert
polymeric materials (examples are PFA or TFM suitably inert polymeric polymers) to contain
acids and samples. For higher pressure capabilities the vessel may be contained within
layers of different microwave transparent materials for strength, durability, and safety. The
vessels internal volume should be at least 45 ml_, capable of withstanding pressures of at
least 30 atm (30 bar or 435 psi), and capable of controlled pressure relief. These
specifications are to provide an appropriate, safe, and durable reaction vessel of which there
are many adequate designs by many suppliers.

CAUTION: The outer layers of vessels are frequently not as acid or reagent resistant as the
liner material and must not be chemically degraded or physically damaged to retain the
performance and safety required. Routine examination of the vessel materials may be
required to ensure their safe use.

CAUTION: The second safety concern relates to the use of sealed containers without pressure
relief devices. Temperature is the important variable controlling the reaction. Pressure is
needed to attain elevated temperatures, but must be safely contained. However, many
digestion vessels constructed from certain suitably inert polymerics may crack, burst, or
explode in the unit under certain pressures. Only suitably inert polymeric (e.g., PFA or TFM)
containers with pressure relief mechanisms or containers with suitably inert polymeric liners
and pressure relief mechanisms are considered acceptable. Users are therefore advised not
to use domestic (kitchen) type microwave ovens or to use inappropriate sealed containers
without pressure relief for microwave acid digestions by this method. Use of laboratory-
grade microwave equipment is required to prevent safety hazards.
5. A rotating turntable is employed to insure homogeneous distribution of microwave radiation
within most systems. The speed of the turntable should be a minimum of 3 rpm.

CAUTION: Laboratories should not use domestic (kitchen) type microwave ovens for this
method. There are several significant safety issues. First, when an acid such as nitric is
used to effect sample digestion in microwave units in open vessel(s), or sealed vessels
equipment, there is the potential for the acid gas vapor released to corrode the safety
devices that prevent the microwave magnetron from shutting off when the door is opened.
This can result in operator exposure to microwave energy. Use of a system with isolated and
corrosion resistant safety devices prevents this from occurring.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 103

4.1.4.2 Supplies

1. Volumetric ware, volumetric flasks, graduated cylinders, 50 & 100 ml_ capacity or equivalent.

2. Filter paper, qualitative or equivalent.

3. Filter funnel, polypropylene, polyethylene or equivalent.

4. Analytical balance, of appropriate capacity, with a ± 0.0001 g or appropriate precision for the
weighing of the sample. Optionally, the vessel with sample and reagents may be weighed,
with an appropriate precision balance, before and after microwave processing to evaluate
the seal integrity in some vessel types.

4.1.5 Reagents

All reagents should be of appropriate purity or high purity (acids for example, should be sub-
boiling distilled where possible) to minimize the blank levels due to elemental contamination. All
references to water in the method refer to reagent water. Other reagent grades may be used,
provided it is first ascertained that the reagent is of sufficient purity to permit its use without
lessening the accuracy of the determination. If the purity of a reagent is questionable, analyze
the reagent to determine the level of impurities. The reagent blank must be less than the MDL in
order to be used.

4.1.6 Procedure

4.1.6.1 General

1. Temperature control of closed vessel microwave instruments provides the main feedback
control performance mechanism for the method. Control requires a temperature sensor in
one or more vessels during the entire decomposition. The microwave decomposition system
should sense the temperature to within ± 2.5 °C and permit adjustment of the microwave
output power within 2 seconds.
2. All digestion vessels and volumetric ware must be carefully acid washed and rinsed with
reagent water. When switching between high concentration samples and low concentration
samples, all digestion vessels (fluoropolymer liners only) should be cleaned by leaching with
hot (1:1) hydrochloric acid (greater than 80°C, but less than boiling) fora minimum of two
hours followed with hot (1:1) nitric acid (greater than 80°C, but less than boiling) for a
minimum of two hours and rinsed with reagent water and dried in a clean environment. This
cleaning procedure should also be used whenever the prior use of the digestion vessels is
unknown or cross contamination from vessels is suspected. Polymeric or glass volumetric
ware (not used with HF) and storage containers should be cleaned by leaching with more
dilute acids (approximately 10% V/V) appropriate for the specific plastics used and then
rinsed with reagent water and dried in a clean environment.

4.1.6.2 Sample Digestion

1. Weigh a well-mixed sample to the nearest 0.001 g into an appropriate vessel equipped with
a pressure relief mechanism. For biological tissues initially use no more than 0.5 g.
2. Add 9 ± 0.1 ml_ concentrated nitric acid and 3 ± 0.1 ml_ concentrated hydrofluoric acid to the
vessel in a fume hood. If the approximate silicon dioxide content of the sample is known, the

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 104

quantity of hydrofluoric acid may be varied from 0-5 ml_ for stoichiometric reasons. Samples
with higher concentrations of silicon dioxide (>70%) may require higher concentrations of
hydrofluoric acid (>3 ml_ HF). Alternatively samples with lower concentrations of silicon
dioxide (< 10% to 0%) may require much less hydrofluoric acid (0.5 ml_ to 0 ml_). Acid
digestion reagent combinations used in the analysis of biological samples is as follows:
Sample HNO2 HF HCj
NIST SRM 2704
Oyster Tissue 900

3. The addition of other reagents with the original acids prior to digestion may permit more
complete oxidation of organic sample constituents, address specific decomposition
chemistry requirements, or address specific elemental stability and solubility problems.

The addition of 2 ± 2 mL concentrated hydrochloric acid to the nitric and hydrofluoric acids is
appropriate for the stabilization of Ba, and Sb and high concentrations of Fe and Al in
solution. The amount of HCI needed will vary depending on the matrix and the concentration
of the analytes. The addition of hydrochloric acid may; however, limit the techniques or
increase the difficulties of analysis. The addition of hydrogen peroxide (30%) in small or
catalytic quantities (such as 0.1 to 2 mL) may aid in the complete oxidation of organic
matter. The addition of water (double deionized) may (0 to 5 mL) improve the solubility of
minerals and prevent temperature spikes due to exothermic reactions.

CAUTION: Only one acid mixture or quantity may be used in a single batch in the microwave to
insure consistent reaction conditions between all vessels and monitored conditions. This
limitation is due to the current practice of monitoring a representative vessel and applying a
uniform microwave field to reproduce these reaction conditions within a group of vessels
being simultaneously heated.
CAUTION: Toxic nitrogen oxide(s), hydrogen fluoride, and toxic chlorine (from the addition of
hydrochloric acid) fumes are usually produced during digestion. Therefore, all steps
involving open or the opening of microwave vessels must be performed in a properly
operating fume ventilation system.

CAUTION: The analyst should wear protective gloves and face protection and must not at any
time permit a solution containing hydrofluoric acid to come in contact with skin or lungs.

CAUTION: The addition of hydrochloric acid must be from concentrated hydrochloric acid and
not from a premixed combination of acids as a buildup of toxic chlorine and possibly other
gases will result from a premixed acid solution. This will over pressurize the vessel due to
the release of these gases from solution upon heating. The gas effect is greatly lessened by
following this suggestion.

CAUTION: When digesting samples containing volatile or easily oxidized organic compounds,
initially weigh no more than 0.10 g and observe the reaction before capping the vessel. If a
vigorous reaction occurs, allow the reaction to cease before capping the vessel. If no
appreciable reaction occurs, a sample weight up to 0.25g can be used.

CAUTION: The addition of hydrogen peroxide should only be done when the reactive
components of the sample are known. Hydrogen peroxide may react rapidly and violently on
easily oxidizable materials and should not be added if the sample may contain large
quantities of easily oxidizable organic constituents.
4. The analyst should be aware of the potential for a vigorous reaction. If a vigorous reaction
occurs upon the initial addition of reagent or the sample is suspected of containing easily

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 105

oxidizable materials, allow the sample to predigest in the uncapped digestion vessel. Heat
may be added in this step for safety considerations (for example the rapid release of carbon
dioxide from carbonates, easily oxidized organic matter, etc.). Once the initial reaction has
ceased, the sample may continue through the digestion procedure.

5. Seal the vessel according to the manufacturer's directions. Properly place the vessel in the
microwave system according to the manufacturer's recommended specifications and
connect appropriate temperature and pressure sensors to vessels according to
manufacturer's specifications.
6. This method is a performance based method, designed to achieve or approach total
decomposition of the sample through achieving specific reaction conditions. The
temperature of each sample should rise to 180 ± 5 °C in approximately 5.5 minutes and
remain at 180 ± 5 °C for 9.5 minutes. The number of samples simultaneously digested is
dependent on the analyst. The number may range from 1 to the maximum number of
vessels that the microwave units magnetron can heat according to the manufacturer's or
literature specifications (the number will depend on the power of the unit, the quantity and
combination of reagents, and the heat loss from the vessels). The pressure should peak
between 5 and 15 minutes for most samples. If the pressure exceeds the pressure limits of
the vessel, the pressure will be reduced by the relief mechanism of the vessel. The total
decomposition of some components of a matrix may require or the reaction kinetics is
dramatically improved with higher reaction temperatures. If microwave digestion systems
and/or vessels are capable of achieving higher temperatures and pressures, the minimum
digestion time of 9.5 minutes at a temperature of at least 180 ± 5°C is an appropriate
alternative. This change will permit the use of pressure systems if the analysis verifies that
180°C is the minimum temperature maintained by these control systems.

For reactive substances, the heating profile may be altered for safety purposes. The
decomposition is primarily controlled by maintaining the reagents at 180 ± 5°C for 9.5
minutes; therefore the time it takes to heat the samples to 180 ± 5°C is not critical. The
samples may be heated at a slower rate to prevent potential uncontrollable exothermic
reactions. The time to reach 180 ± 5 °C may be increased to 10 minutes provided that 180 ±
5 °C is subsequently maintained for 9.5 minutes. The extreme difference in pressure is due
to the gaseous digestion products.

Calibration control is applicable in reproducing this method provided the power in watts
versus time parameters are determined to reproduce the specifications listed. The
calibration settings will be specific to the quantity and combination of reagents, quantity of
vessels, and heat loss characteristics of the vessels. If calibration control is being used, any
vessels containing acids for analytical blank purposes are counted as sample vessels and
when fewer than the recommended number of samples are to be digested, the remaining
vessels should be filled with the same acid mixture to achieve the full complement of
vessels. This provides an energy balance, since the microwave power absorbed is
proportional to the total absorbed mass in the cavity. Irradiate each group of vessels using
the predetermined calibration settings. (Different vessel types should not be mixed).

Pressure control for a specific matrix is applicable if instrument conditions are established
using temperature control. Because each matrix will have a different reaction profile,
performance using temperature control must be developed for every specific matrix type
prior to use of the pressure control system.
7. At the end of the microwave program, allow the vessels to cool for a minimum of 5 minutes
before removing them from the microwave system. When the vessels have cooled to near

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 106

room temperature, determine if the microwave vessels have maintained a seal throughout
the digestion. Due to the wide variability of vessel designs, a single procedure is not
appropriate. For vessels that are sealed as discrete separate entities, the vessel weight may
be taken before and after digestion to evaluate seal integrity. If the weight loss of sample
exceeds 1% of the weight of the sample and reagents, then the sample is considered
compromised. For vessels with burst disks, a careful visual inspection of the disk may
identify compromised vessels. For vessels with resealing pressure relief mechanisms, an
auditory or sometimes a physical sign indicates a vessel has vented.
8. Complete the preparation of the sample by carefully uncapping and venting each vessel in a
fume hood. Vent the vessels using the procedure recommended by the vessel
manufacturer. Transfer the sample to an acid-cleaned bottle. If the digested sample contains
particulates which may clog nebulizers or interfere with injection of the sample into the
instrument, the sample may be centrifuged, allowed to settle, or filtered.

Centrifugation at 2,000 - 3,000 rpm for 10 mins is usually sufficient to clear the supernatant.

Settling: If undissolved material remains such as TiO2, or other refractory oxides, allow the
sample to stand until the supernatant is clear. Allowing a sample to stand overnight will
usually accomplish this. If it does not, centrifuge or filter the sample.

Filtering: If necessary, the filtering apparatus must be thoroughly cleaned and prerinsed with
dilute (approximately 10% V/V) nitric acid. Filter the sample through qualitative filter paper
into a second acid-cleaned container.

9. If the hydrofluoric acid concentration is a consideration in the analysis technique such as
with ICP methods, boric acid may be added to permit the complexation of fluoride to protect
the quartz plasma torch. The amount of acid added may be varied, depending on the
equipment and the analysis procedure. If this option is used, alterations in the measurement
procedure to adjust for the boric acid and any bias it may cause are necessary. This addition
will prevent the measurement of boron as one of the elemental constituents in the sample.
Alternatively, a hydrofluoric acid resistant ICP torch may be used and the addition of boric
acid would be unnecessary for this analytical configuration. All major manufacturers have
hydrofluoric resistant components available for the analysis of solutions containing
hydrofluoric acid.

CAUTION: The traditional use of concentrated solutions of boric acid can cause problems by
turning the digestion solution cloudy or result in a high salt content solution interfering with
some analysis techniques. Dilute solutions of boric acid or other methods of neutralization or
reagent elimination are appropriate to avoid problems with HF and glass sample introduction
devices of analytical instrumentation. Gentle heating often serves to clear cloudy solutions.
Matrix matching of samples and standards will eliminate viscosity differences.

10. The removal or reduction of the quantity of the hydrochloric and hydrofluoric acids prior to
analysis may be desirable. The chemistry and volatility of the analytes of interest should be
considered and evaluated when using this alternative. Evaporation to near dryness in a
controlled environment with controlled pure gas and neutralizing and collection of exhaust
interactions is an alternative where appropriate. This manipulation may be performed in the
microwave system, if the system is capable of this function, or external to the microwave
system in more common apparatus(s). This option must be tested and validated to
determine analyte retention and loss and should be accompanied by equipment validation
possibly using the standard addition method and standard reference materials. This
alternative may be used to alter either the acid concentration and/or acid composition.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 107

NOTE: The final solution typically requires nitric acid to maintain appropriate sample solution
acidity and stability of the elements. Commonly, a 2% (v/v) nitric acid concentration is
desirable. Waste minimization techniques should be used to capture reagent fumes. This
procedure should be tested and validated in the apparatus and on standards before using
on unknown samples.
11. Transfer or decant the sample into volumetric ware and dilute the digest to a known volume.
The digest is now ready for analysis for elements of interest using appropriate elemental
analysis techniques and/or methods.
12. Sample size may be scaled-up from 0.1, 0.25, or 0.5 g to 1.0 g through a series of 0.2 g
sample size increments. Scale-up can produce different reaction conditions and/or produce
increasing gaseous reaction products. Increases in sample size may not require alteration of
the acid quantity or combination, but other reagents may be added to permit a more
complete decomposition and oxidation of organic and other sample constituents where
necessary (such as increasing the HF for the complete destruction of silicates). Each step of
the scale-up must demonstrate safe operation before continuing.

4.1.7 Calculations

The concentrations determined are to be reported on the basis of the actual weight of the
original sample.

4.1.8 Calibration of Microwave Equipment

NOTE: If the microwave unit uses temperature feedback control to follow performance
specifications of the method, then the calibration procedure will not be necessary.
1. Calibration is the normalization and reproduction of microwave field strength to permit
reagent and energy coupling in a predictable and reproducible manner. It balances reagent
heating and heat loss from the vessels and is equipment dependent due to the heat
retention and loss characteristics of the specific vessel. Available power is evaluated to
permit the microwave field output in watts to be transferred from one microwave system to
another. Use of calibration to control this reaction requires balancing output power, coupled
energy, and heat loss to reproduce the temperature heating profile in section 4.1.6.2.6. The
conditions for each acid mixture and each batch containing the same specified number of
vessels must be determined individually. Only identical acid mixtures and vessel models and
specified numbers of vessels may be used in a given batch.
2. For cavity type microwave equipment, this is accomplished by measuring the temperature
rise in 1 kg of water exposed to microwave radiation for a fixed period of time. The analyst
can relate power in watts to the partial power setting of the system. The calibration format
required for laboratory microwave systems depends on the type of electronic system used
by the manufacturer to provide partial microwave power. Few systems have an accurate and
precise linear relationship between percent power settings and absorbed power. Where
linear circuits have been utilized, the calibration curve can be determined by a three-point
calibration method (sec. 4.1.8.4); otherwise, the analyst must use the multiple point
calibration method (sec.4.1.8.3).

3. The multiple point calibration involves the measurement of absorbed power over a large
range of power settings. Typically, for a 600 W unit, the following power settings are
measured; 100, 99, 98, 97, 95, 90, 80, 70, 60, 50, and 40% using the procedure described

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 108

in section 4.1.8.5. This data is clustered about the customary working power ranges.
Nonlinearity has been encountered at the upper end of the calibration. If the system's
electronics are known to have nonlinear deviations in any region of proportional power
control, it will be necessary to make a set of measurements that bracket the power to be
used. The final calibration point should be at the partial power setting that will be used in the
test. This setting should be checked periodically to evaluate the integrity of the calibration. If
a significant change is detected (±10 W) then the entire calibration should be reevaluated.

4. The three-point calibration involves the measurement of absorbed power at three different
power settings. Measure the power at 100% and 50% using the procedure described in
section 4.1.8.5. From the 2-point line calculate the power setting corresponding to the
required power in watts specified in the procedure. Measure the absorbed power at that
partial power setting. If the measured absorbed power does not correspond to the specified
power within ±10 W, use the multiple point calibration in 4.9.3. This point should also be
used to periodically verify the integrity of the calibration.

5. Equilibrate a large volume of water to room temperature (23 ± 2 °C). One kg of reagent
water is weighed (1,000.0 g + 0.1 g) into a suitably inert polymeric beaker or a beaker made
of some other material that does not significantly absorb microwave energy (glass absorbs
microwave energy and is not recommended). The initial temperature of the water should be
23 ± 2 °C measured to ± 0.05 °C. The covered beaker is circulated continuously (in the
normal sample path) through the microwave field for 2 minutes at the desired partial power
setting with the system's exhaust fan on maximum (as it will be during normal operation).
The beaker is removed and the water vigorously stirred. Use a magnetic stirring bar inserted
immediately after microwave irradiation, and record the maximum temperature within the
first 30 seconds to ± 0.05 °C. Use a new sample for each additional measurement. If the
water is reused, both the water and the beaker must have returned to 23 ± 2 °C. Three
measurements at each power setting should be made.

The absorbed power is determined by the following relationship:

P = K CP m AT
t
where:
P = the apparent power absorbed by the sample in watts (W, W = joule sec"1)
K = the conversion factor for thermochemical calories_sec"1 to watts (which is 4.184)
Cp = the heat capacity, thermal capacity, or specific heat (cal g"1 °C"1) of water
m = the mass of the water sample in grams (g)
AT = the final temperature minus the initial temperature (°C)
t = the time in seconds (s)

Using the experimental conditions of 2 minutes and 1 kg of distilled water (heat capacity
at 25 °C is 0.9997 cal g"1 °C"1) the calibration equation simplifies to:

P = 34.86 AT

NOTE: Stable line voltage is necessary for accurate and reproducible calibration and operation.
The line voltage should be within manufacturer's specification, and during measurement and
operation should not vary by more than ±5 V. Electronic components in most microwave
units are matched to the system's function and output. When any part of the high voltage
circuit, power source, or control components in the system have been serviced or replaced,

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 109

it will be necessary to recheck the system's calibration. If the power output has changed
significantly (±10 W) then the entire calibration should be reevaluated.

4.1.9 Quality Control

1. All quality control data must be maintained and available for reference or inspection for a
period determined by all involved parties based on program or project requirements. This
method is restricted to use by, or under supervision of, experienced analysts.
2. Duplicate samples should be processed on a routine basis. A duplicate sample is a sample
brought through the whole sample preparation and analytical process. A duplicate sample
should be processed with each analytical batch or every 20 samples, whichever is the
greater number. A duplicate sample should be prepared for each matrix type.

3. Spiked samples and/or standard reference materials should be included with each group of
samples processed or every 20 samples, whichever is the greater number. A spiked sample
should also be included whenever a new sample matrix is being analyzed.

4. Blank samples should be prepared using the same reagents and quantities used in sample
preparation, placed in vessels of the same type, and processed with the samples.

4.2 METALS IN FISH TISSUE AND SEDIMENT

4.2.1 Inductively Coupled Plasma - Mass Spectrometry

The sensitivity and optimum and linear ranges for each element will vary with the wavelength,
spectrometer, matrix, and operating conditions. Background correction is required for trace
element determination. Background emission must be measured adjacent to analyte lines on
samples during analysis. The position selected for background-intensity measurement, on either
or both sides of the analytical line, will be determined by complexity of the spectrum adjacent to
the analyte line. The position used should be as free as possible from spectral interference and
should reflect the same change in background intensity as occurs at the analyte wavelength
measured. Background correction is not required in cases of line broadening where background
correction measurement would actually degrade the analytical result. The possibility of
additional interferences identified in section 4.2.1.2 should also be recognized and appropriate
corrections made; tests for their presence are described in sections 4.2.1.5.5 and 4.2.1.5.6.
Alternatively, users may choose multivariate calibration methods. In this case, point selections
for background correction are superfluous since whole spectral regions are processed.

4.2.1.1 Summary of Method

This method describes multi-elemental determination of analytes by ICP-MS in environmental
samples (Figure 4.1). The method measures ions produced by a radio-frequency inductively
coupled plasma. Analyte species originating in a liquid are nebulized (See Appendix A for
methodology) and the resulting aerosol is transported by argon gas into the plasma torch. The
ions produced by high temperatures are entrained in the plasma gas and extracted through a
differentially pumped vacuum interface and separated on the basis of their mass-to-charge ratio
by a mass spectrometer. The ions transmitted through the mass spectrometer are quantified by
a channel electron multiplier or Faraday detector and the ion information is processed by the
instrument's data handling system. Interferences must be assessed and valid corrections
applied or the data qualified to indicate problems. Interference correction must include

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 110
compensation for background ions contributed by the plasma gas, reagents, and constituents of
the sample matrix.

4.2.1.2 Interferences
2.
Solvents, reagents, glassware, and other sample processing hardware may yield
artifacts and/or interferences to sample analysis. All these materials must be
demonstrated to be free from interferences under the conditions of the analysis by
analyzing method blanks. Specific selection of reagents and purification of solvents by
distillation in all-glass systems may be necessary.

Interferences must be assessed and valid corrections applied or the data qualified to
indicate problems. Interference correction must include compensation for background
ions contributed by the plasma gas, reagents, and constituents of the sample matrix.
T
Use
appropriate
digestion procedure
(Chapter Thng-e,)
^
f
Initiate
oporat ng
configuration of
instrument
computer,
^
f
f**'t Jp .and
star- i/o
n*-** imont
1
f
Sal up
aperallno conditions
n& rftcomend&d,
^
r
PortorTTi mass
calibration rand
resolution checks,
^
r
Analyze
lower limit QC

M
masses
affect data
recom
^
r
calibrat
covering ;
caltoratio
analy/
approp
sarr
^
anitor all
hat could
csuahly, as
ended,
f
ropare
on cu^vs
p"rrp'in*Pt
i ranrjf* ny
nq the
r-dlH OC
pl«N.
f
Vr-'fy
r^|*i'rt: n* wlh \CV
f 'f-pri'ra from rj
d^'^M^r^ *>1"i ^
soUion. Abo tin«ly^Q
,1 „ i |CV a-inrks

An.^ly/e .1 CCV
L.LCCV, and CCB alter
each 10 sfimpies,

Neb i
sample uni
sEatts a
achieved, C

|i40 erich
1 a stciidy-
gnal ss
ol loci data.
*
/DOCEsV

Determine
problem and correct
it; Recalibrate,
^
T Yes
/' AreV
/ ICV resultsTx
^_

<-
Yes
\ 10% range/
\ ot acluol/
'XV^IMS^'
I No
BefofQ analysis
and ovory 12 hr,
«inply/o inscr1orpnKiU
dhtick solutidri!?! A rand
AR,
i
Hu&h systcsm
beforti ih6 analysis, of
each sample,

Dilute sample
measure a less
abundant isotopes,
Calculate
concent/alien,
Figure 4.1. Inductively Coupled Plasma- Mass Spectrometry
3. Isobaric elemental interferences in ICP-MS are caused by isotopes of different elements
forming atomic ions with the same nominal mass-to-charge ratio (m/z). A data system must

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 111

be used to correct for these interferences. This involves determining the signal for another
isotope of the interfering element and subtracting the appropriate signal from the analyte
isotope signal. Since commercial ICP-MS instruments nominally provide unit resolution at
10% of the peak height, very high ion currents at adjacent masses can also contribute to ion
signals at the mass of interest. Although this type of interference is uncommon, it is not
easily corrected, and samples exhibiting a significant problem of this type could require
resolution improvement, matrix separation, or analysis using another verified and
documented isotope, or use of another method.
Isobaric molecular and doubly-charged ion interferences in ICP-MS are caused by ions
consisting of more than one atom or charge, respectively. Most isobaric interferences that
could affect ICP-MS determinations have been identified. Examples include 75ArCI+ion on
the 75As signal and MoO+ ions on the cadmium isotopes. While the approach used to correct
for molecular isobaric interferences is demonstrated below using the natural isotope
abundances from the literature, the most precise coefficients for an instrument can be
determined from the ratio of the net isotope signals observed for a standard solution at a
concentration providing suitable (<1%) counting statistics.

Because the 35CI natural abundance of 75.77% is 3.13 times the 37CI abundance of 24.23%,
the chloride correction for arsenic can be calculated (approximately) where the 38Ar37CI+
contribution at m/z 75 is a negligible 0.06% of the 40Ar35CI+ signal.

Corrected arsenic signal (using natural isotopes abundances for coefficient approximations)
= (m/z 75 signal) - (3.13) (m/z 77 signal) + (2.73) (m/z 82 signal),

where the final term adjusts for any selenium contribution at 77 m/z.

Note. Arsenic values can be biased high by this type of equation when the net signal at m/z 82
is caused by ions other than 82Se+, (e.g., 81BrH+from bromine wastes).

Corrected cadmium signal (using natural isotopes abundances for coefficient
approximations) = (m/z 114 signal) - (0.027)(m/z 118 signal) - (1.63)(m/z 108 signal)

where last 2 terms adjust for any 114Sn+ or 114MoO+ contributions at m/z 114.

Note. Cadmium values will be biased low by this type of equation when 92ZrO+ions contribute at
m/z 108, but use of m/z 111 for Cd is even subject to direct ^4ZrOH+) and indirect ^ZrO*)
additive interferences when Zr is present.

The accuracy of these types of equations is based upon the constancy of the observed
isotopic ratios for the interfering species. Corrections that presume a constant fraction of a
molecular ion relative to the parent ion have not been found to be reliable, e.g., oxide levels
can vary with operating conditions. If a correction for an oxide ion is based upon the ratio of
parent-to-oxide ion intensities, the correction must be adjusted for the degree of oxide
formation by the use of an appropriate oxide internal standard previously demonstrated to
form a similar level of oxide as the interferent. The use of aerosol desolvation and/or mixed
gas plasmas have been shown to greatly reduce molecular interferences. These techniques
can be used provided that the lower limits of quantitation, accuracy, and precision
requirements for analysis of the samples can be met.

Also, solid phase chelation may be used to eliminate isobaric interferences from element
and molecular sources. An on-line method has been demonstrated for environmental waters
such as sea water, drinking water and acid decomposed samples. The method also
provides a way for preconcentration to enhance quantitation limits simultaneously with
elimination of isobaric interferences. The method relies on chelating resins such as

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 112

imminodiacetate or other appropriate resins and selectively concentrates the elements of
interest while eliminating interfering elements from the sample matrix. By eliminating the
elements that are direct isobaric interferences or those that form isobaric interfering
molecular masses, the mass region is simplified and these interferences cannot occur. The
method has been proven effective for the certification of standard reference materials and
validated using SRMs. The method has the potential to be used on-line or off-line as an
effective sample preparation method specifically designed to address interference problems.

4. Physical interferences are associated with the sample nebulization and transport processes
as well as with ion-transmission efficiencies. Nebulization and transport processes can be
affected if a matrix component causes a change in surface tension or viscosity. Changes in
matrix composition can cause significant signal suppression or enhancement. Dissolved
solids can deposit on the nebulizer tip of a pneumatic nebulizer and on the interface
skimmers (reducing the orifice size and the instrument performance). Total solid levels
below 0.2% (2,000 mg/L) are recommended to minimize solid deposition. An internal
standard can be used to correct for physical interferences, if it is carefully matched to the
analyte so that the two elements are similarly affected by matrix changes. When intolerable
physical interferences are present in a sample, a significant suppression of the internal
standard signals (to < 30% of the signals in the calibrations standard) will be observed.
Dilution of the sample fivefold (1+4) will usually eliminate the problem (see sec. 4.2.1.5.4).

5. Memory interferences or carry-over can occur when there is large concentration differences
between samples or standards which are analyzed sequentially. Sample deposition on the
sampler and skimmer cones, spray chamber design, and the type of nebulizer affect the
extent of observed memory interferences. The rinse period between samples must be long
enough to eliminate significant memory interference.

4.2.1.3 Equipment and Supplies

1. Inductively coupled plasma-mass spectrometer - the system must be capable of providing
resolution, better than or equal to 1.0 amu at 10% peak height is required. The system must
have a mass range from at least 6 to 240 amu and a data system that allows corrections for
isobaric interferences and the application of the internal standard technique. Use of a mass-
flow controller for the nebulizer argon and a peristaltic pump for the sample solution is
recommended.

2. Argon gas supply - High-purity grade (99.99%)

3. Only polyethylene or fluorocarbon (TFE or PFA) containers are recommended for use in this
method.

4.2.1.4 Reagents and Standards

1. Reagent- or trace metals-grade chemicals must be used in all tests. Unless otherwise
indicated, it is intended that all reagents conform to the specifications of the Committee on
Analytical Reagents of the American Chemical Society, where such specifications are
available. Other grades may be used, provided it is first ascertained that the reagent is of
sufficiently high purity to permit its use without lessening the accuracy of the determination.

2. Acids used in the preparation of standards and for sample processing must be of high purity.
Redistilled acids are recommended because of the high sensitivity of ICP-MS. Nitric acid at
less than 2% (v/v) is required for ICP-MS to minimize damage to the interface and to

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 113
    minimize isobaric molecular-ion interferences with the analytes. Concentration of antimony
    between 50-500 ug/l require 1% (v/v) HCI for stability. Consequently, accuracy of analytes
    requiring significant chloride molecular ion corrections (such as As) will degrade.

3.   Reagent water - All references to water in the method refer to distilled water, unless
    otherwise specified. Reagent water must be free of interferences.

4.   Standard stock solutions for each analyte may be purchased or prepared from ultra-high
    purity grade chemicals or metals (99.99 or greater purity). Preapartion procedures are
    outlined in Table 4.2. Recommended internal standards are 6Li, 45Sc, 89Y,  103Rh, 115ln, 15Tb,
    165Ho, 74Ge, and 209Bi. The lithium internal standard should have an enriched abundance of 6Li,
    so interference  from lithium native to the sample is minimized. Other elements may need to
    be used as internal standards when samples contain significant native amounts of the
    recommended internal standards.

Table 4.2. Recommended Stock Solution Preparation Procedures.1
Element
Bismuth
Geranium
Holmium
Indium
Lithium
Rhodium
Scandium
Terbium
Yttrium
Titanium
(interference)
Molybdenum
(interference)
Stock Solution
1 ml = 100 ug of
Bi
1 ml= 100 ug of
Ge
1 ml = 100 ug of
Ho
1 ml = 100 ug of
In
1 ml = 100 ug of
6Li
1 ml = 100 ug of
Rh
1 ml = 100 ug of
Sc
1 ml = 100 ug of
Tb
1 ml = 100 ug of
Y
1 ml = 100 ug of
Ti
1 ml = 100 ug of
Mo
Directions
Dissolve 0.1115 g of Bi2O3 in a minimum amount of dilute HNO3. Add 10 ml of
cone. HMOs and dilute to 1 ,000 ml with reagent water.
Dissolve 0.2954 g of GeCU in a minimum amount of dilute HMOs. Add 1 0 ml of
cone. HNO3 and dilute to 1 ,000 ml with reagent water.
Dissolve 0. 1 757 g of Ho2(CO3)25H2O in 1 0 ml of reagent water and 1 0 ml of
HMOs. After dissolution is complete, warm the solution to degas. Add 10 ml
cone, of HMOs and dilute to 1,000 ml with reagent water.
Dissolve 0.1000 g of indium metal in 10 ml of cone. HNO3. Dilute to 1,000 ml
with reagent water.
Dissolve 0.6312 g of 95-atom-% 6Li, Li2CO3 in 10 ml of reagent water and 10 ml
of HMOs. After dissolution is complete, warm the solution to degas. Add 10 ml
cone, of HNO3 and dilute to 1,000 ml with reagent water.
Dissolve 0.3593 g of ammonium hexachlororhodate (III) (NhUJsRhCle in 10 ml
reagent water. Add 100 ml of cone. HCI and dilute to 1,000 ml with reagent
water.
Dissolve 0.15343 g of Sc2O3 in 10 ml (1 + 1) of hot HNO3. Add 5 ml of cone.
HNO3 and dilute to 1 ,000 ml with reagent water.
Dissolve 0.1828 g of Tb2(CO3)35H2O in 10 m (1 + 1) of HNO3. After dissolution is
complete, warm the solution to degas. Add 5 ml of cone. HNO3 and dilute to
1,000 ml with reagent water.
Dissolve 0. 2316 g of Y2(CO3)33H2O in 10 ml (1 + 1) of HNO3. Add 5 ml cone, of
HNO3 and dilute to 1 ,000 ml with reagent water.
Dissolve 0.4133 g of (NH4)2TiF6in reagent water. Add 2 drops of cone. HF and
dilute to 1 ,000 ml with reagent water.
Dissolve 0.2043 g of (NH4)2MoO4 in reagent water. Dilute to 1,000 ml with
reagent water.
5.   Mixed calibration standard solutions are prepared by diluting the stock-standard solutions to
    levels in the linear range for the instrument in a solvent consisting of 1% (v/v) HMOs in
    reagent water. The calibration standard solutions must contain a suitable concentration of
1 Concentrations are calculated based upon the weight of pure metal added, or with the use of the element fraction and the weight
of the metal salt added. The weight of the analyte is expressed to four significant figures for consistency with the weights below
because rounding to two decimal places can contribute up to 4% error for some of the compounds.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 114

an appropriate internal standard for each analyte. Internal standards may be added on-line
at the time of analysis using a second channel of the peristaltic pump and an appropriate
mixing manifold. Generally, an internal standard should be no more than 50 amu removed
from the analyte.

6. Prior to preparing the mixed standards, each stock solution must be analyzed separately to
determine possible spectral interferences or the presence of impurities. Care must be taken
when preparing the mixed standards to ensure that the elements are compatible and stable
together. Transfer the mixed standard solutions to freshly acid-cleaned FEP fluorocarbon or
previously unused polyethylene or polypropylene bottles for storage. For all intermediate
and working standards, especially low level standards (i.e., <1 ppm), stability must be
demonstrated prior to use. Fresh mixed standards must be prepared as needed with the
realization that concentrations can change on aging.
Three types of blanks are required for analysis. The calibration blank is used to establish the
calibration curve. The method blank is used to monitor for possible contamination resulting
from either the reagents (acids) or the equipment used during sample processing including
filtration. The rinse blank is used to flush the system between all samples and standards.

The calibration blank consists of the same concentration(s) of the same acid(s) used to
prepare the final dilution of the calibrating solutions of the analytes (often 1% HNOs(v/v) in
reagent water) along with the selected concentrations of internal standards such that there
is an appropriate internal standard element for each of the analytes. Use HCI for antimony.

The method blank must contain all of the reagents in the same volumes as used in the
processing of the samples. The method blank must be carried through the complete
procedure and contain the same acid concentration in the final solution as the sample
solution used for analysis.

The rinse blank consists of 1 to 2% of HNOs(v/v) in reagent water. Prepare a sufficient
quantity to flush the system between standards and samples.

7. The interference check solution (ICS) is prepared to contain known concentrations of
interfering elements that will demonstrate the magnitude of interferences and provide an
adequate test of any corrections. Chloride in the ICS provides a means to evaluate software
corrections for chloride-related interferences such as 35CI16O+on 51V+and 40Ar35CI+on 75As+.
Iron is used to demonstrate adequate resolution of the spectrometer for the determination of
manganese. Molybdenum serves to indicate oxide effects on cadmium isotopes. The other
components are present to evaluate the ability of the measurement system to correct for
various molecular-ion isobaric interferences. The ICS is used to verify that the interference
levels are corrected by the data system within quality control limits.
These solutions can be obtained commercially or prepared from ultra-pure reagents by the
procedure outlined in Table 4.3.

Table 4.3. Interference Check Solution Preparation Procedures.
ICS solution
Mixed
solution I
Mixed
solution II
Directions
Add 13.903 g of AI(NO3)39H2O, 2.498 g of CaCO3 (dried at 180 EC for 1 hr before
weighing), 1 .000 g of Fe, 1 .658 g of MgO, 2.305 g of Na2CO3, and 1 .767 g of K2CO3 to
25 ml of reagent water. Slowly add 40 ml of (1+1) HNO3. After dissolution is complete,
warm the solution to degas. Cool and dilute to 1 ,000 ml with reagent water.
Slowly add 7.444 g of 85 % H3PO4, 6.373 g of 96% H2SO4, 40.024 g of 37% HCI, and
1 0.664 g of citric acid C6O7H8 to 1 00 ml of reagent water. Dilute to 1 ,000 ml with reagent

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 115

Mixed
solution III
Working
solution A
Working
solution AB
water.
Add 1 .00 ml each of 1 00-ug/ml As
about 50 ml of reagent water. Add
with reagent water.
, Cd, Se, Cr, Cu, Mn, Ni, and Zn stock solutions to
2.0 ml of concentrated HNO3, and dilute to 100.0 ml
Add 10.0 ml of mixed ICS solution I, 2.0 ml each of 1 00-ug/ml titanium stock solution and
molybdenum stock solution, and 5.0 ml of mixed ICS solution II. Dilute to 100 ml with
reagent water. ICS solution A must be prepared fresh weekly.
Adding 1 0.0 ml of mixed ICS solution I, 2.0 ml each of 1 00-ug/ml titanium stock solution
and molybdenum stock solution, 5.0 ml of mixed ICS solution II and 2.0 ml of mixed ICS
solution III. Dilute to 100 ml with reagent water. ICS solution AB must be prepared fresh
weekly.
The final ICS solution concentrations in Table 4.4 are intended to evaluate corrections for
known interferences.
Table 4.4. Recommended Interference Check Sample Components and Concentrations.
Solution Component
Al
As
Cd
Cr
Cu
Fe
Mn
Ni
Se
Zn
Solution A (mg/L)
100.0
0.0
0.0
0.0
0.0
250.0
0.0
0.0
0.0
0.0
Solution AB (mg/L)
100.0
0.100
0.100
0.200
0.200
250.0
0.200
0.200
0.100
0.100
8.   The initial calibration verification (ICV) standard may be purchased or prepared by
    combining compatible elements from a standard source different from that of the calibration
    standard, and at concentration near the midpoint of the calibration curve.

9.   The continuing calibration verification (CCV) standard should be prepared in the same acid
    matrix using the same standards used for calibration, at a concentration near the mid-point
    of the calibration curve.

10. Mass spectrometer tuning solution. A solution containing elements representing all of the
    mass regions of interest must be prepared to verify that the resolution and mass calibration
    of the instrument are within the required specifications. This solution is also used to verify
    that the instrument has reached thermal stability.

4.2.1.5 Quality Control

1.   Refer to the QAPP for additional guidance on quality assurance and quality control
    protocols. When  inconsistencies exist between QC guidelines, method specific QC criteria
    take precedence. Each lab must work with the Information Management group (Marlys
    Cappaert, Cappaert.Marlys@epamail.epa.gov, 541-754-4467,) to ensure their bench
    sheets and/or data recording spreadsheets are compatible with the electronic deliverables
    the lab will need to submit. The laboratory should also maintain records to document the

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 116

quality of the data generated. All data sheets and quality control data should be maintained
for reference or inspection.
2. Instrument detection limits (IDLs) are useful means to evaluate the instrument noise level
and response changes over time for each analyte from a series of reagent blank analyses
to obtain a calculated concentration. They are not to be confused with the lower limit of
quantitation, nor should they be used in establishing this limit. It may be helpful to compare
the calculated IDLs to the established lower limit of quantitation (see section 4.3.1.6).

IDLs in ug/L can be estimated by calculating the average of the standard deviations of
three runs on three non-consecutive days from the analysis of a reagent blank solution with
seven consecutive measurements per day. Each measurement should be performed as
though it were a separate analytical sample (i.e., each measurement must be followed by a
rinse and/or any other procedure normally performed between the analysis of separate
samples). IDLs should be determined at least every three months or at a project-specific
designated frequency and kept with the instrument log book.

3. Each laboratory must demonstrate initial proficiency. Each laboratory must demonstrate
initial proficiency with each sample preparation and determinative method combination it
utilizes by generating data of acceptable accuracy and precision for target analytes in a
clean matrix. If an autosampler is used to perform sample dilutions, the laboratory should
verify that those dilutions are of equivalent or better accuracy than is achieved by an
experienced analyst performing manual dilutions.

4. Dilute and reanalyze samples that exceed the linear dynamic range or use an alternate,
less sensitive calibration for which quality control data are already established.

5. The intensities of all internal standards must be monitored for every analysis. If the intensity
of any internal standard in a sample falls below 70% of the intensity of that internal
standard in the initial calibration standard, a significant matrix effect must be suspected.
Under these conditions, the established lower limit of quantitation has degraded and the
correction ability of the internal standardization technique becomes questionable. The
following procedure is followed: Make sure the instrument has not drifted by observing the
internal standard intensities in the nearest clean matrix (calibration blank). If the low internal
standard intensities are also seen in the nearest calibration blank, terminate the analysis,
correct the problem, recalibrate, verify the new calibration, and reanalyze the affected
samples. If drift has not occurred, matrix effects need to be removed by dilution of the
affected sample. The sample must be diluted fivefold (1+4) and reanalyzed with the
addition of appropriate amounts of internal standards. If the first dilution does not eliminate
the problem, this procedure must be repeated until the internal-standard intensities rise to
the minimum 70% limit. Reported results must be corrected for all dilutions.

6. To obtain analyte data of known quality, it is necessary to measure more than the analytes
of interest in order to apply corrections or to determine whether interference corrections are
necessary. For example, tungsten oxide moleculars can be very difficult to distinguish from
mercury isotopes. If the concentrations of interference sources (such as C, Cl, Mo, Zr, W)
are such that, at the correction factor, the analyte is less than the limit of quantification and
the concentration of interferents are insignificant, then the data may go uncorrected. Note
that monitoring the interference sources does not necessarily require monitoring the
interferant itself, but that a molecular species may be monitored to indicate the presence of
the interferent. When correction equations are used, all QC criteria must also be met.
Extensive QC for interference corrections is required at all times. The monitored masses
must include those elements whose hydrogen, oxygen, hydroxyl, chlorine, nitrogen, carbon

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 117

and sulfur molecular ions could impact the analytes of interest. Unsuspected interferences
may be detected by adding pure major matrix components to a sample to observe any
impact on the analyte signals. When an interference source is present, the sample
elements impacted must be flagged to indicate (a) the percentage interference correction
applied to the data or (b) an uncorrected interference by virtue of the elemental equation
used for quantitation. The isotope proportions for an element or molecular-ion cluster
provide information useful for quality assurance.

Note: Only isobaric elemental, molecular, and doubly charged interference corrections which
use the observed isotopic-response ratios or parent-to-oxide ratios (provided an oxide
internal standard is used) for each instrument system are acceptable corrections for use.

7. For each batch of samples processed, at least one method blank must be carried
throughout the entire sample preparation and analytical process. If the method blank does
not contain target analytes at a level that interferes with the project-specific DQOs, then the
method blank would be considered acceptable. In the absence of project-specific DQOs, if
the blank is less than 10% of the lower limit of quantitation check sample concentration,
less than 10% of the regulatory limit, or less than 10% of the lowest sample concentration
for each analyte in a given preparation batch, whichever is greater, then the method blank
is considered acceptable. If the method blank cannot be considered acceptable, the
method blank should be re-run once, and if still unacceptable, then all samples after the
last acceptable method blank should be reprepared and reanalyzed along with the other
appropriate batch QC samples. If the method blank exceeds the criteria, but the samples
are all either below the reporting level or below the applicable action level or other DQOs,
then the sample data may be used despite the contamination of the method blank.

8. For each batch of samples processed, at least one laboratory control sample (LCS) must
be carried throughout the entire sample preparation and analytical process. The laboratory
control samples should be spiked with each analyte of interest at the project-specific action
level or, when lacking project specific action levels, at approximately mid-point of the linear
dynamic range. Acceptance criteria should either be defined in the project-specific planning
documents or set at a laboratory derived limit developed through the use of historical
analyses. In the absence of project-specific or historical data generated criteria, this limit
should be set at ± 20% of the spiked value. Acceptance limits derived from historical data
should be no wider that ± 20%. If the laboratory control sample is not acceptable, then the
laboratory control sample should be re-run once and, if still unacceptable, all samples after
the last acceptable laboratory control sample should be reprepared and reanalyzed.

Concurrent analyses of standard reference materials (SRMs) containing known amounts of
analytes in the media of interest are recommended and may be used as an LCS. For solid
SRMs, 80 -120% accuracy may not be achievable and the manufacturer's established
acceptance criterion should be used for soil SRMs.

9. Documenting the effect of the matrix, for a given preparation batch consisting of similar
sample characteristics, should include the analysis of at least one matrix spike and one
duplicate unspiked sample (MS/Dup) or one matrix spike/matrix spike duplicate (MS/MSD)
pair. The decision on whether to prepare and analyze duplicate samples or a matrix
spike/matrix spike duplicate must be based on a knowledge of the samples in the sample
batch or as noted in the project-specific planning documents. If samples are expected to
contain target analytes, then laboratories may use one matrix spike and a duplicate
analysis of an unspiked field sample. If samples are not expected to contain target
analytes, laboratories should use a matrix spike and matrix spike duplicate pair.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 118

For each batch of samples processed, at least one MS/Dup or MS/MSD sample set should
be carried throughout the entire sample preparation and analytical. MS/MSDs are
intralaboratory split samples spiked with identical concentrations of each analyte of interest.
The spiking occurs prior to sample preparation and analysis. An MS/Dup or MS/MSD is
used to document the bias and precision of a method in a given sample matrix.
MS/MSD samples should be spiked at the same level, and with the same spiking material,
as the corresponding laboratory control sample that is at the project-specific action level or,
when lacking project-specific action levels, at approximately mid-point of the linear dynamic
range. Acceptance criteria should either be defined in the project-specific planning
documents or set at a laboratory-derived limit developed through the use of historical
analyses per matrix type analyzed. In the absence of project-specific or historical data
generated criteria, these limits should be set at ± 25% of the spiked value for accuracy and
20 relative percent difference (RPD) for precision. Acceptance limits derived from historical
data should be no wider that ± 25% for accuracy and 20% for precision. If the bias and
precision indicators are outside the laboratory control limits, if the percent recovery is less
than 75% or greater than 125%, or if the relative percent difference is greater than 20%,
then the interference test discussed in Sec. 9.9 should be conducted.

The relative percent difference between spiked matrix duplicate or unspiked duplicate
determinations is to be calculated as follows:

RPD = D1=P2 x100
(0^02)72
where:
RPD = relative percent difference.
D! = first sample value.
D2= second sample value (spiked or unspiked duplicate)

The spiked sample or spiked duplicate sample recovery should be within ± 25% of the
actual value, or within the documented historical acceptance limits for each matrix.

10. If less than acceptable accuracy and precision data are generated, additional quality control
tests below are recommended prior to reporting concentration data for the elements in this
method. At a minimum, these tests should be performed with each batch of samples
prepared/analyzed with corresponding unacceptable data quality results. These tests will
then serve to ensure that neither positive nor negative interferences are affecting the
measurement of any of the elements or distorting the accuracy of the reported values. If
matrix effects are confirmed, the laboratory should consult with the data user when feasible
for possible corrective actions which may include the use of alternative or modified test
procedures so that the analysis is not impacted by the same interference.
Post digestion spike addition. If the MS/MSD recoveries are unacceptable, the same sample
from which the MS/MSD aliquots were prepared should also be spiked with a post digestion
spike. Otherwise, another sample from the same preparation should be used as an alternative.
An analyte spike is added to a portion of a prepared sample, or its dilution, and should be
recovered to within 80% to 120% of the known value. The spike addition should produce a
minimum level of 10 times and a maximum of 100 times the lower limit of quantitation. If this
spike fails, then the dilution test should be run on this sample. If both the MS/MSD and the post
digestion spike fail, then matrix effects are confirmed.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 119

Dilution test. If the analyte concentration is sufficiently high (minimally, a factor of 10 above the
lower limit of quantitation after dilution), an analysis of a 1:5 dilution should agree within ±10% of
the original determination. If not, a chemical or physical interference effect should be suspected.

4.2.1.6 Calibration and Standardization

1. Set up the instrument with proper operating parameters established as detailed below. The
instrument should be allowed to become thermally stable before beginning (usually requiring
at least 30 min of operation prior to calibration).

2. Conduct mass calibration and resolution checks in the mass regions of interest. The mass
calibration and resolution parameters are required criteria which must be met prior to any
samples being analyzed. If the mass calibration differs more than 0.1 amu from the true
value, then the mass calibration must be adjusted to the correct value. The resolution must
also be verified to be less than 0.9 amu full width at 10% peak height.

Sensitivity, instrumental detection limit, precision, linear dynamic range, and interference
corrections need to be established for each individual target analyte on each particular
instrument. All measurements (both target analytes and constituents which interfere with the
target analytes) need to be within the instrument linear range where the correction equations
are valid.

3. The lower limits of quantitation should be established for all isotope masses utilized for each
type of matrix analyzed and for each preparation method used and for each instrument.
These limits are considered the lowest reliable laboratory reporting concentrations and
should be established from the lower limit of quantitation check sample and then confirmed
using either the lowest calibration point or from a low-level calibration check standard.

The lower limit of quantitation check (LLQC) sample should be analyzed after establishing
the lower laboratory reporting limits and on an as needed basis to demonstrate the desired
detection capability. Ideally, this check sample and the low-level calibration verification
standard will be prepared at the same concentrations with the only difference being the
LLQC sample is carried through the entire preparation and analytical procedure. Lower
limits of quantitation are verified when all analytes in the LLQC sample are detected within ±
30% of their true value. This check should be used to both establish and confirm the lowest
quantitation limit.
The lower limits of quantitation determination using reagent water represents a best case
situation and does not represent possible matrix effects of real-world samples. For the
application of lower limits of quantitation on a project-specific basis with established data
quality objectives, low-level matrix specific spike studies may provide data users with a more
reliable indication of the actual method sensitivity and minimum detection capabilities.

4. All masses which could affect data quality should be monitored to determine potential
effects from matrix components on the analyte peaks.
5. Determine calibration curve.

6. All analyses require that a calibration curve be prepared to cover the appropriate
concentration range based on the intended application and prior to establishing the linear
dynamic range. Usually, this means the preparation of a calibration blank and mixed
calibration standard solutions, the highest of which would not exceed the anticipated linear
dynamic range of the instrument. Check the instrument standardization by analyzing
appropriate QC samples as follows.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 120

Individual or mixed calibration standards should be prepared from known primary stock
standards every six months to one year as needed based on the concentration stability as
confirmed from the ICV analyses. The analysis of the ICV, which is prepared from a source
independent of the calibration standards, is necessary to verify the instrument performance
once the system has been calibrated for the desired target analytes. It is recommended that
the ICV solution be obtained commercially as a certified traceable reference material such
that an expiration date can be assigned. Alternately the ICV solution can be prepared from
an independent source on an as needed basis depending on the ability to meet the
calibration verification criteria. If the ICV analysis is outside of the acceptance criteria, at a
minimum the calibration standards must be prepared fresh and the instrument recalibrated
prior to beginning sample analyses. Consideration should also be given to preparing fresh
ICV standards if the new calibration cannot be verified using the existing ICV standard.

Note: This method describes the use of both a low-level and mid-level ICV standard analysis.
For purposes of verifying the initial calibration, only the mid-level ICV needs to be prepared
from a source other than the calibration standards.
The calibration standards should be prepared using the same type of acid or combination of
acids and at similar concentrations as will result in the samples following processing.
The response of the calibration blank should be less than the response of the typical
laboratory lower limit of quantitation for each desired target analyte. Additionally, if the
calibration blank response or continuing calibration blank verification is used to calculate a
theoretical concentration, this value should be less than the level of acceptable blank
contamination as specified in the approved quality assurance project planning documents.
If this is not the case, the reason for the out-of-control condition must be found and
corrected, and the sample analyses may not proceed or the previous ten samples need to
be reanalyzed.
For the initial and daily instrument operation, calibrate the system according to the
instrument manufacturer's guidelines using the mixed calibration standards as noted in
section 4.2.1.4.5. The calibration curve should be prepared daily with a minimum of a
calibration blank and a single standard at the appropriate concentration to effectively
outline the desired quantitation range. Flush the system with the rinse blank between each
standard solution. Use the average of at least three integrations for both calibration and
sample analyses. The resulting curve should then be verified with mid-level and low-level
initial calibration verification standards as outlined below.

Alternatively, the calibration curve can be prepared daily with a minimum of a calibration
blank and three non-zero standards that effectively bracket the desired sample
concentration range. If low-level as compared to mid- or high-level sample concentrations
are expected, the calibration standards should be prepared at the appropriate
concentrations in order to demonstrate the instrument linearity within the anticipated sample
concentration range. For all multi-point calibration scenarios, the lowest non-zero standard
concentration should be considered the lower limit of quantitation.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                              Date: November 2010
	Page 121

    To be considered acceptable, the calibration curve should have a correlation coefficient
    greater than or equal to 0.998. When using a multipoint calibration curve approach, every
    effort should be made to attain an acceptable correlation coefficient based on a linear
    response for each desired target analyte. If the recommended linear response cannot be
    attained using a minimum of three non-zero calibration standards, consideration should be
    given to adding more standards, particularly at the lower concentrations, in order to better
    define the linear range and the lower limit of quantitation.  Conversely, the extreme upper
    and lower calibration points may be removed from the multi-point curve as long as three
    non-zero points remain such that the linear range is narrowed and the non-linear upper
    and/or lower portions are removed. As with the single point calibration option, the multi-point
    calibration should be verified with both a mid- and low-level ICV standard analysis using the
    same 90 -110% and 70 -130% acceptance criteria, respectively.
    Many instrument software packages allow multi-point calibration curves to be "forced"
    through zero. It is acceptable to use  this feature, provided that the resulting calibration
    meets the acceptance criteria, and can be verified by acceptable QC results. Forcing a
    regression through zero should NOT be used as a rationale for reporting results below the
    calibration range defined by the lowest standard in the calibration curve.

    After initial calibration, the calibration curve should be verified by use of an  initial calibration
    verification (ICV) standard analysis. At a minimum, the ICV standard should be prepared
    from an independent (second  source) material at or near the midrange of the calibration
    curve. The acceptance criteria for this mid-range ICV standard  should be ±10% of its true
    value. Additionally, a low-level initial  calibration verification (LLICV) standard should be
    prepared,  using the same source as  the calibration standards, at a concentration expected
    to be the lower limit of quantitation. The suggested acceptance criteria for the LLICV is
    ±30% of its true value. Quantitative sample analyses should not proceed for those analytes
    that fail the second source standard  initial calibration verification, with the exception that
    analyses may continue for those analytes that fail the criteria with an understanding these
    results should be  qualified and would be considered estimated  values. Once the calibration
    acceptance criteria is met, either the lowest calibration standard or the LLICV concentration
    can be used to demonstrate the lower  limit of quantitation and sample results should not be
    quantitated below this lowest standard. In some cases depending on the stated project data
    quality objectives, it may be appropriate to report these results  as estimated, however, they
    should be qualified by noting the results are below the lower limit of quantitation. Therefore,
    the quantitation limit cannot be reported lower than either the LLICV standard used for the
    single point calibration option or the low calibration and/or verification standard used during
    initial multi-point calibration. If the calibration curve cannot be verified within these specified
    limits for the mid-range ICV and LLICV analyses, the cause needs to be determined and the
    instrument recalibrated before samples are analyzed. The analysis data for the initial
    calibration verification analyses should be kept on file with the sample analysis data.
    Both single and multi-point calibration curves must be verified at the end of each analysis
    batch and after every 10 samples by a continuing calibration verification (CCV) standard and
    a continuing calibration blank (CCB). The CCV must be made from the same material as the
    initial calibration standards at or near the mid-range concentration. For the curve to be
    considered valid the acceptance criteria for the CCV standard must be ±10% of its true
    value and the CCB must contain target analytes less than the established lower limit of
    quantitation for the target analyte. If the calibration cannot be verified within the specified
    limits, the sample analysis must be discontinued, the cause determined and the instrument

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 122

recalibrated. All samples following the last acceptable CCV/CCB must be reanalyzed. The
analysis data for the CCV/CCB must be kept on file with the sample analysis data.
The low level continuing calibration verification (LLCCV) standard should also be analyzed
at the end of each analysis batch. A more frequent LLCCV analysis, i.e., every 10 samples
may be necessary if low-level sample concentrations are anticipated and the system stability
at low end of the calibration is questionable. In addition, the analysis of a LLCCV on a more
frequent basis will minimize the number of samples for re-analysis should the LLCCV fail if
only run at the end of the analysis batch. The LLCCV standard should be made from the
same source as the initial calibration standards at the established lower limit of quantitation
as reported by the laboratory. The acceptance criteria for the LLCCV standard should be ±
30% of its true value. If the calibration cannot be verified within these specified limits, the
analysis of samples containing the affected analytes at similar concentrations cannot
continue until the cause is determined and the LLCCV standard successfully analyzed. The
instrument may need to be recalibrated or the lower limit of quantitation adjusted to a
concentration that will ensure a compliant LLCCV analysis. The analysis data for the LLCCV
standard should be kept on file with the sample analysis data.

7. Verify the magnitude of elemental and molecular-ion isobaric interferences and adequacy of
any corrections at the beginning of an analytical run or once every 12 hr, whichever is more
frequent. Do this by analyzing the interference check solutions A and AB. The analyst
should be aware that precipitation from solution AB may occur with some elements.

Note Analysts have noted improved performance in calibration stability if the instrument is
exposed to the interference check solution after cleaning sampler and skimmer cones.
Improved performance is also realized if the instrument is allowed to rinse for 5 or 10 min
before the calibration blank is run.

8. The linear dynamic range is established when the system is first setup, or whenever
significant instrument components have been replaced or repaired, and on an as needed
basis only after the system has been successfully calibrated using either the single or multi-
point standard calibration approach. The upper limit of the linear dynamic range needs to be
established for each wavelength utilized by determining the signal responses from a
minimum of three, preferably five, different concentration standards across the range. The
ranges which may be used for the analysis of samples should be judged by the analyst from
the resulting data. The data, calculations and rationale for the choice of range made should
be documented and kept on file. A standard at the upper limit should be prepared, analyzed
and quantitated against the normal calibration curve. The calculated value should be within
±10% of the true value. New upper range limits should be determined whenever there is a
significant change in instrument response. At a minimum, the range should be checked
every 6 months. The analyst must be aware that if an analyte that is present above its upper
range limit is used to apply a spectral correction, the correction may not be valid and those
analytes where the spectral correction has been applied may be inaccurately reported.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 123

conditions, quantisation is not acceptable above or below the calibration standards.
Additionally, a non-linear curve should be further verified by calculating the actual recovery
of each calibration standard used in the curve. The acceptance criteria for the calibration
standard recovery should be ±10% of its true value for all standards except the lowest
concentration. A recovery of ±30% of its true value should be achieved for the lowest
concentration standard.
9. The analyst should (1) verify that the instrument configuration and operating conditions
satisfy the project-specific analytical requirements and (2) maintain quality control data that
demonstrate and confirm the instrument performance for the reported analytical results.

4.2.1.7 Procedures

1. All samples must be acid digested prior to analysis. Preliminary treatment of matrices is
necessary because of the complexity and variability of sample matrices.

2. Initiate appropriate operating configuration of the instrument's computer and set up the
instrument with the proper operating parameters according to the instrument manufacturer's
instructions.

3. Allow at least 30 min for the instrument to equilibrate before analyzing any samples. This
must be verified by an analysis of the tuning solution at least four integrations with relative
standard deviations of 5% for the analytes contained in the tuning solution.

Note. The instrument should have features to protect it from high ion currents. If not,
precautions must be taken to protect the detector from high ion currents. A channel electron
multiplier or active film multiplier suffers from fatigue after being exposed to high ion
currents. This fatigue can last from several seconds to hours depending on the extent of
exposure. During this time period, response factors are constantly changing, which
invalidates the calibration curve, causes instability, and invalidates sample analyses.
4. Flush the system with the rinse blank solution until the signal levels return to the DQO or
method's levels of quantitation (usually about 30 sec) before the analysis of each sample.
Nebulize each sample until a steady-state signal is achieved (usually about 30 sec) prior to
collecting data. Flow-injection systems may be used as long as they can meet the
performance criteria of this method.

5. Regardless of whether the initial calibration is performed using a single high standard and
the calibration blank or the multi-point option, the laboratory should analyze an LLCCV
(section 4.3.1.6.5). For all analytes and determinations, the laboratory must analyze an ICV
and LLICV immediately following daily calibration. It is recommended that a CCV, LLCCV,
and CCB be analyzed after every ten samples and at the end of the analysis batch.

6. Dilute and reanalyze samples that are more concentrated than the linear range for an
analyte (or species needed for a correction) or measure an alternate but less-abundant
isotope. The linearity at the alternate mass must be confirmed by appropriate calibration.
Alternatively apply solid phase chelation chromatography to eliminate the matrix as
described in section 4.3.1.2.3.

4.2.1.8 Data Analysis and Calculations

1. The quantitative values must be reported in appropriate units, such as micrograms per liter
(ug/L) for aqueous samples and milligrams per kilogram (mg/kg) for solid samples. If

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 124

dilutions were performed, the appropriate corrections must be applied to the sample values.
All results should be reported with up to three significant figures.
2. Calculate results for solids on a dry-weight basis as follows:

(1) A separate determination of percent solids must be performed.

(2) The concentrations determined in the digest are to be reported on the basis of the dry
weight of the sample.
Concentration (dry weight)(mg/kg) = (C x V) / (W x S)

where:
C = Digest Concentration (mg/L)
V = Final volume in liters after sample preparation
W = Weight in kg of wet sample
S = % Sol ids 7100

Calculations must include appropriate interference, internal-standard normalization, and the
summation of signals at 206, 207, and 208 m/z for lead (to compensate for any differences
in the abundances of these isotopes between samples and standards).

3. Results must be reported in units commensurate with their intended use and all dilutions
must be taken into account when computing final results.

4.2.2 Inductively Coupled Plasma - Atomic Emission Spectrometry

The sensitivity and the optimum and linear ranges for each element will vary with the
wavelength, spectrometer, matrix, and operating conditions.

Background correction is required for trace element determination. Background emission must
be measured adjacent to analyte lines on samples during analysis. The position selected for the
background-intensity measurement, on either or both sides of the analytical line, will be
determined by the complexity of the spectrum adjacent to the analyte line. The position used
should be as free as possible from spectral interference and should reflect the same change in
background intensity as occurs at the analyte wavelength measured. Background correction is
not required in cases of line broadening where a background correction measurement would
actually degrade the analytical result.

The possibility of additional interferences identified in section 4.2.2.2 should also be recognized
and appropriate corrections made; tests for their presence are described in sections 4.2.6.5 and
4.2.6.6. Users may instead choose multivariate calibration methods; if used, point selections for
background correction are superfluous since whole spectral regions are processed.

4.2.2.1 Summary of Method

This method describes multielement^! determinations by ICP-AES using sequential or
simultaneous optical systems and axial or radial viewing of the plasma (Figure 4.2). The
instrument measures characteristic emission spectra by optical spectrometry. Samples are
nebulized (Appendix A) and the resulting aerosol is transported to the plasma torch. Element-
specific emission spectra are produced by a radio-frequency inductively coupled plasma. The
spectra are dispersed by a grating spectrometer, and the intensities of the emission lines are
monitored by photosensitive devices.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 125
i
A

Figure 4.2. Inductively Coupled Plasma-Atomic Emission Spectrometry

4.2.2.2 Interferences

Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/
or interferences to sample analysis. All materials must be confirmed free from interferences
under the conditions of the analysis by analyzing method blanks. Specific selection of reagents
and purification of solvents by distillation in all-glass systems may be necessary.

Background and Stray Light

1. Compensation for background emission and stray light can usually be conducted by
subtracting the background emission determined by measurements adjacent to the analyte
wavelength peak. Spectral scans of samples or single element solutions in the analyte
regions may indicate when alternate wavelengths are desirable because of severe spectral
interference. These scans will also show whether the most appropriate estimate of the
background emission is provided by an interpolation from measurements on both sides of
the wavelength peak or by measured emission on only one side. The locations selected for
the measurement of background intensity will be determined by the complexity of the
spectrum adjacent to the wavelength peak. The locations used for routine measurement
must be free of off-line spectral interference (interelement or molecular) or adequately
corrected to reflect the same change in background intensity as occurs at the wavelength
peak. For multivariate methods using whole spectral regions, background scans should be

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 126

included in the correction algorithm. Off-line spectral interferences are handled by including
spectra on interfering species in the algorithm.
2. To determine the appropriate location for off-line background correction, the analyst must
scan the area on either side adjacent to the wavelength and record the apparent emission
intensity from all other method analytes. This spectral information must be documented and
kept on file. The location selected for background correction must be either free of off-line
interelement spectral interference or a computer routine must be used for automatic
correction on all determinations. If a wavelength other than the recommended wavelength is
used, the analyst must determine and document both the overlapping and nearby spectral
interference effects from all method analytes and common elements and provide for their
automatic correction on all analyses. Tests to determine spectral interference must be done
using analyte concentrations that will adequately describe the interference. Normally, 100
mg/L single-element solutions are sufficient. However, for analytes such as iron that may be
found in the sample at high concentration, a more appropriate test would be to use a
concentration near the upper limit of the analytical range.
3. Spectral overlaps may be avoided by using an alternate wavelength or can be compensated
for by equations that correct for interelement contributions. Instruments that use equations
for interelement correction require that the interfering elements be analyzed at the same
time as the element of interest. When operative and uncorrected, interferences will produce
false positive or positively biased determinations. Analysts may apply interelement
correction equations determined on their instruments with tested concentration ranges to
compensate (off-line or on-line) for the effects of interfering elements. For multivariate
calibration methods using whole spectral regions, spectral interferences are handled by
including spectra of the interfering elements in the algorithm.

Note. When using interelement correction equations, the interference may be expressed as
analyte concentration equivalents (i.e., false positive analyte concentrations) arising from
100 mg/L of the interference element. It should be noted that the interference effects must
be evaluated for each individual instrument, since the intensities will vary.

4. Interelement corrections will vary for the same emission line among instruments because of
differences in resolution, as determined by the grating, the entrance and exit slit widths, and
by the order of dispersion. Interelement corrections will also vary depending upon the choice
of background correction points. Selecting a background correction point where an
interfering emission line may appear should be avoided when practical. Interelement
corrections that constitute a major portion of an emission signal may not yield accurate data.
Users should continuously note that some samples may contain uncommon elements that
could contribute spectral interferences.

5. Interference effects must be evaluated for each individual instrument, whether configured as
a sequential or simultaneous instrument. For each instrument, intensities will vary not only
with optical resolution but also with operating conditions (e.g., power, viewing height, argon
flow rate). When using the recommended wavelengths, the analyst is required to determine
and document for each wavelength the effect from referenced interferences as well as any
other suspected interferences that may be specific to the instrument or matrix. The analyst is
encouraged to utilize a computer routine for automatic correction on all analyses.

6. Users of sequential instruments must verify the absence of spectral interference by
scanning over a range of 0.5 nm centered on the wavelength of interest for several samples.
The range for lead, for example, would be from 220.6 to 220.1 nm. This procedure must be
repeated whenever a new matrix is to be analyzed and when a new calibration curve using

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 127

different instrumental conditions is to be prepared. Samples that show an elevated
background emission across the range may be background corrected by applying a
correction factor equal to the emission adjacent to the line or at two points on either side of
the line and interpolating between them. An alternate wavelength that does not exhibit a
background shift or spectral overlap may also be used.
7. If the correction routine is operating properly, the determined apparent analyte(s)
concentration from analysis of each interference solution should fall within a specific
concentration range around the calibration blank. The concentration range is calculated by
multiplying the concentration of the interfering element by the value of the correction factor
being tested and dividing by 10. If after the subtraction of the calibration blank the apparent
analyte concentration falls outside of this range, in either a positive or negative direction, a
change in the correction factor of more than 10% should be suspected. The cause of the
change should be determined and corrected and the correction factor updated. The
interference check solutions should be analyzed more than once to confirm a change has
occurred. Adequate rinse time between solutions and before analysis of the calibration blank
will assist in the confirmation.

8. When interelement corrections are applied, their accuracy should be verified daily, by
analyzing spectral interference check solutions. The correction factors or multivariate
correction matrices tested on a daily basis must be within the 20% criteria for five
consecutive days. All interelement spectral correction factors or multivariate correction
matrices must be verified and updated every six months or when an instrumentation change
occurs, such as one in the torch, nebulizer, injector, or plasma conditions. Standard
solutions should be inspected to ensure that there is no contamination that may be
perceived as a spectral interference.

9. When interelement corrections are not used, verification of absence of interferences is
required.One method to verify the absence of interferences is to use a computer software
routine for comparing the determinative data to established limits for notifying the analyst
when an interfering element is detected in the sample at a concentration that will produce
either an apparent false positive concentration (i.e., greater than the analyte instrument
detection limit), or a false negative analyte concentration (i.e., less than the lower control
limit of the calibration blank defined for a 99% confidence interval).

Another way to verify the absence of interferences is to analyze an interference check
solution which contains similar concentrations of the major components of the samples (>10
mg/L) on a continuing basis to verify the absence of effects at the wavelengths selected.
These data must be kept on file with the sample analysis data. If the check solution confirms
an operative interference that is 320% of the analyte concentration, the analyte must be
determined using (1) analytical and background correction wavelengths (or spectral regions)
free of the interference, (2) by an alternative wavelength, or (3) by another documented test
procedure.

Physical Interferences

Physical Interferences are effects associated with the sample nebulization and transport
processes. Changes in viscosity and surface tension can cause significant inaccuracies,
especially in samples containing high dissolved solids or high acid concentrations. If physical
interferences are present, they must be reduced by diluting the sample, by using a peristaltic
pump, by using an internal standard, or by using a high solids nebulizer. The test described in
section 4.3.2.6.8 will help determine if a physical interference is present.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 128

Note. One problem that can occur with high dissolved solids is salt buildup at the tip of the
nebulizer, affecting aerosol flow rate and causing instrumental drift. The problem can be
controlled by wetting the argon prior to nebulization, by using a tip washer, by using a high
solids nebulizer, or by diluting the sample. Also, it has been reported that better control of the
argon flow rate, especially to the nebulizer, improves instrument performance. This may be
accomplished with the use of mass flow controllers.
Chemical interferences

Chemical interferences include molecular compound formation, ionization effects, and solute
vaporization effects. Normally, these effects are not significant with the ICP technique, but if
observed, can be minimized by careful selection of operating conditions (incident power,
observation position, and so forth), by buffering of the sample, by matrix matching, and by
standard addition procedures. Chemical interferences are highly dependent on matrix type and
the specific analyte element.

Note. An alternative to using the method of standard additions is to use the internal standard
technique, which involves adding one or more elements that are both not found in the samples
and verified to not cause an interelement spectral interference to the samples, standards, and
blanks. Yttrium or scandium is often used. The concentration should be sufficient for optimum
precision, but not so high as to alter the salt concentration of the matrix. The element intensity is
used by the instrument as an internal standard to ratio the analyte intensity signals for both
calibration and quantisation. This technique is very useful in overcoming matrix interferences,
especially in high solids matrices.

Memory interferences

Memory interferences result when analytes in a previous sample contribute to the signals
measured in a new sample. Memory effects can result from sample deposition on the uptake
tubing to the nebulizer and from the build up of sample material in the plasma torch and spray
chamber. The site where these effects occur is dependent on the element and can be
minimized by flushing the system with a rinse blank between samples. The possibility of
memory interferences should be recognized within an analytical run and suitable rinse times
should be used to reduce them.
The rinse times necessary for a particular element must be estimated prior to analysis. This may
be achieved by aspirating a standard containing elements at a concentration ten times the usual
amount or at the top of the linear dynamic range. The aspiration time for this sample should be
the same as a normal sample analysis period, followed by analysis of the rinse blank at
designated intervals. Note the length of time necessary for reducing analyte signals to "equal to"
or "less than" the lower limit of quantitation. Until the required rinse time is established, the rinse
period should be at least 60 sec. between samples and standards. If memory interference is
suspected, the sample must be reanalyzed after a rinse period of sufficient length. Alternate
rinse times may be established by the analyst based upon the project-specific DQOs.

High Salt Concentrations

Users are advised that high salt concentrations can cause analyte signal suppressions and
confuse interference tests. If the instrument does not display negative values, fortify the
interference check solution with the elements of interest at 0.5 to 1 mg/L and measure the
added standard concentration accordingly. Concentrations should be within 20% of the true
spiked concentration or dilution of the samples will be necessary. In the absence of a
measurable analyte, overcorrection could go undetected if a negative value is reported as zero.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 129

4.2.2.3 Equipment and Supplies

1. Inductively coupled argon plasma emission spectrometer
a. Computer-controlled emission spectrometer with background correction.
b. Radio-frequency generator compliant with FCC regulations.
c. Optional mass flow controller for argon nebulizer gas supply.
d. Optional peristaltic pump.
e. Optional autosampler.
2. Argon gas supply - high purity.
3. Volumetric flasks of suitable precision and accuracy.
4. Volumetric pipets of suitable precision and accuracy.

4.2.2.4 Reagents

Reagent- or trace metals-grade chemicals must be used in all tests. Unless otherwise indicated,
it is intended that all reagents conform to the specifications of the Committee on Analytical
Reagents of the American Chemical Society, where such specifications are available. Other
grades may be used, provided it is first ascertained that the reagent is of sufficiently high purity
to permit its use without lessening the accuracy of the determination. If the purity of a reagent is
in question, analyze for contamination. If the concentration of the contamination is less than the
lower limit of quantitation, then the reagent is acceptable.

1. Hydrochloric acid (cone), HCI.
2. Hydrochloric acid HCI (1:1) - Add 500 ml concentrated HCI to 400 ml water and dilute to 1L.
3. Nitric acid (cone), HNO3.
4. Nitric acid, HNO3(1:1) - Add 500 ml concentrated HNO3to 400 ml water and dilute to 1 L
5. Reagent water- Reagent water must be free of interferences.

4.2.2.5 Standards
1. Standard stock solutions may be purchased or prepared from ultra-high purity grade
chemicals or metals (99.99% pure or greater). With several exceptions specifically noted, all
salts must be dried for 1 hr at 105°C. Preparation procedures are contained in Table 4.5.

CAUTION: Many metal salts are extremely toxic if inhaled or swallowed. Wash hands
thoroughly after handling.

Mixed Calibration Standard Solutions

Prepare mixed calibration standard solutions (Table 4.6) by combining appropriate volumes of
the stock solutions above in volumetric flasks. Add the appropriate types and volumes of acids
so that the standards are matrix-matched with the sample digestates. Prior to preparing the
mixed standards, each stock solution should be analyzed separately to determine possible
spectral interference or the presence of impurities. Care should be taken when preparing the
mixed standards to ensure that the elements are compatible and stable together.

Transfer the mixed standard solutions to FEP fluorocarbon or previously unused polyethylene or
polypropylene bottles for storage. For all intermediate and working standards, especially low
level standards (i.e., <1 ppm), stability must be demonstrated prior to use. Freshly-mixed
standards should be prepared, as needed, realizing that concentration can change with age.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
            Page 130
Table 4.5. Ty
Element
Aluminum
Antimony
Arsenic
Cadmium
Chromium
Copper
Iron
Lead
Manganese
Nickel
Selenium
Tin
Zinc
Dical Stock Solution Preparation Procedures
Solution
1 ml = 1000
ug of Al
1 m= 1000
ug of Sb
1 ml = 1000
ug of As
1 ml = 1000
ug of Cd
1 ml = 1000
ug of Cr
1 ml = 1000
ug of Cu
1 ml = 1000
ug of Fe
1 ml = 1000
ug of Pb
1 ml = 1000
ug of Mn
1 ml = 1000
ug of Ni
1 ml = 1000
ug of Se
1 ml = 1000
ug of Sn
1 ml_= 1000
ug of Zn
Directions
Dissolve 1 .000 g of aluminum metal, accurately weighed to at least four significant
figures, in an acid mixture of 4.0 ml of HCI (1:1) and 1.0 ml of concentrated HMOs in a
beaker. Slowly warm the beaker to dissolve the metal. When dissolution is
complete, transfer solution quantitatively to a 1000-ml volumetric flask, add an
additional 10.0ml of HCI (1:1) and dilute to volume with reagent water
Dissolve 2.6673 g of K(SbO)C4H4O6 (element fraction Sb = 0.3749), accurately
weighed to at least four significant figures, in reagent water, add 10 ml of HCI (1:1),
and dilute to volume in a 1 000-ml volumetric flask with reagent water.
Dissolve 1 .3203 g of As2O3 (element fraction As = 0.7574), accurately weighed to at
least four significant figures, in 1 00 ml of reagent water containing 0.4 g of NaOH.
Acidify the solution with 2 ml_ of concentrated HNO3 and dilute to volume in a 1 000-
ml volumetric flask with reagent water.
Dissolve 1.1423 g of CdO (element fraction Cd = 0.8754), accurately weighed to at
least four significant figures, in a minimum amount of (1:1) HMOs. Heat to increase
the rate of dissolution. Add 10.0 ml of concentrated HNO3 and dilute to volume in a
1 000-ml volumetric flask with reagent water.
Dissolve 1 .9231 g of CrO3 (element fraction Cr = 0.5200), accurately weighed to at
least four significant figures, in reagent water. When dissolution is complete, acidify
with 10 ml of concentrated HNO3 and dilute to volume in a 1000-ml volumetric flask
with reagent water.
Dissolve 1 .2564 g of CuO (element fraction Cu = 0.7989), accurately weighed to at
least four significant figures, in a minimum amount of (1:1) HMOs. Add 10.0 ml of
concentrated HNO3 and dilute to volume in a 1 000-ml volumetric flask with reagent
water.
Dissolve 1.4298 g of Fe2O3 (element fraction Fe = 0.6994), accurately weighed to at
least 4 significant figures, in a warm mixture of 20 ml HCI (1:1) and 2 ml of
concentrated HNO3. Cool, add an additional 5.0 ml of concentrated HNO3, and dilute
to volume in a 1 000-ml volumetric flask with reagent water.
Dissolve 1 .5985 g of Pb(NO3)2 (element fraction Pb = 0.6256), accurately weighed to
at least four significant figures, in a minimum amount of (1:1) HNO3. Add 10 ml (1:1)
HNO3 and dilute to volume in a 1 000-ml volumetric flask with reagent water.
Dissolve 1.00 g of manganese metal, accurately weighed to at least four significant
figures, in acid mixture (10 ml of concentrated HCI and 1 ml of concentrated HNO3)
and dilute to volume in a 1 000-ml volumetric flask with reagent water.
Dissolve 1 .000 g of nickel metal, accurately weighed to at least four significant
figures, in 10.0 ml of hot concentrated HNO3, cool, and dilute to volume in a 1000-ml
volumetric flask with reagent water.
Do not dry. Dissolve 1.6332 g of H2SeO3 (element fraction Se = 0.6123), accurately
weighed to at least four significant figures, in reagent water and dilute to volume in a
1 000-ml volumetric flask with reagent water.
Dissolve 1 .000 g of Sn shot, accurately weighed to at least 4 significant figures, in
200 ml of HCI (1:1) with heating to dissolve the metal. Let solution cool and dilute
with HCI (1:1) in a 1000-ml volumetric flask.
Dissolve 1.2447g of ZnO (element fraction Zn=0.8034), accurately weighed to at
least 4 significant figures, in a minimum amount of dilute HNO3. Add 10.0 ml of
concentrated HNO3 and dilute to volume in a 1 000-ml volumetric flask with reagent
water.
Note. Concentrations are calculated based upon the weight of pure metal added, or with the use of the element
fraction and the weight of the metal salt added. The weight of the analyte is expressed to four significant figures for
consistency with the weights below because rounding to two decimal places can contribute up to 4% error for some
of the compounds.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 131

Table 4.6. Mixed Standard Solutions
Solution
I
II
III
IV
VI
Elements
Cd, Mn, Pb, Se
Cu and Fe
As
Al, Grand Ni
P, Sn

and Zn

Blanks

Two types of blanks are required for the analysis. The calibration blank is used in establishing
the analytical curve and the method blank is used to identify possible contamination resulting
from either the reagents (acids) or the equipment used during sample processing including
filtration.
The calibration blank is prepared by acidifying reagent water to the same concentrations of the
acids found in the standards and samples. Prepare a sufficient quantity to flush the system
between standards and samples. The calibration blank will also be used for all initial (ICB) and
continuing calibration blank (CCB) determinations.
The method blank must contain all of the reagents in the same volumes as used in the
processing of the samples. The method blank must be carried through the complete procedure
and contain the same acid concentration in the final solution as the sample solution used for
analysis (section 4.2.2.6.4).

Initial Calibration Standard

The initial calibration verification (ICV) standard may be purchased or prepared by the analyst
(or a purchased second source reference material) by combining compatible elements from a
standard source different from that of the calibration standard and at concentration near the
midpoint of the calibration curve (section 4.2.2.7.7).

Continuing Calibration Standard

The continuing calibration verification (CCV) standard should be prepared in the same acid
matrix using the same standards used for calibration, at a concentration near the mid-point of
the calibration curve (section 4.2.2.7.7).

Interference Check Solution
The interference check solution is prepared to contain known concentrations of interfering
elements that will provide an adequate test of the correction factors. Spike the sample with the
elements of interest, particularly those with known interferences at 0.5 to 1 mg/L. In the
absence of measurable analyte, overcorrection could go undetected because a negative value
could be reported as zero. If the particular instrument will display overcorrection as a negative
number, this spiking procedure will not be necessary.

4.2.2.6 Quality Control

1. Refer to the QAPP for additional guidance on quality assurance and quality control
protocols. When inconsistencies exist between QC guidelines, method specific QC criteria
take precedence. The laboratory should also maintain records to document the quality of
the data generated. All data sheets and quality control data should be maintained for
reference or inspection.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 132

2. Instrument detection limits (IDLs) are useful means to evaluate the instrument noise level
and response changes over time for each analyte from a series of reagent blank analyses to
obtain a calculated concentration. They are not to be confused with the lower limit of
quantitation, nor should they be used in establishing this limit. It may be helpful to compare
the calculated IDLs to the established lower limit of quantitation (section 4.2.2.4).
IDLs in ug/L can be estimated by calculating the average of the standard deviations of three
runs on three non-consecutive days from the analysis of a reagent blank solution with seven
consecutive measurements per day. Each measurement should be performed as though it
were a separate analytical sample (i.e., each measurement must be followed by a rinse
and/or any other procedure normally performed between the analysis of separate samples).
IDLs should be determined at least every three months or at a project-specific designated
frequency and kept with the instrument log book.
3. Each laboratory must demonstrate initial proficiency with each sample.

Note. If an autosampler is used to perform sample dilutions, the laboratory should verify that
those dilutions are of equivalent or better accuracy than is achieved by an experienced
analyst performing manual dilutions.
4. Dilute and reanalyze samples that exceed the linear dynamic range or use an alternate,
less sensitive calibration for which quality control data are already established.

5. For each batch of samples processed, at least one method blank must be carried
throughout the entire sample preparation and analytical process. If the method blank does
not contain target analytes at a level that interferes with the project-specific DQOs, then the
method blank would be considered acceptable. In the absence of project-specific DQOs, if
the blank is less than 10% of the lower limit of quantitation check sample concentration, less
than 10% of the regulatory limit, or less than 10% of the lowest sample concentration for
each analyte in a given preparation batch, whichever is greater, then the method blank is
considered acceptable. If the method blank cannot be considered acceptable, the method
blank should be re-run once, and if still unacceptable, then all samples after the last
acceptable method blank should be reprepared and reanalyzed along with the other
appropriate batch QC samples. If the method blank exceeds the criteria, but the samples
are all either below the reporting level or below the applicable action level or other DQOs,
then the sample data may be used despite the contamination of the method blank.

6. For each batch of samples processed, at least one laboratory control sample (LCS) must be
carried throughout the entire sample preparation and analytical process. The laboratory
control samples should be spiked with each analyte of interest at the project-specific action
level or, when lacking project specific action levels, at approximately mid-point of the linear
dynamic range. Acceptance criteria should either be defined in the project-specific planning
documents or set at a laboratory derived limit developed through the use of historical
analyses. In the absence of project-specific or historical data generated criteria, this limit
should be set at ± 20% of the spiked value. Acceptance limits derived from historical data
should be no wider that ± 20%. If the laboratory control sample is not acceptable, then the
laboratory control sample should be re-run once and, if still unacceptable, all samples after
the last acceptable laboratory control sample should be reprepared and reanalyzed.
7. Concurrent analyses of standard reference materials (SRMs) containing known amounts of
analytes in the media of interest are recommended and may be used as an LCS. For solid
SRMs, 80 -120% accuracy may not be achievable and the manufacturer's established
acceptance criterion should be used for soil SRMs.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
_ Page 133

8. Documenting the effect of the matrix, for a given preparation batch consisting of similar
sample characteristics, should include the analysis of at least one matrix spike and one
duplicate unspiked sample (MS/Dup) or one matrix spike/matrix spike duplicate (MS/MSD)
pair. The decision on whether to prepare and analyze duplicate samples or a matrix
spike/matrix spike duplicate must be based on knowledge of the samples in the sample
batch or as noted in the project-specific planning documents. If samples are expected to
contain target analytes, then laboratories may use one matrix spike and a duplicate analysis
of an unspiked field sample. If samples are not expected to contain target analytes,
laboratories should use a matrix spike and matrix spike duplicate pair.

MS/MSD samples should be spiked at the same level, and with the same spiking material,
as the corresponding laboratory control sample that is at the project-specific action level or,
when lacking project-specific action levels, at approximately mid-point of the linear dynamic
range. Acceptance criteria should either be defined in the project-specific planning
documents or set at a laboratory-derived limit developed through the use of historical
analyses per matrix type analyzed. In the absence of project-specific or historical data
generated criteria, these limits should be set at ± 25% of the spiked value for accuracy and
20 relative percent difference (RPD) for precision. Acceptance limits derived from historical
data should be no wider that ± 25% for accuracy and 20% for precision. If the bias and
precision indicators are outside the laboratory control limits, if the percent recovery is less
than 75% or greater than 125%, or if the relative percent difference is greater than 20%,
then the interference test discussed in Sec. 4.2.2.9.9 should be conducted.

The relative percent difference between spiked matrix duplicate or unspiked duplicate
determinations is to be calculated as follows:
x100
(0^02)72
where:
RPD = relative percent difference.
D! = first sample value.
D2= second sample value (spiked or unspiked duplicate)
The spiked sample or spiked duplicate sample recovery should be within ± 25% of the actual
value, or within the documented historical acceptance limits for each matrix.
9. If less than acceptable accuracy and precision data are generated, additional quality control
tests below are recommended prior to reporting concentration data for the elements in this
method. At a minimum, these tests should be performed with each batch of samples
prepared/analyzed with corresponding unacceptable data quality results. These tests will
then serve to ensure that neither positive nor negative interferences are affecting the
measurement of any of the elements or distorting the accuracy of the reported values. If
matrix effects are confirmed, the laboratory should consult with the data user when feasible
for possible corrective actions which may include the use of alternative or modified test
procedures so that the analysis is not impacted by the same interference.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 134

Post digestion spike addition. If the MS/MSD recoveries are unacceptable, the same sample
from which the MS/MSD aliquots were prepared should also be spiked with a post digestion
spike. Otherwise, another sample from the same preparation should be used as an
alternative. An analyte spike is added to a portion of a prepared sample, or its dilution, and
should be recovered to within 80% to 120% of the known value. The spike addition should
produce a minimum level of 10 times and a maximum of 100 times the lower limit of
quantitation. If this spike fails, then the dilution test should be run on this sample. If both the
MS/MSD and the post digestion spike fail, then matrix effects are confirmed.
Dilution test. If the analyte concentration is sufficiently high (minimally, a factor of 10 above
the lower limit of quantitation after dilution), an analysis of a 1:5 dilution should agree within
± 10% of the original determination. If not, then a chemical or physical interference effect
should be suspected.

Note. If spectral overlap is suspected, then the use of computerized compensation, an alternate
wavelength, or comparison with an alternate method is recommended.

4.2.2.7 Calibration and Standardization

2. Sensitivity, instrumental detection limit, precision, linear dynamic range, and interference
corrections need to be established for each individual target analyte on each particular
instrument. All measurements (both target analytes and constituents which interfere with the
target analytes) need to be within the instrument linear range where the correction equations
are valid.
3. The lower limits of quantitation should be established for all isotope masses utilized for each
type of matrix analyzed and for each preparation method used and for each instrument.
These limits are considered the lowest reliable laboratory reporting concentrations and
should be established from the lower limit of quantitation check sample and then confirmed
using either the lowest calibration point or from a low-level calibration check standard.

Lower limit of quantitation check sample:

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 135
4.  Specific recommended wavelengths are listed in Table 4.7 below. Other wavelengths may
   be substituted (e.g., in the case of an interference) if they provide the needed sensitivity and
   are treated with the same corrective techniques for spectral interference.

Table 4.7. Recommended Wavelengths and Estimated Instrumental Detection Limits
Element
Aluminum
Antimony
Arsenic
Cadmium
Chromium
Copper
Iron
Lead
Manganese
Nickel
Selenium
Tin
Zinc
Wavelength (nm)
308.215
206.833
193.696
226.502
267.716
324.754
259.940
220.353
257.610
231 .604 (2nd order)
196.026
189. 980 (2nd order)
21 3. 856 (2nd order)
Estimated Detection
Limit (|jm)
30
21
35
2.3
4.7
3.6
4.1
28.0
0.93
10
50
17
1.2
For radial viewed plasma, operating conditions for aqueous solutions usually vary from:
       1100 to 1200 watts forward power,
       14 to 18 mm viewing height,
       15 to 19 L/min argon coolant flow,
       0.6 to 1.5 L/min argon nebulizer flow,
       1 to 1.8 mL/min sample pumping rate with a 1 minute preflush time and measurement
       time near 1 sec per wavelength peak for sequential instruments and a rinse time of 10
       sec per replicate with a 1  sec per replicate read time for simultaneous instruments.

For axial viewed plasma, the conditions will usually vary from:

       1100 to 1500 watts forward power,
       15 to 19 L/min argon coolant flow,
       0.6 to 1.5 L/min argon nebulizer flow,
       1 to 1.8 mL/min sample pumping rate with a 1 minute preflush time and measurement
       time near 1 sec per wavelength peak for sequential instruments and a rinse time of 10
       sec per replicate with a 1  sec per replicate read time for simultaneous instruments.

    One recommended way  to achieve repeatable interference correction factors is to adjust the
    argon aerosol flow to reproduce the Cu/Mn intensity ratio at 324.754 nm and 257.610 nm
    respectively. This can be performed before daily calibration and after the instrument warm-
    up period.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 136

5. Plasma optimization

The plasma operating conditions need to be optimized prior to use of the instrument. The
purpose of plasma optimization is to provide a maximum signal to background ratio for
some of the least sensitive elements in the analytical array. The use of a mass flow
controller to regulate the nebulizer gas flow or source optimization software greatly
facilitates the procedure. This routine is not required on a daily basis, it is only required
when first setting up a new instrument, or following a change in operating conditions. The
following procedure is recommended or follow the manufacturer's recommendations.
Ignite the radial plasma and select an appropriate incident radio frequency (RF) power.
Allow the instrument to become thermally stable before beginning, about 30 to 60 minutes
of operation. While aspirating a 1000 ug/L solution of yttrium, follow the instrument
manufacturer's instructions and adjust the aerosol carrier gas flow rate through the
nebulizer so a definitive blue emission region of the plasma extends approximately from 5
to 20 mm above the top of the load coil. Record the nebulizer gas flow rate or pressure
setting for future reference. The yttrium solution can also be used for coarse optical
alignment of the torch by observing the overlay of the blue light over the entrance slit to the
optical system.

After establishing the nebulizer gas flow rate, determine the solution uptake rate of the
nebulizer in mL/min by aspirating a known volume of a calibration blank for a period of at
least three minutes. Divide the volume aspirated by the time in minutes and record the
uptake rate. Set the peristaltic pump to deliver that rate in a steady even flow.

Profile the instrument to align it optically as it will be used during analysis. The following
procedure is written for vertical optimization in the radial mode, but it also can be used for
horizontal optimization. Aspirate a solution containing 10 ug/L of several selected elements.
As, Se, Tl, and Pb are the least sensitive of the elements and most in need of optimization.
Other elements may be used, based on project-specific protocols. (Cr, Cu, and Mn also
have been used with success.) Collect intensity data at the wavelength peak for each
analyte at 1 mm intervals from 14 to 18 mm above the load coil. (This region of the plasma
is referred to as the analytical zone.) Repeat the process using the calibration blank.
Determine the net signal to blank intensity ratio for each analyte for each viewing height
setting. Choose the height for viewing the plasma that provides the best net intensity ratios
for the elements analyzed or the highest intensity ratio for the least sensitive element. For
optimization in the axial mode, follow the instrument manufacturer's instructions.

The instrument operating conditions finally selected as being optimum should provide the
most appropriate instrument responses that correlate to the desired target analyte
sensitivity while meeting the minimum quality control criteria noted in this method or as
specified in the project-specific planning documents.

If the instrument operating conditions, such as incident power or nebulizer gas flow rate,
are changed, or if a new torch injector tube with a different orifice internal diameter is
installed, then the plasma and viewing height should be re-optimized.

After completing the initial optimization of operating conditions, and before analyzing
samples, the laboratory should establish and initially verify an interelement spectral
interference correction routine to be used during sample analysis with interference check
standards that closely match the anticipated properties of the expected sample matrices,
i.e., for saltwater type matrices the interference check standard should contain components
that match the salinities of the proposed sample matrix. A general description of spectral

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 137

interferences and the analytical requirements for background correction, in particular, are
discussed in section 4.2.2.2.
Before daily calibration, and after the instrument warm-up period, the nebulizer gas flow
rate should be reset to the determined optimized flow. If a mass flow controller is being
used, it should be set to the recorded optimized flow rate. In order to maintain valid spectral
interelement correction routines, the nebulizer gas flow rate should be the same (<2%
change) from day to day.

6. For operation with organic solvents, the use of the auxiliary argon inlet is recommended, as
is the use of solvent-resistant tubing, increased plasma (coolant) argon flow, decreased
nebulizer flow, and increased RF power, to obtain stable operation and precise
measurements.

7. Determine calibration curve.

All analyses require that a calibration curve be prepared to cover the appropriate
concentration range based on the intended application and prior to establishing the linear
dynamic range. Usually, this means the preparation of a calibration blank and mixed
calibration standard solutions, the highest of which would not exceed the anticipated linear
dynamic range of the instrument. Check the instrument standardization by analyzing
appropriate QC samples as follows.
Individual or mixed calibration standards should be prepared from known primary stock
standards every six months to one year as needed based on the concentration stability as
confirmed from the ICV analyses. The analysis of the ICV, which is prepared from a source
independent of the calibration standards, is necessary to verify the instrument performance
once the system has been calibrated for the desired target analytes. It is recommended
that the ICV solution be obtained commercially as a certified traceable reference material
such that an expiration date can be assigned. Alternately the ICV solution can be prepared
from an independent source on an as needed basis depending on the ability to meet the
calibration verification criteria. If the ICV analysis is outside of the acceptance criteria, at a
minimum the calibration standards must be prepared fresh and the instrument recalibrated
prior to beginning sample analyses. Consideration should also be given to preparing fresh
ICV standards if the new calibration cannot be verified using the existing ICV standard.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 138

For the initial and daily instrument operation, calibrate the system according to the
instrument manufacturer's guidelines using the mixed calibration standards as noted in
section 4.2.2.5.3. The calibration curve should be prepared daily with a minimum of a
calibration blank and a single standard at the appropriate concentration to effectively
outline the desired quantitation range. Flush the system with the rinse blank between each
standard solution. Use the average of at least three integrations for both calibration and
sample analyses. The resulting curve should then be verified with mid-level and low-level
initial calibration verification standards as outlined in section 4.2.2.7.8.
Alternatively, the calibration curve can be prepared daily with a minimum of a calibration
blank and three non-zero standards that effectively bracket the desired sample
concentration range. If low-level as compared to mid- or high-level sample concentrations
are expected, the calibration standards should be prepared at the appropriate
concentrations in order to demonstrate the instrument linearity within the anticipated
sample concentration range. For all multi-point calibration scenarios, the lowest non-zero
standard concentration should be considered the lower limit of quantitation.

Note. Regardless of whether the instrument is calibrated using only a minimum number of
standards or with a multi-point curve, the upper limit of the quantitation range may exceed
the highest concentration calibration point and can be defined as the "linear dynamic"
range, while the lower limit can be identified as the "lower limit of quantitation limit" (LLQL)
and will be either the concentration of the lowest calibration standard (for multi-point
curves) or the concentration of the low level ICV/CCV check standard. Results reported
outside these limits would not be recommended unless they are qualified as estimated. See
Sec. 4.2.2.10.4.4 for recommendations on how to determine the linear dynamic range,
while the guidance in this section and section 4.2.2.7.8 provide options for defining the
lower limit of quantitation.
To be considered acceptable, the calibration curve should have a correlation coefficient
greater than or equal to 0.998. When using a multipoint calibration curve approach, every
effort should be made to attain an acceptable correlation coefficient based on a linear
response for each desired target analyte. If the recommended linear response cannot be
attained using a minimum of three non-zero calibration standards, consideration should be
given to adding more standards, particularly at the lower concentrations, in order to better
define the linear range and the lower limit of quantitation. Conversely, the extreme upper
and lower calibration points may be removed from the multi-point curve as long as three
non-zero points remain such that the linear range is narrowed and the non-linear upper
and/or lower portions are removed. As with the single point calibration option, the multi-
point calibration should be verified with both a mid- and low-level ICV standard analysis
using the same 90 -110% and 70 -130% acceptance criteria, respectively.

Many instrument software packages allow multi-point calibration curves to be "forced"
through zero. It is acceptable to use this feature, provided that the resulting calibration
meets the acceptance criteria, and can be verified by acceptable QC results. Forcing a
regression through zero should NOT be used as a rationale for reporting results below the
calibration range defined by the lowest standard in the calibration curve.
After initial calibration, the calibration curve should be verified by use of an initial calibration
verification (ICV) standard analysis. At a minimum, the ICV standard should be prepared
from an independent (second source) material at or near the midrange of the calibration
curve. The acceptance criteria for this mid-range ICV standard should be ±10% of its true
value. Additionally, a low-level initial calibration verification (LLICV) standard should be

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 139

prepared, using the same source as the calibration standards, at a concentration expected
to be the lower limit of quantitation. The suggested acceptance criteria for the LLICV is
±30% of its true value. Quantitative sample analyses should not proceed for those analytes
that fail the second source standard initial calibration verification, with the exception that
analyses may continue for those analytes that fail the criteria with an understanding these
results should be qualified and would be considered estimated values. Once the calibration
acceptance criteria is met, either the lowest calibration standard or the LLICV concentration
can be used to demonstrate the lower limit of quantitation and sample results should not be
quantitated below this lowest standard. In some cases depending on project data quality
objectives, it may be appropriate to report these results, as estimated; however, they
should be qualified by noting the results are below the lower limit of quantitation. Therefore,
the quantitation limit cannot be reported lower than either the LLICV standard used for the
single point calibration option or the low calibration and/or verification standard used during
initial multi-point calibration. If the calibration curve cannot be verified within these specified
limits for the mid-range ICV and LLICV analyses, the cause needs to be determined and
the instrument recalibrated before samples are analyzed. The analysis data for the initial
calibration verification analyses should be kept on file with the sample analysis data.

Both the single and multi-point calibration curves should be verified at the end of each
analysis batch and after every 10 samples by use of a continuing calibration verification
(CCV) standard and a continuing calibration blank (CCB). The CCV should be made from
the same material as the initial calibration standards at or near the mid-range
concentration. For the curve to be considered valid the acceptance criteria for the CCV
standard should be ±10% of its true value and the CCB should contain target analytes less
than the established lower limit of quantitation for any desired target analyte. If the
calibration cannot be verified within the specified limits, the sample analysis must be
discontinued, the cause determined and the instrument recalibrated. All samples following
the last acceptable CCV/CCB must be reanalyzed. The analysis data for the CCV/CCB
should be kept on file with the sample analysis data.

The low level continuing calibration verification (LLCCV) standard should also be analyzed
at the end of each analysis batch. A more frequent LLCCV analysis, i.e., every 10 samples
may be necessary if low-level sample concentrations are anticipated and the system
stability at low end of the calibration is questionable. In addition, the analysis of a LLCCV
on a more frequent basis will minimize the number of samples for re-analysis should the
LLCCV fail if only run at the end of the analysis batch. The LLCCV standard should be
made from the same source as the initial calibration standards at the established lower limit
of quantitation as reported by the laboratory. The acceptance criteria for the LLCCV
standard should be ± 30% of its true value. If the calibration cannot be verified within these
specified limits, the analysis of samples containing the affected analytes at similar
concentrations cannot continue until the cause is determined and the LLCCV standard
successfully analyzed. The instrument may need to be recalibrated or the lower limit of
quantitation adjusted to a concentration that will ensure a compliant LLCCV analysis. The
analysis data for the LLCCV standard should be kept on file with the sample analysis data.
8. The linear dynamic range is established when the system is first setup, or whenever
significant instrument components have been replaced or repaired, and on an as needed
basis only after the system has been successfully calibrated using either the single or multi-
point standard calibration approach. The upper limit of the linear dynamic range needs to
be established for each wavelength utilized by determining the signal responses from a
minimum of three, preferably five, different concentration standards across the range. The

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 140

ranges which may be used for the analysis of samples should be judged by the analyst
from the resulting data. The data, calculations and rationale for the choice of range made
should be documented and kept on file. A standard at the upper limit should be prepared,
analyzed and quantitated against the normal calibration curve. The calculated value should
be within 10% (±10%) of the true value. New upper range limits should be determined
whenever there is a significant change in instrument response. At a minimum, the range
should be checked every six months. The analyst should be aware that if an analyte that is
present above its upper range limit is used to apply a spectral correction, the correction
may not be valid and those analytes where the spectral correction has been applied may
be inaccurately reported.

Note. Some metals may exhibit non-linear response curves due to ionization and self-
absorption effects. These curves may be used if the instrument allows it, however the
effective range must be checked and the second order curve fit should have a correlation
coefficient of 0.998 or better. Third order fits are not acceptable. These non-linear response
curves should be revalidated and/or recalculated on a daily basis using the same
calibration verification QC checks as a linear calibration curve. Since these curves are
much more sensitive to changes in operating conditions than the linear lines, they should
be checked whenever there have been moderate equipment changes. Under these
calibration conditions, quantisation is not acceptable above or below the calibration
standards. Additionally, a non-linear curve should be further verified by calculating the
actual recovery of each calibration standard used in the curve. The acceptance criteria for
the calibration standard recovery should be ±10% of its true value for all standards except
the lowest concentration. A recovery of ±30% of its true value should be achieved for the
lowest concentration standard.

9. The analyst should verify that the instrument configuration and operating conditions satisfy
the project-specific analytical requirements and maintain quality control data that
demonstrate and confirm the instrument performance for the reported analytical results.

4.2.2.8 Procedure

1. Preliminary acid digestion of matrices is required. All associated QC samples (i.e., method
blank, LCS and MS/MSD) must undergo the same procedures. Samples which are not
digested must either use an internal standard or be matrix-matched with the standards.

2. Profile and calibrate the instrument according to the instrument manufacturer's
recommended procedures, using the typical mixed calibration standard solutions described
in section 4.2.2.5.2. Flush the system with the calibration blank (Sec 4.2.2.5.3) between
each standard or as the manufacturer recommends. (Use the average intensity of multiple
exposures for both standardization and sample analysis to reduce random error.) The
calibration curve should be prepared as detailed in section 4.2.2.7.7.
3. Regardless of whether the initial calibration is performed using a single high standard and
the calibration blank or the multi-point option, the laboratory should analyze an LLCCV. For
all analytes and determinations, the laboratory must analyze an ICV and LLICV
immediately following daily calibration. It is recommended that a CCV, LLCCV, and CCB be
analyzed after every ten samples and at the end of the analysis batch (section 4.2.2.7.7).

4. Rinse the system with the calibration blank solution before the analysis of each sample.
The rinse time will be one minute. Each laboratory may establish a reduction in this rinse
time through a suitable demonstration. Analyze the samples and record the results.

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 141


 4.2.2.9 Data Analysis and Calculations

 1.   The quantitative values must be reported in appropriate units, such as micrograms per liter
     (ug/L) for aqueous samples and milligrams per kilogram (mg/kg) for solid samples. If
     dilutions were performed, the appropriate corrections must be applied to the sample values.
     All results should be reported with up to three significant figures.

 2.   Calculate results for solids on a dry-weight basis as follows:

       (1) A separate determination of percent solids must be performed.

       (2) The concentrations determined in the digest are to be reported on the basis of the
       dry weight of the sample.

              Concentration (dry weight)(mg/kg) =   (Cx V)
                                                (WxS)
              where:

              C = Digest Concentration (mg/L)
              V = Final volume in liters after sample preparation
              W = Weight in kg of wet sample
              S = % Sol ids 7100
     Calculations must include appropriate interference, internal-standard normalization, and the
     summation of signals at 206, 207, and 208 m/z for lead (to compensate for any differences
     in the abundances of these isotopes between samples and standards).
 3.   Results must be reported in units commensurate with their intended use and all dilutions
     must be taken into account when computing final results.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 142

4.3 MERCURY IN FISH TISSUE AND SEDIMENTS

4.3.1 Scope of Application

1. This method may be used for both saltwater and freshwater samples.
2. This procedure measures total mercury (organic v. inorganic) in fish tissue and sediments.
3. The range of the method is 0.2 - 5 ug/g. The range may be extended beyond the normal
range by increasing or decreasing sample size or through instrument and recorder control.

4.3.2 Summary of Method

1. A weighed portion of the sample is digested in aqua regia for 2 minutes at 95°C, followed
by oxidation with potassium permanganate. Mercury in the digested sample is then
measured by the conventional cold vapor technique.
2. An alternate digestion involving the use of an autoclave is described in section 4.3.8.3.

4.3.3 Sample Handling and Preservation

1. Because of the extreme sensitivity of the procedure and the omnipresence of mercury, care
must be taken to avoid extraneous contamination. Sampling devices and sample jars
should be ascertained to be free of mercury; the sample should not be exposed to any
condition in the laboratory that may result in contact or air-borne mercury contamination.
2. While the sample may be analyzed without drying, it has been found to be more convenient
to analyze a dry sample. Moisture may be driven off in a drying oven at a temperature of
60°C. No mercury losses have been observed by using this drying step. The dry sample
should be pulverized and thoroughly mixed before the aliquot is weighed.

4.3.4 Interferences

1. The same types of interferences that may occur in water samples are also possible with
fish tissue and sediments, i.e., sulfides, high copper, high chlorides, etc.
2. Volatile materials which absorb at 253.7 nm will cause a positive interference. In order to
remove any interfering volatile materials, the dead air space in the BOD bottle should be
purged before the addition of stannous sulfate.

4.3.5 Apparatus

1. Atomic Absorption Spectrophotometer. Any atomic absorption unit having an open sample
presentation area in which to mount the absorption cell is suitable. Instrument settings
recommended by the particular manufacturer should be followed.
2. Mercury Hollow Cathode Lamp. Westinghouse WL-22847, argon filled, or equivalent.
3. Any multi-range variable speed recorder that is compatible with the UV detection system.
4. Standard spectrophotometer cells 10 cm long, having quartz end windows may be used.
Suitable cells may be constructed from plexiglass tubing, 1" O.D. X 4-1/2". The ends are
ground perpendicular to the longitudinal axis and quartz windows (1" diameter X 1/16"
thickness) are cemented in place. Gas inlet and outlet ports (also of plexiglass but 1/4"
O.D.) are attached approximately 1/2" from each end. The cell is strapped to a burner for
support and aligned in the light beam to give the maximum transmittance.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 143

Note. Two 2"X2" cards with one inch diameter holes may be placed over each end of the cell
to assist in positioning the cell for maximum transmittance.
5. Air Pump. Any peristaltic pump capable of delivering 1 liter of air per minute may be used.
A Masterflex pump with electronic speed control has been found to be satisfactory.
Regulated compressed air can be used in an open one-pass system.
6. Flowmeter. Flowmeter must be capable of measuring an air flow of 1 liter per minute.
7. Aeration Tubing. Tygon tubing is used for passage of the mercury vapor from the sample
bottle to the absorption cell and return. Straight glass tubing terminating in a coarse porous
frit is used for sparging air into the sample.
8. Drying Tube. 6" X 3/4" diameter tube containing 20 g of magnesium perchlorate.
Note. In place of the magnesium perchlorate drying tube, a small reading lamp with 60 W bulb
may be used to prevent condensation of moisture inside the cell. The lamp is positioned to
shine on the absorption cell maintaining the air temperature in the cell about 10°C above
ambient.

4.3.6 Reagents

1. Aqua Regia. Prepare immediately before use by carefully adding three volumes of cone. HCI
to one volume of cone. HNO3.
2. Sulfuric Acid, 0.5 N. Dilute 14.0 ml of cone, sulfuric acid to 1 liter.
3. Stannous Sulfate. Add 25 g stannous sulfate to 250 ml of the 0.5 N sulfuric acid. This
mixture is a suspension and should be stirred continuously during use.
4. Sodium Chloride-Hydroxylamine Sulfate Solution. Dissolve 12 g of sodium chloride and 12 g
of hydroxylamine sulfate in distilled water and dilute to 100 ml.
Note. A 10% solution of stannous chloride may be substituted for the stannous sulfate and
hydroxylamine hydrochloride may be used in place of hydroxylamine sulfate in the sodium
chloride-hydroxylamine sulfate solution.
5. Potassium Permanganate. 5% solution, w/v. Dissolve 5 g of potassium permanganate in
100 ml of distilled water.
6. Stock Mercury Solution. Dissolve 0.1354 g of mercuric chloride in 75 ml of distilled water.
Add 10 ml of cone, nitric acid and adjust the volume to 100.0 ml. 1.0 ml = 1.0 mg Hg.
7. Working Mercury Solution. Make successive dilutions of the stock mercury solution to obtain
a working standard containing 0.1 ug/ml. This working standard and the dilution of the stock
mercury solutions should be prepared fresh daily. Acidity of the working standard should be
maintained at 0.15% nitric acid. This acid should be added to the flask as needed before the
addition of the aliquot.

4.3.7 Calibration

1. Transfer 0, 0..5, 1.0, 2.0, 5.0 and 10 ml aliquots of the working mercury solution containing 0
to 1.0 ug of mercury to a series of 300 ml BOD bottles. Add enough distilled water to each
bottle to make a total volume of 10 ml. Add 5 ml of aqua regia and heat for 2 minutes in a
water bath at 95°C.
2. Allow sample to cool; add 50 ml distilled water and 15 ml of KMnO4 solution to each bottle
and return to water bath for 30 mins. Cool and add 6 ml of sodium chloride-hydroxylamine
sulfate solution to reduce the excess permanganate. Add 50 ml distilled water.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 144

3. Treating each bottle individually, add 5 ml of stannous sulfate solution and immediately
attach the bottle to the aeration apparatus. At this point, the sample is allowed to stand
quietly without manual agitation. The circulating pump, which has previously been adjusted
to rate of 1 liter per minute, is allowed to run continuously. The absorbance, as exhibited
either on the spectrophotometer or the recorder, will increase and reach maximum within 30
seconds. As soon as the recorder pen levels off, approximately 1 minute, open the bypass
value and continue the aeration until the absorbance returns to its minimum value. Close the
bypass value, remove the fritted tubing from the BOD bottle and continue the aeration.
Proceed with the standards and construct a standard curve by plotting peak height versus
micrograms of mercury.
Note. Because of the toxic nature of mercury vapor precaution must be taken to avoid its
inhalation. Therefore, a bypass has been included in the system to either vent the mercury
vapor into an exhaust hood or pass the vapor through some absorbing media, such as:
a) equal volumes of 0.1 N KmnO4 and 10% H2SO4
b) 0.25% iodine in a 3% Kl solution.
Specially treated charcoal that will absorb mercury vapor is also commercially available.

4.3.8 Procedure

1. Weigh triplicate 0.2 g portions of dry sample and place in bottom of a BOD bottle. Add 5 ml
of distilled water and 5 ml of aqua regia. Heat for 2 mins in a water bath at 95°C. Cool, add
50 ml distilled water and 15 ml potassium permanganate solution to each sample bottle. Mix
and place in the water bath for 30 mins at 95°C. Cool and add 6 ml of sodium chloride-
hydroxylamine sulfate to reduce the excess permanganate. Add 55 ml of distilled water.
2. Treating each bottle individually, add 5 ml of stannous sulfate and immediately attach the
bottle to the aeration apparatus. Continue as described under section 4.2.7.3 above.
3. An alternate digestion procedure employing an autoclave may also be used. In this method
5 ml of cone. H2SO4 and 2 ml of cone. HNO3 are added to the 0.2 g of sample. Add 5 ml of
saturated KMnO4 solution and the bottle covered with a piece of aluminum foil. The samples
are autoclaved at 121°C and 15 Ibs. for 15 minutes. Cool, make up to a volume of 100 ml
with distilled water and add 6 ml of sodium chloride hydroxylamine sulfate solution to reduce
the excess permanganate. Purge the dead air space and continue as described under
section 4.3.7.3.

4.3.9 Calculation

1. Measure the peak height of the unknown from the chart and read the mercury value from
the standard curve.
2. Calculate the mercury concentration in the sample by the formula:

ug Hg/g = UQ Hg in the aliquot
wt. of the aliquot (g)

3. Report mercury concentrations as follows:
Below 0.1 ug/g, <0.1; between 0.1 and 1 ug/g, to the nearest 0.01 ug;
Between 1 and 10 ug/g, to nearest 0.1 ug;
Above 10 ug/g, to nearest ug.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 145

4.4 SAMPLE PREPARATION FOR ORGANIC COMPOUNDS IN FISH TISSUE AND
SEDIMENTS

4.4.1 Ultrasonic Extraction

The extraction procedure is divided into two sections, based on the expected concentration of
organics in the sample (Figure 4.3). The low concentration method (individual organic
components of less than or equal to 20 mg/kg) uses a larger sample size and a more rigorous
extraction procedure (lower concentrations are more difficult to extract). The medium/high
concentration method (individual organic components of greater than 20 mg/kg) is much simpler
and therefore faster.

4.4.2 Apparatus and Materials

1. Apparatus for grinding dry waste samples.

2. Ultrasonic preparation - A horn-type device equipped with a titanium tip, or a device that will
give equivalent performance, shall be used.

Ultrasonic Disrupter - The disrupter must have a minimum power wattage of 300 watts, with
pulsing capability. A device designed to reduce the cavitation sound is recommended.
Follow the manufacturer's instructions for preparing the disrupter for extraction of samples
with low and medium/high concentration.
Use a 3/4" horn for the low concentration method and a 1/8" tapered microtip attached to a
1/2" horn for the medium/high concentration method.

3. Sonabox - Recommended with above disrupters for decreasing cavitation sound
4. Drying oven - capable of maintaining 105°C.

5. Desiccator.

6. Vacuum or pressure filtration apparatus.

7. Kuderna-Danish (K-D) apparatus.
Concentrator tube - 10-ml, graduated Snyder column - Two-ball micro
Evaporation flask - 500-ml Springs -1/2 inch
Snyder column - Three-ball macro
8. The following glassware is recommended for the purpose of solvent recovery during the
concentration procedures requiring the use of Kuderna-Danish evaporative concentrators.
Incorporation of this apparatus may be required by State or local municipality regulations
that govern air emissions of volatile organics.

Solvent vapor recovery system
Boiling chips - Solvent-extracted, approximately 10/40 mesh
Water bath - Heated, with concentric ring cover, capable of temperature control (± 5°C). The
batch should be used in a hood.
Balance - Top-loading, capable of accurately weighing to the nearest 0.01 g.
Vials - 2-ml, for GC autosampler, with PTFE-lined screw caps or crimp tops.
Glass scintillation vials - 20-ml, with PTFE-lined screw caps.
Spatula - Stainless steel or PTFE

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 146

Syringe - 5-ml
Drying column - 20-mm ID Pyrex chromatographic column with Pyrex glass wool at the bottom.

Note. Fritted glass discs are difficult to decontaminate after highly contaminated extracts have
been passed through. Columns without frits may be purchased. Use a small pad of Pyrex glass
wool to retain the adsorbent. Prewash the glass wool pad with 50 ml of acetone followed by 50
ml of elution solvent prior to packing the column with adsorbent.

4.4.3 Reagents

1. Reagent-grade or pesticide-grade chemicals must be used in all tests. Unless otherwise
indicated, it is intended that all reagents conform to specifications of the Committee on
Analytical Reagents of the American Chemical Society. Other grades may be used,
provided it is first ascertained that the reagent is of sufficiently high purity to permit its use
without lessening the accuracy of the determination. Reagents should be stored in glass to
prevent the leaching of contaminants from plastic containers.

2. Organic-free reagent water.

3. Sodium sulfate (granular, anhydrous), Na2SO4. Purify by heating at 400°C for 4 hours in a
shallow tray, or by precleaning the sodium sulfate with methylene chloride. If the sodium
sulfate is precleaned with methylene chloride, a method blank must be analyzed,
demonstrating that there is no interference from the sodium sulfate.

4. Samples must be extracted using a solvent that gives optimum, reproducible recovery of the
analytes of interest from the sample matrix. Solvents must be pesticide grade in quality or
equivalent, and each lot of solvent should be determined to be free of phthalates.

Semivolatile organics may be extracted with acetone/methylene chloride (1:1,
v/v), CH3COCH3/CH2Cl2 or acetone/hexane (1:1, v/v), CH3COCH3/C6H14.
Organochlorine pesticides may be extracted with acetone/hexane (1:1, v/v),
CH3COCH3/C6H14, or acetone/methylene chloride (1:1,v/v), CH3COCH3/CH2Cl2
Exchange solvents - All solvents must be pesticide quality or equivalent.

Hexane, C6H14 Acetonitrile, CH3CN
Propanol, (CH3)2CHOH Methanol, CH3OH
Cyclohexane, C6H12
5. Solvents used in the extraction and cleanup procedures include n-hexane, diethyl ether,
methylene chloride, acetone, ethylacetate, and isooctane (2,2,4-trimethylpentane) and the
solvents must be exchanged to n-hexane or isooctane prior to analysis. Therefore, the use
of n-hexane and isooctane will be required in this procedure.

4.4.4 Procedure
1. Decant and discard any water layer on a sample. Mix sample thoroughly, especially
composited samples. Discard any foreign objects such as sticks, leaves, and rocks.

2. Determination of percent dry weight. When sample results are to be calculated on a dry
weight basis, a second portion of sample should be weighed out at the same time as the
portion used for analytical determination.

Caution. The drying oven should be contained in a hood or vented. Significant laboratory
contamination may result from drying a heavily contaminated sample.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 147

Immediately after weighing the sample for extraction, weigh 5-10 g of the sample into a
recorded crucible. Dry this aliquot overnight at 105°C. Allow to cool in a desiccator before
weighing. Calculate the % dry weight as follows:

% dry weight = g of dry sample x100
g of sample

4.4.4.1 Extraction Method for Samples Expected to Contain Low Concentrations of
Organics and Pesticides (less than or equal to 20 mg/kg)

The following steps should be performed rapidly to avoid loss of the more volatile extractables.

Weigh approximately 30 g of sample into a 400-ml beaker. Record weight to the nearest 0.1 g.

Note. Nonporous or wet samples (e.g., clay type) that do not have a free-flowing sandy texture
must be mixed with 60 g of anhydrous sodium sulfate, using a spatula. If required, more sodium
sulfate may be added. After addition of sodium sulfate, the sample should be free flowing.
Add 1.0 ml of the surrogate standard solution to all samples, spiked samples, QC samples, and
blanks. For the sample in each batch selected for spiking, add 1 .Oml of matrix spiking solution.

Note. If gel permeation cleanup will be used, the analyst should either add twice the volume of
the surrogate spiking solution (and matrix spiking solution, where applicable), or concentrate the
final extract to half the normal volume, to compensate for the half of the extract that is lost due
to loading of the GPC column.

Immediately add 100 ml of the appropriate/recommended extraction solvent or solvent mixture.
Place the bottom surface of the tip of the 3/4 inch disrupter horn about 1/2 inch below the
surface of the solvent, but above the tissue or sediment layer. Be sure the horn is properly
tuned according to the manufacturer's instructions.
Extract ultrasonically for 3 minutes, with output control knob set at 10 (full power) and with mode
switch on Pulse (pulsing energy rather than continuous energy) and percent-duty cycle knob set
at 50% (energy on 50% of time and off 50% of time). Do not use microtip probe.
Decant the extract and filter it through Whatman No. 41 filter paper in a Buchner funnel that is
attached to a clean 500-ml filtration flask. Alternatively, decant the extract into a centrifuge bottle
and centrifuge at low speed to remove particles.

Repeat the extraction two or more times with two additional 100 ml portions of solvent. Decant
off the solvent after each ultrasonic extraction. On the final ultrasonic extraction, pour the entire
sample into the Buchner funnel and rinse with extraction solvent. Apply a vacuum to the filtration
flask, and collect the solvent extract. Continue filtration until all visible solvent is removed from
the funnel, but do not attempt to completely dry the sample, as the continued application of a
vacuum may result in the loss of some analytes.

Alternatively, if centrifugation is used, transfer the entire sample to the centrifuge bottle.
Centrifuge at low speed, and then decant the solvent from the bottle.
Assemble a Kuderna-Danish (K-D) concentrator (if necessary) by attaching a 10-ml
concentrator tube to a 500-mL evaporator flask. Attach the solvent vapor recovery glassware
(condenser and collection device) to the Snyder column of the Kuderna-Danish apparatus
following manufacturer's instructions. Transfer filtered extract to a 500-ml evaporator flask.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 148

Add 1 - 2 clean boiling chips to the evaporation flask, and attach a three-ball Snyder column.
Prewet the Snyder column by adding about 1 ml methylene chloride to the top. Place the K-D
apparatus on a hot water bath (80 - 90°C) so that the concentrator tube is partially immersed in
the hot water and the entire lower rounded surface of the flask is bathed with hot vapor. Adjust
the vertical position of the apparatus and the water temperature, as required, to complete the
concentration in 10 -15 min. At the proper rate of distillation the balls of the column will actively
chatter, but the chambers will not flood with condensed solvent. When the apparent volume of
liquid reaches 1 ml, remove the K-D apparatus and allow it to drain and cool for at least 10 min.
If a solvent exchange is required, momentarily remove the Snyder column, add 50 ml of the
exchange solvent and a new boiling chip, and re-attach the Snyder column. Concentrate the
extract as described below, raising the temperature of the water bath, if necessary, to maintain
proper distillation. When the apparent volume again reaches 1-2 ml, remove the K-D apparatus
and allow it to drain and cool for at least 10 minutes.

Remove the Snyder column and rinse the flask and its lower joints into the concentrator tube
with 1-2 ml of methylene chloride or exchange solvent. The extract may be further concentrated
by using the technique outlined below or adjusted to 10.0 ml with the solvent last used. If further
concentration is indicated, either micro Snyder column technique or nitrogen blowdown
technique may be used to adjust the extract to the final volume required.
Micro Snyder column technique

Add a clean boiling chip and attach a two-ball micro Snyder column to the concentrator tube.
Prewet the column by adding approximately 0.5 ml of methylene chloride, or exchange solvent
through the top. Place the apparatus in the hot water bath. Adjust the vertical position and the
water temperature, as required, to complete the concentration in 5-10 minutes. At the proper
rate of distillation the balls of the column will actively chatter, but the chambers will not flood.
When the liquid reaches an apparent volume of approximately 0.5 ml, remove the apparatus
from the water bath, allow to drain and cool for at least 10 minutes. Remove the micro Snyder
column and rinse its lower joint with approximately 0.2 ml of appropriate solvent and add to the
concentrator tube. Adjust the final volume to the volume required for cleanup.

Nitrogen blowdown technique
Place the concentrator tube in a warm water bath (approximately 35°C) and evaporate the
solvent volume to the required level using a gentle stream of clean, dry nitrogen (filtered through
a column of activated carbon). The internal wall of the tube must be rinsed down several times
with the appropriate solvent during the operation. During evaporation, the solvent level in the
tube must be positioned to prevent water from condensing into the sample (i.e., the solvent level
should be below the level of the water bath). Under normal operating conditions, the extract
should not be allowed to become dry. When the volume of solvent is reduced below 1 ml,
semivolatile analytes may be lost.

Caution. Do not use plasticized tubing between the carbon trap and the sample, since it may
introduce contaminants.

4.4.4.2 Extraction Method for Samples Expected to Contain High Concentrations of
Organics (greater than 20 mg/kg).

Transfer approximately 2 g of sample to a 20-ml vial. Wipe the mouth of the vial with a tissue to
remove any sample material. Record the exact weight of sample taken. Cap the vial before
proceeding with the next sample to avoid any cross contamination.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 149

Add 2 g of anhydrous sodium sulfate to sample in the 20-ml vial and mix well.

Surrogates are added to all samples, spikes, and blanks. Add 1.0 ml of surrogate spiking
solution to sample mixture. For the sample in each analytical batch selected for spiking, add 1.0
ml of the matrix spiking standard. If gel permeation cleanup is to be used, the analyst should
either add twice the volume of the surrogate spiking solution (and matrix spiking solution, where
applicable), or concentrate the final extract to half the normal volume, to compensate for the half
of the extract that is lost due to loading of the GPC column.

Immediately add whatever volume of solvent is necessary to bring the final volume to 10.0 ml
considering the added volume of surrogates and matrix spikes. Disrupt the sample with the 1/8
in. tapered microtip ultrasonic probe for 2 minutes at output control setting 5 and with mode
switch on pulse and percent duty cycle at 50%. For nonpolar compounds (i.e., organochlorine
pesticides, PCBs), use hexane or appropriate solvent. For other semivolatile organics, use
methylene chloride.

Loosely pack a disposable Pasteur pipette with 2 to 3 cm of glass wool. Filter the sample extract
through the glass wool and collect the extract in a suitable container. The entire 10 ml of
extraction solvent cannot be recovered from the sample. Therefore, the analyst should collect a
volume appropriate for the sensitivity of the determinative method.

4.4.5 Extract Cleanup

1. The specific cleanup procedure used will depend on the nature of the sample to be analyzed
and the data quality objectives for the measurements. General guidance for sample extract
cleanup is provided in this section.

2. For biological (i.e., fish tissue), or samples containing high molecular weight materials, use
of GPC - pesticide option is recommended. Frequently an adsorption chromatographic
cleanup (alumina, silica gel, or Florisil®) may also be necessary following the GPC cleanup.

3. Alumina may be used to remove phthalate esters.

4. Florisil® may be used to separate organochlorine pesticides from aliphatic compounds,
aromatics, and nitrogen-containing compounds.
5. Silica gel may be used to separate single component organochlorine pesticides from some
interferants.

6. Sulfur, which may be present in certain samples, interferes with the electron capture gas
chromatography of certain pesticides and should be removed with either copper or
tetrabutylammonium sulfite. The mixture is shaken and the extract is removed from the
sulfur cleanup reagent.

4.4.6 Sample Handling

If analysis of the extract will not be performed immediately, stopper the concentrator tube and
refrigerate. If the extract will be stored longer than 2 days, it should be transferred to a vial with
a PTFE-lined cap and labeled appropriately.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 150
4.5    ORGANOCHLORINE PESTICIDES IN FISH TISSUE AND SEDIMENTS

4.5.1   Scope and Application

1.   This method may be used for both saltwater and freshwater fish tissue and sediments

2.   This method is used to determine concentrations of organochlorine pesticides in extracts
    from solid and liquid matrices, using fused-silica, open tubular, capillary columns with
    electron capture detectors or electrolytic conductivity detectors. The following 20 indicator
    compounds (Table 4.8) are determined using either single- or dual-column analysis system:

Table 4.8.  Indicator List of Organchlorine Pesticides
Compound
Aldrin
Y-BHC (Lindane)
a-Chlordane
2,4'-DDD
4,4'-DDD
2,4'-DDE
4,4'-DDE
2,4'-DDT
4,4'-DDT
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan sulfate
Endrin
Heptachlor
Heptachlorepoxide
Hexachlorobenzene
Mi rex
Toxaphene
frans-Nonachlor
Chemical Abstract Service
(CAS) Registry No.
309-00-2
58-89-9
5103-71-9
53-19-0
72-54-8
3424-82-6
72-55-9
789-02-6
50-29-3
60-57-1
959-98-8
33213-65-9
1031-07-8
72-20-8
76-44-8
1024-57-3
118-74-1
2385-85-5
8001-35-2
39765-80-5
3.  This method no longer includes Polychlorinated biphenyls (PCBs) as in the list of target
   analytes.  A separate method for the analysis of PCBs is included in Section 4.6.

4.5.2   Summary of Method

A measured volume or weight of liquid or solid sample is extracted using an ultrasonic
extraction technique. The ultrasonic process ensures intimate contact of the sample matrix with
the extraction solvent.  Cleanup steps are applied to the extract, depending on the nature of the
matrix interferences and the target analytes. Suggested cleanups include alumina, Florisil, silica
gel, gel permeation chromatography, and sulfur.  Finally the extract is analyzed by injecting a
measured aliquot into a gas chromatograph equipped with either a narrow-bore or wide-bore
fused-silica capillary column and either an electron capture detector (GC/ECD), or an
electrolytic conductivity detector (GC/ELCD).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 151

4.5.3 Interferences

1. Solvents, reagents, glassware, and other sample processing hardware may yield artifacts
and/or interferences to sample analysis. All materials must be demonstrated to be free from
interferences under conditions of analysis by analyzing method blanks. Specific selection of
reagents and purification of solvents by distillation in all-glass systems is necessary.

Cross-contamination of clean glassware routinely occurs when plastics are handled during
extraction steps, especially when solvent-wetted surfaces are handled. Glassware must be
scrupulously cleaned. Clean all glassware as soon as possible after use by rinsing with the
last solvent used. This should be followed by detergent washing with hot water, and rinses
with tap water and organic-free reagent water. Drain the glassware and dry it in an oven at
130 °C for several hours, or rinse with methanol and drain. Store dry glassware in a clean
environment. Other appropriate glassware cleaning procedures may be employed.

2. Interferences co-extracted from the samples will vary considerably from sample to sample.
While general cleanup techniques are referenced or provided as part of this method,
unique samples may require additional cleanup approaches to achieve desired degrees of
discrimination and quantitation.

3. Interferences by phthalate esters introduced during sample preparation can pose a major
problem in pesticide determinations. Interferences from phthalate esters can best be
minimized by avoiding contact with any plastic materials and checking all solvents and
reagents for phthalate contamination. This includes common flexible plastics contain
varying amounts of phthalate esters which are easily extracted or leached from such
materials during laboratory operations. Exhaustive cleanup of solvents, reagents and
glassware may be necessary to eliminate background phthalate ester contamination.

4. The presence of sulfur will result in broad peaks that interfere with the detection of early-
eluting organochlorine pesticides. Sulfur contamination should be expected with tissue or
sediment samples. Sulfur should be removed prior to anlaysis.

5. Waxes, lipids, and other high molecular weight materials can be removed by gel
permeation chromatography (GPC) cleanup.
6. Other halogenated pesticides or industrial chemicals may interfere with the analysis of
pesticides. Certain coeluting organophosphorus pesticides may be eliminated using GPC ~
pesticide option. Coeluting chlorophenols may be eliminated by using silica gel, Florisil, or
alumina. Polychlorinated biphenyls (PCBs) also may interfere with the analysis of the
organochlorine pesticides. The problem may be most severe for the analysis of
multicomponent analytes such as chlordane, toxaphene, and Strobane.
7. Coelution among the many target analytes in this method can cause interference problems.
The following target analytes may coelute on the GC columns listed, when using the single-
column analysis scheme:

DB 608 Trifluralin/diallate isomers
PCNB/dichlone/lsodrin

DB1701 Captafol/mirex
Methoxychlor/endosulfan sulfate

8. The following compounds may coelute using the dual-column analysis scheme. In general,
the DB-5 column resolves fewer compounds than the DB-1701.

DB-5 Permethrin/heptachlor epoxide

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 152

Endosulfan l/c/s-chlordane
Perthane/endrin
Endosulfan ll/chloropropylate/chlorobenzilate
4,4'-DDT/endosulfan sulfate
Methoxychlor/dicofol

DB-1701 Chlorothalonil/p-BHC
5-BHC/DCPA/permethrin
c/s-Chlordane/frans-nonachlor

Nitrofen, dichlone, carbophenothion, and dichloran exhibit extensive peak tailing on both
columns. Simazine and atrazine give poor responses on the ECD detector. Triazine
compounds should be analyzed using anitrogen-phosphorus detector (NPD) option.

4.5.4 Equipment and Supplies

1. Gas chromatograph (GC) - An analytical system complete with gas chromatograph suitable
for on-column and split-splitless injection and all necessary accessories including syringes,
analytical columns, gases, electron capture detectors (ECD), and recorder/integrator or data
system. Electrolytic conductivity detectors (ELCD) may also be employed if appropriate for
project needs. If the dual-column option is employed, the gas chromatograph must be
equipped with two detectors.

2. GC columns. The single-column approach involves one analysis to determine that a
compound is present, followed by a second analysis to confirm the identity of the compound
(section 4.5.11) The single-column approach may employ either narrow-bore (#0.32-mm ID)
columns or wide-bore (0.53-mm ID) columns. The dual-column approach generally employs
a single injection that is split between two columns that are mounted in a single gas
chromatograph. The dual-column approach generally employs wide-bore (0.53-mm ID)
columns, but columns of other diameters may be employed if the analyst can demonstrate
and document acceptable performance for the intended application. A third alternative is to
employ dual columns mounted in a single GC, but with each column connected to a
separate injector and a separate detector.

Note. The listing of these columns in this method is not intended to exclude the use of other
columns that are available or that may be developed. Laboratories may use these columns
or other columns provided that the laboratories document method performance data (e.g.,
chromatographic resolution, analyte breakdown, and sensitivity) that are appropriate for the
intended application.
Narrow-bore columns for single-column analysis (use both columns to confirm compound
identifications unless another confirmation technique such as GC/MS is employed). Narrow-
bore columns should be installed in split/splitless (Grob-type) injectors.
30-m x 0.25-mm or 0.32-mm ID fused-silica capillary column chemically bonded
with SE-54 (DB-5 or equivalent), 1-um film thickness.
30-m x 0.25-mm ID fused-silica capillary column chemically bonded with 35
percent phenyl methylpolysiloxane (DB-608, SPB-608, or equivalent), 2.5 urn
coating thickness, 1-um film thickness.
Wide-bore columns for single-column analysis (use two of the three columns listed to
confirm compound identifications unless another confirmation technique such as GC/MS is

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 153

employed). Wide-bore columns should be installed in 1/4-inch injectors, with deactivated
liners designed specifically for use with these columns.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 35 percent
phenyl methylpolysiloxane (DB-608, SPB-608, RTx-35, or equivalent), 0.5-um or 0.83-
um film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 50 percent
phenyl methylpolysiloxane (DB-1701, or equivalent), 1.0- urn film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 95 percent
dimethyl - 5 percent diphenyl polysiloxane (DB-5, SPB-5, RTx-5, or equivalent), 1.5-
um film thickness.

Wide-bore columns for dual-column analysis ~ The two pairs of recommended columns are
listed below.

Column pair 1:
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54 (DB-5,
SPB-5, RTx-5, or equivalent), 1.5-um film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 50 percent
phenyl methylpolysiloxane (DB-1701, or equivalent), 1.0-um film thickness.
Column pair 1 is mounted in a press-fit Y-shaped glass 3-way union or a Y-shaped
fused-silica connector, or equivalent.

Note. When connecting columns to a press-fit Y-shaped connector, a better seal may be
achieved by first soaking the ends of the capillary columns in alcohol for about 10 sec to soften
the polyimide coating.

Column pair 2:
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54 (DB-5,
SPB-5, RTx-5, or equivalent), 0.83-um film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 50 percent
phenyl methylpolysiloxane (DB-1701, or equivalent), 1.0-um film thickness.

Column pair 2 is mounted in an 8-inch deactivated glass injection tee.
3. Column rinsing kit - Bonded-phase column rinse kit

4. Volumetric flasks, 10-ml and 25-ml, for preparation of standards.

4.5.5 Reagents and Standards

2. Organic-free reagent water.
3. Solvents used for the preparation of GC standards include acetone, (CH3)2CO and toluene,
C6H5CH3. All solvent lots must be pesticide grade in quality or equivalent and should be
determined to be free of phthalates.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 154

Note. Store the standard solutions (stock, composite, calibration, internal, and surrogate) at
<6°C in polytetrafluoroethylene (PTFE)-sealed containers, in the dark. When a lot of
standards is prepared, aliquots of that lot should be stored in individual small vials. All stock
standard solutions must be replaced after one year, or sooner if routine QC (see section
5.7.7) indicates a problem. All other standard solutions must be replaced after six months,
or sooner, if routine QC indicates a problem.
4. Stock standard solutions

Stock standard solutions (1000 mg/L) may be prepared from pure standard materials or
purchased as certified solutions. Prepare stock standard solutions by accurately weighing
0.0100 g of pure compound. Dissolve the compound in isooctane or hexane and dilute to
volume in a 10-ml volumetric flask. If compound purity is 96 percent or greater, the weight
can be used without correction to calculate the concentration of the stock standard solution.

Note. /3-BHC, dieldrin, and some other standards may not be adequately soluble in isooctane. A
small amount of acetone or toluene should be used to dissolve these compounds during
the preparation of the stock standard solutions.
5. Composite stock standard

Prepared from individual stock solutions. For composite stock standards containing less
than 25 components, take exactly 1 ml of each individual stock solution at a concentration
of 1000 mg/L, add solvent, and mix the solutions in a 25-ml volumetric flask. This
composite solution can be further diluted to obtain the desired concentrations. For
composite stock standards containing more than 25 components, use volumetric flasks of
the appropriate volume (e.g., 50-ml, 100-ml), and follow the procedure described above.

6. Calibration standards
Calibration standards should be prepared at a minimum of five different concentrations by
dilution of the composite stock standard with isooctane or hexane. The concentrations
should correspond to the expected range of concentrations found in real samples and
should bracket the linear range of the detector.

Although all single component analytes can be resolved on a new 35 percent phenyl methyl
silicone column (e.g., DB-608), two calibration mixtures should be prepared for the single
component analytes of this method. This procedure is established to minimize potential
resolution and quantitation problems on confirmation columns or on older 35 percent
phenyl methyl silicone (e.g. DB-608) columns and to allow determination of endrin and DDT
breakdown for instrument quality control (section 4.6.7).

Separate calibration standards are necessary for each multi-component target analyte
(e.g., toxaphene and chlordane). The analysts should evaluate the specific toxaphene
standard carefully. Some toxaphene components, particularly the more heavily chlorinated
components, are subject to dechlorination reactions. As a result, standards from different
vendors may exhibit marked differences which could lead to possible false negative results
or to large differences in quantitative results.

7. Internal standard (optional)
Pentachloronitrobenzene is suggested as an internal standard for the single-column
analysis, when it is not considered to be a target analyte. 1-Bromo-2-nitrobenzene may
also be used. Prepare a solution of 5000 mg/L (5000 ng/ul) of pentachloronitrobenzene or
1-bromo-2-nitrobenzene. Spike 10 ul of this solution into each 1 ml of sample extract.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 155

Bromo-2-nitrobenzene is suggested as an internal standard for the dual-column analysis.
Prepare a solution of 5000 mg/L (5000 ng/ul) of 1-bromo-2-nitrobenzene. Spike 10 ul of this
solution into each 1 ml of sample extract.

8. Surrogate standards. The performance of the method should be monitored using surrogate
compounds. Surrogate standards are added to all samples, method blanks, matrix spikes,
and calibration standards. The following compounds are recommended as possible
surrogates. Other surrogates may be used, provided that the analyst can demonstrate and
document performance appropriate for the data quality needs of the particular application.
9. Decachlorobiphenyl and tetrachloro-m-xylene have been found to be a useful pair of
surrogates for both the single-column and dual-column configurations.

Chloro-3-nitrobenzotrifluoride may also be useful as a surrogate if the chromatographic
conditions of the dual-column configuration cannot be adjusted to preclude coelution of a
target analyte with either of the surrogates. However, this compound elutes early in the
chromatographic run and may be subject to other interference problems. A recommended
concentration for this surrogate is 500 ng/u. Use a spiking volume of 100 ul for a 1-L
aqueous sample. Other surrogate concentrations may be used, as appropriate for the
intended application.

Store surrogate spiking solutions at <6 °C in PTFE-sealed containers in the dark.

4.5.6 Gas Chromotography Specifications

1. This method allows the analyst to choose between a single-column and a dual-column
configuration in the injector port. The list of columns in this method is not intended to
exclude the use of other columns that are available or that may be developed. Wide-bore or
narrow-bore columns may be used with either option. Laboratories may use these or other
capillary columns or columns of other dimensions, provided that the laboratories document
method performance data (e.g., chromatographic resolution, analyte breakdown, and
sensitivity) that are appropriate for the intended application.

2. Single-column analysis
This capillary GC/ECD method allows the analyst the option of using 0.25 or 0.32-mm ID
capillary columns (narrow-bore) or 0.53-mm ID capillary columns (wide-bore). Narrow-bore
columns generally provide greater chromatographic resolution than wide-bore columns,
although narrow-bore columns have a lower sample capacity. As a result, narrow-bore
columns may be more suitable for relatively clean samples or for extracts that have been
prepared with one or more of the clean-up options referenced in the method. Wide-bore
columns (0.53-mm ID) may be more suitable for more complex environmental and waste
matrices. However, the choice of the appropriate column diameter is left to professional
judgement of the analyst.

Each laboratory must determine retention times and retention time windows for their
specific application of the method.

3. Dual-column analysis

The dual-column/dual-detector approach recommends the use of two 30-m x 0.53-mm ID
fused-silica open-tubular columns of different polarities, thus of different selectivities toward
the target analytes. The columns are connected to an injection tee and separate electron
capture detectors or to both separate injectors and separate detectors. However, the choice
of the appropriate column dimensions is left to the professional judgement of the analyst.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 156

Each laboratory must determine retention times and retention time windows for their specific
application of the method.

4.5.7 Quality Control and Assurance

1. Refer to the QAPP for guidance on quality assurance (QA) and quality control (QC)
protocols. When inconsistencies exist between QC guidelines, method-specific QC criteria
take precedence over both technique-specific criteria and technique-specific QC criteria take
precedence over the criteria in the QAPP. Each laboratory should maintain a formal quality
assurance program. Each lab must work with the Information Management group (Marlys
Cappaert, Cappaert.Marlys@epamail.epa.gov, 541-754-4467,) to ensure their bench sheets
and/or data recording spreadsheets are compatible with the electronic deliverables the lab
will need to submit. The laboratory should also maintain records to document the quality of
the data generated. All data sheets and quality control data should be maintained for
reference or inspection.
2. Include a calibration standard after each group of 20 samples, (however it is recommended
that a calibration standard be included after every 10 samples to minimize the number of
repeat injections) in the analysis sequence as a calibration check. Thus, injections of
method blank extracts, matrix spike samples, and other non-standards are counted in the
total. Solvent blanks, injected as a check on cross-contamination, need not be counted in
the total. The response factors for the calibration verification standard must be within ±20%
of the initial calibration (Section 4.5.9). When this calibration verification standard falls out of
this acceptance window, the laboratory should stop analyses and take corrective action.

Whenever quantitation is accomplished using an internal standard, internal standards must
be evaluated for acceptance. The measured area of the internal standard must be no more
than 50 percent different from the average area calculated during initial calibration. When
the internal standard peak area is outside the limit, all samples that fall outside the QC
criteria must be reanalyzed. The retention times of the internal standards must also be
evaluated. A retention time shift of >30 sec necessitates reanalysis of the affected sample.
DDT and endrin are easily degraded in the injection port. Breakdown occurs when the
injection port liner is contaminated with high boiling residue from sample injection or when
the injector contains metal fittings. Check for degradation problems by injecting a standard
containing only 4,4'-DDT and endrin. Presence of 4,4'-DDE, 4,4'-DDD, endrin ketone or
endrin indicates breakdown. If degradation of either DDT or endrin exceeds 15%, take
corrective action before proceeding with calibration. Unless otherwise specified in an
approved project plan, this test should be performed even when DDT and endrin are not
target analytes for a given project, as a test of the inertness of the analytical system.

Calculate percent breakdown as follows:

% breakdown of DDT = sum of degradation peak areas (ODD + DDE) x 100
sum of all peak areas (DDT + DDE + ODD)

% breakdown of endrin = sum of degradation peak areas (aldehyde + ketone)
x 100 sum of all peak areas (endrin + aldehyde + ketone)

The breakdown of DDT and endrin should be measured before samples are analyzed and
at the beginning of each 12-hr shift. Injector maintenance and recalibration should be
completed if the breakdown is greater than 15% for either compound.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 157

Whenever silica gel or Florisil® cleanups are used, the analyst must demonstrate that the
fractionation scheme is reproducible. Batch to batch variation in the composition of the
silica gel or Florisil® or overloading the column may cause a change in the distribution
patterns of the organochlorine pesticides. When compounds are found in two fractions, add
the concentrations found in the fractions, and correct for any additional dilution.
3. Initial demonstration of proficiency. Each laboratory must demonstrate initial proficiency
with each sample preparation and determinative method combination it utilizes, by
generating data of acceptable accuracy and precision for target analytes in a clean matrix.
If an autosampler is used to perform sample dilutions, before using the autosampler to
dilute samples, the laboratory should satisfy itself that those dilutions are of equivalent or
better accuracy than is achieved by an experienced analyst performing manual dilutions.

It is suggested that the QC reference sample concentrate contain each analyte of interest
at 10 mg/l in the concentrate. A 1-ml spike of this concentrate into 1 L of reagent water will
yield a sample concentration of10 ug/l. If this method is to be used for analysis of chlordane
or toxaphene only, the QC reference sample concentrate should contain the most
representative multicomponent mixture at a suggested concentration of 50 mg/L in
acetone. Other concentrations may be used, as appropriate for the intended application.

Calculate the average recovery and the standard deviation of the recoveries of the analytes
in each of the four QC reference samples.

4. Initially, before processing any samples, the analyst should demonstrate that all parts of the
equipment in contact with the sample and reagents are interference-free. This is
accomplished through the analysis of a method blank. As a continuing check, each time
samples are extracted, cleaned up, and analyzed, and when there is a change in reagents,
a method blank should be prepared and analyzed for the compounds of interest as a
safeguard against chronic laboratory contamination. If a peak is observed within the
retention time window of any analyte that would prevent the determination of that analyte,
determine the source and eliminate it, if possible, before processing the samples. The
blanks should be carried through all stages of sample preparation and analysis. When new
reagents or chemicals are received, the laboratory should monitor the preparation and/or
analysis blanks associated with samples for any signs of contamination. It is not necessary
to test every new batch of reagents or chemicals prior to sample preparation if the source
shows no prior problems. However, if reagents are changed during a preparation batch,
separate blanks need to be prepared for each set of reagents.

5. Sample quality control for preparation and analysis. The laboratory must also have
procedures for documenting the effect of the matrix on method performance (precision,
accuracy, method sensitivity). At a minimum, this should include the analysis of QC
samples including a method blank, a matrix spike, a duplicate, and a laboratory control
sample (LCS) in each analytical batch and the addition of surrogates to each field sample
and QC sample when surrogates are used. Any method blanks, matrix spike samples, and
replicate samples should be subjected to the same analytical procedures (section 5.7.9) as
those used on actual samples.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 158

duplicate analysis of an unspiked field sample. If samples are not expected to contain target
analytes, the laboratories should use a matrix spike and matrix spike duplicate pair.
A laboratory control sample (LCS) should be included with each analytical batch. The LCS
consists of an aliquot of a clean (control) matrix similar to the sample matrix and of the same
weight or volume. The LCS is spiked with the same analytes at the same concentrations as
the matrix spike, when appropriate. When the results of the matrix spike analysis indicate a
potential problem due to the sample matrix itself, the LCS results are used to verify that the
laboratory can perform the analysis in a clean matrix.
6. 6 Surrogate recoveries. If surrogates are used, the laboratory should evaluate surrogate
recovery data from individual samples versus the surrogate control limits developed by the
laboratory. Procedures for evaluating the recoveries of multiple surrogates and the
associated corrective actions should be defined in an approved project plan.

7. It is recommended that the laboratory adopt additional quality assurance practices for use
with this method. The specific practices that are most productive depend upon the needs of
the laboratory and the nature of the samples. Whenever possible, the laboratory should
analyze standard reference materials and participate in relevant performance evaluation
studies.

4.5.8 Calibration and Standardization

1. Prepare calibration standards using the procedures in Section 4.5.5.6. Refer to Section
4.5.7.2 of this method for proper calibration techniques for both initial calibration and
calibration verification. The procedure for either internal or external calibration may be used.
In most cases, external standard calibration is used with this method because of the
sensitivity of the electron capture detector and the probability of the internal standard being
affected by interferences. Because several of the pesticides may coelute on any single
column, the analysts should use two calibration mixtures. The specific mixture should be
selected to minimize the problem of peak overlap.

Note. Because of the sensitivity of the electron capture detector, always clean the injection port
and column prior to performing the initial calibration.

The analysis of the multi-component analytes should employ a single-point calibration. A
single calibration standard near the mid-point of the expected calibration range of each
multi-component analyte is included with the initial calibration of the single component
analytes for pattern recognition, so that the analyst is familiar with the patterns and retention
times on each column. The calibration standard may be at a lower concentration than the
mid-point of the expected range, if appropriate for the project.

For calibration verification (each 12-hr shift), all target analytes specified in the project plan
must be injected.

2. Establish the GC operating conditions appropriate for the configuration (single-column or
dual column). Optimize the instrumental conditions for resolution of the target analytes and
sensitivity. An initial oven temperature of < 140 -150 °C may be necessary to resolve the
four BHC isomers. A final temperature of between 240 °C and 270 °C may be necessary to
elute decachlorobiphenyl. The use of injector pressure programming will improve the
chromatography of late eluting peaks.

Note. Once established, the same operating conditions must be used for both calibrations and
sample analyses.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
_ Page 159

3. A 2-|jl injection volume of each calibration standard is recommended. Other injection
volumes may be employed, provided that the analyst can demonstrate adequate sensitivity
for the compounds of interest.

4. Because of the low concentration of pesticide standards injected on a GC/ECD, column
adsorption may be a problem when the GC has not been used for a day or more. Therefore,
the GC column should be primed (or deactivated) by injecting a pesticide standard mixture
approximately 20 times more concentrated than the midconcentration standard. Inject this
standard mixture prior to beginning the initial calibration or calibration verification.

Note. Several analytes, including aldrin, may be observed in the injection just following this
system priming because of carry-over. Always run an acceptable blank prior to running any
standards or samples.

5. Calibration factors

When external standard calibration is employed, calculate the calibration factor for each analyte
at each concentration, the mean calibration factor, and the relative standard deviation (RSD) of
the calibration factors, using the formulae below. If internal standard calibration is employed,
refer to Method 8000 for the calculation of response factors.

Calculate the calibration factor for each analyte at each concentration as:
CF = Peak Area (or Height) of the compound in the Standard
Mass of the Compound Injected (ng)

Calculate the mean calibration factor for each analyte as:
_
mean CF = CF = ^ -
n
where n is the number of standards analyzed.

Calculate the standard deviation (SD) and the RSD of the calibration factors for each
analyte as:
3D =
n-1
RSD = — x 100
cT
If the RSD for each analyte is < 20%, then the response of the instrument is considered
linear and the mean calibration factor may be used to quantitate sample results. If the RSD
is greater than 20%, the analyst should consider other calibration options, which may
include either a linear calibration not through the origin or a non-linear calibration model
(e.g., a polynomial equation).
6. Retention time windows. Absolute retention times are generally used for compound
identification. When absolute retention times are used, retention time windows are crucial
to the identification of target compounds. Retention time windows are established to
compensate for minor shifts in absolute retention times as a result of sample loadings and
normal chromatographic variability. The width of the retention time window should be
carefully established to minimize the occurrence of both false positive and false negative
results. Tight retention time windows may result in false negatives and/or may cause

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 160

unnecessary reanalysis of samples when surrogates or spiked compounds are erroneously
not identified. Overly wide retention time windows may result in false positive results that
cannot be confirmed upon further analysis. Other approaches to compound identification
may be employed, provided that the analyst can demonstrate and document that the
approaches are appropriate for the intended application.
Before establishing the retention time windows, make sure that the gas chromatographic
system is operating within optimum conditions. The widths of the retention time windows
should be determined by the experienced analyst.

4.5.9 Analytical Procedure and Analysis

1. The same GC operating conditions used for the initial calibration must be employed for the
analysis of samples.

2. Verify calibration at least once each 12-hr shift by injecting calibration verification standards
prior to conducting any sample analyses. Analysts should alternate the use of high and low
concentration mixtures of single-component analytes and multicomponent analytes for
calibration verification. A calibration standard must also be injected at intervals of not less
than once every twenty samples (after every 10 samples is recommended to minimize the
number of samples requiring re-injection when QC limits are exceeded) and at the end of
the analysis sequence. See section 4.5.7.2 for additional guidance on the frequency of the
standard injections.

The calibration factor for each analyte should not exceed a ±20 percent difference from the
mean calibration factor calculated for the initial calibration.

If the calibration does not meet the ±20% limit on the basis of each compound, check the
instrument operating conditions, and if necessary, restore them to the original settings, and
inject another aliquot of the calibration verification standard. If the response for the analyte
is still not within ±20%, then a new initial calibration must be prepared. The effects of a
failing calibration verification standard on sample results are discussed in section 4.5.9.

3. Compare the retention time of each analyte in the calibration standard with the absolute
retention time windows. Each analyte in each subsequent standard run during the 12-hr
period must fall within its respective retention time window. If not, the gas chromatographic
system must either be adjusted so that a second analysis of the standard does result in all
analytes falling within their retention time windows, or a new initial calibration must be
performed and new retention time windows established. As noted, other approaches to
compound identification may be employed, provided that the analyst can demonstrate and
document that the approaches are appropriate for the intended application.

4. Inject a measured aliquot of the concentrated sample extract. A 2-ul aliquot is suggested,
however, the same injection volume should be used for both the calibration standards and
the sample extracts, unless the analyst can demonstrate acceptable performance using
different volumes or conditions. Record the volume injected and the resulting peak size in
area units.

5. Confirmation. Tentative identification of an analyte (either single-component or multi-
component) occurs when a peak from a sample extract falls within the daily retention time
window. Confirmation is necessary when the sample composition is not well characterized.
Confirmatory techniques such as gas chromatography with a dissimilar column or a mass
spectrometer should be used. See section 4.5.11 of this method for information on the use
of GC/MS as a confirmation technique.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 161

When results are confirmed using a second GC column of dissimilar stationary phase, the
analyst should check the agreement between the quantitative result on both columns once
the identification has been confirmed.

6. When using the external calibration procedure, determine the quantity of each component
peak in the sample chromatogram which corresponds to the compounds used for
calibration purposes, as follows. The appropriate selection of a baseline from which the
peak area or height can be determined is necessary for proper quantitation.

For aqueous samples:

Concentration (ug/l) = (AxKVtKD)
(CF)(Vi)(Vs)
where:

Ax = Area (or height) of the peak for the analyte in the sample.
Vt = Total volume of the concentrated extract (ul).
D = Dilution factor, if the sample or extract was diluted prior to analysis. If no dilution
was made, D = 1. The dilution factor is always dimensionless.
CF = Mean calibration factor from the initial calibration (area/ng).
Vi = Volume of the extract injected (ul). The injection volume for samples and
calibration standards should be the same, unless the analyst can demonstrate
acceptable performance using different volumes or conditions.
Vs = Volume of the aqueous sample extracted in ml. If units of liters are used for this
term, multiply the results by 1000.
Using the units given here for these terms will result in a concentration in units of
ng/ml, which is equivalent to ug/l.

For non-aqueous samples:

Concentration (ug/kg) = (Ax)(Vt)(D)
(CF)(Vi)(Ws)
where:

Ax, Vt, D, CF, and Vi are the same as for aqueous samples
Ws = Weight of sample extracted (g). The wet weight or dry weight may be used,
depending upon the specific application of the data. If units of kilograms are used
for this term, multiply the results by 1000.
Using the units given here for these terms will result in a concentration in units of ng/g,
which is equivalent to ug/kg.

If the responses exceed the calibration range of the system, dilute the extract and
reanalyze. Peak height measurements are recommended over peak area integration when
overlapping peaks cause errors in area integration.

If partially overlapping or coeluting peaks are found, change GC columns or try GC/MS
quantitation.

7. Each sample analysis employing external standard calibration must be bracketed with an
acceptable initial calibration, calibration verification standards (each 12- hr analytical shift),
or calibration standards interspersed within the samples. The results from these bracketing
standards must meet the calibration verification criteria.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 162

Although analysis of a single mid-concentration standard (standard mixture or multi-
component analyte) will satisfy the minimum requirements, analysts are urged to use
different calibration verification standards during organochlorine pesticide analyses. Also,
multi-level standards (mixtures or multi-component analytes) are highly recommended to
ensure that the detector response remains stable for all analytes over the calibration range.
When a calibration verification standard fails to meet the QC criteria, all samples that were
injected after the last standard that last met the QC criteria must be evaluated to prevent
misquantitations and possible false negative results, and reinjection of the sample extracts
may be necessary. More frequent analyses of standards will minimize the number of sample
extracts that would have to be reinjected if the QC limits are violated for standard analysis.

However, if the standard analyzed after a group of samples exhibits a response for an
analyte that is above the acceptance limit, i.e., >20%, and the analyte was not detected in
the specific samples analyzed during the analytical shift, then the extracts for those samples
do not need to be reanalyzed, as the verification standard has demonstrated that the analyte
would have been detected were it present. In contrast, if an analyte above the QC limits was
detected in a sample extract, then reinjection is necessary to ensure accurate quantitation. If
an analyte was not detected in the sample and the standard response is more than 20%
below the initial calibration response, then reinjection is necessary to ensure that the
detector response has not deteriorated to the point that the analyte would not have been
detected even though it was present (i.e., a false negative result).

8. Sample injections may continue for as long as the calibration verification standards and
standards interspersed with the samples meet instrument QC requirements. It is
recommended that standards be analyzed after every 10 samples (required after every 20
samples and at the end of a set) to minimize the number of samples that must be re-injected
when the standards fail the QC limits. The sequence ends when the set of samples has
been injected or when qualitative and/or quantitative QC criteria are exceeded.

9. The use of internal standard calibration techniques does not require that all sample results
be bracketed with calibration verification standards. However, when internal standard
calibration is used, the retention times of the internal standards and the area responses of
the internal standards should be checked for each analysis. Retention time shifts of >30 sec
from the retention time of the most recent calibration standard and/or changes in internal
standard areas of more than -50 to +100% are cause for concern and must be investigated.
10. If the peak response is less than 2.5 times the baseline noise level, the validity of the
quantitative result may be questionable. Consult with the source of the sample to determine
whether further concentration of the sample is warranted.
11. Use the calibration standards analyzed during the sequence to evaluate retention time
stability. Each subsequent injection of a standard during the 12-hr analytical shift (i.e., those
standards injected every 20 samples, or more frequently) must be checked against retention
time windows. If any of these subsequent standards fall outside their absolute retention time
windows, the GC system is out of control. Determine the cause of the problem and correct it.
If the problem cannot be corrected, a new initial calibration must be performed.

12. The identification of mixtures (i.e., chlordane and toxaphene) is not based on a single peak,
but rather on the characteristic peaks that comprise the "fingerprint" of the mixture, using
both the retention times and shapes of the indicator peaks. Quantitation is based on the
areas of the characteristic peaks as compared to the areas of the corresponding peaks at
the same retention times in the calibration standard, using either internal or external

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 163

calibration procedures. See section 4.5.11 for information on the use of GC/MS as a
confirmation technique.
13. If compound identification or quantitation is precluded due to interference (e.g., broad,
rounded peaks or ill-defined baselines), cleanup of the extract or replacement of the
capillary column or detector is warranted. Rerun the sample on another instrument to
determine if the problem results from analytical hardware or the sample matrix.

4.5.10 Quantitation of Multi-Component Analytes

Multi-component analytes present problems in measurement. Suggestions are offered in the
following sections for handling toxaphene, Strobane, chlordane, BHC, and DDT.

1. Toxaphene is manufactured by the chlorination of camphenes, and Strobane results from
the chlorination of a mixture of camphenes and pinenes. Quantitation of toxaphene or
Strobane is difficult, but reasonable accuracy can be obtained. To calculate toxaphene from
GC/ECD results:

Adjust the sample size so that the major toxaphene peaks are 10 - 70% of full-scale
deflection (FSD). Inject a toxaphene standard that is estimated to be within ±10 ng of the
sample amount. Quantitate toxaphene using the total area of the toxaphene pattern or
using 4 to 6 major peaks.
While toxaphene contains a large number of compounds that will produce well resolved
peaks in a GC/ECD chromatogram, it also contains many other components that are not
chromatographically resolved. This unresolved complex mixture results in the "hump" in the
chromatogram that is characteristic of this mixture. Although the resolved peaks are
important for the identification of the mixture, the area of the unresolved complex mixture
contributes a significant portion of the area of the total response.

To measure total area, construct the baseline of toxaphene in the sample chromatogram
between the retention times of the first and last eluting toxaphene components in the
standard. In order to use the total area approach, the pattern in the sample chromatogram
must be compared to that of the standard to ensure that all of the major components in the
standard are present in the sample. Otherwise, the sample concentration may be
significantly underestimated.

Toxaphene may also be quantitated on the basis of 4 to 6 major peaks. A collaborative
study of a series of toxaphene residues evaluated several approaches to quantitation of
this compound, including the use of the total area of the peaks in the toxaphene
chromatogram and the use of a subset of 4 to 6 peaks. That study indicated that the use of
4 to 6 peaks provides results that agree well with the total peak area approach and may
avoid difficulties when interferences with toxaphene peaks are present in the early portion
of the chromatogram from compounds such as DDT. Whichever approach is employed
should be documented and available to the data user, if necessary.

When toxaphene is determined using the 4 to 6 peaks approach, the analyst must take
care to evaluate the relative areas of the peaks chosen in the sample and standard
chromatograms. It is highly unlikely that the peaks will match exactly, but the analyst should
not employ peaks from the sample chromatogram whose relative sizes or areas appear to
be disproportionally larger or smaller in the sample compared to the standard.

The heights or areas of the 4 to 6 peaks that are selected should be summed together and
used to determine the toxaphene concentration. Alternatively, use each peak in the

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 164

standard to calculate a calibration factor for that peak, using the total mass of toxaphene in
the standard. These calibration factors are then used to calculate the concentration of each
corresponding peak in the sample chromatogram and the 4 to 6 resulting concentrations
are averaged to provide the final result for the sample.

2. Technical chlordane is a mixture of at least 11 major components and 30 or more minor
components that have been used to prepare specific pesticide formulations. The exact
percentages of c/s-chlordane and frans-chlordane in technical material are not completely
defined, and are not consistent from batch to batch. Moreover, changes may occur when
the technical material is used to prepare specific pesticide formulations. The approach used
for evaluating and reporting chlordane results will depend on the end use of the results and
the analyst's skill in interpreting this multicomponent pesticide residue. The following
sections discuss three options: reporting technical chlordane (CAS number 12789-03-6),
reporting chlordane (not otherwise specified, or n.o.s., CAS number 57-74-9), and reporting
the individual chlordane components that can be identified under individual CAS numbers.

When the GC pattern of the residue resembles that of technical chlordane, the analyst may
quantitate chlordane residues by comparing the total area of the chlordane chromatogram
using three to five major peaks or the total area. If the heptachlor epoxide peak is relatively
small, include it as part of the total chlordane area for calculation of the residue. If
heptachlor and/or heptachlor epoxide are much out of proportion, calculate these
separately and subtract their areas from the total area to give a corrected chlordane area.

Note. Octachloro epoxide, a metabolite of chlordane, can easily be mistaken for heptachlor
epoxide on a nonpolar GC column.
To measure the total area of the chlordane chromatogram, inject an amount of a technical
chlordane standard which will produce a chromatogram in which the major peaks are
approximately the same size as those in the sample chromatograms. Construct the
baseline of technical chlordane in the standard chromatogram between the retention times
of the first and last eluting chlordane components. Use this area and the mass of technical
chlordane in the standard to calculate a calibration factor. Construct a similar baseline in
the sample chromatogram, measure the area, and use the calibration factor to calculate the
concentration in the sample.

The GC pattern of a chlordane residue in a sample may differ considerably from that of the
technical chlordane standard. In such instances, it may not be practical to relate a sample
chromatogram back to the pesticide active ingredient technical chlordane. Therefore,
depending on the objectives of the analysis, the analyst may choose to report the sum of all
the identifiable chlordane components as "chlordane (n.o.s.)" under CAS number 57-74-9.
The third option is to quantitate the peaks of c/s-chlordane, frans-chlordane, and heptachlor
separately against the appropriate reference materials, and report these individual
components under their respective CAS numbers.

To measure the total area of the chlordane chromatogram, inject an amount of a technical
chlordane standard which will produce a chromatogram in which the major peaks are
approximately the same size as those in the sample chromatograms.

3. Hexachlorocyclohexane is known as BHC, from the former name, benzene hexachloride.
Technical grade BHC is a cream colored amorphous solid with a very characteristic musty
odor. It consists of a mixture of six chemically distinct isomers and one or more
heptachlorocyclohexanes and octachlorocyclohexanes. Commercial BHC preparations may

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 165

show a wide variance in the percentage of individual isomers present. Quantitate each
isomer (a, p, y, and 5) separately against a standard of the respective pure isomer.
4. Technical DDT consists primarily of a mixture of 4,4'-DDT (approximately 75%) and 2,4'-
DDT. As DDT weathers, 4,4'-DDE, 2,4'-DDE, 4,4'-DDD, and 2,4'-DDD are formed. Since
the 4,4'-isomers of DDT, DDE, and ODD predominate in the environment, and these are
the isomers normally regulated by EPA, sample extracts should be quantitated against
standards of the respective pure isomers of 4,4'-DDT, 4,4'-DDE, and 4,4'-DDD.

4.5.11 GC/MS Confirmation

GC/MS confirmation may be used in conjunction with either single-column or dual-column
analysis if the concentration is sufficient for detection by GC/MS.
1. Full-scan GC/MS will normally require a concentration of approximately 10 ng/uL in the final
extract for each single-component compound. Ion trap or selected ion monitoring will
normally require a concentration of approximately 1 ng/uL.
2. The GC/MS must be calibrated for the specific target pesticides when it is used for
quantitative analysis. If GC/MS is used only for confirmation of the identification of the
target analytes, then the analyst must demonstrate that those pesticides identified by
GC/ECD can be confirmed by GC/MS. This demonstration may be accomplished by
analyzing a single-point standard containing the analytes of interest at or below the
concentrations reported in the GC/ECD analysis.

3. GC/MS is not recommended for confirmation when concentrations are below 1 ng/uL in the
extract, unless a more sensitive mass spectrometer is employed.

4. GC/MS confirmation should be accomplished by analyzing the same extract that is used for
GC/ECD analysis and the extract of the associated method blank.

5. If a base/neutral/acid extraction of an aqueous sample was performed for an analysis of
semivolatile organics, then that extract and the associated blank may be used for GC/MS
confirmation if the surrogates and internal standards do not interfere and if it is
demonstrated that the analyte is stable during acid/base partitioning. However, if the
compounds are not detected in the base/neutral/acid extract, then GC/MS analysis of the
pesticide extract should be performed.

6. When system performance does not meet the established QC requirements,
Chromatographic system maintenance as corrective action is required, and may include the
application of splitter connections as described below.

For dual-columns which are connected using a press-fit Y-shaped glass splitter or a Y-shaped
fused-silica connector, clean and deactivate the splitter port insert or replace with a cleaned and
deactivated splitter. Break off the first few centimeters (up to 30 cm) of the injection port side of
the column. Remove the columns and solvent backflush according to the manufacturer's
instructions. If these procedures fail to eliminate the degradation problem, it may be necessary
to deactivate the metal injector body and/or replace the columns.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 166
4.6    POLYCHLORINATED BIPHENOLS (PCBs) IN FISH TISSUE AND SEDIMENTS

4.6.1   Scope and Application

   1.   This method may be used for both saltwater and freshwater fish tissue and sediments

   2.   This method may be used to determine the concentrations of polychlorinated biphenyls
       (PCBs) or as individual  PCB congeners in extracts using open-tubular, capillary columns
       with electron capture detectors (ECD) or electrolytic conductivity detectors (ELCD). The
       21 PCB congeners listed below (Table 4.9) have been determined by this method, using
       either a single- or dual column analysis system.

Table 4.9. Indicator List of Polychlorinated Biphenyls (PCBs)
Compound
2,4'-Dichlorobiphenyl
2,2',5-Trichlorobiphenyl
2,4,4'-Trichlorobiphenyl
2,2',3,5'-Tetrachlorobiphenyl
2,2',5,5'-Tetrachlorobiphenyl
2,3',4,4'-Tetrachlorobiphenyl
3,3',4,4'-Tetrachlorobiphenyl
2,2',4,5,5'-Pentachlorobiphenyl
2,3,3',4,4'-Pentachlorobiphenyl
2,3,3',4',6-Pentachlorobiphenyl
2, 3,4, 4', 5- Pentachlorobipheny
3, 3,4, 4', 5- Pentachlorobiphenyl
2,2',3,3',4,4'-Hexachlorobiphenyl
2,2',3,4,4',5'-Hexachlorobiphenyl
2,2',4,4',5,5'-Hexachlorobiphenyl
2,2',3,3',4,4',5-Heptachlorobiphenyl
2,2',3,4,4',5,5'-Heptachlorobiphenyl
2,2',3,4',5,5',6-Heptachlorobiphenyl
2,2',3,3',4,4',5,6-Octachlorobiphenyl
2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl
2,2',3,3',4,4',5,5',6,6'-Decachlorobiphenyl
IUPAC
(PCB) No.
8
18
28
44
52
66
77
101
105
110
118
126
128
138
153
170
180
187
195
206
209
Chemical Abstract
Service (CAS) Registry No.
34883-43-7
37680-65-2
7012-37-5
41464-39-5
35693-99-3
32598-10-0
32598-13-3
37680-73-2
32598-14-4
38380-03-9
31508-00-6
57465-28-8
38380-07-3
35065-28-2
35065-27-1
35065-30-6
35065-29-3
52663-68-0
52663-78-2
40486-72-9
2051-24-3
4.6.2   Summary of Method

Fish tissue or sediment samples are extracted with hexane-acetone (1:1) or methylene chloride-
acetone (1:1) using the ultrasonic extraction technique described in Sec. 4.4. Extracts for PCB
analysis are subjected to a sequential sulfuric acid/potassium permanganate cleanup designed
specifically for these analytes. This cleanup technique will remove many single component
organochlorine or organophosphorus pesticides. Therefore, this method is not applicable to the
analysis of those compounds. The extract is analyzed by injecting a measured aliquot into a
gas chromatograph equipped with a narrow- or wide-bore fused-silica capillary column and an
electron capture detector (GC/ECD) or an electrolytic conductivity detector (GC/ELCD).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 167

4.6.3 Interferences

1. Solvents, reagents, glassware, and other sample processing hardware may yield artifacts
and/or interferences to sample analysis. All of these materials must be demonstrated to be
free from interferences under the conditions of the analysis by analyzing method blanks.
Specific selection of reagents and purification of solvents by distillation in all-glass systems
may be necessary.

2. Interferences co-extracted from the samples will vary considerably from matrix to matrix.
While general cleanup techniques are referenced or provided as part of this method,
unique samples may require additional cleanup approaches to achieve desired degrees of
discrimination and quantitation. Sources of interference in this method can be grouped into
four broad categories, as follows:
Contaminated solvents, reagents, or sample processing hardware.
Contaminated GC carrier gas, parts, column surfaces, or detector surfaces.
Compounds extracted from the sample matrix to which the detector will respond, such as
single-component chlorinated pesticides, including DDT analogs (DDT, DDE, and ODD).

Note. A standard of the DDT analogs should be injected to determine which of the PCB peaks
may be subject to interferences on the analytical columns used.
Coelution of related analytes ~ All PCB congeners cannot be separated using the GC
columns and procedures described in this method. If this procedure is expanded to
encompass other congeners, then the analyst must either document the resolution of the
congeners in question or establish procedures for reporting the results of coeluting
congeners that are appropriate for the intended application.

3. Interferences by phthalate esters introduced during sample preparation can pose a major
problem in PCB determinations. Interferences from phthalate esters are minimized by
avoiding contact with any plastic materials and checking all solvents and reagents for
phthalate contamination.

4. Cross-contamination of clean glassware can routinely occur when plastics are handled
during extraction steps, especially when solvent-wetted surfaces are handled. Glassware
must be scrupulously cleaned. Clean all glassware as soon as possible after use by rinsing
with the last solvent used. This should be followed by detergent washing with hot water,
and rinses with tap water and organic-free reagent water. Drain the glassware, and dry it in
an oven at 130 °C for several hours, or rinse with methanol and drain.

Note. Oven-drying of glassware used for PCB analysis can increase contamination because
PCBs are readily volatilized in the oven and spread to other glassware. Therefore, exercise
caution, and do not dry glassware from samples containing high concentrations of PCBs
with glassware that may be used for trace analyses.

5. Sulfur (S8) is readily extracted from soil samples and may cause chromatographic
interferences in the determination of PCBs. Sulfur contamination should be expected with
samples and should be removed with either copper or tetrabutylammonium sulfite. The
mixture is shaken and the extract is removed from the sulfur cleanup reagent.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 168

4.6.4 Equipment and Supplies

1. Gas chromatograph. An analytical system complete with GC suitable for on-column and
split-splitless injection and necessary accessories including syringes, analytical columns,
gases, electron capture detectors, and recorder/integrator or data system. Electrolytic
conductivity detectors may also be employed if appropriate for project needs. If the dual-
column option is employed, the GC must be equipped with two separate detectors.

2. GC columns. The method describes procedures for single- and dual-column analyses. The
single-column approach involves one analysis to determine that a compound is present,
and a second analysis to confirm the identity of the compound. The single-column
approach may employ either narrow-bore (< 0.32-mm ID) columns or wide-bore (0.53-mm
ID) columns. The dual-column approach generally employs a single injection that is split
between two columns that are mounted in a single GC. The dual-column approach
generally employs wide-bore (0.53-mm ID) columns, but columns of other diameters may
be employed if the analyst can demonstrate and document acceptable performance for the
intended application. A third alternative is to employ dual columns mounted in a single GC,
but with each column connected to a separate injector and a separate detector.

The listing of these columns is not intended to exclude the use of other columns that are
available or that may be developed. Laboratories may use these columns or other columns
provided that the laboratories document method performance data (e.g., chromatographic
resolution, analyte breakdown, and sensitivity) appropriate for the intended application.

Narrow-bore columns for single-column analysis (use both columns to confirm compound
identifications unless another confirmation technique such as GC/MS is employed).
Narrow-bore columns should be installed in split/splitless (Grob-type) injectors.

30-m x 0.25-mm or 0.32-mm ID fused-silica capillary column chemically bonded with
SE-54, 1-um film thickness.
30-m x 0.25-mm ID fused-silica capillary column chemically bonded with 35 percent
phenyl methylpolysiloxane, 2.5 urn coating thickness, 1-um film thickness.

Wide-bore columns for single-column analysis (use two of the three columns listed to
confirm compound identifications unless another confirmation technique such as GC/MS is
employed). Wide-bore columns should be installed in 1/4-inch injectors, with deactivated
liners designed specifically for use with these columns.

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 35 percent
phenyl methylpolysiloxane, 0.5-um or 0.83-um film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 14%
cyanopropylmethylpolysiloxane , 1.0-um film thickness.

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54, 1.5-
um film thickness.

Wide-bore columns for dual-column analysis.

Column pair 1:

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54, 0.5-
um film thickness.

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 14%
cyanopropylmethylpolysiloxane, 1.0-um film thickness.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 169

Column pair 1 is mounted in a press-fit Y-shaped glass 3-way union or a Y-shaped fused-
silica connector or equivalent. When connecting columns to a press-fit Y-shaped
connector, a better seal may be achieved by first soaking the ends of the capillary columns
in alcohol for about 10 sec to soften the polyimide coating.

Column pair 2:

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54,
0.83-um film thickness.

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 14%
cyanopropylmethylpolysiloxane1.0-um film thickness.
Column pair 2 is mounted in an 8-in. deactivated glass injection tee or equivalent.

Column pair 3:

30-m x 0.53-mm ID fused-silica capillary column chemically bonded with SE-54,
1.5-um film thickness.
30-m x 0.53-mm ID fused-silica capillary column chemically bonded with 35
percent phenyl methylpolysiloxane, 0.5-um film thickness.

Column pair 3 is mounted in separate injectors and separate detectors.
3. Column rinsing kit - Bonded-phase column rinse kit

4. Volumetric flasks ~ 10-mL and 25-mL, for preparation of standards.

5. Analytical balance, capable of weighing to 0.0001 g.

4.6.5 Reagents and Standards

Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it is intended
that all reagents conform to specifications of the Committee on Analytical Reagents of the
American Chemical Society, where such specifications are available. Other grades may be
used, provided it is first ascertained that the reagent is of sufficiently high purity to permit its use
without lessening the accuracy of the determination.

Reagents should be stored in glass to prevent the leaching of contaminants from plastic
containers. Store the standard solutions (stock, composite, calibration, internal, and surrogate)
at <6 °C in polytetrafluoroethylene (PTFE)-sealed containers in the dark. When a lot of
standards is prepared, aliquots of that lot should be stored in individual small vials. All stock
standard solutions must be replaced after one year, or sooner if routine QC (see section 4.6.7)
indicates a problem. All other standard solutions must be replaced after six months or sooner if
routine QC indicates a problem.

1. Organic-free reagent water.

2. Standard stock solutions
Stock standard solutions (1000 mg/L). May be prepared from pure standard materials or
can be purchased as certified solutions. Prepare stock standard solutions by accurately
weighing 0.0100 g of pure compound. Dissolve the compound in isooctane or hexane and
dilute to volume in a 10-ml volumetric flask. If compound purity is 96 percent or greater, the
weight can be used without correction to calculate the concentration of the stock standard
solution. Commercially-prepared stock standard solutions may be used at any
concentration if they are certified by the manufacturer or by an independent source.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 170

3. Calibration standards for PCB congeners

If results are to be determined for individual PCB congeners, then standards for the pure
congeners must be prepared. This procedure may be appropriate for other congeners as
well, but the analyst must either document the resolution of the congeners in question or
establish procedures for reporting the results of coeluting congeners that are appropriate
for the intended application.

Stock standards may be prepared or may be purchased as commercially-prepared
solutions. Prepare a minimum of five calibration standards containing equal concentrations
of two conengers by dilution of the stock standard with isooctane or hexane. The
concentrations should correspond to the expected range of concentrations found in real
samples and should bracket the linear range of the detector

4. Internal standard

When PCB congeners are to be determined, the use of an internal standard is highly
recommended. Decachlorobiphenyl may be used as an internal standard, added to each
sample extract prior to analysis, and included in each of the initial calibration standards.
5. Surrogate standards

The performance of the method should be monitored using surrogate compounds.

Surrogate standards are added to all samples, method blanks, matrix spikes, and
calibration standards. The choice of surrogate compounds will depend on analysis mode
chosen, e.g., congeners. The following compounds are recommended as surrogates. Other
surrogates may be used, provided that the analyst can demonstrate and document
performance appropriate for the data quality needs of the particular application.

When PCB congeners are to be determined, decachlorobiphenyl is recommended for use
as an internal standard, and therefore it cannot also be used as a surrogate. Tetrachloro-m-
xylene may be used as a surrogate for PCB congener analysis. The recommended spiking
solution concentration is 5 mg/L in acetone.
If decachlorobiphenyl is a target congener for the analysis, 2,2',4,4',5,5'- hexabromo-
biphenyl may be used as an internal standard or a surrogate.

6. DDT Analog standard.
Used to determine if the commonly found DDT analogs (DDT, DDE, and ODD) elute at the
same retention times as any of the target analytes (congeners). A single standard
containing all three compounds should be sufficient. The concentration of the standard is
left to the judgement of the analyst.

4.6.6 GC Specifications

This method allows the analyst to choose between a single-column and a dual-column
configuration in the injector port. The columns listed in this section were the columns used to
develop the method performance data. Listing these columns in this method is not intended to
exclude the use of other columns that are available or that may be developed. Wide-bore or
narrow-bore columns may be used with either option. Laboratories may use either the columns
listed in this method or other capillary columns or columns of other dimensions, provided that
the laboratories document method performance data (e.g., chromatographic resolution, analyte
breakdown, and sensitivity) that are appropriate for the intended application.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 171

Single-column analysis

This capillary GC/ECD method allows the analyst the option of using 0.25-mm or 0.32-mm ID
capillary columns (narrow-bore) or 0.53-mm ID capillary columns (wide-bore). Narrow-bore
columns generally provide greater chromatographic resolution than widebore columns, although
narrow-bore columns have a lower sample capacity. As a result, narrow-bore columns may be
more suitable for relatively clean samples or for extracts that have been prepared with one or
more of the clean-up options referenced in the method. Wide-bore columns (0.53-mm ID) may
be more suitable for more complex environmental and waste matrices. However, the choice of
the appropriate column diameter is left to the professional judgement of the analyst.

Dual-column analysis

The dual-column/dual-detector approach recommends the use of two 30-m x 0.53-mm ID fused-
silica open-tubular columns of different polarities, thus, different selectivities towards the target
analytes. The columns may be connected to an injection tee and separate electron capture
detectors, or to both separate injectors and separate detectors. However, the choice of the
appropriate column dimensions is left to the professional judgement of the analyst.

GC temperature programs and flow rates.
Establish the GC temperature program and flow rate necessary to separate the
analytes. When determining PCBs as congeners, difficulties may be encountered with coelution
of congener 153 and other sample components. Each laboratory must determine retention times
and retention time windows for their specific application of the method. Once established, the
same operating conditions must be used for the analysis of samples and standards.

4.6.7 Quality Control and Assurance

2. Include a calibration standard after each group of 20 samples, (however it is recommended
that a calibration standard be included after every 10 samples to minimize the number of
repeat injections) in the analysis sequence as a calibration check. Thus, injections of
method blank extracts, matrix spike samples, and other non-standards are counted in the
total. Solvent blanks, injected as a check on cross-contamination, need not be counted in
the total. The response factors for the calibration verification standard should be within
±20% of the initial calibration. When this calibration verification standard falls out of this
acceptance window, the laboratory should stop analyses and take corrective action.
Whenever quantitation is accomplished using an internal standard, internal standards must
be evaluated for acceptance. The measured area of the internal standard must be no more
than 50 percent different from the average area calculated during initial calibration. When
the internal standard peak area is outside the limit, all samples that fall outside the QC

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 172

criteria must be reanalyzed. The retention times of the internal standards must also be
evaluated. A retention time shift of >30 sec necessitates reanalysis of the affected sample.
3. The lab must demonstrate initial proficiency with each sample prep and determinative
method combination it utilizes, by generating data of acceptable accuracy and precision for
target analytes in a clean matrix. If an autosampler is used to perform sample dilutions,
before using the autosampler to dilute samples, the lab should satisfy itself that those
dilutions are of equivalent or better accuracy than is achieved by an experienced analyst
performing manual dilutions. It is suggested that the QC reference sample concentrate
contain PCBs as congeners at 10-50 mg/l in the concentrate. A 1-ml spike of this
concentrate into 1 L of reagent water will yield a sample concentration of 10 ug/l.

4. Calculate the average recovery and the standard deviation of the recoveries of the analytes
in each of the four QC reference samples.

5. Before processing samples, the analyst must demonstrate that all parts of the equipment in
contact with the sample and reagents are interference-free through the analysis of a
method blank. Each time samples are extracted, cleaned up, and analyzed, and when
there is a change in reagents, a method blank must be prepared and analyzed for the
compounds of interest as a safeguard against chronic contamination. If a peak is observed
within the retention time window of any analyte that would prevent the determination of that
analyte, determine the source and eliminate it, if possible, before processing the samples.
The blanks should be carried through all stages of sample preparation and analysis. When
new reagents or chemicals are received, the lab should monitor the preparation and/or
analysis blanks associated with samples for any signs of contamination. It is not necessary
to test every new batch of reagents or chemicals prior to sample preparation if the source
shows no prior problems. However, if reagents are changed during a preparation batch,
separate blanks need to be prepared for each set of reagents.
6. Sample quality control for preparation and analysis. The lab must also have procedures for
documenting the effect of the matrix on method performance (precision, accuracy, method
sensitivity). At a minimum, this should include the analysis of QC samples including a
method blank, a matrix spike, a duplicate, and a laboratory control sample (LCS) in each
analytical batch and the addition of surrogates to each field sample and QC sample when
surrogates are used. Any method blanks, matrix spike samples, and replicate samples
should be subjected to the same analytical procedures as those used on actual samples.
Documenting the effect of the matrix should include the analysis of at least one matrix spike
and one duplicate unspiked sample or one matrix spike/matrix spike duplicate pair. The
decision on whether to prepare and analyze duplicate samples or a matrix spike/matrix
spike duplicate must be based on knowledge of the samples in the sample batch. If
samples are expected to contain target analytes, the laboratories should use a matrix spike
and matrix spike duplicate pair, spiked with a congener mixture. However, when specific
congeners are known to be present or expected in samples, the specific congener should
be used for spiking.

A laboratory control sample (LCS) should be included with each analytical batch. The LCS
consists of an aliquot of a clean (control) matrix similar to the sample matrix and of the
same weight or volume. The LCS is spiked with the same analytes at the same
concentrations as the matrix spike, when appropriate. When the results of the matrix spike
analysis indicate a potential problem due to the sample matrix itself, the LCS results are
used to verify that the laboratory can perform the analysis in a clean matrix.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 173

7. Surrogate recoveries. If surrogates are used, the laboratory should evaluate surrogate
recovery data from individual samples versus the surrogate control limits developed by the
laboratory. Procedures for evaluating the recoveries of multiple surrogates and the
associated corrective actions should be defined in an approved project plan.

8. It is recommended that the laboratory adopt additional quality assurance practices with this
method. The specific practices depend upon the needs of the laboratory and the nature of
the samples. Whenever possible, the laboratory should analyze standard reference
materials and participate in relevant performance evaluation studies.

4.6.8 Calibration and Standardization

1. Prepare calibration standards using the procedures in section 4.6.5. Refer to section
4.6.7.2 for proper calibration techniques for both initial calibration and calibration
verification. When PCBs are to be determined as congeners, the use of internal standard
calibration is highly recommended. Therefore, the calibration standards must contain the
internal standard (see section 4.6.5.4) at the same concentration as the sample extracts.

Note. Because of the sensitivity of the electron capture detector, always clean the injection port
and column prior to performing the initial calibration.

2. When PCBs are to be quantitatively determined as congeners, an initial multi-point
calibration must be performed that includes standards for all target analytes (congeners).

3. Establish the GC operating conditions appropriate for the configuration (single-column or
dual column). Optimize the instrumental conditions for resolution of the target compounds
and sensitivity. A final temperature of between 240 °C and 275 °C may be needed to elute
decachlorobipheny. The use of injector pressure programming will improve the
chromatography of late eluting peaks. Once established, the same operating conditions
must be used for both calibrations and sample analyses.

4. A 2-ul injection of each calibration standard is recommended. Other injection volumes may
be employed, provided that the analyst can demonstrate adequate sensitivity for the
compounds of interest.

5. Record the peak area (or height) for each congener peak to be used for quantitation.
6. When determining PCB congeners by the internal standard procedure, calculate the
response factor (RF) for each congener in the calibration standards relative to the internal
standard, decachlorobiphenyl, using the equation that follows.

RF =

where:
As= Peak area (or height) of the analyte or surrogate.
Ais= Peak area (or height) of the internal standard.
Cs= Concentration of the analyte or surrogate, in ug/l.
Cis= Concentration of the internal standard, in ug/l.

7. The response factors or calibration factors from the initial calibration are used to evaluate
the linearity of the initial calibration, if a linear calibration model is used. This involves the
calculation of the mean response or calibration factor, the standard deviation, and the
relative standard deviation (RSD) for each congener peak.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 174

8. Retention time windows. Absolute retention times are generally used for compound
identification. When absolute retention times are used, retention time windows are crucial
to the identification of target compounds, and must be established. Retention time windows
are established to compensate for minor shifts in absolute retention times as a result of
sample loadings and normal chromatographic variability. The width of the retention time
window must be carefully established to minimize the occurrence of both false positive and
false negative results. Tight retention time windows may result in false negatives and/or
may cause unnecessary reanalysis of samples when surrogates or spiked compounds are
erroneously not identified. Overly wide retention time windows may result in false positive
results that cannot be confirmed upon further analysis. Other approaches to compound
identification may be employed, provided that the analyst can demonstrate and document
that the approaches are appropriate for the intended application. When PCBs are
determined as congeners by an internal standard technique, absolute retention times may
be used in conjunction with relative retention times (relative to the internal standard).

When conducting congener analysis, it is important to determine that common single-
component pesticides such as DDT, ODD, and DDE do not elute at the same retention
times as the target congeners. Therefore, in conjunction with determining the retention time
windows of the congeners, the analyst should analyze a standard containing the DDT
analogs. This standard need only be analyzed when the retention time windows are
determined. It is not considered part of the routine initial calibration or calibration
verification steps in the method, nor is there any performance criteria associated with the
analysis of this standard. If PCB congener analysis is performed and any of the DDT
analogs elute at the same retention time as a PCB congener of interest, then the analyst
must adjust the GC conditions to achieve better resolution.

4.6.9 Gas Chromatography Analasis of Sample Extracts

1. The same GC operating conditions used for the initial calibration must be employed for the
analysis of samples.
2. Verify calibration at least once each 12-hr shift by injecting calibration verification standards
prior to conducting any sample analyses. A calibration standard must also be injected at
intervals of not less than once every twenty samples (after every 10 samples is
recommended to minimize the number of samples requiring reinjection when QC limits are
exceeded) and at the end of the analysis sequence.

The calibration factor for each analyte calculated from the calibration verification standard
(CFV) should not exceed a difference of more than ±20 percent when compared to the
mean calibration factor from the initial calibration curve.

UF - TF
% Difference = — >! * 100
CF

When internal standard calibration is used for PCB congeners, the response factor
calculated from the calibration verification standard (RFV) should not exceed a ±20 percent
difference when compared to the mean response factor from the initial calibration.

pp _ DC
% Difference = ———- * 100
W

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 175

3. Inject a measured aliquot of the concentrated sample extract. A 2-ul aliquot is suggested,
however, other injection volumes may be employed, provided that the analyst can
demonstrate adequate sensitivity for the compounds of interest. The same injection volume
should be used for both the calibration standards and the sample extracts, unless the
analyst can demonstrate acceptable performance using different volumes or conditions.
Record the volume injected and the resulting peak size in area units.

4. Qualitative identifications of target analytes are made by examination of the sample
chromatograms, as described in section 4.6.10.1.

5. Quantitative results are determined for each identified analyte (congener), using the
procedures described in sections 4.6.10 and 4.6.11 for either the internal or the external
calibration procedure. If the responses in the sample chromatogram exceed the calibration
range of the system, dilute the extract and reanalyze. Peak height measurements are
recommended over peak area when overlapping peaks cause errors in area integration.

6. Each sample analysis employing external standard calibration must be bracketed with an
acceptable initial calibration, calibration verification standard(s) (each 12-hr analytical shift),
or calibration standards interspersed within the samples. The results from these bracketing
standards must meet the calibration verification criteria discussed in #2 above.

Multi-level standards (mixtures or multi-component analytes) are highly recommended to
ensure that detector response remains stable for all analytes over the calibration range.
When a calibration verification standard fails to meet the QC criteria, all samples that were
injected after the last standard that met the QC criteria must be evaluated to prevent
misquantitations and possible false negative results, and reinjection of the sample extracts
may be required. More frequent analyses of standards will minimize the number of sample
extracts that would have to be reinjected if the QC limits are violated for the standard
analysis. However, if the standard analyzed after a group of samples exhibits a response
for an analyte that is above the acceptance limit, i.e., >20%, and the analyte was not
detected in the specific samples analyzed during the analytical shift, then the extracts for
those samples do not need to be reanalyzed, since the verification standard has
demonstrated that the analyte would have been detected if it were present. In contrast, if an
analyte above the QC limits was detected in a sample extract, then reinjection is necessary
to ensure accurate quantitation.
If an analyte was not detected in the sample and the standard response is more than 20%
below the initial calibration response, then reinjection is necessary. The purpose of this
reinjection is to ensure that the analyte could be detected, if present, despite the change in
the detector response, e.g., to protect against a false negative result.

7. Sample injections may continue for as long as the calibration verification standards and
standards interspersed with the samples meet instrument QC requirements. It is
recommended that standards be analyzed after every 10 samples (required after every 20
samples and at the end of a set) to minimize the number of samples that must be re-
injected when the standards fail the QC limits. The sequence ends when the set of samples
has been injected or when qualitative or quantitative QC criteria are exceeded.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 176

8. The use of internal standard calibration techniques does not require that all sample results
be bracketed with calibration verification standards. However, when internal standard
calibration is used, the retention times of the internal standards and the area responses of
the internal standards should be checked for each analysis. Retention time shifts of more
than 30 sec. from the retention time of the most recent calibration standard and/or changes
in internal standard areas of more than -50 to +100% are cause for concern and must be
investigated.

9. If the peak response is less than 2.5 times the baseline noise level, the validity of the
quantitative result may be questionable. The analyst should consult with the source of the
sample to determine whether further concentration of the sample is warranted.

10. Use the calibration standards analyzed during the sequence to evaluate retention time
stability. If any of the standards fall outside their daily retention time windows, the system is
out of control. Determine the cause of the problem and correct it.

11. If compound identification or quantitation is precluded due to interferences (e.g., broad,
rounded peaks or ill-defined baselines are present), corrective action is warranted. Cleanup
of the extract or replacement of the capillary column or detector may be necessary. The
analyst may begin by rerunning the sample on another instrument to determine if the
problem results from analytical hardware or the sample matrix.

4.6.10 Qualitative Identification

1. The identification of PCBs as congeners using this method with an electron capture
detector is based on agreement between the retention times of peaks in the sample
chromatogram with the retention time windows established through the analysis of
standards of the target analytes.
Tentative identification of an analyte occurs when a peak from a sample extract falls within
the established retention time window for a specific target analyte. Confirmation is
necessary when the sample composition is not well characterized. See section 4.6.12.4 of
this method for information on the use of GC/MS as a confirmation technique.
When results are confirmed using a second GC column of dissimilar stationary phase, the
analyst should check the agreement between the quantitative results on both columns once
the identification has been confirmed.
2. When simultaneous analyses are performed from a single injection (the dual-column GC
configuration described in section 4.6.6), it is not practical to designate one column as the
analytical (primary) column and the other as the confirmation column. Since the calibration
standards are analyzed on both columns, both columns must meet the calibration
acceptance criteria. If the retention times of the peaks on both columns fall within the
retention time windows on the respective columns, then the target analyte identification has
been confirmed.
3. The results of a single column/single injection analysis may be confirmed on a second,
dissimilar, GC column. In order to be used for confirmation, retention time windows must
have been established for the second GC column, and the analyst must demonstrate the
sensitivity of the second column analysis. This demonstration must include the analysis of
a standard of the target analyte at a concentration at least as low as the concentration
estimated from the primary analysis. That standard may be individual congeners.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 177

4.6.11 Quantitative Identification

PCBs as Congeners

1. The quantitation of PCB congeners is accomplished by the comparison of the sample
chromatogram to those of the PCB congener standards, using the internal standard
technique. Calculate the concentration of each congener.

2. Depending on project requirements, the PCB congener results may be reported as
congeners, or may be summed and reported as total PCBs.

3. The analytical procedures for these congeners may be appropriate for the analysis of other
congeners not specifically included in this method and may be used as a template for the
development of such a procedure. However, all PCB congeners cannot be separated using
the GC columns and procedures described in this method. If this procedure is expanded to
encompass other congeners, then the analyst must either document the resolution of the
congeners in question or establish procedures for report.

4.6.12 Confirmation

1. Tentative identification of an analyte occurs when a peak from a sample extract falls within
the daily retention time window. Confirmation is necessary when the sample composition is
not well characterized. Confirmatory techniques such as gas chromatography with a
dissimilar column or a mass spectrometer should be used.

2. When results are confirmed using a second GC column of dissimilar stationary phase, the
analyst should check the agreement between the quantitative results on both columns once
the identification has been confirmed.

3. When the dual-column approach is employed, the target phenols are identified and
confirmed when they meet the identification criteria on both columns.
4. GC/MS confirmation. GC/MS confirmation may be used in conjunction with either single-or
dual-column analysis if the concentration is sufficient for detection by GC/MS.

Full-scan quadrupole GC/MS will normally require a higher concentration of the analyte of
interest than full-scan ion trap or selected ion monitoring techniques. The concentrations
will be instrument-dependent, but values for full-scan quadrupole GC/MS may be as high
as10 ng/ul in the final extract, while ion trap or SIM may only be a concentration of 1 ng/ul.

The GC/MS must be calibrated for the target analytes when it is used for quantitative
analysis. If GC/MS is used only for confirmation of the identification of the target analytes,
then the analyst must demonstrate that those PCBs identified by GC/ECD can be
confirmed by GC/MS. This demonstration may be accomplished by analyzing a single-point
standard containing the analytes of interest at or below the concentrations reported in the
GC/ECD analysis.

GC/MS confirmation should be accomplished by analyzing the same extract used for
GC/ECD analysis and the extract of the associated blank.

5. GC/AED confirmation may be used in conjunction with either single-column or dual-column
analysis if the concentration is sufficient for detection by GC/AED.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 178
   4.7 POLYNUCLEAR AROMATIC HYDROCARBONS (PAHS) IN SEDIMENTS ONLY

4.7.1   Scope and Application

1.  This method may be used for both saltwater and freshwater sediments.

2.  This method is used to determine the concentration of 25 semivolatile organic compounds
   (Table 4.10) in extracts prepared from sediment samples.

Table 4.10. Indicator List of Polynuclear Aromatic Hydrocarbons (PAHs)
Compound
Acenaphthene
Acenaphthylene
Anthracene
Benz(a)anthracene
Benzo(b)fluoranthene
Benzo(k)fluoranthene
Benzo(g,h,i)perylene
Benzo(a)pyrene
Benzo(e)pyrene
Biphenyl
Chrysene
Dibenz(a,h)anthracene
Dibenzothiophene
2,6-dimethylnaphthalene
Fluoranthene
Fluorene
lndeno(1 ,2,3-c,d)pyrene
1 -methylnaphthalene
2-methylnaphthalene
1 -methylphenanthrene
Naphthalene
Phenanthrene
Perylene
Pyrene
2,3,5-trimethylnaphthalene
Chemical Abstract Service
(CAS) Registry No.
83-32-9
208-96-8
120-12-7
56-55-3
205-99-2
207-08-9
191-24-2
50-32-8
1 92-97-2
92-52-4
218-01-09
53-70-3
1 32-65-0
581-42-0
206-44-0
86-73-7
1 93-39-5
90-12-9
91-57-6
832-69-9
91-20-3
85-01-8
77392-71-3
129-00-0
2245-38-7
3.   This method can be used to quantitate most neutral, acidic, and basic organic compounds
    that are soluble in methylene chloride (or other suitable solvents provided that the desired
    performance data can be generated) and capable of being eluted, without derivatization, as
    sharp peaks from a gas chromatographic fused-silica capillary column coated with a slightly
    polar silicone. Such compounds include polynuclear aromatic hydrocarbons.

4.   In most cases, this method is not appropriate for the quantitation of multicomponent
    analytes, e.g., Toxaphene,  Chlordane, Aroclor, etc., because of limited sensitivity for those
    analytes (see sections 4.5 and 4.6).

5.   The lower limit of quantitation for this method when determining an individual compound is
    approximately 660 ug/kg (wet weight) for sediment samples. Lower limits of quantitation will
    be proportionately higher for sample extracts that require dilution to avoid saturation of the
    detector.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 179

4.7.2 Summary of Method

The semivolatile compounds are introduced into the GC/MS by injecting the sample extract into
a gas chromatograph (GC) equipped with a narrow-bore fused-silica capillary column. The GC
column is temperature-programmed to separate the analytes, which are then detected with a
mass spectrometer (MS) connected to the gas chromatograph. Analytes eluted from the
capillary column are introduced into the mass spectrometer via a jet separator or a direct
connection. Identification of target analytes is accomplished by comparing their mass spectra
with the electron impact (or electron impact-like) spectra of authentic standards. Quantitation is
accomplished by comparing the response of a major (quantitation) ion relative to an internal
standard using an appropriate calibration curve for the intended application.

4.7.3 Interferences

2. Raw GC/MS data from all blanks, samples, and spikes must be evaluated for interferences.
Determine if the source of interference is in the preparation and/or cleanup of the samples
and take corrective action to eliminate the problem.

3. Contamination by carryover can occur whenever high-concentration and low-concentration
samples are sequentially analyzed. To reduce carryover, the sample syringe must be
rinsed with solvent between sample injections. Whenever an unusually concentrated
sample is encountered, it should be followed by the analysis of solvent to check for cross
contamination.

4.7.4 Equipment and Supplies

1. Gas chromatograph
An analytical system equipped with a temperature-programmable gas chromatograph
suitable for splitless injection and all required accessories, including syringes, analytical
columns, and gases. The capillary column should be directly coupled to the source.

2. Column
30-m x 0.25-mm ID (or 0.32-mm ID) 0.25, 0.5, or 1-um film thickness silicone-coated fused-
silica capillary column. The columns listed in this section were the columns used in
developing the method. The listing of these columns in this method is not intended to
exclude the use of other columns that may be developed. Laboratories may use these
columns or other capillary columns provided that the laboratories document method
performance data (e.g., chromatographic resolution, analyte breakdown, and sensitivity)
that are appropriate for the intended application.
3. Mass spectrometer
Capable of scanning from 35 to 500 amu every 1 sec or less, using 70 volts (nominal)
electron energy in the electron impact ionization mode. The mass spectrometer must be
capable of producing a mass spectrum for decafluorotriphenylphosphine (DFTPP) which
meets the criteria as outlined in section 4.7.10.1.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 180

An ion trap mass spectrometer may be used if it is capable of axial modulation to reduce
ion-molecule reactions and can produce electron impact-like spectra that match those in
the EPA/NIST Library. The mass spectrometer must be capable of producing a mass
spectrum for DFTPP which meets the criteria alos outlined in section 4.7.10.1

4. GC/MS interface
Any GC-to-MS interface may be used that gives acceptable calibration points for each
compound of interest and achieves acceptable tuning performance criteria. For a narrow-
bore capillary column, the interface is usually capillary-direct into the mass spectrometer
source.

5. Data system
A computer system should be interfaced to the mass spectrometer. The system must allow
the continuous acquisition and storage on machine-readable media of all mass spectra
obtained throughout the duration of the chromatographic program. The computer should
have software that can search any GC/MS data file for ions of a specific mass and that can
plot such ion abundances versus time or scan number. This type of plot is defined as an
Extracted Ion Current Profile (EICP). Software should also be available that allows
integrating the abundances in any EICP between specified time or scan-number limits. The
most recent version of the EPA/NIST Mass Spectral Library should also be available.
6. Guard column (optional) - (deactivated fused-silica, 0.25-mm ID x 6-m) between the
injection port and the analytical column joined with column connectors

7. Syringe. 10-uL.

8. Volumetric flasks, Class A ~ Appropriate sizes equipped with ground-glass stoppers.

9. Balance. Analytical, capable of weighing 0.0001 g.
10. Bottles - Glass with polytetrafluoroethylene (PTFE)-lined screw caps or crimp tops.

4.7.5 Reagents and Standards

1. Reagent-grade chemicals must be used in all tests. Unless otherwise indicated, it is
intended that all reagents conform to the specifications of the Committee on Analytical
Reagents of the American Chemical Society, where such specifications are available.
Other grades may be used, provided it is first ascertained that the reagent is of sufficiently
high purity to permit its use without lessening the accuracy of the determination. Reagents
should be stored in glass to prevent the leaching of contaminants from plastic containers.
2. Organic-free reagent water

3. Solvents. Acetone, hexane, methylene chloride, isooctane, carbon disulfide, toluene, and
other appropriate solvents. All solvents should be pesticide quality or equivalent. Solvents
should be degassed prior to use.

4. Stock standard solutions (1000 mg/L)
Standard solutions may be prepared from pure standard materials or purchased.
Commercially-prepared stock standards may be used at any concentration if they are
certified by the manufacturer or by an independent source.

Prepare stock standard solutions by accurately weighing about 0.0100 g of pure material.
Dissolve the material in pesticide quality acetone or other suitable solvent and dilute to
volume in a 10-mL volumetric flask. Larger volumes can be used at the convenience of the

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 181

analyst. When compound purity is assayed to be 96% or greater, the weight may be used
without correction to calculate the concentration of the stock standard.
Transfer the stock standard solutions into bottles equipped with PTFE lined screw-caps.
Store, protected from light, at <6 °C or as recommended by the standard manufacturer.
Stock standard solutions should be checked frequently for signs of degradation or
evaporation, especially just prior to preparing calibration standards from them.

Stock standard solutions must be replaced after 1 year or sooner if comparison with quality
control check samples indicates a problem.
It is recommended that nitrosamine compounds be placed together in a separate
calibration mix and not combined with other calibration mixes. When using a premixed
certified standard, consult the manufacturer's instructions for additional guidance.

Mixes with hydrochloride salts may contain hydrochloric acid, which can cause analytical
difficulties. When using a premixed certified standard, consult the manufacturer's
instructions for additional guidance.

5. Internal standard solutions
The internal standards recommended are 1,4-dichlorobenzene-cf4, naphthalene-cf8,
acenaphthene-cf™, phenanthrene-cf™, chrysene-cf^, and perylene-cf^. Other compounds
may be used as internal standards as long as the criteria in section 4.7.10.1.2 are met.
Dissolve 0.200 g of each compound with a small volume of carbon disulfide. Transfer to a
50-mL volumetric flask and dilute to volume with methylene chloride so that the final solvent
is approximately 20% carbon disulfide. Most of the compounds are also soluble in small
volumes of methanol, acetone, or toluene, except for perylene-cf^- The resulting solution
will contain each standard at a concentration of 4,000 ng/uL. Each 1-mL sample extract
undergoing analysis should be spiked with 10 uL of the internal standard solution, resulting
in a concentration of 40 ng/uL of each internal standard. Store away from any light source
at <6°C when not in use (-10°C is recommended). When using premixed certified solutions,
store according to the manufacturer's documented holding time and storage temperature
recommendations.

If a more sensitive mass spectrometer is employed to achieve lower quantitation levels, a
more dilute internal standard solution may be required. Area counts of the internal standard
peaks should be between 50-200% of the area of the target analytes in the mid-point
calibration analysis.
6. GC/MS tuning standard
A methylene chloride solution containing 50 ng/uL of decafluorotriphenylphosphine
(DFTPP) should be prepared. The standard should also contain 50 ng/uL each of 4,4'-DDT,
pentachlorophenol, and benzidine to verify injection port inertness and GC column
performance. Alternate concentrations may be used to compensate for different injection
volumes if the total amount injected is 50 ng or less. Store away from any light source at
<6°C when not in use (-10°C is recommended). If a more sensitive mass spectrometer is
employed to achieve lower quantitation levels, a more dilute tuning solution may be
necessary. When using premixed certified solutions, store according to the manufacturer's
documented holding time and storage temperature recommendations.
7. Calibration standards
A minimum of five calibration standards should be prepared at different concentrations. At
least one of the calibration standards should correspond to a sample concentration at or
below that necessary to meet the data quality objectives of the project. The remaining

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 182

standards should correspond to the range of concentrations found in actual samples but
should not exceed the working range of the GC/MS system. Each standard and/or series of
calibration standards prepared at a given concentration should contain all the desired
project-specific target analytes for which quantitation and quantitative results are to be
reported by this method.
It is the intent of EPA that all target analytes for a particular analysis be included in the
calibration standard(s). The laboratory shall not report a quantitative result for a target
analyte that was not included in the calibration standard(s).
Each 1-mL aliquot of calibration standard should be spiked with 10 uL of the internal
standard solution prior to analysis. All standards should be stored away from any light
source at <6 °C when not in use (-10 °C is recommended), and should be freshly prepared
once a year, or sooner if check standards indicate a problem. The calibration verification
standard should be prepared, as necessary, and stored at <6 °C. When using premixed
certified solutions, store according to the manufacturer's documented holding time and
storage temperature recommendations.
8. Surrogate standards
The recommended surrogates are phenol-cf6, 2-fluorophenol, 2,4,6-tribromophenol,
nitrobenzene-cfs, 2-fluorobiphenyl, and p-terphenyl-cfM.

NOTE: In the presence of samples containing residual chlorine, phenol-d6 has been known to
react to form chlorinated phenolic compounds that are not detected as the original spiked
surrogate. Sample preservation precautions should be used when residual chlorine is
known to be present in order to minimize degradation of deuterated phenols or any other
susceptible target analyte.
Surrogate standard check. Determine what the appropriate concentration should be for the
blank extracts after all extraction, cleanup, and concentration steps. Inject the concentration
into the GC/MS to determine recovery of surrogate standards. It is recommended that this
check be done whenever a new surrogate spiking solution is prepared.

If a more sensitive mass spectrometer is employed to achieve lower quantitation levels, a
more dilute surrogate solution may be necessary.
9. Matrix spike and laboratory control standards
The same standard may be used as the laboratory control standard (LCS) and the spiking
solution should be the same source as used for the initial calibration standards to restrict
the influence of standard accuracy on the determination of recovery through preparation
and analysis.

Matrix spike check. Determine what concentration should be in the blank extracts after all
extraction, cleanup, and concentration steps. Inject this concentration into the GC/MS to
determine recovery. It is recommended that this check be done whenever a new matrix
spiking solution is prepared.

If a more sensitive mass spectrometer is employed to achieve lower quantitation levels, a
more dilute matrix and LCS spiking solution may be necessary.

4.7.6 Quality Control

1. Refer to the QAPP for guidance on quality assurance and quality control protocols. When
inconsistencies exist between QC guidelines, method-specific QC criteria take precedence
over both technique-specific criteria and those criteria given the QAPP, and technique-

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 183

specific QC criteria take precedence over the criteria in the QAPP. Any effort involving the
collection of analytical data should include development of a structured and systematic
planning document, such as a Sampling and Analysis Plan (SAP), which translates project
objectives and specifications into directions for those that will implement the project and
assess the results. Each lab should maintain a formal quality assurance program. Each lab
must work with the Information Management group (Marlys Cappaert, 541-754-4467
Cappaert.Marlys@epamail.epa.gov) to ensure their bench sheets and/or data recording
spreadsheets are compatible with the electronic deliverables the lab will need to submit.
The laboratory should also maintain records to document the quality of the data generated.
All data sheets and quality control data should be maintained for reference or inspection.

2. Quality control procedures necessary to evaluate the GC system operation include
evaluation of retention time windows, calibration verification and chromatographic analysis
of samples. In addition, discussions regarding the instrument QC requirements listed below
can be found in the referenced sections of this method,

The GC/MS must be tuned to meet the recommended DFTPP criteria prior to the initial
calibration and for each 12-hr period during which analyses are performed.

There must be an initial calibration of the GC/MS system as described in section 4.7.10.1.
In addition, the initial calibration curve should be verified immediately after performing the
standard analyses using a second source standard (prepared using standards different
from the calibration standards). The suggested acceptance limits for this initial calibration
verification analysis are 70 -130%. Alternative acceptance limits may be appropriate based
on the desired project-specific data quality objectives. Quantitative sample analyses should
not proceed for those analytes that fail the second source standard initial calibration
verification. However, analyses may continue for those analytes that fail the criteria with an
understanding these results could be used for screening purposes and would be
considered estimated values.

The GC/MS system must meet the calibration verification acceptance criteria in section
4.7.10.2 each 12 hrs. The RRTof the sample component must fall within the RRT window
of the standard component provided in section 4.7.11.2.
3. Initial demonstration of proficiency. Each laboratory must demonstrate initial proficiency
with each sample preparation and determinative method combination it utilizes by
generating data of acceptable accuracy and precision for target analytes in a clean matrix.
The laboratory must also repeat the following operations whenever new staff members are
trained or significant changes in instrumentation are made.

4. Before processing samples, the analyst must demonstrate that all parts of the equipment in
contact with the sample and reagents are interference-free. This is accomplished through
the analysis of a method blank. Each time samples are extracted, cleaned up, and
analyzed, a method blank must be prepared and analyzed for the compounds of interest as
a safeguard against chronic laboratory contamination. If a peak is observed within the
retention time window of any analyte that would prevent the determination of that analyte,
determine the source and eliminate it, if possible, before processing the samples. The
blanks should be carried through all stages of sample preparation and analysis. When new
reagents or chemicals are received, the lab should monitor the preparation and/or analysis
blanks associated with samples for any signs of contamination. It is not necessary to test
every new batch of reagents or chemicals prior to sample preparation if the source shows
no prior problems. However, if reagents are changed during a preparation batch, separate
blanks need to be prepared for each set of reagents.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 184

5. The laboratory must also have procedures for documenting the effect of the matrix on
method performance (precision, accuracy, method sensitivity). At a minimum, this should
include the analysis of QC samples including a method blank, a matrix spike, a duplicate,
and a laboratory control sample (LCS) in each analytical batch and the addition of
surrogates to each field sample and QC sample when surrogates are used. Any method
blanks, matrix spike samples, and replicate samples should be subjected to the same
analytical procedures as those used on actual samples.

Documenting the effect of the matrix should include the analysis of at least one matrix spike
and one duplicate unspiked sample or one matrix spike/matrix spike duplicate pair. The
decision on whether to prepare and analyze duplicate samples or a matrix spike/matrix
spike duplicate must be based on knowledge of the samples in the sample batch. If
samples are expected to contain target analytes, laboratories may use a matrix spike and a
duplicate analysis of an unspiked field sample. If samples are not expected to contain
target analytes, then laboratories should use a matrix spike and matrix spike duplicate pair.
Consult Method 8000 for information on developing acceptance criteria for the MS/MSD.
A laboratory control sample (LCS) should be included with each analytical batch. The LCS
consists of an aliquot of a clean (control) matrix similar to the sample matrix and of the
same weight or volume. The LCS is spiked with the same analytes at the same
concentrations as the matrix spike, when appropriate. When the results of the matrix spike
analysis indicate a potential problem due to the sample matrix itself, the LCS results are
used to verify that the laboratory can perform the analysis in a clean matrix.

6. Surrogate recoveries. If surrogates are used, the laboratory should evaluate surrogate
recovery data from individual samples versus the surrogate control limits developed by the
laboratory. Procedures for evaluating the recoveries of multiple surrogates and the
associated corrective actions should be defined in an approved project plan.

4.7.7 Calibration and Standardization

4.7.7.1 Initial Calibration

1. Establish the GC/MS operating conditions as follows:

Mass range: 35-500 amu
Scan time: <1 sec/scan
Initial temperature: 40 °C, hold for 4 min
Temperature program: 40-320 °C at 10 °C/min
Final temperature: 320 °C, hold until 2 min after benzo[g,h,i]perylene elutes
Injector temperature: 250-300 °C
Transfer line temperature: 250-300 °C
Source temperature: According to manufacturer's specifications
Injector: Grob-type, splitless
Injection volume: 1-2 uL
Carrier gas: Hydrogen at 50 cm/sec or helium at 30 cm/sec
Set axial modulation, manifold temperature, and emission current to manufacturer's
recommendations

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 185

Split injection is allowed if the sensitivity of the mass spectrometer is sufficient.

2. The GC/MS system must be hardware-tuned such that injecting 50 ng or less of DFTPP
meets the manufacturer's specified acceptance criteria. The tuning criteria may depend on
the type of instrumentation, e.g., Time-of-Flight, Ion Trap, etc. In these cases it would be
appropriate to follow the manufacturer's tuning instructions or some other consistent tuning
criteria. However, no matter which tuning criteria is selected, the system calibration must not
begin until the tuning acceptance criteria are met with the sample analyses performed under
the same conditions as the calibration standards.

In the absence of specific recommendations on how to acquire the mass spectrum of
DFTPP from the instrument manufacturer, the following approach should be used: Three
scans (the peak apex scan and the scans immediately preceding and following the apex)
are acquired and averaged. Background subtraction is required, and must be accomplished
using a single scan acquired within 20 scans of the elution of DFTPP. The background
subtraction should be designed only to eliminate column bleed or instrument background
ions. Do not subtract part of the DFTPP peak or any other discrete peak that does not
coelute with DFTPP.

Use the DFTPP mass intensity criteria in the manufacturer's instructions as primary tuning
acceptance criteria. Alternatively, other documented tuning criteria may be used provided
that method performance is not adversely affected. The analyst is always free to choose
criteria that are tighter than those included in this method or to use other documented
criteria provided they are used consistently throughout the initial calibration, calibration
verification, and sample analyses.

Note. All subsequent standards, samples, MS/MSDs, and blanks associated with a DFTPP
analysis must use the identical mass spectrometer instrument conditions.
The GC/MS tuning standard solution should also be used to assess GC column
performance and injection port inertness. Degradation of DDT to DDE and ODD should not
exceed 20%.

If degradation is excessive and/or poor chromatography is noted, the injection port may
require cleaning. It may also be necessary to break off the first 6 to12 in. of the capillary
column. The use of a guard column (section 4.7.4.6) between the injection port and the
analytical column may help prolong analytical column performance life.
3. The internal standards selected should permit most of the components of interest in a
chromatogram to have retention times of 0.80-1.20 relative to one of the internal standards.
Use the base peak ion from the specific internal standard as the primary ion for quantitation.
If interferences are noted, use the next most intense ion as the quantitation ion (e.g., for 1,4-
dichlorobenzene-cf4, use m/z 150 for quantitation).

4. Analyze 1-2 uL of each calibration standard (containing the compounds for quantitation and
the appropriate surrogates and internal standards) and tabulate the area of the primary ion
against concentration for each target analyte. A set of at least five calibration standards is
necessary (see section 4.7.5.7). Alternate injection volumes may be used if the applicable
quality control requirements for using this method are met. The injection volume must be the
same for all standards and sample extracts.

5. Initial calibration calculations

6. Calculate response factors (RFs) for each target analyte relative to one of the internal
standards as follows:

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 186

RF = _AsXCjs_
Ajs X Lrs
where,
As= Peak area (or height) of the analyte or surrogate.
Ais= Peak area (or height) of the internal standard.
Cs= Concentration of the analyte or surrogate, in ug/L.
Cis= Concentration of the internal standard, in ug/L.

Calculate the mean response factor and the relative standard deviation (RSD) of the
response factors for each target analyte using the following equations. The RSD must be
<20% for each target analyte. It is recommended that a minimum response factor for the
most common target analytes be demonstrated for each individual calibration level to ensure
that these compounds are behaving as expected. In addition, meeting minimum response
factor criteria for the lowest calibration standard is critical in establishing and demonstrating
the desired sensitivity. Due to the large number of compounds that may be analyzed by this
method, some compounds will fail to meet these criteria. For these occasions, it is
acknowledged that the failing compounds may not be critical to the specific project and
therefore they may be used as qualified data or estimated values for screening purposes.
mean RF = RF= n
SD= V— RSD==x100
n-\ RF

Where:
RFi = RF for each of the calibration standards

RF= mean RF for each compound from the initial calibration
n = number of calibration standards, e.g., 5
If more than 10% of the compounds included with the initial calibration exceed the 20% RSD
limit and do not meet the minimum correlation coefficient (0.99) for alternate curve fits, then
the chromatographic system is considered too reactive for analysis to begin. Clean or
replace the injector liner and/or capillary column, then repeat the calibration procedure.
7. Evaluation of retention times. The relative retention time (RRT) of each target analyte in
each calibration standard should agree within 0.06 RRT units. Late-eluting target analytes
usually have much better agreement.

RRT = Retention time of the anlaysis
Retention time of the internal standard

8. Linearity of target analytes. If the RSD of any target analyte is 20% or less, then the relative
response factor is assumed to be constant over the calibration range, and the average
relative response factor may be used for quantitation (section 4.7.12.2).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 187

If the RSD of any target analyte is greater than 20%. One of the options must be applied to
GC/MS calibration in this situation, or a new initial calibration must be performed. The
average RF should not be used for compounds that have an RSD greater than 20% unless
the concentration is reported as estimated.

When the RSD exceeds 20%, the plotting and visual inspection of a calibration curve can be
a useful diagnostic tool. The inspection may indicate analytical problems, including errors in
standard preparation, the presence of active sites in the chromatographic system, analytes
that exhibit poor chromatographic behavior, etc.
Due to the large number of compounds that may be analyzed by this method, some
compounds may fail to meet either the 20% RSD, minimum correlation coefficient criteria
(0.99). Any calibration method but it should be used consistently. It is considered
inappropriate once the calibration analyses are completed to select an alternative calibration
procedure in order to pass the recommended criteria on a case-by-case basis. If
compounds fail to meet these criteria, the associated concentrations may still be determined
but they must be reported as estimated. In order to report nondetects, it must be
demonstrated that there is adequate sensitivity to detect the failed compounds at the
applicable lower quantitation limit.

4.7.7.2 GC/MS Calibration Verification

1. Prior to the analysis of samples or calibration standards, inject 50 ng or less of the DFTPP
standard into the GC/MS system. The resultant mass spectrum for DFTPP must meet the
criteria as outlined in section 4.7.10.1 before sample analysis begins. These criteria must be
demonstrated each 12-hr shift during which samples are analyzed.

2. The initial calibration function for each target analyte should be checked immediately after
the first occurrence in the region of the middle of the calibration range with a standard from
a source different from that used for the initial calibration. The value determined from the
second source check should be within 30% of the expected concentration. An alternative
recovery limit may be appropriate based on the desired project-specific data quality
objectives. Quantitative sample analyses should not proceed for those analytes that fail the
second source standard initial calibration verification. However, analyses may continue for
those analytes that fail the criteria with an understanding these results could be used for
screening purposes and would be considered estimated values.

3. The initial calibration for each compound of interest should be verified once every 12 hrs
prior to sample analysis, using the introduction technique and conditions used for samples.
This is accomplished by analyzing a calibration standard (containing all the compounds for
quantitation) at a concentration either near the midpoint concentration for the calibrating
range of the GC/MS or near the action level for the project. The results must be compared
against the most recent initial calibration curve and should meet the verification acceptance
criteria below.

Note. The DFTPP and calibration verification standard may be combined into a single standard
as long as both tuning and calibration verification acceptance criteria for the project can be
met without interferences.
4. A method blank is analyzed prior to sample analyses in order to ensure that the total system
(introduction device, transfer lines and GC/MS system) is contaminant-free. If the method
blank indicates contamination, then it may be appropriate to analyze a solvent blank to
demonstrate that the contamination is not a result of carryover from standards or samples.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 188

5. Calibration verification standard criteria. Each of the most common target analytes in the
calibration verification standard should meet the minimum response factors. This criterion is
particularly important when the common target analytes are also critical project-required
compounds. This is the same check that is applied during the initial calibration.

If the minimum response factors are not met, the system should be evaluated, and
corrective action should be taken before sample analysis begins. Possible problems include
standard mixture degradation, injection port inlet contamination, contamination at the front
end of the analytical column, and active sites in the column or chromatographic system.
All target compounds of interest must be evaluated using a 20% criterion. Use percent
difference when performing the average response factor model calibration. Use percent drift
when calibrating using a regression fit model. If the percent difference or percent drift for a
compound is less than or equal to 20%, then the initial calibration for that compound is
assumed to be valid. Due to the large numbers of compounds that may be analyzed by this
method, it is expected that some compounds will fail to meet the criterion. If the criterion is
not met (i.e., greater than 20% difference or drift) for more than 20% of the compounds
included in the initial calibration, then corrective action must be taken prior to the analysis of
samples. In cases where compounds fail, they may still be reported as non-detects if it can
be demonstrated that there was adequate sensitivity to detect the compound at the
applicable quantitation limit. For situations when the failed compound is present, the
concentrations must be reported as estimated values.

Problems similar to those listed under initial calibration could affect the ability to pass the
calibration verification standard analysis. If the problem cannot be corrected by other
measures, a new initial calibration must be generated. The calibration verification criteria
must be met before sample analysis begins.

The method of linear regression analysis has the potential for a significant bias to the lower
portion of a calibration curve, while the relative percent difference and quadratic methods of
calibration do not have this potential bias. When calculating the calibration curves using the
linear regression model, a minimum quantitation check on the viability of the lowest
calibration point should be performed by re-fitting the response from the low concentration
calibration standard back into the curve. It is not necessary to re-analyze a low
concentration standard, rather the data system can recalculate the concentrations as if it
were an unknown sample. The recalculated concentration of the low calibration point should
be within ± 30% of the standard's true concentration. Other recovery criteria may be
applicable depending on the project's data quality objectives and for those situations the
minimum quantitation check criteria should be outlined in a laboratory standard operating
procedure, or a project-specific Quality Assurance Project Plan. Analytes which do not meet
the minimum quantitation calibration re-fitting criteria should not be considered and
corrective action such as redefining the lower limit of quantitation and/or reporting those
specific target analytes as estimated when the concentration is at or near the lowest
calibration point may be appropriate.

6. Internal standard retention time. The retention times of the internal standards in the
calibration verification standard must be evaluated immediately after or during data
acquisition. If the retention time for any internal standard changes by more than 30 sec from
that in the mid-point standard level of the most recent initial calibration sequence, then the
chromatographic system must be inspected for malfunctions and corrections must be made,
as required. When corrections are made, reanalysis of samples analyzed while the system
was malfunctioning is required.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 189

7. Internal standard response. If the EICP area for any of the internal standards in the
calibration verification standard changes by a factor of two (-50% to +100%) from that in the
mid-point standard level of the most recent initial calibration sequence, the mass
spectrometer must be inspected for malfunctions and corrections must be made, as
appropriate. When corrections are made, reanalysis of samples analyzed while the system
was malfunctioning is required.

4.7.8 Procedures

1. Samples are prepared using ultrasonic extraction method described in section 4.4.

2. Cleanup procedures may not be necessary for a relatively clean sample matrix, but most
extracts from environmental and waste samples will require additional preparation before
analysis. The specific cleanup procedure used will depend on the nature of the sample to be
analyzed and the data quality objectives for the measurements.

4.7.8.1 GC/MS Analysis of Samples

1. It is highly recommended that sample extracts be screened on a GC/FID or GC/PID using
the same type of capillary column used in the GC/MS system. This will minimize
contamination of the GC/MS system from unexpectedly high concentrations of organic
compounds.

2. Allow the sample extract to warm to room temperature. Just prior to analysis, add 10 uL of the
internal standard solution to the 1 ml_ of concentrated sample extract obtained from sample
preparation.

3. Inject an aliquot of the sample extract into the GC/MS system, using the same operating
conditions that were used for the calibration. The volume to be injected should include an
appropriate concentration that is within the calibration range of base/neutral and acid
surrogates using the surrogate solution as noted in section 4.7.5.8. The injection volume
must be the same volume that was used for the calibration standards.
4. If the response for any quantitation ion exceeds the initial calibration range of the GC/MS
system, the sample extract must be diluted and reanalyzed. Additional internal standard
solution must be added to the diluted extract to maintain the same concentration as in the
calibration standards (usually 40 ng/uL, or other concentrations as appropriate, if a more
sensitive GC/MS system is being used). Secondary ion quantitation should be used only
when there are sample interferences with the primary ion.
Monitor internal standard retention times in all samples, spikes, blanks, and standards to
effectively check drifting, method performance, poor injection execution, and anticipate the
need for system inspection and/or maintenance. Internal standard responses (area counts)
must be monitored in all samples, spikes, blanks for similar reasons. If the EICP area for any
of the internal standards in samples, spikes and blanks changes by a factor of two (-50% to
+100%) from the areas determined in the continuing calibration analyzed that day, corrective
action must be taken. The samples, spikes or blanks should be reanalyzed or the data
should be qualified.

When ions from a compound in the sample saturate the detector, this analysis should be
followed by the analysis of an instrument blank consisting of clean solvent. If the blank
analysis is not free of interferences, then the system must be decontaminated. Sample
analysis may not resume until the blank analysis is demonstrated to be free of interferences.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 190

Contamination from one sample to the next on the instrument usually takes place in the
syringe. If adequate syringe washes are employed, then carryover from high concentration
samples can usually be avoided.

All dilutions should keep the response of the major constituents (previously saturated peaks)
in the upper half of the linear range of the curve.

5. The use of selected ion monitoring (SIM) is acceptable for applications requiring quantitation
limits below the normal range of electron impact mass spectrometry. However, SIM may
provide a lesser degree of confidence in the compound identification, since less mass
spectral information is available. Using the primary ion for quantitation and the secondary
ions for confirmation set up the collection groups based on their retention times. The
selected ions are nominal ions and most compounds have small mass defect, usually less
than 0.2 amu, in their spectra. These mass defects should be used in the acquisition table.
The dwell time may be automatically calculated by the laboratory's GC/MS software or
manually calculated using the following formula. The total scan time should be less than
1,000 msec and produce at least 5 to 10 scans per chromatographic peak. The start and
stop times for the SIM groups are determined from the full scan analysis using the formula
below:

Dwell Time for the Group = Scan Time (msec.)
Total Ions in the Group

4.7.8.2 Analyte identification

The qualitative identification of compounds determined by this method is based on retention
time and on comparison of the sample mass spectrum, after background correction, with
characteristic ions in a reference mass spectrum. The reference mass spectrum must be
generated by the laboratory using the conditions of this method. The characteristic ions from the
reference mass spectrum are defined as the three ions of greatest relative intensity, or any ions
over 30% relative intensity, if less than three such ions occur in the reference spectrum.
Compounds are identified when the following six criteria are met.

1. The intensities of the characteristic ions of a compound must maximize in the same scan or
within one scan of each other. Selection of a peak by a data system target compound
search routine where the search is based on the presence of a target chromatographic
peak containing ions specific for the target compound at a compound-specific retention
time will be accepted as meeting this criterion.
2. The RRT of the sample component is within ± 0.06 RRT units of the RRT of the standard
component.

3. The relative intensities of the characteristic ions agree within 30% of the relative intensities
of these ions in the reference spectrum. (Example: For an ion with an abundance of 50% in
the reference spectrum, the corresponding abundance in a sample spectrum can range
between 20 and 80%.) Use professional judgement in interpretation where interferences are
observed.
4. Structural isomers that produce very similar mass spectra should be identified as individual
isomers if they have sufficiently different GC retention times. Sufficient GC resolution is
achieved if the height of the valley between two isomer peaks is <50% of the average of the
two peak heights. Otherwise, structural isomers are identified as isomeric pairs. The
resolution should be verified on the mid-point concentration of the initial calibration as well

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 191

as the laboratory designated continuing calibration verification level if closely eluting isomers
are to be reported (e.g., benzo(b)fluoranthene and benzo(k)fluoranthene).
5. Identification is hampered when sample components are not resolved chromatographically
and produce mass spectra containing ions contributed by more than one analyte. When gas
chromatographic peaks obviously represent more than one sample component (i.e., a
broadened peak with shoulder(s) or a valley between two or more maxima), appropriate
selection of analyte spectra and background spectra is important.

6. Examination of extracted ion current profiles of appropriate ions can aid in the selection of
spectra and in qualitative identification of compounds. When analytes coelute (i.e., only one
chromatographic peak is apparent), the identification criteria may be met, but each analyte
spectrum will contain extraneous ions contributed by the coeluting compound.

4.7.9 Quantitation

1. Once a target compound has been identified, the quantitation of that compound will be
based on the integrated abundance of the primary characteristic ion from the EICP. It is
highly recommended to use the integration produced by the software if the integration is
correct because the software should produce more consistent integrations. However,
manual integrations may be necessary when the software does not produce proper
integrations because baseline selection is improper; the correct peak is missed; a coelution
is integrated; the peak is partially integrated; etc. The analyst is responsible for ensuring that
the integration is correct whether performed by the software or done manually.

Manual integrations should not be substituted for proper maintenance of the instrument or
setup of the method (e.g. retention time updates, integration parameter files, etc). The
analyst should seek to minimize manual integration by properly maintaining the instrument,
updating retention times, and configuring peak integration parameters.

2. If the RSD of a compound's response factor is 20% or less, the concentration in the extract
may be determined using the average response factor (RF) from initial calibration data.

3. Where applicable, the concentration of any non-target analytes identified in the sample
should be estimated. The same formula as in section 4.8.10.1.5 should be used with the
following modifications: The areas Ax and Ais should be from the total ion chromatograms,
and the RF for the compound should be assumed to be 1.

4. The resulting concentration should be reported indicating that the value is an estimate. Use
the nearest internal standard free of interferences.
5. Structural isomers that produce very similar mass spectra must be quantitated as individual
isomers if they have sufficiently different GC retention times. Sufficient GC resolution is
achieved if the height of the valley between two isomer peaks is <50% of the average of the
two peak heights. Otherwise, structural isomers are identified as isomeric pairs. The
resolution should be verified on the mid-point concentration of the initial calibration as well
as the laboratory designated continuing calibration verification level if closely eluting isomers
are to be reported (e.g., benzo(b)fluoranthene and benzo(k)fluoranthene).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 192

5.0 SEDIMENTS

5.1 SEDIMENT GRAIN SIZE AND CHARACTERIZATION

Suggested analytical methods for sediment grain size are described in section 5.1. However,
some laboratories participating in the survey may choose to employ other analytical methods.
Labs engaged by EPA or the State may use a different analytical method as long as the lab is
able to achieve the same performance requirements as the standard methods. Performance
data must be submitted to EPA prior to initiating any analyses. Methods performance
requirements for this program identify detection limit, precision and accuracy objectives for each
indicator. Method performance requirements for sediment grain size are shown in Table 5.1

Table 5.1. Laboratory method performance requirements for sediment grain size.
Indicator/Data Type Maximum Allowable
Accuracy (Bias) Goal
Particle Size NA
Maximum Allowable
Precision Goal
10%
Completeness
Goal
95%
5.1 .1 Scope of Application
Silts and Clays are those particles that pass through a 63 urn mesh sieve. Materials retained on
the sieve are primarily sands (63 urn - 2 mm) and occasionally small amounts of gravel (2 mm -
64 mm). This method is suitable for both saltwater and freshwater sediments.

5.1.2 Sample Storage and Equipment

Sediment samples must be chilled at 4-5°C prior to processing. Samples must not be allowed to
dry before grain size analyses are conducted. Sieves used to determine sediment grain size will
not be used for other purposes (e.g., benthic sorting). All wet sieving procedures are to be
carried out using stainless steel screens. Fine screens (63 urn mesh) will be cleaned with
copious amounts of water to prevent clogging of mesh openings. Screens must not be cleaned
with brushes, which may distort openings. Sediments will not be forced through screens.

An analytical balance accurate to 0.1 mg will be used for all weighing. Prior to each use, the
balance will be zeroed, and its calibration will be checked using a standard weight. The same
standard weight (each standard is numbered) will be used for all weight measurements for a
particular batch of samples.

5.1.3 Procedures for Silt-Clay Content Determination

5.1.3.1 Sediment Preparation
1. Sediment samples will be retrieved from cold storage and brought to room temperature.
Sample numbers will be recorded on a silt-clay analysis data sheet.

2. Sediments will be removed from storage bags, placed in a clean 250 ml glass beaker and
homogenized by stirring the sediment with a small spatula with a small amount of deionized
water added for lubrication (if necessary) for at least three minutes. After stirring, rinse
sediment from the spatula back into beaker using deionized water.

3. The amount of sediment to be processed depends upon sediment type. With more sample,
the grains interfere with each other too much during settling and may flocculate; with too
little sample, the experimental error in weighing becomes large with respect to sample size.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
_ Page 193

4. For sandy sediments, -45-50 g wet weight are removed from the 250 ml glass beaker and
placed in a clean 100 ml glass beaker for wet sieving. For muddy sediments, -20-25 g wet
weight are removed from the 250 ml glass beaker to a 100 ml glass beaker for wet sieving.

5. The remaining unused sediment will be returned to the original storage bag and held in cold
storage until all QA/QC checks for this sample have been passed.

5.1.3.2 Dispersion of Sediment Clay Fraction and Wet Sieving the Sample

1 . Make-up a 5 g/L stock solution of dispersant. Add 5 grams of sodium exametaphosphate
"Calgon"to 1 liter of deionized water.

2. Add 20 ml of dispersant solution (100 mg hexametaphosphate) and 30 ml distilled water to
the sample. Stir, using a magnetic stirrer for 1 to 5 mins to break-up sediment aggregrates.

3. After stirring, the sample will be wet sieved through a 63 urn mesh sieve into a large
evaporation dish and wash all fines into the sieve using as little distilled water as possible.

4. The volume of sediment + water in the evaporation dish must be < 900 ml to allow for
rinsing the sample into a 1000 ml graduated cylinder.
5.1.3.3 Treatment of the Silt and Clay Fraction (particles <
1. Carefully transfer mud in evaporation dish to a 1000 ml graduated cylinder. Rinse the mud
(generally medium-coarse silt-size (16-63 urn particles) found at the bottom of the dish into
the graduated cylinder using deionized water, being careful not to exceed 1000 ml mark.

2. Set the sediment fraction remaining in the sieve aside.

3. Fill the cylinder with deionized water up to the 1000 ml mark. Using a metal stirring rod,
vigorously stir the water column from bottom to top, using short strokes, starting at the base
of the column and working upwards. Keep stirring until the material is distributed uniformly
throughout the column. End up stirring with long, smooth strokes the full length of the
column. Be careful not to break the water surface as material could be lost. Place a beaker
with tap water next to the cylinder and insert a thermometer to record water temperature.

4. Immediately (<20 sec) after stirring, withdraw 40 ml of sample using a 40 ml volumetric
pipette. Expel sample into a recorded 50 ml glass beaker. Rinse pipette with a small volume
of deionized water, and add the rinse to the 50 ml beaker. If the sample is taken in two parts
(i.e., two 20-ml samples), the cylinder will be stirred between extractions and samples
withdrawn after each stirring and added to the beaker.

5. The 50 ml glass beaker will be placed in an oven at 100°C until dry (typically 24 hours). A
randomly selected subsample of each batch will be reweighed after an additional 24 hour
drying period, as a check for the stability of the dry weight measurement.

5.1.3.4 Treatment of Sand (>63 mm) Fraction Retained on Sieve
If necessary, remove shell and shells fragments, pieces of wood and algae. Air dry and record
weight on the biomass data sheet.

The sand fraction (>63 urn) is transferred to a recorded 50 ml glass beaker and placed in an
oven at 100°C until dry (typically for 24 hrs). A randomly selected subsample of each batch is
reweighed after an additional 24-hour drying period to check for stability of the dry weight
measurement.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 194

5.1.3.5 Analysis of Samples

1. After drying, remove beaker from oven and let equilibrate with the atmosphere for 1.5 hr
before weighing. Weigh each fraction sample to nearest 0.001 grams and record weight.

2. Calculate weight of silt-clay fraction using correction factor as follows:

Silt-Clay wt. = Net wt. x (total volume in cylinder) - dispersant wt.
(sample volume from cylinder)

Note: The total volume in cylinder is 1000 ml. The sample volume from cylinder is 40 ml. Using
the prescribed methods, this results in a dispersant weight of 4 mg.

3. Calculate percent silt-clay fraction

% silt-clay = silt-clay wt. x100
(sand wt. + silt-clay wt.)

4. Calculate percent sand

% sand = 100 - % silt-clay

Note: (100 - % mud) is not, in all cases, equal to the percent sand, since gravel sized
particles (>2mm but < 64 mm) may be present in some samples

5.1.4 Procedures for Percent Water Content
The percent water content of the sample is needed to correct sediment dry weights for salt
content, since salts are left behind during the drying process.

5.1.4.1 Sample Preparation
1. Retrieve sediment samples from cold storage and bring to room temperature. Record
sample numbers on data sheet. Samples that have dried cannot be used.
2. Remove sediment from storage bag, place in a 250 ml beaker and homogenize.
Homogenization will be accomplished by stirring the sediment with a small spatula for at
least three minutes. Do not rinse the spatula and do not add water to the beaker during the
homogenization process.

3. Place approximately 5-10 grams wet weight of sediment into a clean recorded 50 ml glass
beaker and weight immediately. The sample must not be allowed to stand at room
temperature for more than a few minutes since evaporation will affect the water content
measurement. Store any unused sediment from each sample at 4°-5°C for QA/QC analysis
and other sediment analyses.

4. Place the sample in a drying oven at 100°C until dry (typically 24 hours). Store dry samples
in a dessicator containing hydrous silica gel until cooled (1 hour).

Note: dry samples may absorb moisture from wet sample, thus dry samples should be
removed before placing moist samples in the oven.

5. All weight is recorded on data sheets to the nearest 0.001 gram. Ten percent of randomly
selected subsamples of each batch are reweighed after an additional 24 hour drying period
as a check for stability for the dry weight measurement (a change of <0.1% is expected).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 195

5.1.4.2 Calculation of Sediment Water Content

Water loss (ml) = Gross wet wt. - Gross dry wt.
% water = (Gross wet wt. - Gross dry wt.) x 100
Net wet wt.

For saline sediments a correction factor must be applied because dry salts are included in the
dry sediment weight. For this value use the bottom salinity recorded during field measurements:
Salt wt. (g) = Water loss (ml) x Salinity (mg/ml or ppt)

Corrected dry weight = Gross dry wt. - Salt wt.

Corrected percent water = (Gross wet wt. - Corrected dry wt.) x 100
Net wet wt.

Note: Assume a water density of 1g/ml for the (fresh) water that evaporates

5.1.5 Procedures for Sediment Grain Size Distribution
This procedure is used to determine the percent by weight of soil and clays in sediment
samples. It provides a method for determination of weight percent quantiles for sediments as
well as the quantile deviation of skewness.

5.1.5.1 Sample Preparation
1. Retrieve sediment samples from cold storage and bring to room temperature. Record
sample numbers on data sheet. Return unused portion to storage.

2. For sandy sediments, remove -45-50 g wet weight. For muddy sediment remove -20-25 g
wet weight. Place sediment into a 250 ml beaker for homogenization.

3. If the sample is <20% silt-clay, skip the next step and proceed to step 5 of this section.
4. If the sample is greater than 20% silt-clay, the organics in the sample must be removed as
follows: Initially, add enough deionized water to cover the sample. Add small quantities of
30% H2O2 to the sample, stirring until any effervescence ceases. Cover beaker with large
watch glass cover if frothing is excessive. If the solution heats excessively, cool the beaker
in a water bath. Continue adding hbChuntil frothing ceases, then slowly heat to 60°-70°C
(hhO2 decomposes above 70°C). Observe for 10 minutes to ensure that the possibility of a
strong reaction has passed. Add hbCh until no further reaction occurs. An accepted
alternative method, such as the combustion method, may also be used so long as it has
been documented and does not impact the QA.
5. Homogenize the sample by stirring with a spatula and a small amount of deionized water
for at least three minutes.

5.1.5.2 Dispersion of Clay Fraction of Sediments and Wet Sieving the Sample
1. Make-up a 5g/L stock solution of dispersant. Add 5 grams of sodium exametaphosphate
"Calgon"to 1 liter of deionized water.

2. Add 20 ml of the dispersant solution (100 mg of hexametaphosphate) and 30 ml of distilled
water to the sample. Stir, using a magnetic stirrer for one to five minutes to break-up
sediment aggregrates.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
                                                                 Date: November 2010
                                                                           Page 196
After stirring, the sample will be wet sieved through a 63 urn mesh sieve into a large
evaporation dish and wash all fines into the sieve using as little distilled water as possible.
The volume of sediment + water in the evaporation dish must be < 900 ml to allow for
rinsing the sample into a 1000 ml graduated cylinder.
3.
4.
5.1.5.3 Treatment of the Silt and Clay Fraction (particles <
1.   Carefully transfer mud in evaporation dish to a 1000 ml graduated cylinder. Rinse the mud
    (generally medium-coarse silt-size (16-63 urn particles) found at the bottom of the dish into
    the graduated cylinder using deionized water, being careful not to exceed 1000 ml mark.

2.   Set the sediment fraction remaining in the sieve aside.

3.   Fill the cylinder with deionized water up to the 1000 ml mark. Using a metal stirring rod,
    vigorously stir the water column from bottom to top, using short strokes, starting at the base
    of the column and working upwards. Keep stirring  until the material is distributed uniformly
    throughout the column. End up stirring with long, smooth strokes the full length of the
    column. Be careful not to break the water surface as material could be lost. Place a beaker
    with tap water next to the cylinder and insert a thermometer to record water temperature.

5.1.5.4 Analysis of the Silt and Clay (<63jjm) Fraction

1 .   Stir the cylinder to suspend the sample. As soon as the stir rod emerges for the last time,
    start the timer. At the end of 20 seconds, insert the pipette to a depth of 20 cm and
    withdraw exactly 20 ml. This is  the most important single step in this exercise as
    subsequent analyses are based on the calculation of the total mud weight.

2.   Transfer the pipette sample fractions to separate recorded 50 ml glass beakers. Each
    pipette withdrawal should be rinsed with a small volume of deionized water which is then
    added to the 50 ml sample beaker.

3.   Continue to withdraw 20-ml samples with the 20 ml volumetric pipette at the depths and
    times indicated in Table 5.2 for the recorded water  temperature.

Table 5.2.  Sampling Time Intervals

EH
nrs.MT.TFH


4.0
4.5

5. it
5.5
6.0
6.3
7,0
7.5
*.D
R.S
!?.!)

EXA.VETER
Wfcmqiri


62.5
44

31
22
16
11
7,8
5.5
3.$
2.8
2.0
IfJfSl'Ki
SAMFLNG
71FFTK
fun)
Sl.irlTimc:
20
10
R«Stir«:ifJl:
10
JO
10
JO
10
10
10
s
?

IS
4w:l!i
0:18:^
0:33;42
1:04:44
2:05:47
?.:(vj:M
3:56:26
TTYichta'ulu
21
dyrccaC

0:00: on
0:UO:2I)
0:Ul:53
n:rj3:on
0:04:54
O.Ofi.46
0:!0:07
0:?s:oi
0:32:5fi
1:03:13
2:02:45
1:59'. 10
3:50.41
•.ULOl.LllUJJ
22
i*at,v

0:00:00
0:00:10
0:01:50
0:0^:00
0:04:51
0:06:^10
0:OD:57
0:17:4?.
0:32:13
1:01:46
1:59:5J
LS6:23
3.45:13
ii5,inii-jlcs,ra
13
decree; C

0:00:00
0:00:20
0:01:4S
0:03:00
0:04:4*
0:06.? 5
0:OB:47
0:17:z;
0;3I:32
1:00:24
1:*7:D9
1:53.43
3:40 .D]
dn«airiE(b:ra
24
i1u>imi n

0:00:00
(t:(IO;2(>
0:01:45
0 03:00
0:tM:46
0.06.30
0:OU:38
0.17:0]
0-30;53
tt:51>:05
1:54:32
1:51:11
3:35:03
IH:M).
25
dwrK-if

0 OC:00
0:00:20
0.01:^3
0 03:00
0:04:44
0.0«:26
0:0'J:2'J
0.1fi:4?
0-3fl;lS
0:57:^
1:52:02
1.4<:i«
3. 3ft: IS

26
fry-***

0.00:00
D-OO-IO
0*11:40
003:00
0:04:41
0.06:31
O.O'J:!';
0:16:2?
0-19:3(i
Ll_^6:30
1:49:23
1.46.12
3.25 Ifi

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                              Date: November 2010
	Page 197

 4.   Each beaker should be placed in an oven at 100°C for 24 hrs. All weights are to be
     recorded on the data sheet.

 5.1.5.5 Calculations for Determining Sample Withdrawal Times for Pipette Analysis

 Sample withdrawal times for pipette analysis are based upon Stoke's law written as:

                           T=    Depth
                              1500*A*(d2)
       where:
       T is time in minutes,
       Depth is in centimeters,
       A is a constant, and
       d is the  particle diameter in millimeters.

 The A value is a function of temperature, gravity, and density of particles. Assume a density of
 2.65 (associated with quartz  or clay minerals). The following table relates various temperatures
 to the constant A.
Temp. (°C)
A
20
3.57
21
3.66
22
3.75
23
3.84
24
3.93
25
4.02
26
4.12
 5.1.5.6 Removal Of Carbonates (if warranted)
 1.  If a sediment contains >50%, by weight, of calcareous material, the sample is described as
    a carbonate sediment.  Process carbonate sediments as follows.

 2.  Record the weight of the sand fraction to 0.001 grams.  A 10% (by volume) HCI solution will
    be added to the dried and weighed sediment. Cover the sediment completely with HCI and
    let sit for four hours. Additional acid will be added and if foaming is apparent, the sample will
    be left to stand for several more hours. This process will be repeated until no further reaction
    occurs with subsequent additions of HCI.

 3.  The sample will be transferred to a 63 urn sieve and washed using copious amounts of
    deionized water. This will remove any salts formed during the acidification step.

 4.  Transfer the  sample to a 100 ml glass beaker, dry, and weigh to 0.001 grams.

 5.  Calculate the sediment carbonate content
       % carbonate = (wt. of sand (before acid) - wt. of sand  (after acid))
                                 wt. of sand (before acid)

 5.1.5.7 Treatment Of The Sand (>63jjm) Fraction

 1.   1 Transfer the >63 urn sand fraction to a 250 ml glass  beaker and place in a drying oven at
     100°C until dry (typically 24 hrs).

 2.   Transfer the dried sediment into the top of a stack of clean, stainless steel sieves
     composed of 500 urn (1.0 0), 355 urn (1.5 0), 250 urn (2.0 0), 180 urn (2.5 0), and 125
     urn (3.0 0),  90 urn (-3.5 0) and 63 urn (4.0 0) sieve with a closed pan on the bottom.
     Shake on a  rotary tapper (Ro-tap) for 15 minutes.

 3.   Weigh each sieved fraction as follows: Tare a 100 ml beaker to zero; add the 500 urn (1.0
     0) sediment fraction and weigh to 0.001 grams. Next add the 355 urn (1.5 0) fraction to the

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                             Date: November 2010
	Page 198
     beaker. Proceed to add subsequent fractions until all weighed. Record the individual and
     cumulative weights of the sand fraction to 0.001 grams.
 5.1.6  Calculations for Sediment Grain Size Distributions
 1.   Calculate total weight of mud (silt-clay) in sample (obtained at 20 sec withdrawal intervals).
              Total mud weight (g) = (sample wt. x 50) - dispersant weight
              Dispersant weight = 0.1 gram
     Note: the amount of mud in each 20 ml withdrawal is equal to 1/50 of the total amount of
     mud remaining in the 1000 ml cylinder at the withdrawal time and at the withdrawal depth.
 2.   Calculate the total sample weight
              Total sample weight (g) = total mud weight + total sand weight
 3.   Determine cumulative percentages for each mud (<63 urn size fraction
     Each pipette sample represents material in the column finer than a certain grain size. First
     multiply each size fraction by 50, subtract the weight of dispersant.
     Fraction wt. (g) for each 0 size = (Wt. of sand for each 0 size X 50) - Dispersant wt. (g)
     Then divide each fraction by the total sample weight, subtract from 1, and multiply the
     product by 100 to determine cumulative percentages for each sand (<63 urn) fraction.
       Cumulative % (for each 0)  =   1  - Fraction wt.  (g) for each 0 size   x 100
                                         Total sample weight (g)
 4.   Determine cumulative percentages for each sand  (>63 urn) size fraction
       % Wt. of each sand fraction = Wt. of sand for each 0 size   x 100
                                     Total sample weight
       Add the percentages  incrementally to obtain cumulative weight percentages.
 5.1.7  Determination  of Statistical Parameters Of Grain Size
 1.   Plot the cumulative curve of the sample and  read the 0 values which correspond to the
     24th (025), 50th (i.e., median (Md0)) and 75th (075) percentiles by linear interpolation.
 2.   Calculate the Phi Quartile Deviation (QD0 and Phi Quartile Skewness (Skq0). Record the
     Md0, QD0, and Skq0 for each sample.
       Phi Quartile  Deviation = (075 - 025) / 2
       Phi Quartile  Skewness = (0™+ 0^) - (2 x Md a)

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 199

5.2 ASSESSING SEDIMENT TOXICITY USING ESTUARINE AND MARINE AMPHIPODS

Suggested analytical methods for sediment toxicity are described in sections 5.2 and 5.3. Some
laboratories participating in the survey may choose to employ other analytical methods. Labs
engaged by EPA or the State may use a different analytical method as long as the lab is able to
achieve the same performance requirements as the standard methods. Performance data must
be submitted to EPA prior to initiating any analyses. Methods performance requirements for this
program identify detection limit, precision and accuracy objectives for each indicator. Method
performance requirements for sediment toxicity are shown in Table 5.3.

Table 5.3. Laboratory method performance requirements for sediment toxicity.
Indicator/Data Type
Sediment toxicity
Maximum Allowable
Accuracy (Bias) Goal
NA
Maximum Allowable
Precision Goal
NA
Completeness
Goal
95%
5.2.1 Scope of Application

This method is for use with sediments from oligohaline to fully marine environments. Procedures
are described for testing estuarine and marine amphipod crustaceans in the laboratory to
evaluate the toxicity of contaminants associated with whole sediments. This method may only
be used for salt water sediments.

5.2.2 Summary of Method
Sediments are collected from nearshore coastal sites using a modified Van Veen or ponar grab
sampler. A toxicity method is outlined for Leptocheirus plumulosus, an Atlantic coast estuarine
sediment-burrowing amphipod species. The toxicity test is conducted for 10 d in 1 L glass
chambers containing 175 mL of sediment and 800 mL of overlying seawater. Exposure is static
(i.e., water is not renewed), and the animals are not fed over the 10 d exposure period.
Sediment tests include control sediment (sometimes called a negative control). The endpoint in
the toxicity test is survival. Procedures are described for use with sediments with pore water
salinity ranging from > 0%o to fully marine.

5.2.3 Interferences
1. Interferences are characteristics of a sediment or sediment test system that can potentially
confound interpretation of test results. There are three categories of interfering factors:
those characteristics of sediments affecting survival independent of chemical concentration
(i.e., non-contaminant factors); changes in chemical bioavailability as a function of sediment
manipulation or storage; and the presence of indigenous organisms.

2. There are a number of non-contaminant factors that may influence amphipod survival in
these tests. The most important and variable factors include sediment particle size, pore
water salinity, and pore water ammonia. The physico-chemical properties of each test
sediment must be within the tolerance limits of the test organism.

3. Sediment collection, handling, and storage may alter contaminant bioavailablity and
concentration by changing the physical, chemical, or biological characteristics of the
sediment. These manipulation processes are generally thought to increase availability of
organic compounds because of disruption of the equilibrium with organic carbon in the pore
water per particle system. Similarly, oxidation of anaerobic sediments increases the
availability of certain metals. Because the availability of contaminants may be a function of

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 200

the degree of manipulation, it is recommended that handling, storage, and preparation of the
sediment for actual testing be as consistent as possible.
Sediment samples should not be sieved to remove indigenous organisms unless there is a
good reason to believe indigenous organisms may influence the response of the test
organism. However, large indigenous organisms and large debris can be removed using
forceps. Reynoldson et al. (1994) observed reduced growth of amphipods, midges, and
mayflies in sediments with elevated numbers of oligochaetes and recommended sieving
sediments suspected to have high numbers of indigenous oligochaetes. If sediments must
be sieved, it may be desirable to analyze samples before and after sieving (e.g., pore-water
metals, DOC, and AVS) to document the influence of sieving on sediment chemistry.

5. Depletion of aqueous and sediment-sorbed contaminants resulting from uptake by an
organism or test chamber may also influence availability. In most cases, the organism is a
minor sink for contaminants relative to the sediment. However, within the burrow of an
organism, sediment desorption kinetics may limit uptake rates. Desorption of a particular
compound from sediment may range from easily reversible (labile; within minutes) to
irreversible (non-labile: within days or months).

6. Salinity of the overlying water is an additional factor that can affect the bioavailability of
metals. Some metals (e.g., cadmium) are more bioavailable at lower salinities. Therefore, if
a sediment sample from a low salinity location is tested with overlying waters of high salinity,
there is the potential that metal toxicity may be reduced.

5.2.4 Equipment and Supplies
1. The facility should include separate areas for culturing and testing to reduce the possibility
of contamination by test materials and other substances, especially volatile compounds.
Holding, acclimation, and culture chambers should not be in a room where sediment tests
are conducted, stock solutions or sediments are prepared, or where equipment is cleaned.

2. Equipment and supplies that contact stock solutions, sediments or overlying water should
not contain substances that can be leached or dissolved in amounts that adversely affect
the test organisms. In addition, equipment and supplies that contact sediment or water
should be chosen to minimize sorption of test materials from water. Glass, type 316
stainless steel, nylon, high-density polyethylene, polycarbonate and fluorocarbon plastics
should be used whenever possible to minimize leaching, dissolution, and sorption. High-
density plastic containers are recommended for holding, acclimation, and culture chambers.

3. Environmental chamber (or equivalent) with photoperiod and temperature control of 5-25°C.

4. Water purification system capable of producing at least 1 mega-ohm water.

5. Analytical balance capable of accurately weighing to 0.01 mg.
6. One liter glass containers (beakers or wide-mouthed jars) with an internal diameter of 10 cm
serve as test chambers. Each test chamber should have a cover. Acceptable test chamber
covers include watch glasses, plastic lids, and 9 cm diameter glass culture dishes. It may be
necessary to drill a hole in each cover to allow for the insertion of a pipette for aeration. A
full list of equipment and supplies is in Table 5.4 below.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 201
Table 5.4. Equipment and Supplies for Culturing and Testing Estuarine and Marine Amphipods.
A. Biological
Supplies
Brood stock of test organisms
TetraMin®
dried wheat leaves
dried alfalfa leaves Neo-Novum®
Algae (e.g., Pseudoisochrysis paradnxa
and Phaeoductylum tricornutum
[optional])
B. Glassware
Culture chambers (30 cm x 45 cm x 15 cm
plastic wash bin)
Test chambers (1 L glass jar or beaker)
Glass bowls
Wide-bore pipets (4 to 6 mm ID)
Glass disposable pipets
Graduated cylinders (assorted sizes, 10
ml to 4 L)
C. Instruments
and Equipment
Dissecting microscope
Stainless-steel sieves (e.g., U.S.
Standard No. 25, 30, 35, 10, 50 mesh)
Photoperiod timers
Light meter
Temperature controllers
Thermometer
Continuous recording thermometer
Photoperiod timer
Dissolved oxygen meter
pH meter
Selective ion meter
Ammonia electrode (or ammonia kit)
Salinity meter/temperature compensating
salinity refractometer
Drying oven
Desiccator
Balance (0.01 mg sensitivity)
Refrigerator
Freezer
Light box
Hemacytometer
Mortar and pestle or blender
D.
Miscellaneous
Air supply and air stones (oil free and
regulated)
Glass hole-cutting bits
Glass glue
Aluminum weighing pans
Deionized water
Sieve cups (mesh size 10.5 mm)
Dissecting probes
E. Chemicals
Acetone (reagent grade)
Hexane (reagent grade)
Hydrochloric acid (reagent grade)
Reagents for preparing synthetic seawater
(reagent grade): CaCI2»2 H2O, KBr,
KCI, MgCI2«6 H2O, Na2B4O7'IO H2O,
NaCI, NaHCO3, Na2SO4, SrCI2«6 H2O
Formalin
Ethanol
Rose bengal
Cadmium chloride
Sodium dodecyl sulfate
Copper sulfate
Detergent (non-phosphate)
5.2.5 Reagents and Water

5.2.5.1 Reagents

See Table 5.2. Section E above for a list of chemicals and reagents. All reagents should be at
least reagent grade, unless a test on formulation commercial product, technical-grade, or use-
grade material is specified.

5.2.5.2 Sea water

Sea water used to test and culture organisms should be uniform in quality. Acceptable sea
water should allow satisfactory survival, growth, or reproduction of the test organisms. Test
organisms should not show signs of disease or apparent stress (e.g., discoloration, unusual
behavior). If problems are observed in the culturing or testing of organisms, it is desirable to
evaluate the characteristics of the water.

Reconstituted sea water is prepared by adding specified amounts of reagent grade chemicals to
high-purity distilled or deionized water. Suitable salt reagents can be reagent grade chemicals,
commercial sea salts, such as Forty Fathoms®, Instant Ocean®, or HW Marinemix®. Pre-
formulated brine (e.g., 60 to 90%) prepared with dry ocean salts or heat-concentrated natural
sea water can also be used. Acceptable high-purity water can be prepared using deionization,

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 202

distillation, or reverse-osmosis units. Test water can also be prepared by diluting natural water
with deionized water.

5.2.6 Sample Manipulation

1. Homogenization

Sediment samples tend to settle during shipment. Water above the sediment is not discarded,
but is mixed back into the sediment during homogenization. Sediment samples should not be
sieved to remove indigenous organisms unless there is a good reason to believe indigenous
organisms may influence the response of the test organism (See Section 5.3.3, #3).

2. Analytical Methodology

The precision, accuracy, and bias of each analytical method used should be determined in the
appropriate matrix: that is, sediment or water. Reagent blanks and analytical standards should
be analyzed and recoveries should be calculated.
Measurement of test material(s) concentration in water can be accomplished by pipeting water
samples from about 1 to 2 cm above the sediment surface in the test chamber. Overlying water
samples should not contain any surface debris, any material from the sides of the test chamber,
or any sediment. Measurement of test material(s) concentration in sediment at the end of a test
can be taken by siphoning most of the overlying water without disturbing the surface of the
sediment, then removing appropriate aliquots of the sediment for chemical analysis.

5.2.7 Quality Control

1. Before a sediment test is conducted in any test facility, the analyst should conduct a "non-
toxicant" test with the potential test species in which all test chambers contain a control
sediment (sometimes called the negative control), and clean overlying water for the
amphipod species to be tested. Survival of the test organism will demonstrate whether
facilities, water, control sediment, and handling techniques are adequate to achieve
acceptable species-specific control survival. Evaluations may also be conducted of the
magnitude of the within- and between-chamber variance in a test. For the test to be
acceptable, survival at 10 d must equal or exceed 90% in the control sediments.
2. If the supplier has not conducted five reference toxicity tests with the test organism, the
testing laboratory must do so before starting a sediment test. Intralaboratory precision,
expressed as a coefficient of variation of the range in response for each type of test to be
used in a laboratory should be determined by performing five or more tests with different
batches of test organisms, using the same reference toxicant, at the same concentrations,
with the same test conditions (e.g., the same test duration, type of water, age of test
organisms) and same data analysis methods. A reference toxicant concentration series (0.5
or higher) should be selected that will consistently provide partial mortalities at two or more
concentrations of the test chemical (section 5.3.10).

Reference toxicants such as cadmium (available as cadmium chloride (CdCI2)), copper
(available as copper sulfate (CuSO4)), and sodium dodecyl sulfide (SDS) are available for
use. No one reference toxicant can be used to measure the condition of test organisms in
respect to another toxicant with a different mode of action. Test conditions for conducting
reference-toxicity tests with L. plumulosus are outlined in Table 5.5.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 203
Table 5.5.  Recommended Test Conditions for Conducting Reference-Toxicity Tests
Parameter
1. Test type:
2. Dilution series:
3. Toxicant:
4. Temperature:
5. Salinity:
6. Light quality:
7. Photoperiod:
8. Renewal of water:
9. Age and size of test organisms:
10. Test chamber:
11. Volume of water:
12. Number of organisms per chamber:
1 3. Number of replicate chamber per
/treatment:
14. Aeration:
15. Dilution water:
16. Test duration:
17. Endpoint:
18. Test acceptability:
Conditions
Water-only test
Control and at least 5 test concentrations ((0.5 dilution factor)
Cd, Cu, Sodium dodecyl sulfate (SDS)
25°C for L. plumulosus
20%o
Chambers should be kept in dark or colored with opaque
material
24 hD
None
L. plumulosus 2 - 4 mm (no mature males or females)
1 L glass beaker or jar
800 ml (minimum)
n = 20 if 1 per replicate; n = 1 0 (minimum) if >1 per replicate
1 minimum; 2 recommended
Recommended: but not necessary if >90% dissolved oxygen
saturation can be achieved without aeration
Culture water, surface water, site water, or reconstituted
water
96 h
Survival (LC50);
90% control survival
3.   Before conducting tests with contaminated sediment, the laboratory should conduct five
    exposures in control sediment. It is recommended that these five exposures with control
    sediment be conducted concurrently with the five reference toxicity tests described above.

4.   Each lab must work with the Information Management group (Marlys Cappaert,
    Cappaert.Marlys@epamail.epa.gov,  541-754-4467,) to ensure their bench sheets and/or
    data recording spreadsheets are compatible with the electronic deliverables the lab will
    need to submit.

5.2.8   Culturing and Maintaining Test  Organisms

1.  The quality of test organisms obtained from an outside source, regardless of whether they
   are from culture or collected from the  field, must be verified  by conducting a reference-
   toxicity test concurrently with the sediment test. For cultured organisms, the supplier should
   provide data with the shipment describing the history of the  sensitivity of organisms from the
   same source culture. For field-collected organisms, the supplier  should provide data with the
   shipment describing the collection location, the time and date of collection, the water salinity
   and temperature at the time of collection, and collection site sediment for holding and
   acclimation purposes.

2.  L. plumulosus used in tests should be pre-reproductive animals that are 2 to 4 mm in length.
   To obtain animals in this size range, sediment from culture chambers containing mixed-size

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 204

amphipods should be poured over a sieve series that consists of the following sequence of
mesh sizes: 0.71 mm, 0.50 mm, and 0.25 mm. Animals retained on the 0.50 mm mesh
screen should be washed into a shallow glass pan. The smaller animals from this group
should be selected for toxicity testing. Gravid females should be avoided.

Alternatively, test animals within a narrow size range are obtained by isolating the smallest
amphipods which are allowed to grow until they reach a testable size. To obtain the smallest
amphipods, transfer sediment from culture chambers containing mixed-size amphipods over
a sieve series that consists of the following sequence of mesh sizes: 1.0 mm, 0.5 mm, and
0.25 mm. Animals retained on the 0.25 mm mesh screen should be small juveniles that are
1.1 to 2.0 mm in length. They will take ~2 weeks to reach testable size after isolation. The
amphipods retained on the 0.25 mm screen are washed into a culture chamber that is set up
as a normal culture chamber, i.e., containing a thin (-1 cm) sediment layer and maintained
under culture conditions for the 2-week interim period. By the end of the 2-week grow-out
period, the animals should be of testable size and be within a narrow size and age range.

3. Holding and Acclimation
Density. Amphipods should be held and acclimated (if necessary) in containers (4 to 8 L
volume) that contain a 2 to 4 cm layer of collection site sediment that has been sieved
through a 0.5 mm mesh screen. Approximately 350 amphipods should be added to each 8 L
container. Amphipod density should not exceed 1 amphipod/cm2.

Duration. Depending on temperature and salinity at the collection site, amphipods may have
to be acclimated to standard test conditions. If necessary, changes in temperature or salinity
to bring amphipods from the collection site conditions to the test conditions should be made
gradually. Once test conditions are achieved, amphipods should be maintained at these
conditions for at least two days before testing to allow for acclimation. Amphipods held for
more than ten days should not be used for testing.

Temperature. Overlying water temperature must not be changed by more than 3 °C per day
during acclimation to the test temperature. Once the test temperature is reached,
amphipods must be maintained at that temperature for a minimum of 2 days.

Salinity. The target test salinity for L. plumulosus is 20%o. It is likely that the collection site
salinity will be considerably lower than this. Upon arrival in the laboratory, the water used to
hold the organisms should be adjusted to 20%o by adjusting the salinity in the holding
container at a rate that must not exceed 5%o per 24 h. The amphipods should be
maintained at 20%o for 2 days before testing.

5.2.9 Procedure

1. The 10-d sediment toxicity test with L. plumufosus must be conducted at the species-
specific temperature and salinity with a 24 h light photoperiod at an illuminance of about 500
to 1000 lux. Test chambers are 1 L glass chambers containing 175 ml_ of sediment and 800
ml_ of overlying reconstituted seawater. Five replicates are recommended for routine testing.
Exposure is static (i.e., water is not renewed), and the animals are not fed over the 10 d
exposure period. Conditions for conducting a 10-d sediment toxicity test are summarized in
Table 5.6.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 205
Table 5.6. Test Conditions for Conducting a10-d Sediment Toxicity Test
Parameter
1 . Test type:
2. Temperature:
3. Salinity
4. Light quality:
5. Illuminance:
6. Photoperiod:
7. Test chamber:
8. Sediment volume:
9. Overlying water volume:
10. Renewal of overlying water:
1 1 . Size and life stage of amphipods:
12. Number of organisms per chamber:
13. Number of replicate
chambers/treatment:
14. Feeding:
15. Aeration:
16. Overlying water:
17. Overlying water quality:
18. Test duration:
19. Endpoints:
20. Test acceptability:
Conditions
Whole sediment toxicity test, static
25°Cforl_. plumulosus
20%o
Wide-spectrum fluorescent lights
500 -1000 lux
24LOD
1 L glass beaker or jarwith ~10 cm I. D.
1 75 ml (2 cm)
800 ml
None
L. plumulosus: 2-4 mm (no mature males or females)
20 per test chamber
4 (minimum); 5 (recommended)
None
Water in each test chamber should be aerated overnight
before start of test and throughout the test aeration at rate that
maintains >90% saturation of dissolved oxygen concentration
Clean sea water, natural or reconstituted water
Temperature daily; pH, ammonia, salinity, and DO at test start
and end.
10 d
Survival
Minimum mean control survival of 90%
2.  Introduction of Sediment (Day -1).  One day prior to the addition of amphipods, field
   collected test sediment is homogenized by stirring in the sediment storage container or by
   using a rolling mill, feed mixer, or other suitable apparatus. Control and reference sediments
   are included.  Excess water on the surface of the sediment can indicate separation of solid
   and liquid components.

   A 175ml_ aliquot of thoroughly homogenized sediment is added to each test chamber. It is
   important that an identical volume be added to each replicate test chamber: at a minimum
   the volume added should equate to a depth of 2 cm in the test chamber.

3.  Addition of Overlying Water (Day -1). To  minimize disruption of sediment as test seawater is
   added, a turbulence reducer should be used. The turbulence reducer may be either a disk
   cut from polyethylene, nylon,  or Teflon® sheeting (4 to 6 mil), or a glass petri dish attached
   (open face up) to a glass pipette. If a disk is used as the turbulence reducer, it should fit the
   inside diameter of the test chamber and have attached a length of nylon monofilament (or
   nontoxic equivalent) line. The turbulence reducer is positioned just above the sediment
   surface and raised as sea water is added to the 750-mL mark on the side of the test
   chamber. The turbulence reducer is removed and rinsed with test sea water between
   replicates of a treatment. A separate turbulence reducer is used for each treatment. The

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 206

test chambers should be covered, placed in a temperature controlled water bath and gently
aerated.
4. Addition of Amphipods (Day 0). On the day of the test, add amphipods to the test chambers.
Approximately one-third more amphipods than are needed for the test should be sieved
from the culture or control sediment in the holding container and transferred to a sorting tray.
L. plumulosus should be isolated using a 0.5 mm sieve. Sieving should be conducted with
sea water of the same temperature and salinity as the holding and test water.

Once isolated, active amphipods should be randomly selected using a transfer pipette or
other suitable tool (not forceps), and distributed among dishes or cups containing
approximately 150 mL of test sea water until each container has twenty amphipods. The
distribution of amphipods to the test chambers must be executed in a randomized fashion.

Amphipods should be added to test chambers without disruption of the sediment by placing
a 6-mil polyethylene, nylon, or Teflon® disk on the water surface and gently pouring the
water and amphipods from the sorting container over the disk into the test chamber. The
disk should be removed once the amphipods have been introduced. Alternatively,
amphipods from the sorting container can be poured into a sieve cup (mesh size <0.5 mm)
and gently washed into the test chamber with test sea water. Any amphipods remaining in
the sorting container should be gently washed into the test chamber using test sea water.
The water level should be brought up to the 950 mL mark, the test chamber covered, and
aeration continued.

After the addition of the animals, the test chambers should be examined for animals that
may have been injured or stressed during the processes. Injured or stressed animals should
be removed. Allow 5 to 10 min for animals to bury into the test sediment. Amphipods that
have not burrowed within this time should be replaced with animals from the same sieved
population, unless they are repeatedly burrowing into the sediment and immediately
emerging in an apparent avoidance response. In that case, the amphipod is not replaced.
Record the number of amphipods that are removed.

5. Ending the Test (Day 10). The contents of the test chambers must be sieved (> 0.5 mm
mesh) to isolate the test animals. Test water should be used for sieving. Material retained on
the sieve should be washed into a sorting tray with clean test sea water. The sieve should
be slapped forcefully against the surface of the water to ensure that all amphipods are
dislodged from the screen
Material that has been washed from the sieve into the sorting tray should be carefully
examined for the presence of amphipods. Numbers of live, missing, and dead amphipods
should be recorded for each test chamber. Missing animals are assumed to have died
should be considered dead in calculations of the percent survival for each replicate
treatment. Amphipods that are inactive but not obviously dead must be observed using a
low-power dissecting microscope or a hand-held magnifying glass. Any animal that fails to
exhibit movement (i.e. neuromuscular twitch of pleopods or antennae) upon gentle prodding
with a probe should be considered dead.

6. A summary of the procedure is contained in Table 5.7 below.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 207
Table 5.7. General Activity Schedule for Conducting 10-d Sediment Toxicity Test
Days
Days -1 0 to -3
Days -9 to -2
Day-1
Day 0
Day 1
Day 2
Days 3 to 7
Day8
Day 9
Day 10
Activity
Collect or receive amphipods from supplier and place into collection site sediment.
Alternatively, separate 2-4 mm L. plumulosus from culture.
Acclimate and observe amphipods to species-specific test conditions.
Observe amphipods, monitor water conditions. Add sediment to each test chamber.
Place chambers into exposure system, and start aeration.
Measure temperature of overlying water in test chambers. Transfer 20 amphipods
into each test chamber. Archive 20 test organisms for length determination.
Measure temperature. Observe behavior of test organisms and ensure that each test
chamber is receiving air. Measure dissolved oxygen in test chambers to which
aeration has been cut-off.
Measure total water quality (pH, temperature, dissolved oxygen, salinity, total
ammonia) of overlying water. Observe behavior of test organisms and ensure that
each test chamber is receiving air.
Same as Day 1 .
Same as Day 2.
Same as Day 1
Measure temperature. End the test by collecting the amphipods with a sieve.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 208

5.3 SEDIMENT TOXICITY USING FRESHWATER AMPHIPODS

5.3.1 Scope of Application
1. This method is for use with sediments from freshwater aquatic environments. Procedures
are described for testing amphipod crustaceans in the laboratory to evaluate the toxicity in
terms of survival rate from contaminants associated with whole sediments.
2. This method is used for freshwater sediments. See Section 5.2 for method for assessing
salt water sediments.

5.3.2 Summary of Method

This section describes procedures for testing freshwater organisms in the laboratory to evaluate
the potential short-term toxicity of sediments. Sediments are collected from the field and the
toxicity method will be applied to the amphipod Hyalella azteca. The method described here is
for conducting a 10-d acute toxicity test. The test is conducted for 10 days in 300-mL chambers
containing 100 ml_ of sediment and 175 ml_ of overlying water. Overlying water is added daily
and test organisms are fed during the toxicity tests. The endpoint in the 10-d toxicity test with H.
azteca is survival.

5.3.3 Interferences
1. Interferences are characteristics of a sediment or sediment test system that can potentially
confound interpretation of test results. There are three categories of interfering factors:
those characteristics of sediments affecting survival independent of chemical concentration
(i.e., non-contaminant factors); changes in chemical bioavailability as a function of sediment
manipulation or storage; and the presence of indigenous organisms.

4. Testing sediments at temperatures different from that in the field might affect contaminant
solubility, partitioning coefficients, or other physical and chemical characteristics. Interaction
between sediment and overlying water and the ratio of sediment to overlying water may
influence bioavailability.
5. Depletion of aqueous and sediment-sorbed contaminants resulting from uptake by an
organism or test chamber may also influence availability. In most cases, the organism is a
minor sink for contaminants relative to the sediment. However, within the burrow of an
organism, sediment desorption kinetics may limit uptake rates. Desorption of a particular
compound from sediment may range from easily reversible (labile; within minutes) to
irreversible (non-labile: within days or months).

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 209

6. Sediment samples should not be sieved to remove indigenous organisms unless there is a
good reason to believe indigenous organisms may influence the response of the test
organism. However, large indigenous organisms and large debris can be removed using
forceps. Reynoldson et al. (1994) observed reduced growth of amphipods, midges, and
mayflies in sediments with elevated numbers of oligochaetes and recommended sieving
sediments suspected to have high numbers of indigenous oligochaetes. If sediments must
be sieved, it may be desirable to analyze samples before and after sieving (e.g., pore-water
metals, DOC, and AVS) to document the influence of sieving on sediment chemistry.

5.3.4 Equipment and Supplies

1. The facility should include separate areas for culturing and testing to reduce the possibility
of contamination by test materials and other substances, especially volatile compounds.
Holding, acclimation, and culture chambers should not be in a room where sediment tests
are conducted, stock solutions or sediments are prepared, or where equipment is cleaned.
2. Equipment and supplies that contact stock solutions, sediments or overlying water should
not contain substances that can be leached or dissolved in amounts that adversely affect
the test organisms. In addition, equipment and supplies that contact sediment or water
should be chosen to minimize sorption of test materials from water. Glass, type 316
stainless steel, nylon, high-density polyethylene, polycarbonate and fluorocarbon plastics
should be used whenever possible to minimize leaching, dissolution, and sorption. High-
density plastic containers are recommended for holding, acclimation, and culture chambers.
3. Environmental chamber or equivalent with photoperiod and temperature control of 20-25°C.

4. Water purification system capable of producing at least 1 mega-ohm water.

5. The water-delivery system used in water-renewal testing can be one of several designs.
The system should be capable of delivering water to each replicate test chamber. Diluter
systems have been successfully modified for sediment testing. The water-delivery system
should be calibrated before the test by determining the flow rate of the overlying water. At
any particular time during the test, flow rates through any two test chambers should not
differ by more than 10%.

6. Test chambers may be constructed in several ways depending on the contaminants of
interest. Clear silicone adhesives, suitable for aquaria, sorb some organic compounds that
might be difficult to remove. Therefore, as little adhesive as possible should be in contact
with the test material. Extra beads of adhesive should be on the outside of the test
chambers rather than on the inside. To leach potentially toxic compounds from the
adhesive, all new test chambers constructed using silicone adhesives should be held at
least 48 h in overlying water before use in a test. A full list of equipment and supplies is in Table
5.8 below.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
Page 210
Table 5.8. Equipment and Supplies forCulturing and Testing the Freshwater Amphipod H. azteca
A. Biological
Supplies
Brood stock of test organisms
Active dry yeast
Cerophyl® (dried cereal leaves)
Trout food pellets
Diatoms (e.g., Navicula sp.)
B. Glassware
Culture chambers
Test chambers (300-mL high-form
lipless beaker)
Juvenile holding beakers (1 L)
Wide-bore pipets (4- to 6-mm ID)
Glass disposable pipets
Burettes (for hardness and alkalinity
determinations)
Graduated cylinders (assorted sizes, 10
ml to 2 L)
C. Instruments
and Equipment
Dissecting microscope
Stainless-steel sieves (e.g., U.S.
Standard No. 25, 30, 35, 10, 50 mesh)
Photoperiod timers
Light meter
Temperature controllers
Thermometer
Continuous recording thermometer
Photoperiod timer
Dissolved oxygen meter
pH meter
Selective ion meter
Ammonia electrode (or ammonia kit)
Salinity meter/temperature
compensating salinity refractometer
Drying oven
Desiccator
Balance (0.01 mg sensitivity)
Blender
Refrigerator
Freezer
Light box
Hemacytometer
D. Miscellaneous
Ventilation system for test chambers
Air supply and airstones (oil free and
regulated)
Cotton surgical gauze or cheese cloth
Stainless-steel screen (no. 60 mesh, for
test chambers)
Glass hole-cutting bits
Plastic mesh (110-um mesh opening;
Nytex®110)
Aluminum weighing pans
Fluorescent light bulbs
Nalgene bottles (500 mL and 1000 mL)
Airline tubing
White plastic dish pan
Coiled-web material
Shallow pans (plastic (light-colored),
glass, stainless steel)
Silicon adhesive caulking
E. Chemicals
Detergent (non-phosphate)
Acetone (reagent grade)
Hexane (reagent grade)
Hydrochloric acid (reagent grade)
Copper Sulfate, Potassium Chloride
Reagents for reconstituting water
Formalin (or Notox®)
Sucrose
5.3.5 Reagents and Water

5.3.5.1 Reagents

All reagents should be at least reagent grade, unless a test on formulation commercial product,
technical-grade, or use-grade material is specified.

5.3.5.2 Water

1. Water used to test and culture organisms should be uniform in quality. Acceptable
freshwater should allow satisfactory survival, growth, or reproduction of the test organisms.
Test organisms should not show signs of disease or apparent stress (e.g., discoloration.
unusual behavior). If problems are observed in the culturing or testing of organisms, it is
desirable to evaluate the characteristics of the water.

2. Reconstituted water should be prepared by adding specified amounts of reagent-grade
chemicals to high-purity distilled or deionized water. Deionized water should be obtained
from a system capable of producing at least 1 mega-ohm water. Conductivity, pH, hardness,
dissolved oxygen, and alkalinity should be measured on each batch of reconstituted water.
Reconstituted water should be aerated before use to adjust pH and dissolved oxygen to the

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 211
   acceptable ranges. It is recommended replenishing reconstituted water every 2 weeks.
   Below is a summary of salts to be added to 100 liters of deionized water to make up
   reconstituted water for use in Hyalella azteca sediment toxicity testing (Borgman 1996).
Salt
KCI
NaHC03
MgSO4
CaCI2
NaBr
MW
74.54
84
120.3
110.9
102.8
g/100L
0.373
8.4
3.0075
11.09
0.103
5.3.6   Sample Manipulation
1.  Sediment samples tend to settle during shipment.  Water above the sediment should not be
   discarded, but should be mixed back into the sediment during homogenization.

2.  Analytical Methodology:  The precision, accuracy, and bias of each analytical method used
   should be determined in the appropriate matrix: that is, sediment or water. Reagent blanks
   and analytical standards should be analyzed and recoveries should be calculated.

   Measurement of test material(s) concentration  in water can be accomplished by pipeting
   water samples from about 1 to 2 cm above the sediment surface in the test chamber.
   Overlying water samples should  not contain any surface debris, any material from the sides
   of the test chamber, or any sediment.  Measurement of test material(s) concentration in
   sediment at the end of a test can be taken by siphoning most of the overlying water without
   disturbing the surface of the sediment, then removing appropriate aliquots of the sediment
   for chemical analysis.

5.3.7   Quality Control
1.  Before a sediment test is conducted, the analyst must conduct a "non-toxicant" test with
   each potential test species in which all test chambers contain a control sediment (sometimes
   called the negative control), and  clean overlying water for each amphipod species to  be
   tested. Survival of the test organism will demonstrate whether facilities, water,  control
   sediment, and handling techniques are adequate to achieve acceptable species-specific
   control survival. Evaluations may also be conducted of the magnitude of the within- and
   between-chamber variance in a test. For the test to be acceptable, survival  at 10 d must
   equal or exceed 90% for amphipod species in the control sediments.

2.  If the supplier has not conducted five reference toxicity tests with the test organism, the
   testing laboratory must do so before starting a sediment test. Intralaboratory precision,
   expressed as a coefficient of variation of the range in response for each type of test to be
   used in a laboratory should be determined by performing five or more tests with different
   batches of test organisms, using the same reference toxicant, at the same concentrations,
   with the same test conditions (e.g., the same test duration,  type of water, age of test
   organisms) and same data analysis methods. A reference toxicant concentration series (0.5
   or higher) should be selected that will consistently provide partial mortalities at two or more
   concentrations of the test chemical. Reference toxicants such as sodium (in the form of
   sodium chloride (NaCI)), potassium (as potassium chloride (KCI)), cadmium  (available as
   cadmium chloride (CdCI2)), and copper (available as copper sulfate (CuSO4)) are available

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 212
   for use. No one reference toxicant can be used to measure the condition of test organisms in
   respect to another toxicant with a different mode of action. Test conditions for conducting reference-
   toxicity tests are outlined in Table 5.9.

Table 5.9. Recommended Test Conditions for Conducting Reference-Toxicity Tests
Parameter
1. Test type:
2. Dilution series:
3. Toxicant:
4. Temperature:
5. Light quality:
6. Illuminance:
7. Photoperiod:
8. Renewal of water:
9. Age of test organisms:
10. Test chamber:
11. Volume of water:
12. Number of organisms per chamber:
13. Replicate chambers/treatment
14. Feeding:
15. Substrate:
16. Aeration:
17. Dilution water:
18. Test duration:
19. Endpoint:
20. Test acceptability:
Conditions
Water-only test
Control and at least 5 test concentrations (0.5 dilution factor)
Na, K, Cd, orCu,
23°±1°C
Wide-spectrum fluorescent lights
100 to 1000 lux
16L8D
None
7-14 d old (1-2 d range in age)
30 ml plastic cups (covered with glass or plastic)
20 ml
10
4 minimum
0.1 ml YCT (1 800 mg/L stock) on Day 0 and Day 2
Nitex® screen (1 1 0 mesh)
None
Cultured, well water, surface water, site water, or reconstituted
96 h
Survival (LC50);
90% control survival
3.   Before conducting tests with contaminated sediment, the laboratory should demonstrate its
    ability to conduct tests by conducting five exposures in control sediment. It is recommended
    that these five exposures with control sediment be conducted concurrently with the five
    reference toxicity tests described above.
4.   The lab must work with the Information Management group (Marlys Cappaert,541-754-4467,
    Cappaert.Marlys@epamail.epa.gov,) to ensure their bench sheets and/or data recording
    spreadsheets are compatible with the electronic deliverables the lab will need to submit.

5.3.8   Culturing and Maintaining Test Organisms
5.3.8.1 General
All organisms in a test must be from the same source. Organisms may be obtained from
laboratory cultures or from  commercial or government sources. The test organism used should
be identified using an appropriate taxonomic key, and verification should be documented.
Obtaining organisms from wild populations should be avoided.
A group of organisms should not be used for a test if they appear to be unhealthy, discolored, or
otherwise stressed (e.g., >20% mortality for 48 h before the start of a test). If the organisms fail

-------
 National Coastal Condition Assessment
 Laboratory Methods Manual                                              Date: November 2010
	Page 213

 to meet these criteria, the entire batch should be discarded and a new batch should be
 obtained. All organisms should be as uniform as possible in age and life stage. Test organisms
 should be handled as little as possible.

 Organisms can be cultured using either static or renewal procedures. Renewal is recommended
 to limit loss of the culture organisms from a drop in dissolved oxygen or a buildup of waste
 products. In renewal systems, there should be at least one volume addition/d of culture water to
 each chamber.  In static systems, the overlying water volume should be changed at least weekly
 by siphoning down to a level just above the substrate and  slowly adding fresh water. Extra care
 should be taken to ensure that proper water quality is maintained in static systems. For
 example, aeration is needed in static systems to maintain dissolved oxygen at >2.5 mg/L.

 It may be desirable for laboratories to periodically perform 96-h water-only reference-toxicity
 tests to assess  the sensitivity of culture organisms. Data from these reference-toxicity tests
 could be used to assess genetic strain or life-stage sensitivity to select chemicals.

 5.3.8.2 Culturing Procedures for Hyalella azteca
 1.  The culturing  procedure must produce 7- to  14-d-old amphipods to start a 10-d sediment
    test. The  10-d test with should start with a narrow range in size or age (1- to 2-d range in
    age) to reduce potential variability in growth  at the end of the 10-d test. This narrower range
    would be easiest to obtain using known age  organisms instead of sieving the cultures to
    obtain similar sized amphipods (i.e., amphipods within a range of 1- to 2-d old will be more
    uniform in size than organisms within the range of 7 d).

 2.  The following procedure can be used to obtain known age amphipods to start a test. Mature
    amphipods  (50 organisms >30-d old at 23°C) are held in 2-L glass beakers containing 1 L of
    aerated culture water and cotton gauze as a substrate. Amphipods are fed 10 mL of a yeast-
    Cerophyl®-trout chow (YCT) mixture and 10 mL of the green algae Selenastrum
    capricornutum (about 3.5 x 107 cells/mL). Five mL of each  food is added to each culture
    daily, except for renewal days, when 10 mL  of each  food is added.

    Water in the culture chambers is changed weekly. Survival of adults and juveniles and
    production of young amphipods should be measured at this time. The contents of the culture
    chambers are poured into a translucent white plastic or white enamel pan. After the adults
    are removed, the remaining amphipods will range in age from <1- to 7-d old. Young
    amphipods  are transferred with a pipet into a 1-L beaker containing culture water and are
    held for one week before starting a toxicity test. Organisms are fed 10 mL of YCT and 10 mL
    of green algae on start-up day and 5 mL of each food each following day. Survival of young
    amphipods  should be >80% during this one-week holding period. Some of the adult
    amphipods  can be expected to die in the culture chambers, but mortality greater than about
    50% should be cause for concern. Reproductive rates  in culture chambers containing 60
    adults can be as high as 500 young per week.  A decrease in reproductive rate may be
    caused by a change in water quality, temperature, food quality, or brood stock health. Adult
    females will continue to reproduce for several months.
 3.  Laboratories that use mixed-age amphipods for testing must demonstrate that the procedure
    used to isolate amphipods will produce test organisms that are 7- to 14-d old. For example,
    amphipods  passing through a #35 sieve (500 urn), but stopped by a #45 sieve (355 urn) will
    average 1.54 mm (SD 0.09) in length. The mean length of these sieved organisms
    corresponds to that of 6-d-old amphipods. After holding for 3 d before testing to eliminate
    organisms injured during sieving, these amphipods would be about 9 d old (length 1.84 mm,
    SD 0.11)  at the start of a toxicity test.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 214
   In a different but similar method, smaller amphipods are isolated from larger amphipods
   using a stack of sieves: #30 (600 urn), #40 (425 urn), and #60 (250 urn). Sieves should be
   held under water to isolate the amphipods. Amphipods may float on the surface of the water
   if they are exposed to air. Artificial substrate or leaves are placed in the #30 sieve. Culture
   water is rinsed through the sieves and small amphipods stopped by the #60 sieve are
   washed into a collecting pan. Larger amphipods in the #30 and #40 sieves are returned to
   the culture chamber. The smaller amphipods are then placed in 1-L beakers containing
   culture water  and food (about 200 amphipods per beaker) with gentle aeration.
   Amphipods should be held and fed at a rate similar to the mass cultures for at least 2 d
   before the start of a test to eliminate animals injured during handling.

5.3.9   Procedure
1.  The recommended 10-d sediment toxicity test with H. azteca must be conducted at 23°C
   with a 16L8D photoperiod at an illuminance of about 100 to  1000 lux . Test chambers are
   300-mL high-form lipless beakers containing 100 ml_ of sediment and 175 ml_ of overlying
   water. Ten 7- to 14-d-old amphipods per replicate are used to start a test. The 10-d test
   should start with a narrow range in size or age of H. azteca (i.e., 1- to 2-d range in age) to
   reduce potential variability in growth at the end of a 10-d test. Four replicates are
   recommended for routine testing. Amphipods in each test chamber are fed 1.0 ml_ of YCT
   food daily. Each chamber receives 2 volume additions/d of overlying water. Water renewals
   may be manual or automated. Conditions for conducting a 10-d sediment toxicity test are
   summarized in Table 5.10 below.

Table 5.10. Recommended Test Conditions for Conducting  10-d Sediment Toxicity Tests
Parameter
1 . Test type:
2. Temperature:
3. Light quality:
4. Illuminance:
5. Photoperiod:
6. Test chamber:
7. Sediment volume
8. Overlying water volume:
9. Renewal of overlying water:
10. Age of organisms:
1 1 . Number of organisms/ chamber:
12. Replicate chambers/treatment:
13. Feeding:
14. Aeration:
15. Test duration:
16. Endpoint:
17. Test acceptability:
Conditions
Whole-sediment toxicity test with renewal of overlying
water
23°±1°C
Wide-spectrum fluorescent lights
100 to 1000 lux
16L8D
300 mL high-form lipless beaker
100mL
175mL
2 volume additions/d; continuous or intermittent (e.g.,
addition every 12 h)
7- to 1 4-d old at the start of the test (1 - to 2-d range in
1 volume
age)
10
4 recommended
YCT food, fed 1 .0 mL daily (1800 mg/L stock) to each
test chamber.
None unless DO in overlying water drops below 2.5 mg/L
10d
Survival
Min. mean control survival of 80%.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 215

2. Introduction of Sediment (Day -1). The day before the sediment test is started each
sediment should be thoroughly homogenized and added to the test chambers. Each test
chamber should contain the same amount of sediment, determined either by volume or by
weight. Overlying water is added to the chambers on Day -1 in a manner that minimizes
suspension of sediment. This can be accomplished by gently pouring water along the sides
of the chambers or by pouring water onto a baffle (e.g., a circular piece of Teflon® with a
handle attached) placed above the sediment to dissipate the force of the water. A test
begins when the organisms are added to the test chambers (Day 0).
3. Addition of Amphipods (Day 0). Amphipods should be introduced into the overlying water
below the air-water interface. Test organisms can be pipetted directly into overlying water.
The size of the test organisms at the start of the test should be measured using the same
measure (length or weight) that will be used to assess their size at the end of the test. For
length, a minimum of 20 organisms should be measured. For weight measurement, a larger
sample size (e.g., 80) may be desirable because of the relative small mass of the
organisms. Test organisms should be handled as little as possible.
4. Feeding (Day 0 to Day 9). For each beaker, 1.0 ml_ of YCT is added from Day 0 to Day 9.
Suspensions of food should be thoroughly mixed before aliquots are taken. In some
instances, the addition of the food may alter the availability of the contaminants in the
sediment. Furthermore, if too much food is added to the test chamber or if the mortality of
test organisms is high, fungal or bacterial growth may develop on the sediment surface.
Therefore, the amount of food added to the test chambers is kept to a minimum.

5. Ending a Test (Day 10).Any of the surviving amphipods in the water column or on the
surface of the sediment can be pipetted from the beaker before sieving the sediment.
Immobile organisms isolated from the sediment surface or from sieved material should be
considered dead. A #40 sieve (425-um mesh) can be used to remove amphipods from
sediment. Alternatively, sieving of sediment can be accomplished as follows: (1) pour about
half of the overlying water through a #50- (300-um) mesh sieve, (2) swirl the remaining
water to suspend the upper 1 cm of sediment, (3) pour this slurry through the #50-mesh
sieve and wash the contents of the sieve into an examination pan, (4) rinse the coarser
sediment remaining in the test chamber through a #40- (425-um) mesh sieve and wash the
contents of this second sieve into a second examination pan. Surviving test organisms are
removed from the two pans and counted. A summary of the procedure is in Table 5.11.

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
           Page 216
Table 5.11.  General Activity Schedule for Conducting 10-d Sediment Toxicity Test
Day
Day -7
Days -6 to -2
Day-1
Day 0
Daysl to 8
Day 9
Day 10
Activity
Separate known-age amphipods from the cultures and place in holding chambers. Begin
preparing food for the test. There should be a 1- to 2-d range in age of amphipods used
to start the test.
Feed and observe isolated amphipods, monitor water quality (e.g., temperature and
dissolved oxygen).
Feed and observe isolated amphipods, monitor water quality. Add sediment into each
test chamber, place chambers into exposure system, and start renewing overlying water.
Measure total water quality (pH, temperature, dissolved oxygen, hardness, alkalinity,
conductivity, ammonia). Transfer 10 7- to 14-day-old amphipods into each test chamber.
Release organisms under the surface of the water. Add 1.0 ml of YCT into each test
chamber. Archive 20 test organisms for length determination or archive 80 test
organisms for dry weight determination. Observe behavior of test organisms.
Add 1 .0 ml of YCT food to each test chamber. Measure temperature and dissolved
oxygen. Observe behavior of test organisms.
Measure total water quality.
Measure temperature and dissolved oxygen. End the test by collecting the amphipods
with a sieve. Count survivors and prepare organisms for weight or length measurements.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 217

6.0 INFAUNAL BENTHIC MACROINVERTEBRATE COMMUNITIES

6.1 SCOPE AND APPLICATION

This method describes the laboratory procedures used to measure the species composition and
abundance of macroinvertebrate fauna found in estuarine or freshwater sediments. The
procedure is designed to produce data of consistent quality meeting the measurement quality
objectives (MQOs) of 10% total error for the extraction of organisms and 10% total error for the
identification and enumeration of extracted fauna (Section 6.6). Upon request to the EPA, and
subsequent approval of alternate methods, performance-based evaluations may also be utilized
to qualify participating labs.

6.2 SUMMARY OF METHOD

Sediment grab samples will be sieved (0.5 mm mesh; 1.0 mm mesh for CA, OR, and WA) in the
field and preserved in 10% buffered formalin prior to shipping to the laboratory. Preserved
samples are sorted, identified, and enumerated to the lowest practical taxonomic level (genus or
species) using standardized keys and references.

6.3 SAMPLE STORAGE AND TREAMENT

Samples will be preserved in 10% buffered formalin.

Samples should be stored in a cool, dry area away from direct sunlight.

6.4 SORTING

1. Sort all samples under a minimum of 6X dissecting microscope.

2. Remove the macroinvertebrates with forceps and place in internally labeled vials containing
70-80% denatured ethanol. Remove taxa such as polychaetes, oligochaetes, bivalves,
gastropods, crustaceans, etc. Do not remove empty snail or bivalve sheels; surface-
dwelling or strictly pelagic arthropod taxa such as Collembola, Veliidae, Gerridae,
Notonectidae, Corixidae, Cladocera, or Copepoda; or terrestrial taxa. Also, do not remove
fragments such as legs, antennae, gills, or wings.

3. Once the QC check of the material in the pan has been completed, remove the material
from the pan and place it in a separate container with preservative (70-80% ethanol). The
container must be appropriately labeled both inside and outside. The lid must be tightly
closed and the container archived until all necessary QC checks are completed. Do not
discard any sample portion until instructed by the QC Officer and the Project Manager.

6.5 PROCEDURE

6.5.1 Identification and Enumeration - General

1. Empty one sample vial at a time into a Petri dish. Add preservative to keep organisms
covered. View the sample under the stereo dissecting microscope.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 218

2. Identify organisms to the lowest practical taxonomic level. Specimens that are difficult to
identify will be set aside in vials and preserved for later. These specimens may require
further processing (see Section 6.5.2) or a taxonomist with different area of expertise.

3. Count the number of taxa in each sample. Specimens will be identified, counted and
removed from Petri dish one at a time. Remove similar organisms to other dishes to be
placed in vials.

4. Specimens that can be identified only to genus, family or order will also be included in the
total number of taxa in each sample (e.g., organisms identified to be within the family
Spionidae will be counted as one taxon). If a specimen identified to genus, family or order
can be identified as one of several taxa already identified in the sample, that organism will
not be counted as an additional taxa. For example, if an organism is identified as far as
belonging to Tellinidae, and the taxonomist believes it could be either Macoma balthica or
Macoma mitchelli, both of which are present in the sample, the specimen would be
recorded as Tellinidae and would not be included in the taxa count for that sample.

5. If organisms can be identified, they are counted ONLY if:

The fragment included the head,
The mollusk shell (bivalve or gastropod) is occupied by a specimen, or

The specimen is the sole representative of a taxon in the sample.

6. If early instar or juvenile specimens can be identified, they are counted:
As the same taxon if, with confidence, they can be associated with one or more mature
specimens that have a more developed morphology.

As a separate taxon if the specimen is the sole representative of a taxon in the sample.

6.5.2 Subsampling

If the laboratory believes that subsampling for a particular organism (such as Oligochaetes and
Chironomids) may be appropriate for a given sample, they should contact Treda Grayson at
U.S. EPA Office of Water, 202-566-0916, Grayson.Treda@epamail.epa.gov.

If the number of specimens is greater than 400, proceed as follows:

Distribute the sample on a gridded tray as evenly as possible. Select grids randomly until at
least 200 specimens are identified to be mounted. Any remaining specimens in the last grid will
also be identified so the total number of specimens in these samples will be slightly >200.
1. Specimens in the remaining grids will be enumerated.

2. Prepare slide mounts using appropriate media and view organisms under compound
microscope.

6.6 QUALITY ASSURANCE AND QUALITY CONTROL

6.6.1 Sorting QC

1. Experienced QC Officers will use 6-1 Ox microscopes to check all sorted material from
the first five samples processed by a sorter to ensure that each meets the 90% sorting
efficiency (SE). This will not only apply to inexperienced sorters, but also to those initially

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 219

deemed as "experienced." Qualification will only occur when sorters achieve > 90%
sorting efficiency for five samples consecutively.
2. The QC Officer will calculate percent sorting efficiency (PSE) for each sample as follows:

A
PSE = xlOO
A + B

where A = number of organisms found by the primary sorter, and B = number of recoveries
(organisms missed by the primary sort and found by the QC check).

If the SE for each of these five consecutive samples is > 90% for an individual, this individual
is considered "experienced" and can serve as a QC Officer. In the event that an individual
fails to achieve > 90% SE, they will be required to sort an additional five samples to continue
to monitor their SE. However, if they show marked improvement in their SE prior to
completion of the next five samples, whereby they acquire the > 90% SE, the QA Officer
may, at his/her discretion, consider this individual "experienced." SE should not be
calculated for samples processed by more than one individual.

3. After individuals qualify, 10% (1 of 10, randomly selected) of their samples will be checked.

4. If an "experienced" individual fails to maintain a > 90% sorting efficiency as determined by
QC checks, an internal lab QC Officer will perform QC checks on every grid of five
consecutive samples until a > 90% sorting efficiency is achieved on all five. During this time,
that individual will not be able to perform QC checks.

5. Randomly select 10% of the sample pickates (sort residue) for an internal lab QC check for
missed specimens. If samples contain more than 10% of the original number of organisms
found in the sample, a project facilitator will make a determination as to whether more of the
samples need to be resorted (upon closer examination of the data).

6.6.2 Taxonomic QC

1. On receipt of the samples, the project facilitator will randomly select 10% of the samples to
be sent to the QC taxonomist, another experienced taxonomist who did not participate in the
original identifications. A chain-of-custody form will be completed and sent with the samples.

2. The QC taxonomist will perform whole-sample re-identifications, with care taken to ensure
inclusion of all slide-mounted specimens, completing a separate copy of the taxonomic
bench sheet for each sample. Each lab must work with the Information Management group
(Marlys Cappaert, Cappaert.Marlys@epamail.epa.gov, 541-754-4467,) to ensure their
bench sheets are compatible with the electronic deliverables the lab will need to submit.
Each bench sheet must be labeled with the term "QC Re-ID." As each bench sheet is
completed, it must be faxed to the project facilitator.

3. The project facilitator will compare the taxonomic results (counts AND identifications)
generated by the primary and QC taxonomists for each sample and calculate percent
disagreement in enumeration (PDE) and percent taxonomic disagreement (PTD) as
measures of taxonomic precision (Stribling et al. 2003) as follows:

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 220

\n\-n2\
PDE = - LxlOO
n\ + n2

where n1 is the number of specimens counted in a sample by the first taxonomist and n2
is the number of specimens counted by the QC taxonomist.
PTD =
xlOO
where compos is the number of agreements (positive comparisons) and N is the total
number of specimens in the larger of the two counts.

4. Unless otherwise specified by project goals and objectives, the measurement quality
objective for enumerations will be a mean PDE less than or equal to 5 and a mean PTD
less than or equal to 15, calculated from all the samples in the 10% set sent to the QC
taxonomist. Results greater than these values will be investigated and logged for
indication of error patterns or trends, but all values will generally be considered
acceptable for further analysis, unless the investigation reveals significant problems.

5. Corrective action will include determining problem areas (taxa) and consistent
disagreements, addressing problems through taxonomist interactions. Disagreements
resulting from identification to a specific taxonomic level, creating the possibility to
double-count "unique" or "distinct" taxa will also be rectified through corrective actions.

6. The project facilitator will prepare a report or technical memorandum. This document will
quantify both aspects of taxonomic precision, assess data acceptability, highlight
taxonomic problem areas, and provide recommendations for improving precision. This
report will be submitted to the project manager, with copies sent to the primary and QC
taxonomists and another copy maintained in the project file.

6.8 REFERENCES

Borgmann, U. 1996. Systematic Analysis of Aqueous Ion Requirements of Hyalella azteca: A
Standard Artificial Medium Including the Essential Bromide Ion. Archives of Environmental
Contamination and Toxicology. 30: 356-363.

USEPA. 1994. Methods for Assessing the Toxicity of Sediment-associated Contaminants with
Estuarine and Marine Amphipods. EPA/600/R-94/025. U.S. Environmental Protection
Agency, Office of Research and Development, Narragansett, Rl.

USEPA. 1995. Environmental Monitoring and Assessment Program (EMAP) Laboratory Methods
Manual Estuaries. Volume 1 - Biological and Physical Analyses. Section 5 Sediment Silt-
Clay Content, Sediment Grain Size Distribution, Total Organic Carbon Concentrations
Laboratory Procedures. EPA/620/R-95/008. U.S. Environmental Protection Agency, Office
of Research and Development, Narragansett, Rl.

USEPA. 1995. Environmental Monitoring and Assessment Program (EMAP) Laboratory
Methods Manual Estuaries. Volume 1 - Biological and Physical Analyses. Section 3.
Benthic Macroinvertebrates. EPA/620/R-95/008. U.S. Environmental Protection
Agency, Office of Research and Development, Narragansett, Rl

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 221

USEPA. 1997. Determination of Carbon and Nitrogen in Sediments and Particulates of
Estuarine/Coastal Water Using Elemental Analysis in Methods for Determination of
chemical substances in Marine and Estuarine Environmental matrices -2nd Edition.
EPA/600/R-97/072. U.S. Environmental Protection Agency, Office of Research and
Development, Washington, DC.

USEPA. 2000. Methods for Measuring the Toxicity and Bioaccumulation of Sediment-
associated Contaminants with Freshwater Invertebrates (2nd edition). EPA/600/R-
99/064. U.S. Environmental Protection Agency, Office of Research and Development.
Duluth, MN and Office of Water, Washington, DC.

USEPA, 2000. Guidance for Assessing Chemical Contaminant Data for Use In Fish Advisories,
Volume 1: Fish Sampling and Analysis - Third Edition. EPA/823/B-00-007. U.S.
Environmental Protection Agency, Office of Water, Office of Science and Technology.
Washington, DC.

USEPA. 2006. Survey of the Nation's Lakes: Laboratory Manual. EPA/841/B-06/005. U.S.
Environmental Protection Agency, Office of Water, Office of Environmental Information.
Washington, DC.
APPENDIX A

[The following appendix, "APPENDIX J: RECOMMENDED PROCEDURES FOR PREPARING
WHOLE FISH COMPOSITE HOMOGENATE SAMPLES", has been excerpted from "Guidance
for Assessing Chemical Contaminant Data for Use in Fish Advisories. Volume 1: Fish Sampling
and Analysis." Third Edition. EPA 823-B-00-007. USEPA, Office of Water. November 2000.]

Note: For NCA purposes, the sections referring to fish sex and age determinations and
assessment of morphological abnormalities, as well as additional references to these in the text,
are not relevant and may be ignored.

-------
                                                                       APPENDIX J
APPENDIX J


RECOMMENDED PROCEDURES FOR PREPARING WHOLE
FISH COMPOSITE HOMOGENATE SAMPLES

J.1      GENERAL GUIDELINES

              Laboratory processing to prepare whole fish composite samples (diagrammed in
              Figure J-1) involves

                  Inspecting individual fish for foreign material on the surface and rinsing if
                  necessary
              •   Weighing individual fish
                  Examining each fish for morphological abnormalities (optional)
                  Removing scales or otoliths for age determination (optional)
                  Determining the sex of each fish  (optional)
                  Preparing individual whole fish homogenates
                  Preparing a composite whole fish homogenate.

              Whole fish should be shipped on wet or blue ice from the field to the  sample
              processing laboratory if next-day delivery is assured. Fish samples arriving in this
              manner (chilled but not frozen) should be weighed, scales and/or  otoliths
              removed, and  the  sex of each fish  determined within 48 hours of  sample
              collection.  The grinding/homogenization procedure may  be carried out more
              easily and efficiently if the sample has been frozen previously (Stober, 1991).
              Therefore, the samples should then be frozen (<-20 °C) in the laboratory prior to
              being homogenized.

              If the fish samples arrive frozen (i.e., on  dry ice) at the sample processing
              laboratory, precautions should be taken during weighing, removal of scales and/or
              otoliths, and sex determination to ensure that any liquid formed  in  thawing
              remains with the sample.  Note:    The liquid will  contain target  analyte
              contaminants and lipid material that should be included in the sample for analysis.

              The thawed or partially thawed whole fish  should  then be homogenized
              individually, and equal weights of each  homogenate should be combined to form
              the composite sample.  Individual homogenates and/or composite homogenates
              may be frozen; however, frozen individual homogenates must be rehomogenized
              before compositing, and frozen composite homogenates must be rehomogenized
              before aliquotting for analysis. The maximum holding time from sample collection
              to analysis for mercury is 28 days at <-20 °C; for all other  analytes, the holding
              time is 1 year at <-20 °C (Stober, 1991).  Recommended container materials,


                                                                               J-3

-------
                                                                                               APPENDIX J
                           Log in fish samples using COG procedures

                                         V
                              Unwrap and inspect individual fish

                                         V
                                   Weigh individual fish

                                         V
               Remove and archive  scales and/or otoliths for age determination (optional)

                                         V
                 Determine sex (optional); note morphological abnormalities (optional)
                                         V
      Remove scales from all scaled fish

                V
Remove skin from scaleless fish (e.g., catfish)

                V
                                          V
                                         Fillet fisr

                                          V
                                      Weigh fillets (g)

                                         V
                                     Homogenize fillets
                       Divide homogenized sample into quarters, mix opposite
                             quarters, and then mix halves (3 times)
                                                            Optional
                               Composite equal weights (g) of
                              homogenized fillet tissues from the
                               selected number of fish (200-g)
                              Seal and label (200-g) composite
                            homogenate in appropriate container(s)
                            and store at <-20 °C until analysis (see
                             Table 7-1 for recommended container
                                materials and holding times).
                  Save remainder of fillet
                 homogenate from each fish
                Seal and label individual fillet
                homogenates in appropriate
                 container(s) and archive at
                 <-20 °C (see Table 7-1 for
                  recommended container
                materials and holding times).
 COG = Chain of custody.
Figure J-1.  Laboratory sample preparation and handling for whole fish
                          composite homogenate samples.
                                                                                                           J-4

-------
APPENDIX J
preservation temperatures, and holding times are given in Table J-1. Note:
Holding times in Table J-1 are maximum times recommended for holding samples
from the time they are received at the laboratory until they are analyzed. These
holding times are based on guidance that is sometimes administrative rather than
technical in nature; there are no promulgated holding time criteria fortissues (U.S.
EPA, 1995b). If states choose to use longer holding times, they must
demonstrate and document the stability of the target analyte residues over the
extended holding times.

J.2 SAMPLE PROCESSING PROCEDURES

Fish sample processing procedures are discussed in more detail in the sections
below. Each time custody of a sample or set of samples is transferred from one
person to another during processing, the Personal Custody Record of the chain-
of-custody (COC) form that originated in the field (Figure 6-8) must be completed
and signed by both parties so that possession and location of the samples can be
traced at all times (see Section 7.1). As each sample processing procedure is
performed, it should be documented directly in a bound laboratory notebook or
on standard forms that can be taped or pasted into the notebook. The use of a
standard form is recommended to ensure consistency and completeness of the
record. Several existing programs have developed forms similar to the sample
processing record for whole fish composite samples shown in Figure J-2.

J.2.1 Sample Inspection

Individual fish received for filleting should be unwrapped and inspected carefully
to ensure that they have not been compromised in any way (i.e., not properly
preserved during shipment). Any specimen deemed unsuitable for further
processing and analysis should be discarded and identified on the sample
processing record.

J.2.2 Sample Weighing

A wet weight should be determined for each fish. All samples should be weighed
on balances that are properly calibrated and of adequate accuracy and precision
to meet program data quality objectives. Balance calibration should be checked
at the beginning and end of each weighing session and after every 20 weighings
in a weighing session.

Fish shipped on wet or blue ice should be weighed directly on a foil-lined balance
tray. To prevent cross contamination between individual fish, the foil lining should
be replaced after each weighing. Frozen fish (i.e., those shipped on dry ice)
should be weighed in clean, tared, noncontaminating containers if they will thaw
before the weighing can be completed. Liquid from the thawed sample must be
J-5

-------
                                                                               APPENDIX J
 Table J-1.  Recommendations for Container Materials, Preservation, and Holding
 Times for Fish, Shellfish, and Turtle Tissues from Receipt at Sample Processing
                                Laboratory to Analysis
Analyte
Mercury
Other metals
Organics

Metals and
organics
Matrix
Tissue (whole specimens,
homogenates)
Tissue (whole specimens,
homogenates)
Tissue (whole specimens,
homogenates)

Tissue (whole specimens,
homogenates)
Sample
container
Plastic,
borosilicate
glass, quartz,
and PTFE
Plastic,
borosilicate
glass, quartz,
and PTFE
Borosilicate
glass, quartz,
PTFE, and
aluminum foil
Borosilicate
glass, quartz,
and PTFE
Storage
Preservation Holding time3
Freeze at <-20 °C 28 days"
Freeze at <-20 °C 6 months0
Freeze at <-20 °C 1 yeard

Freeze at <-20 °C 28 days
(mercury);
6 months (for
    Lipids      Tissue (whole specimens,
               homogenates)
   Plastic,
 borosilicate
glass, quartz,
   PTFE
Freeze at <-20 °C
other metals);
and 1 year (for
  organics)

    1 year
PTFE = Polytetrafluoroethylene for Teflon.
 Maximum holding times recommended by U.S. EPA (1995b).
 This maximum holding time is also recommended by the Puget Sound Estuary Program  (1990). The
 California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program
 (Crawford and Luoma, 1993) recommend a maximum holding time of 6 months for all metals, including
 mercury.
 This maximum holding time is also recommended by the California Department of Fish and Game (1990),
 the 301(h) monitoring program (U.S. EPA, 1986), and the USGS National Water Quality Assessment
 Program (Crawford and Luoma, 1993). The Puget Sound Estuary Program (1990) recommends a  maximum
 holding time of 2 years.
 This maximum holding time is also recommended by the Puget Sound Estuary Program (1990).  The
 California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program
 (Crawford and Luoma, 1993) recommend a more conservative maximum holding time of 6 months. EPA
 (1995a) recommends a maximum holding time of 1  year at <-10 °C for dioxins and dibenzofurans.
                                                                                        J-6

-------
                                                                                     APPENDIX J
     Sample Processing Record for Fish Contaminant Monitoring Program—Whole Fish Composites
     Project No..
                               Sampling Date and Time:.
     STUDY PHASE:  Screening
     SITE LOCATION
     Site Name/Number
     County/Parish:
     State WaterbooV Segment Number .
                                            LatTLong.:	
                                            Waterbody Type:.
     Bottom Feeder - Species Name:.
     Composite Sample #:	
     Fish*
     001
     002
     003
     004
     005
     006
     007
     008
     009
     010
     Analyst
     Initials/Date
Weight (g)
Scales/Otoliths
 Removed (/)
  Number of Individuals:	
 Sex       Homogenate
(M, F)       Prepared (/)
                          Weight of homogenate
                          taken for composite (g)
                                             Total Composite Homogenate Weight
     Predator - Species Name:
     Composite Sample #:	
                                 Number of Individuals:.
     Fish*
     001
     002
     003
     004
     005
     006
     007
     008
     009
     010
     Analyst
     Initials/Date
Weight (g)
Scales/Otoliths
 Removed (/)
 Sex
(M,F)
           Homogenate
           Prepared (/)
Weight of homogenate
taken for composite (g)
                                             Total Composite Homogenate Weight
     Notes:
Figure J-2. Example of a sample processing record for fish contaminant monitoring
                             program—whole fish composites.
                                                                                               J-7

-------
APPENDIX J
kept in the container as part of the sample because it will contain lipid material
that has separated from the tissue (Stober, 1991).

All weights should be recorded to the nearest gram on the sample processing
record and/or in the laboratory notebook.

J.2.3 Age Determination

Age provides a good indication of the duration of exposure to pollutants (Versar,
1982). A few scales or otoliths (Jearld, 1983) should be removed from each fish
and delivered to a fisheries biologist for age determination. For most warm water
inland gamefish, 5 to 10 scales should be removed from below the lateral line and
behind the pectoral fin. On softrayed fish such as trout and salmon, the scales
should be taken just above the lateral line (WDNR, 1988). For catfish and other
scaleless fish, the pectoral fin spines should be clipped and saved (Versar, 1982).
The scales, spines, or otoliths may be stored by sealing them in small envelopes
(such as coin envelopes) or plastic bags labeled with, and cross-referenced by,
the identification number assigned to the tissue specimen (Versar, 1982).
Removal of scales, spines, or otoliths from each fish should be noted (by a check
mark) on the sample processing record.

J.2.4 Sex Determination (Optional)

To determine the sex of a fish, an incision should be made on the ventral surface
of the body from a point immediately anterior to the anus toward the head to a
point immediately posterior to the pelvic fins. If necessary, a second incision
should be made on the left side of the fish from the initial point of the first incision
toward the dorsal fin. The resulting flap should be folded back to observe the
gonads. Ovaries appear whitish to greenish to golden brown and have a granular
texture. Testes appear creamy white and have a smooth texture (Texas Water
Commission, 1990). The sex of each fish should be recorded on the sample
processing record.

J.2.5 Assessment of Morphological Abnormalities (Optional)

Assessment of gross morphological abnormalities in finfish is optional. This
assessment may be conducted in the field (see Section 6.3.1.5) or during initial
inspection at the central processing laboratory prior to filleting. States interested
in documenting morphological abnormalities should consult Sinderman (1983) and
review recommended protocols for fish pathology studies used in the Puget
Sound Estuary Program (1990).

J.2.6 Preparation of Individual Homogenates

To ensure even distribution of contaminants throughout tissue samples, whole fish
must be ground and homogenized prior to analyses.
J-8

-------
APPENDIX J
Smaller whole fish may be ground in a hand crank meat grinder (fish < 300 g) or
a food processor (fish 300-1,000 g). Larger (>1,000 g) fish may be cut into
2.5-cm cubes with a food service band saw and then ground in either a small or
large homogenizer. To avoid contamination by metals, grinders and homo-
genizers used to grind and blend tissue should have tantalum or titanium blades
and/or probes. Stainless steel blades and probes have been found to be a
potential source of nickel and chromium contamination (due to abrasion at high
speeds) and should be avoided.

Grinding and homogenization of biological tissue, especially skin from whole fish
samples, is easier when the tissue is partially frozen (Stober, 1991). Chilling the
grinder/homogenizer briefly with a few chips of dry ice will reduce the tendency
of the tissue to stick to the grinder.

The ground sample should be divided into quarters, opposite quarters mixed
together by hand, and the two halves mixed back together. The grinding,
quartering, and hand mixing should be repeated two more times. If chunks of
tissue are present at this point, the grinding/homogenizing should be repeated.
No chunks of tissue should remain because these may not be extracted or
digested efficiently. If the sample is to be analyzed for metals only, the ground
tissue may be mixed by hand in a polyethylene bag (Stober, 1991). Homogeni-
zation of each individual fish should be noted on the sample processing record.
At this time, individual whole fish homogenates may be either composited or
frozen and stored at <-20 °C in cleaned containers that are noncontaminating for
the analyses to be performed (see Table J-1).

J.2.7 Preparation of Composite Homogenates

Composite homogenates should be prepared from equal weights of individual
homogenates. If individual whole fish homogenates have been frozen, they
should be thawed partially and rehomogenized prior to compositing. Any
associated liquid should be maintained as a part of the sample. The weight of
each individual homogenate that is used in the composite homogenate should be
recorded, to the nearest gram, on the sample processing record.

Each composite homogenate should be blended by dividing it into quarters,
mixing opposite quarters together by hand, and mixing the two halves together.
The quartering and mixing should be repeated at least two more times. If the
sample is to be analyzed only for metals, the composite homogenate may be
mixed by hand in a polyethylene bag (Stober, 1991). At this time, the composite
homogenate may be processed for analysis or frozen and stored at <-20 °C (see
Table J-1).

The remainder of each individual homogenate should be archived at <-20 °C with
the designation "Archive" and the expiration date recorded on the sample label.
The location of the archived samples should be indicated on the sample
processing record under "Notes."
J-9

-------
APPENDIX J
It is essential that the weights of individual homogenates yield a composite
homogenate of adequate size to perform all necessary analyses. Weights of
individual homogenates required for a composite homogenate, based on the
number of fish per composite and the weight of composite homogenate
recommended for analyses of all screening study target analytes (see Table 4-1),
are given in Table J-2. The total composite weight required for intensive studies
may be less than in screening studies if the number of target analytes is reduced
significantly.

The recommended sample size of 200 g for screening studies is intended to
provide sufficient sample material to (1) analyze for all recommended target
analytes (see Table 4-1) at appropriate detection limits, (2) meet minimum QA
and QC requirements for the analyses of replicate, matrix spike, and duplicate
matrix spike samples (see Section 8.3.3.4), and (3) allow for reanalysis if the QA
and QC control limits are not met or if the sample is lost. However, sample size
requirements may vary among laboratories and the analytical methods used.
Therefore, it is the responsibility of each program manager to consult with the
analytical laboratory supervisor to determine the actual weights of composite
homogenates required to analyze for all selected target analytes at appropriate
detection limits.

J.3 REFERENCES

California Department of Fish and Game. 1990. Laboratory Quality Assurance
Program Plan. Environmental Services Division, Sacramento, CA.

Crawford, J.K., and S.N. Luoma. 1993. Guidelines for Studies of Contaminants
in Biological Tissues for the National Water-Quality Assessment Program.
USGS Open-File Report 92-494. U.S. Geological Survey, Lemoyne, PA.

Jearld, A. 1983. Age determination, pp. 301-324. In: Fisheries Techniques.
L.A. Nielsen and D. Johnson (eds.). American Fisheries Society, Bethesda,
MD.

Puget Sound Estuary Program. 1990 (revised). Recommended protocols for fish
pathology studies in Puget Sound. Prepared by PTI Environmental Services,
Bellevue, WA. In: Recommended Protocols and Guidelines for Measuring
Selected Environmental Variables in Puget Sound. Region 10, U.S.
Environmental Protection Agency, Seattle, WA. (Looseleaf)

Sinderman, C. J. 1983. An examination of some relationships between pollution
and disease. Rapp. P. V. Reun. Cons. Int. Explor. Mer. 182:37-43.

Stober, Q. J. 1991. Guidelines for Fish Sampling and Tissue Preparation for
Bioaccumulative Contaminants. Environmental Services Division, Region 4,
U.S. Environmental Protection Agency, Athens, GA.
J-10

-------
                                                          APPENDIX J
Texas Water Commission.  1990.  Texas Tissue Sampling Guidelines. Texas
    Water Commission, Austin, TX.

U.S. EPA (U.S. Environmental  Protection Agency).  1986.   Bioaccumulation
    Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants
    and 301(h) Pesticides in  Tissues from Marine and Estuarine Organisms.
    EPA-503/6-90-002. Office of Marine and Estuarine Protection, Washington,
    DC.

U.S. EPA (U.S. Environmental  Protection Agency).  1995a.   Method  1613b.
    Tetra- through  Octa-Chlorinated Dioxins and Furans by Isotope Dilution
    HRGC/HRMS.   Final Draft.   Office of Water, Office  of Science  and
    Technology, Washington,  DC.

U.S. EPA (Environmental Protection Agency). 1995b.  QA/QC Guidance for
    Sampling and Analysis of  Sediments,  Water, and Tissues for  Dredged
    Material Evaluations—Chemical Evaluations.  EPA 823-B-95-001.  Office of
    Water, Washington, DC, and Department of the Army, U.S. Army Corps of
    Engineers, Washington, DC.

Versar,  Inc.  1982.  Sampling Protocols for Collecting Surface  Water,  Bed
    Sediment, Bivalves and Fish for Priority Pollutant Analysis-Final Draft Report.
    EPA Contract  68-01-6195.   Prepared  for  U.S. EPA Office  of Water
    Regulations and Standards.  Versar, Inc., Springfield, VA.

WDNR (Wsconsin Department of Natural Resources). 1988.  Fish Contaminant
    Monitoring Program—Field and Laboratory Guidelines (1005.1).  Madison,
    Wl.
                                                                 J-11

-------
National Coastal Condition Assessment
Laboratory Methods Manual                                          Date: November 2010
                                                                        Page 222
                                 APPENDIX B
                                Statement of Work
         Determination of Parent and Alkyl Polycyclic Aromatic Hydrocarbons
                  In Environmental Samples Related to BP Oil Spill

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 223

The Environmental Protection Agency (EPA) is currently responding to multiple environmental
issues as a result of the oil spill and dispersant usage in the Gulf of Mexico. Of particular
concern are the environmental impacts of crude oil to the ecological systems along the Gulf
coast from Texas to Florida, and possibly to upper continental coastline along the eastern
United States. Potentially thousands of environmental samples will be collected and analyzed
for volatile organic compounds (VOCs), metals, and semi-volatile organic compounds (SVOCs),
including Polycyclic Aromatic Hydrocarbons (PAH). The SVOC analysis, to date, have only
included the parent PAH compounds typically analyzed and reported by such analytical
protocols as found in EPA's Solid Waste Guidance document SW-846, METHOD 8270D
SEMIVOLATILE ORGANIC COMPOUNDS BY GAS CHROMATOGRAPHY/MASS
SPECTROMETRY (GC/MS). The EPA has now determined a need to evaluate the toxic effects
of the spilled crude oil on the aquatic ecosystems in the greater Gulf area, and must expand the
list of SVOCs to include alkyl substituted PAH compounds.

1.0 PURPOSE

There are limited analytical methods currently published that address the analysis of alkyl PAH.
SW-846 Method 8272, which is a new method to be released in the next SW-846 update, and
EPA's Contract Laboratory Program's (CLP) "Modified Analysis" protocol based upon the
existing CLP SVOC method, both address analysis of alkyl PAHs. Also ASTM Method D7363-
07 presents a method for determination of parent and alkyl PAH in sediment pore waters. This
method, however, uses solid-phase microextraction GC/MS techniques that may not be
appropriate for sea water and oily waters.

The purpose of this Statement of Work (SOW) is to provide an analytical laboratory an analytical
protocol for the sample preparation and analysis of parent PAH and alkyl PAH compounds in
water and sediment. The SOW is a combined protocol derived from SW-846, CLP and ASTM.
Web-link references to these published methods are included in this SOW. Reporting protocols
will also be incorporated into this SOW to allow the laboratory to deliver chromatograms to EPA
for the purpose of "fingerprinting" in case correlation analysis needs to be made between
environmental samples and the raw crude oil spilling from the BP oil platform in the Gulf.

2.0 SPECIAL REQUIREMENTS

The laboratory performing the analyses presented in this SOW must have demonstrated
experience in the analysis of SVOCs, including the parent PAH and alkyl PAH compounds listed
in Table 1. The laboratory should also have demonstrated experience in oil correlation analysis
via GC/MS fingerprinting. The laboratory must also demonstrate the existence of a formal
laboratory Quality System. This may be demonstrated by showing proof of an existing
accreditation certificate issued by NELAC or proof of an existing contract issued through the
EPA CLP. The laboratory must certify in writing that they have current GC/MS capability and
capacity to provide for analytical results within 72 hours of sample receipt at a frequency of
approximately 20 samples per day.

3.0 QUALITY ASSURANCE (QA) REQUIREMENTS

Laboratory must perform all analysis under a formal Laboratory Quality Assurance Program. At
a minimum, the laboratory must have a Quality Program that address items in Sections 1.0
through 7.0 of Exhibit E taken from the EPA CLP SOM1.1 found at the following:
http://www.epa.gov/superfund/programs/clp/som1.htm. The laboratory must have a QA/QC

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 224
program in place to assess the quality of the data. EPA requires that recipients of funds for work
involving environmental data comply with the American National Standard ANSI/ASQC E4-
1994, "Specifications and Guidelines for Quality Systems for Environmental Data Collection and
Environmental Technology Programs." Information concerning quality specifications for non-
EPA organizations is available at http://www.epa.gov/quality/exmural.html. EPA reserves the
right to inspect all laboratory Quality Assurance documents, including method specific Standard
Operating Procedures (SOPs). EPA also reserves the right to inspect all raw data and to
perform on-site inspections of the laboratory during the period of performance of any contract
agreed upon for this project.

4.0    DETAILED TASK DESCRIPTION

Table 1 lists the target analytes required for this SOW.  Analyses are to be performed using the
Selected Ion Monitoring (SIM) technique. Table 1 lists the specific ions that are to be monitored
and quantified.

Table 1. Primary Ions Monitored for Each Target Analyte during GC/MS Analysis
Compound
n-alkanes(C10-C35)*
Naphthalene-d8 (IS)
Naphthalene P
2-Methylnaphthalene P
1-Methylnaphthalene P
C2-naphthalenes
CS-naphthalenes
C4-naphthalenes
2-Methylnaphthalene-d1 0(MS)
Acenaphthene-d10 (IS)
Acenaphthylene P
Acenaphthene P
Acenaphthylene-d8 (S)
Fluorene-dlO(IS)
Fluorene P
C1-fluorenes
C2-f lucre nes
C3-fluorenes
Phenanthrene-d10
Phenanthrene P
C1 -phenanthrenes/anthracenes
C2-phenanthrenes/anthracenes
C3-phenanthrenes/anthracenes
C4-phenanthrenes/anthracenes
2,3-Dimethylanthracene R
1-Methylphenanthrene R
Anthracene P
Anthracene-d10 (S)
Fluoranthene-d10 (IS)
Fluoranthene P
Ion
57
136
128
142
142
156
170
184
152
162
152
153
160
176
166
180
194
208
188
178
192
206
220
234
206
192
178
188
212
202
Compound
Pyrene P
C1 -Fluoranthenes/Pyrenes
C2-Fluoranthenes/Pyrenes
C3-Fluoranthenes/Pyrenes
1-Methylpyrene R
2-methylfluoranthene R
Pyrene-d10 (S)
Chrysene-d12(IS)
Chrysene P
C1-chrysenes
C2-chrysenes
C3-chrysenes
C4-chrysenes
6-methylchrysene R
Benzo (a) anthracene P
Benzo (a) pyrene-d12 (S)
Perylene-d12(IS)
Benzo(b)fluoranthene P
Benzo(k)fluoranthene P
Benzo(e)pyrene P
Benzo(a)pyrene P
Perylene P
lndeno(1,2,3-cd)pyrene P
Dibenzo(a,h)anthracene P
Benzo(g,h,i)perylene P
Hopanes (191 family)*
Steranes(217family)*
Steranes(218family)*


Ion
202
216
230
244
216
216
212
240
228
242
256
270
284
242
228
264
264
252
252
252
252
252
276
278
276
191
217
218


* = Not to be quantified. For fingerprinting analysis only; IS = Internal Standard; S = Surrogate Compound;
P = Parent PAH; MS = Matrix Spike Compound; R = Reference Compound

-------
National Coastal Condition Assessment
Laboratory Methods Manual                                              Date: November 2010
                                                                                Page 225

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 226

4.1 Sample Preparation

It is to be expected that water and sediment samples will be analyzed, and there is the potential
that oil from the spill may be analyzed as well. Sample preparation procedures to follow for
samples received under this SOW are as follows:

Water

Water samples and water samples containing slight oil contamination (visible sheen, but very
little water - oil interface) should be extracted using a continuous liquid-liquid extraction
technique detailed in EPA's CLP protocols for SVOCs. This can be found in Exhibit D of
SOM1.1 at http://www.epa.gov/superfund/programs/clp/som1 .htm, with the following exceptions:
Do not use the internal standard and deuterated monitoring compounds (DMC) listed in the
SOM protocol. Instead use the internal standards and surrogate compounds listed in Table 1.
All references to SMO (sample management office) shall be ignored. Instead for any
consultation of protocol or issues, discuss with the EPA personnel designated in the SOW.
Also, there is concern that the ASTM protocol for using solid phase microextraction may not be
applicable for the oil spill, and therefore the micorextraction technique is not to be used.

For samples having a definite water - oil interface, the laboratory should contact the EPA
Regional sample requestor for instructions, e.g. shake sample and extract as a whole sample,
or separate and analyze water and oil as separate phases.

Sediment

The laboratory should follow the Soil/Sediment protocols detailed in the EPA CLP SOM 1.1. Any
of the three listed techniques for sample extraction many be used. For extracts that appear
"clean" after concentration, the GPC procedure given in SOM1.1 may be skipped. For sample
extracts with obvious high levels of oil matrix, the alumina cleanup procedure detailed in 40 CFR
Part 300, Appendix C, Section 4.6.3.1 may be used. The surrogates listed above shall still be
used if cleanup is performed via the CFR protocol.

Oil

Samples received that are obvious oil samples may be analyzed using a direct dilution process.
A measured aliquot of the oil sample is placed in a 10 mL volumetric flask and brought to
volume with methylene chloride. The amount of oil diluted shall be at the laboratory's discretion,
but the dilution shall be such that the best detection levels may be achieved while preventing
undue contamination of the analytical GC column and GC/MS. The sample and methylene
chloride shall be shaken and then allowed to settle until all obvious non-soluble material has
settled to the bottom. An aliquot of the oil/methylene chloride extract shall be then analyzed by
GC/MS. Surrogates are to be added to the oil sample aliquot before methylene chloride is
added.

4.2 Instrumental Analysis

All analysis are to be made on GC/MS instrumentation and are to be made in the selected Ion
Mode (SIM). The laboratory should follow the protocols given in the EPA SOM1.1 method for
SVOC SIM analyses with the exception that the analytes, internal standard compounds and
surrogates listed in Table 1 are to be analyzed instead of those listed in SOM 1.1. All parent

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 227
PAH, alkyl PAH, and matrix spike compounds must be calculated using the internal standard
method in which response factors are calculated. The laboratory must tune each GC/MS
instrument to meet the criteria listed in SOM 1.1. The laboratory must also calibrate target
analytes and surrogates using a minimum of a 5-point calibration curve, as detailed in SOM 1.1.
The laboratory must calibrate target analytes and surrogates at the concentrations listed in
Table 2. The 5-point calibration analysis is to be performed before any samples are analyzed
under this SOW.  Each  parent PAH and surrogate listed in Table 1  is to be calibrated against
the internal standard listed directly above it in Table 1.  For example, anthracene is to be
calibrated using phenanthrene as the internal standard. The % RSD should be calculated for
each PAH using the instructions given in SOM 1.1. Each parent PAH should have a %RSD no
greater than 20%. After 15 samples, including QC samples, have been analyzed, or in
situations in which the GC/MS has been inoperative for 6 hours or longer, a calibration
verification analysis  must be made to ensure the applicability of the 5-point calibration curve.
The lab must analyze the single point calibration verification at a concentration level consistent
with either Level 3 or Level 4 in Table 2. After the calibration verification analysis, the laboratory
should  calculate the %Difference according to instructions in SOM 1.1.  The % Difference must
be no greater than +/- 30%.  If the % Difference is greater than 30%, the laboratory may
reanalyze the calibration verification again.  If the instrument can still not meet the %Difference
criteria, the laboratory must perform instrument maintenance and analyze another 5-point
calibration analysis.

There are no 5-point calibration or continuing calibration criteria for the alkyl PAH compounds.
Alkyl PAH compounds are calculated against the internal standard listed directly above  the alkyl
PAH in Table 1. The same response factor used for the parent PAH is to be used for the alkyl
PAH associated with the parent PAH. Specifically:  C2, C3, and C4 naphthalenes will use the
same response factor as naphthalene; C1, C2, and C3 fluorenes will use the same response
factor as fluorene; C1, C2, C3 and C4 phenanthrenes/anthracenes shall use the same response
factor as phenanthrene; C1, C2, and C3 fluoranthenes/pyrenes shall use the same response
factor as fluoranthene; C1, C2, C3 and C4 chrysenes shall use the same response factor as
chrysene.

** Reference compounds have been added to the list of analytes that are to be included in
calibration analysis.  A response factor for each of the reference compounds is to be calculated
at each calibration level. The response factor will be used for informational purposes only.  The
reference compound is  not to be specifically analyzed for in the environmental samples. For
example, 1-methylpyrene  should be included in the calibration analysis and a response factor is
to be determined for the compounds using Fluoranthene-d10 as the internal standard. When
the environmental samples have are analyzed only the total C1-Fluoranthenes/Pyrenes will be
calculated using the response factor that was calculated for pyrene.

Table 2. Calibration Concentrations for Target PAH and Surrogate Compounds: Initial 5-Point Calibration
Compound Class
PAH
Surrogates
Internal Standards
Reference Compounds
Calibration
Level 1
(ng/uL)
0.10
0.10
0.40
0.10
Calibration
Level 2
(ng/uL)
0.20
0.20
0.40
0.20
Calibration
Level 3
(ng/uL)
0.40
0.40
0.40
0.40
Calibration
Level 4
(ng/uL)
0.80
0.80
0.40
0.80
Calibration
Level 5
(ng/uL)
1.0
1.0
0.40
1.0
SOW Required Detection Limits:

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 228
For this SOW, the instrument and sample preparation scheme must be such that a Reporting
limit of 0.1 ug/L can be achieved for water samples. Sediment samples must meet a reporting
limit of 4 ug/kg. For oily samples requiring only dilution with methylene chloride for sample
preparation, the laboratory should strive to reach as low of a reporting limit as feasible.

4.3 Special Quality Control Analyses

The laboratory must ensure their analytical systems are in control. For each batch of 15
samples, the laboratory must analyze reagent blanks for each matrix analyzed. The laboratory
shall ensure that no PAH constituents are present in the laboratory blank analysis. If the blank
samples are contaminated with PAH, the laboratory must re-analyze any samples associated
with the blanks, at no additional cost to the government.

For every batch of 15 samples, a matrix spike (MS) and matrix spike duplicate (MSD) must be
analyzed. The MS/MSD pair shall consist of 2-methylnaphthalene-d10. The concentration of
the compound is left to the discretion of the laboratory, but should be at a concentration that
falls within the mid-range level of the 5-point calibration curve. Percent recovery is calculated
for each MS and MSD. The MS and MSD percent recoveries must lie between 50 - 150%
recovery. If this criterion is not met, all samples associated with the MS/MSD shall be
reanalyzed at no additional cost to the government.

5.0 DELIVERABLES

Copies of all raw and calculated analytical data associated with this SOW shall remain in
possession of the laboratory at least until 365 days from the time results have been submitted to
EPA.

A copy of calculated results for the parent PAH and alkyl PAH compounds shall be submitted
via electronic spreadsheet to each EPA recipient listed in the Task Order directive from EPA.
These results must contain at a minimum the results of the PAH analysis, as well as results for
surrogate analysis. A copy of the 5-point calibration analysis must be included with the
tabulated results of the sample analysis. This must include the calculated response factors for
each parent PAH, each surrogate, and each reference compound. Also, electronic pdf copies of
various chromatograms shall also be submitted with the calculated results. The chromatograms
required are the following:

(a) Total ion chromatogram in which all compounds present in the sample, including internal
standards and surrogates are shown.
(b) Extracted ion chromatogram detailing the fingerprint of the m/z 57 mass ion, which is
characteristic of normal and branched alkanes. The chromatogram retention time
window must be such that normal alkanes from C10 through 35 are displayed in a single
chromatogram.
(c) Extracted ion chromatogram detailing the fingerprint of the m/z 57 mass ion
characteristic of the normal alkanes C17 and C18, and the branched alkanes, Pristane
and Phytane. The chromatogram retention time window must be set to start 0.5 minutes
before the elution of C17 and end 0.5 minutes after the elution of Phytane.
(d) Extracted ion chromatogram detailing the fingerprint of the m/z 156 mass ion, which is
characteristic of the C2- naphthalenes. These compounds should lie retention time wise

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 229

between C14 and C15 normal alkanes. For the pattern recognition of these compounds
see page 16 of the ASTM D7363-07 method.
(e) Extracted ion chromatogram detailing the fingerprint of the m/z 170 mass ion, which is
characteristic of the C3- naphthalenes. These compounds should lie retention time wise
between C15 and C16 normal alkanes. For the pattern recognition of these compounds
see page 16 of the ASTM D7363-07 method.
(f) Extracted ion chromatogram detailing the fingerprint of the m/z 184 mass ion, which is
characteristic of the C4- naphthalenes. These compounds should lie retention time wise
between C16 and C17 normal alkanes. For the pattern recognition of these compounds
see page 16 of the ASTM D7363-07 method. If an internal standard or surrogate
compound containing mass 184 also elutes in this time frame, normalize the height of
the chromatogram on the C4 naphthalenes.
(g) Extracted ion chromatogram detailing the fingerprint of the m/z 180 mass ion, which is
characteristic of the C1-fluorenes. For the pattern recognition of these compounds see
page 19 of the ASTM D7363-07 method.
(h) Extracted ion chromatogram detailing the fingerprint of the m/z 194 mass ion, which is
characteristic of the C2-fluorenes. For the pattern recognition of these compounds see
page 19 of the ASTM D7363-07 method.
(i) Extracted ion chromatogram detailing the fingerprint of the m/z 208 mass ion, which is
characteristic of the C3-fluorenes. For the pattern recognition of these compounds see
page 19 of the ASTM D7363-07 method. If an internal standard or surrogate compound
containing mass 208 also elutes in this time frame, normalize the height of the
chromatogram on the C3-fluorenes.
(j) Extracted ion chromatogram detailing the fingerprint of the m/z 192 mass ion, which is
characteristic of the C1-phenanthrenes. These compounds should lie retention time
wise between C19 and C20 normal alkanes. For the pattern recognition of these
compounds see page 20 of the ASTM D7363-07 method.
(k) Extracted ion chromatogram detailing the fingerprint of the m/z 206 mass ion, which is
characteristic of the C2-phenanthrenes. These compounds should lie retention time
wise between C20 and C21 normal alkanes. For the pattern recognition of these
compounds see page 20 of the ASTM D7363-07 method.
(I) Extracted ion chromatogram detailing the fingerprint of the m/z 220 mass ion, which is
characteristic of the C3-phenanthrenes. These compounds should lie retention time
wise between C21 and C22 normal alkanes. For the pattern recognition of these
compounds see page 20 of the ASTM D7363-07 method.
(m) Extracted ion chromatogram detailing the fingerprint of the m/z 234 mass ion, which is
characteristic of the C4-phenanthrenes. These compounds should lie retention time
wise between C22 and C23 normal alkanes. For the pattern recognition of these
compounds see page 20 of the ASTM D7363-07 method.
(n) Extracted ion chromatogram detailing the fingerprint of the m/z 216 mass ion, which is
characteristic of the C1-Fluoranthenes.
(o) Extracted ion chromatogram detailing the fingerprint of the m/z 230 mass ion, which is
characteristic of the C2-Fluoranthenes.
(p) Extracted ion chromatogram detailing the fingerprint of the m/z 244 mass ion, which is
characteristic of the C3-Fluoranthenes.
(q) Extracted ion chromatogram detailing the fingerprint of the m/z 242 mass ion, which is
characteristic of the C1-chrysenes.

-------
National Coastal Condition Assessment
Laboratory Methods Manual Date: November 2010
Page 230

(r) Extracted ion chromatogram detailing the fingerprint of the m/z 256 mass ion, which is
characteristic of the C2-chrysenes.
(s) Extracted ion chromatogram detailing the fingerprint of the m/z 270 mass ion, which is
characteristic of the C3-chrysenes.
(t) Extracted ion chromatogram detailing the fingerprint of the m/z 284 mass ion, which is
characteristic of the C4-chrysenes.
(u) Extracted ion chromatogram detailing the fingerprint of the m/z 191 mass ion, which is
characteristic of the 27 through 34 carbon hopane family.
(v) Extracted ion chromatogram detailing the fingerprint of the m/z 217 mass ion, which is
characteristic of the 27 through 29 carbon alpha, beta sterane family.
(w) Extracted ion chromatogram detailing the fingerprint of the m/z 218 mass ion, which is
characteristic of the 27 through 29 carbon alpha, beta sterane family.

One should reference a journal citation or petroleum geochemistry for the retention time location
of the sterane and hopane families. As an example: Baling and Faksness: Environmental
Forensics (2002) 3, pp. 263-278; Improved and Standardized Methodology for Oil Spill
Fingerprinting.

A reference grade crude oil sample shall be diluted in methylene chloride and analyzed using
the exact GC and GC/MS conditions that are used to detect, quantify, and fingerprint the
analytes given above. The laboratory must use the South Louisiana Sweet Crude oil sample
provided by RT Corporation, Laramie, WY. If the laboratory does not possess this crude oil,
they must procure a sample from RTC. This reference sample shall be used to determine exact
retention times and patterns for the PAH homologues and hopane and sterane families.

Every effort should be made to ensure that the chromatograms are presented in such a manner
as to be able to easily distinguish each individual homologue of the alkylated PAH series and
the hopane and sterane families.

5.1 Laboratory Deliverable Turnaround times

Quantified spreadsheet results must be delivered to EPA within 72 hours of receipt of sample.
The required fingerprint chromatograms must be delivered to EPA within 5 days of receipt of
samples.

-------
National Coastal Condition Assessment
Laboratory Methods Manual                                         Date: November 2010
                                                                        Page 231
                                APPENDIX C


             Summary of EPA Analytical Methods for Dispersant Analysis

-------
National Coastal Condition Assessment
Laboratory Methods Manual
Date: November 2010
          Page 232
                Summary of EPA Analytical Methods for Dispersant Analysis
Compound

Propylene Glycol
2-Butoxyethanol
Di(Propylene Glycol)
Butyl Ether
2-Ethylhexanol
Dioctylsulfosuccinate,
sodium salt
CAS

57-55-6
111-76-2
29911-28-
2
104-76-7
577-11-7
EPA Method
ID

EPA SW 846
Modified
8270
EPA R5/6
LC
EPA R5/6
LC
EPA SW 846
Method 8260
EPA RAM-
DOSS
Technology

Direct Inject
GC/MS
Direct Inject
LC/MS/MS
Direct Inject
LC/MS/MS
Heated
purge
GC/MS
LC/MS/MS
Reporting
Limits

500 ug/L
125 ug/L
1 ug/L
10 ug/L
20 ug/L
EPA
Benchmark

500,000 ug/L
165 ug/L
ND
ND
40 ug/L
CAS:  Chemical Abstract Service number
ND: Not determined at this time
SW846: "Test Methods for Evaluating Solid Waste, Physical/Chemical Methods"
          See (http://www.epa.gov/epawaste/hazard/testmethods/sw846/index.htm')
LC/MS/MS: Liquid Chromatograph with Tandem MassSpectrometry

-------