New Preservation Techniques for Nutrient Samples

In Reply Refer To:                               August 5, 1994
Mail Stop 412

OFFICE OF WATER QUALITY TECHNICAL MEMORANDUM 94.16

Subject:  New Preservation Techniques for Nutrient Samples

                        SUMMARY

Beginning October 1, 1994, the Water Resources Division will
discontinue the use of mercuric chloride to preserve ambient
samples collected for analysis of nutrients.  Instead, the
Division will preserve ambient water samples to be analyzed
for nutrients by chilling only, with mailing within 3 days
to the National Water Quality Laboratory (NWQL).  Some
Division projects may be collecting nutrient samples in
cooperation with regulatory agencies wherein the samples are
defined as compliance (collected for compliance with the
Clean Water Act or the Safe Drinking Water Act) rather than
ambient.  The U.S. Environmental Protection Agency (EPA)
and many State regulatory agencies require sulfuric acid
preservation of such compliance nutrient samples.  Therefore,
on October 1, 1994, the NWQL will also begin analyzing sulfuric
acid-preserved samples.

This memorandum identifies a number of actions required to
implement the noted changes.  Table 1 summarizes the actions
and associated implementation dates.

Table 1.  Actions and Implementation Dates Required to Change
          Nutrient Preservation Techniques
_______________________________________________________________________
Implementation Date                           Action
_______________________________________________________________________

October 1, 1994            o Discontinue use of mercuric chloride
                             to preserve nutrient samples.
                           o Adopt chilling only to preserve
                             ambient nutrient samples.
                           o NWQL begins analysis of chilled only
                             nutrient samples.*
                           o All schedules are changed and mercuric
                             chloride-preserved NWQL codes are
                             replaced with chilled only NWQL codes.*
                           o NWQL also begins analysis of nutrient
                             samples preserved with sulfuric acid
                             collected for compliance purposes.
                           o Begin 3-month grace period for NWQL
                             analysis of nutrient samples preserved
                             with mercuric chloride to assist
                             Division projects that have special
                             need for these data.

December 31, 1994          o End 3-month grace period for analysis
                             of nutrient samples preserved with
                             mercuric chloride.

January 1, 1995            o Lab codes for nutrient samples
                             preserved with mercuric chloride
                             become invalid and can no longer be
                             requested.

February 1, 1995           o Begin 1-month period in which the NWQL
                             will accept unused mercuric chloride
                             ampoules for disposal.

February 28, 1995          o End 1-month period in which the NWQL
                             will accept unused mercuric chloride
                             ampoules for disposal.
_____________________________________________________________________
  *The term "chilled only" means chilling and maintaining all samples
at 4 degrees Celsius (without freezing).  Thus, for unfiltered nutrient
constituents, field processing of chilled only samples includes filling
and chilling the nutrient bottle.  In contrast, for the so-called
dissolved nutrient constituents, field processing of chilled only samples
includes filtration through a 0.45-micrometer filter into a nutrient
bottle followed by chilling.

                     BASIS FOR THE DECISION

In 1992-93, Charles Patton and Earl Truitt of the NWQL compared the
effects of three preservation techniques--mercuric chloride plus

chilling, sulfuric acid plus chilling, and chilling only--on nutrients
in 11 surface-water and 3 ground-water samples from around the United
States.  Table 2 shows the ranges of nutrient constituent
concentrations for the samples.

Table 2.  Constituent Concentration Ranges of Samples in the Nutrient
          Preservation Experiment
______________________________________________________________________

            Constituent(s)*                 Range of Concentration
                                                   (mg/L)
______________________________________________________________________
                                        Surface Water    Ground Water
                                           n = 11          n = 3
______________________________________________________________________

Ammonia, filtered                        0.02-5.5         0.02-0.04
Nitrate + nitrite, filtered              0.04-8.7         2.4 -56
Nitrite, filtered                        0.00-1.9         0.00-0.01
Organic nitrogen + ammonia, filtered     0.1 -6.9         0.05-1.3
Organic nitrogen + ammonia, unfiltered   0.2 -9.8         0.04-1.4
Orthophosphate, filtered                 0.01-3.9         0.00-0.03
Phosphorus, filtered                     0.01-3.9         0.00-0.08
Phosphorus, unfiltered                   0.03-4.8         0.00-0.2
______________________________________________________________________
  *The term "filtered" replaces the previously used term "dissolved"
and "unfiltered" replaces "whole water recoverable."

The data show that the samples encompassed a wide range of
concentrations for each constituent, representing conditions from
pristine to contaminated.

The comparative preservation tests were run for a minimum of 31
days on each sample.  The overall results indicated that within
analytical and test precision, chilling only provides equivalent
preservation to mercuric chloride plus chilling or to sulfuric
acid plus chilling.  As expected, the tests also showed that
sulfuric acid systematically destroys nitrite in samples.  In
contrast to expectations, the 31-day stabilities of orthophosphate
in all acid-preserved samples were comparable to those for
mercuric chloride-preserved and chilled only samples.

A summary of the nutrient preservation experiment is attached to
the electronic copy of this technical memorandum.  To access the
memorandum and the attachment, one must be registered on QVARSA.
After login, enter the command TECH_MEMO and follow the
instructions given for the menu-driven system.  In addition,
Charles Patton is preparing a detailed report explaining the
experimental design of the study and the results.  District and
Regional Water-Quality Specialists will be notified by EDOC when
this document is available.

In actual Division use, as in the NWQL experiment, all samples to
be analyzed for filtered nutrient constituents (previously referred
to as dissolved) are passed through a 0.45-micrometer filter at the
collection site.  This step is known to remove most bacteria and
other microorganisms from filtrates.  Therefore, microorganisms
present in original samples should have negligible effect on the
stability of filtered nutrient constituents.  In the case of
unfiltered samples, nitrogen and phosphorus metabolized by
microorganisms will be released during the rigorous digestion step
included in the analysis (with the exception of nitrate plus nitrite
nitrogen).

Thus, based on experimental results, plus knowledge of the effects
of filtration and the analytical methods, the Division selected
chilling only as the standard technique for preserving ambient
samples for nutrient analysis.  Chilling only was preferable to
sulfuric acid plus chilling to (a) avoid widespread shipment, use,
and disposal of sulfuric acid ampoules, (b) achieve the lowest
possible costs, and (c) minimize the number of bottles that
projects must fill and ship to the NWQL for each nutrient sample.
With chilling only, two bottles are required for all nutrient
species.  With sulfuric acid plus chilling, a separate, third
bottle, using chilling only, is required for the nitrite and
orthophosphate determinations.

                   THREE-MONTH GRACE PERIOD

Some Division projects may be in the middle of long-term
collection of nutrient data using mercuric chloride plus chilling
for preservation.  To assist these projects, the NWQL will
continue to offer analysis of samples preserved with mercuric
chloride through December 31, 1994.  Projects can also use this
period to send split nutrient samples to the NWQL (one preserved
with mercuric chloride, the other chilled only) if a project-
specific need exists for such a comparison.  For split sample
work, projects will be responsible for all analytical costs and
interpretation of the resultant data.

         DISPOSAL OF UNUSED MERCURIC CHLORIDE AMPOULES

The NWQL will accept unused mercuric chloride ampoules for
disposal February 1-28, 1995.  This timeframe allows projects
about 6 months to locate and prepare unused ampoules for shipping.
The NWQL must notify regulatory authorities in advance of the
extra disposal of mercuric chloride during February.  Therefore,
no ampoules can be accepted before February 1, 1995.  After
February, the NWQL will not be permitted to dispose of waste water
which has been treated to remove mercuric chloride.  Therefore,
unused ampoules received after February 28, 1995, will be returned
to the senders who will have to arrange for disposal at a licensed
disposal facility within their respective States.

Mercuric chloride ampoules are a hazardous material and must be
packed and shipped according to Department of Transportation (DOT)
regulations.  Unused ampoules MUST be shipped in their ORIGINAL
cardboard containers with the foam liner or in a cooler with the
ampoules double bagged in trash bags with packing material.  The
original cardboard containers are DOT approved, but other
cardboard boxes are not approved and cannot be used.  The unused
ampoules should be shipped via United Parcel Service (UPS) ground
transportation.  Federal Express and the Postal Service should not
be used.  Please write "ATTENTION CARLOS AROZARENA -- UNUSED HGCL2
AMPOULES" on each shipping container.  Contact Will Lanier, NWQL,
(WDLANIER), at telephone number (303) 467-8065, if you have
problems either preparing the unused ampoules for shipping, or
coordinating your shipment with the local UPS office.

Before shipping, please send an EDOC to Carlos Arozarena (USERID -
SAFETY) stating that a specified number of unused ampoules will be
sent on a given date.  This forewarning is required by UPS in case
there is an accident.  Please include the phone number of the NWQL
guard [(303) 467-8064] in the shipping information so that if a
problem occurs, the NWQL will be contacted and can then work to
resolve the problem.

IMPORTANT:  Please note that the NWQL is offering disposal only of
unused mercuric chloride ampoules.  Do not send other hazardous
materials for disposal.  Other hazardous materials sent will be
returned, or if return is illegal, the NWQL will bill the sender
for the full cost of disposal.

Elimination of mercuric chloride as a preservative is a change
that has been widely sought for a number of years by Division
personnel.  To investigate options, the OWQ and NWQL expended a
large amount of funds.  Also, costs associated with handling and
disposal of the unused ampoules will be borne by the NWQL.  For
these reasons, no refunds will be made for unused mercuric
chloride ampoules sent to the NWQL.

  COORDINATION WITH THE U.S. ENVIRONMENTAL PROTECTION AGENCY

The NWQL and OWQ have shared the experimental results of the tests
with the EPA under the auspices of the Methods Comparability
Council of the Intergovernmental Task Force on Monitoring Water
Quality.  We believe this effort will lead to EPA signing a memo
acknowledging that chilling only is acceptable as the
preservation technique for ambient water samples collected for
nutrient analysis.  The EPA will continue to require that sulfuric
acid be used in conjunction with chilling to preserve nutrient
samples collected for compliance with the Clean Water Act and the
Safe Drinking Water Act.

                 SULFURIC ACID-PRESERVED SAMPLES

The NWQL will begin offering analysis of sulfuric acid-preserved
nutrient samples on October 1, 1994.  The purpose of offering analysis
for sulfuric acid-preserved samples is to enable Division projects to
meet regulatory agency requirements for chemical preservation of
compliance samples.

Sulfuric acid ampoules to preserve nutrient samples (in the
standard 125-milliliter bottle) are available through the Ocala
Quality of Water Service Unit and can be ordered through the Ocala
Supply Orders ID (OCALAMAN).  The cost is $35 per lot of twenty-
four 1-milliliter ampoules containing 25-percent sulfuric acid.

         FIELD PREPARATION INSTRUCTIONS AND TURNAROUND TIMES
                       FOR NUTRIENT SAMPLES

                   Ambient Samples (Chill Preserved)

As in the past, ambient samples to be analyzed for filtered nutrient
constituents must be passed through a 0.45-micrometer filter into a
brown 125-milliliter polyethylene bottle and immediately packed in
ice at the collection site.  Designate these samples as "FCC" to
differentiate them from filtered, chilled samples preserved with
mercuric chloride, which will continue to be designated "FC"
until December 31, 1994.  Omit the filtration step for ambient
samples to be analyzed for unfiltered nutrient constituents.
Designate these samples as "RCC" to differentiate them from raw
chilled samples preserved with mercuric chloride, which will continue
to be designated "RC" until December 31, 1994.  Within 3 days of
collection, ship the chilled samples to the NWQL with next-day-delivery
priority.  The NWQL will analyze nutrient samples within 14 days
of receipt.  These timeframes will insure that analytical
determinations are performed well within the 31-day holding times
supported by the experimental data.

               Compliance Samples (Sulfuric Acid Preserved)

Beginning on October 1, 1994, the NWQL will analyze nutrient
samples collected to assure compliance with requirements of the
Federal Clean Water Act and the Safe Drinking Water Act.  Field
processing and maximum holding times prior to analysis for such
samples are specified in Table II, paragraph 136.3, Volume 40 of
the Code of Federal Regulations.  Specifically, samples to be
analyzed for filtered nutrient constituents must be passed through
a 0.45-micrometer filter into a brown 125-milliliter polyethylene
bottle, acidified to pH < 2 with sulfuric acid, and immediately
packed in ice at the collection site.  Designate these samples as
"FCA" (filtered, chilled, acidified).  Omit the filtration step
for compliance samples to be analyzed for unfiltered nutrient
constituents.  Designate these samples as "RCA" (raw, chilled,
acidified).  Within 3 days of collection, ship the acidified,
chilled samples to the NWQL with next-day-delivery priority.
The NWQL will analyze nutrient samples within 14 days of receipt.
These timeframes will insure that analytical determinations are
performed well within the 28-day holding times specified in the
Code of Federal Regulations.

Compliance monitoring samples to be analyzed for nitrite and
orthophosphate must be passed through a 0.45-micrometer filter
into a brown 125-milliliter polyethylene bottle, and immediately
packed in ice at the collection site.  Sulfuric acid MAY NOT be
added to these samples, which must be analyzed within 48 hours of
collection.  While field processing for "FCC" samples as described
above is appropriate for such samples, the 48-hour holding time
requirement is difficult to achieve when sampling locations are
distant from the NWQL.  Therefore, for compliance monitoring
samples, it is most practical to arrange for analytical
determinations of nitrite and orthophosphate at a laboratory near
the collection site.  In some cases, it may be possible to make
special arrangements at the NWQL to determine nitrite and
orthophosphate in compliance samples.  Special arrangements for
such samples would include MANDATORY, 1-week advance notice to
affected NWQL service units and Monday, Tuesday, or Wednesday
arrival days at the NWQL.  To make special arrangements, contact
the Chief, Inorganic Program (MSHOCKEY) at telephone number
303/467-8101.

   INFORMATION FOR SUBMITTING NUTRIENT SAMPLES TO THE NWQL

To implement the described changes, the NWQL will change
schedules, delete and create lab codes, and create new bottle
types.  Projects need to observe the upcoming changes to eliminate
sample mix-ups and analytical errors.  If District personnel wish
to run comparison studies between chilled only and chilled plus
chemically preserved samples, sample splits should be treated as
duplicates and submitted on different Analytical Services Request
(ASR) forms using different times.  Unless this is done, the
electronic data system will report only the second analytical
value; the first will be lost.

On October 1, 1994, mercuric chloride lab codes will be deleted
from all schedules and replaced with new lab codes for the chilled
only preservation technique.  If mercuric chloride lab codes are
needed during the 3-month grace period (October 1-December 31,
1994) they must be recorded as an "add" on the ASR form.  On
January 1, 1995, all mercuric chloride-related lab codes will
become invalid and may not be requested.

The NWQL will prepare new schedules for sulfuric acid-preserved
samples.  The schedule numbers will be released in September
1994.

IMPORTANT:  After February 28, 1995, the NWQL will no longer be
able to dispose of mercuric chloride-preserved samples.
Furthermore, the NWQL will not be running standards for mercuric
chloride-preserved samples.  Therefore, analytical results will be
incorrect for samples designated "filtered-chilled only" ("FCC"
bottle designation) or "raw-chilled only ("RCC" bottle
designation) if they are actually preserved with mercuric
chloride.

Table 3 summarizes information that will be needed for personnel
submitting samples to the NWQL for nutrient analyses beginning
October 1, 1994.

Table 3.  Information for Submitting Samples to the NWQL for Nutrient
          Analysis Beginning on October 1, 1994
______________________________________________________________________________

                                    Standard Range Analyses

-------------------------------------------------------------------------------
                             HgCl2       | Chilled Only**    |    H2SO4**
                        NWQL       M Bot | NWQL       M Bot | NWQL       M Bot
CONSTITUENT(S)*         Code PCode C Typ | Code PCode C Typ | Code PCode C Typ
                                         |                  |
-------------------------------------------------------------------------------
Nitrogen, Ammonia,      0301 00608 B FC  | 1976 00608 F FCC | 1991 00608 G FCA
  filtered                               |                  |
Nitrogen, Nitrate +     0228 00631 B FC  | 1975 00631 E FCC | 1990 00631 F FCA
  Nitrite, filtered                      |                  |
Nitrogen, Nitrite,      0160 00613 B FC  | 1973 00613 F FCC |        NA
  filtered                               |                  |
Nitrogen, Organic +     1687 00623 C FC  | 1985 00623 D FCC | 1994 00623 E FCA
  Ammonia, filtered                      |                  |
Nitrogen, Total         1570 00602 B FC  | 1989 00602 C FCC |       NA
  Species, filtered                      |                  |
Nitrogen, Organic +     1688 00625 C RC  | 1986 00625 D RCC | 1995 00625 E RCA
  Ammonia, unfiltered                    |                  |
Phosphorus,             0162 00671 B FC  | 1974 00671 H FCC |       NA
  Orthophosphate,                        |                  |
  filtered                               |                  |
Phosphorus, filtered    1685 00666 C FC  | 1983 00666 D FCC | 1992 00666 E FCA
Phosphorus, unfiltered  1686 00665 C RC  | 1984 00665 D RCC | 1993 00665 E RCA
Phosphorus,             0282 00678 A RC  | 1987 00678 B RCC |       NA
  Orthophosphate +                       |                  |
  hydrolyzable,                          |                  |
  unfiltered                             |                  |
Phosphorus,             0279 00677 A FC  | 1988 00677 B FCC |       NA
  Orthophosphate +                       |                  |
  hydrolyzable,                          |                  |
  filtered                               |                  |

-----------------------------------------------------------------------------

                                       Low-Level Analyses

------------------------------------------------------------------------------
                             HgCl2       | Chilled Only**  |      H2SO4
                        NWQL       M Bot | NWQL       M Bot | NWQL       M Bot
CONSTITUENT(S)*         Code PCode C Typ | Code PCode C Typ | Code PCode C Typ
                                         |                  |
------------------------------------------------------------------------------
Nitrogen, Ammonia,      0830 00608 A FC  | 1980 00608 H FCC |       NA
  filtered                               |                  |
Nitrogen, Nitrite +     0826 00631 A FC  | 1979 00631 G FCC |       NA
  Nitrate, filtered                      |                  |
Nitrogen, Nitrite       0827 00613 A FC  | 1977 00613 H FCC |       NA
Phosphorus, filtered    0828 00671 A FC  | 1978 00671 I FCC |       NA
  Orthophosphate                         |                  |
Phosphorus, filtered    0829 00666 A FC  | 1981 00666 F FCC |       NA
Phosphorus, unfiltered  0837 00665 A RC  | 1982 00665 F RCC |       NA

------------------------------------------------------------------------------

             Existing "Unpreserved" Lab Codes
        Which Will Be Deleted To Avoid Confusion

-----------------------------------------------------------------------------
                        NWQL       M Bot
                        Code PCode C Typ

-----------------------------------------------------------------------------
Nitrogen, Ammonia-LL    1278 00608 C FU
Nitrogen, Nitrite +     1578 00631 C FU
  Nitrate
Phosphorus-LL,          1277 00671 D FU

-----------------------------------------------------------------------------

               EXPLANATION OF SYMBOLS AND ABBREVIATIONS

 * - The term "filtered" replaces the previously used term "dissolved"
     and "unfiltered" replaces "whole water recoverable."
 ** - Lab codes for chilled only and sulfuric acid will not be
      initialized in the system until October 1, 1994
 HgCl2 - Samples preserved by mercuric chloride plus chilling
 Chilled only - Samples preserved by chilling only
 H2SO4 - Samples preserved by sulfuric acid plus chilling
 NWQL Code - Same as Lab Code in the NWQL Laboratory Services Catalog
 PCode - Parameter Code
 MC - Method Code
 Bot Typ - Bottle Type
 NA - No analyses available

                          BOTTLE TYPES

FC  - Filtered Chilled (Preserved with Mercuric Chloride)
RC  - Raw Chilled (Preserved with Mercuric Chloride)
FCC - Filtered, Chilled Only (No Preservatives Added)
RCC - Raw, Chilled Only (No Preservatives Added)
FCA - Filtered, Chilled, (Preserved with Sulfuric Acid 25%)
RCA - Raw, Chilled, (Preserved with Sulfuric Acid 25%)
FU  - Filtered, untreated
________________________________________________________________________________

  IF YOU HAVE QUESTIONS ABOUT PRESERVATION OF AMBIENT SAMPLES

Most nutrient samples collected by Division projects for submittal
to the NWQL are categorized as ambient rather than compliance.
As described in this memorandum, the Division requirement is for
ambient samples to be preserved by chilling only, with sulfuric
acid plus chilling to be used only for compliance samples.
However, projects may occasionally need to collect ambient samples
for nutrient analysis that have special characteristics that warrant
concern regarding preservation.  When such cases arise, please
contact Charles Patton at the NWQL [(303) 467-8084] or your Regional
Water-Quality Specialist to discuss how to preserve the samples.

                                   David A. Rickert
                                   Chief, Office of Water Quality

(Attachment in electronic OWQ Technical Memorandum
system but not included with hard copy)

This memorandum supersedes Office of Water Quality Technical
Memorandums 80.26 and 85.07 and modifies Office of Water Quality
Technical Memorandums 92.08 and 92.11.

Key Words:     Mercuric chloride, sample preservation, nutrients,
               ambient sample, compliance sample

Distribution:  A, B, S, FO, PO, AH

                                                      ATTACHMENT

Draft Summary of OWQ/NWQL Nutrient Preservation Experiment

What follows is a draft that outlines:  1)  questions raised in
the planning phase of this work about the ability of three widely
applied field treatments to stabilize nutrient concentrations in
samples prior to laboratory analysis; 2) the experimental design
that evolved to answer these questions; 3)  differences between
this study and some previous ones; 4)  field and laboratory sample
processing protocols; 5)  preliminary interpretation of data, and;
6) a list of data analysis tasks that still need to be completed.
Ed Gilroy performed most of the statistical analysis on the data
set and he will be a co-author on reports and journal articles
resulting from this study.
============================================================

DRAFT          DRAFT
QUESTIONS ADDRESSED
1.   How do field treatments --
     *    addition of 40 mg mercury(II)/L and chilling (USGS)
     *    addition of sulfuric acid (reduce sample pH to < 2) and
          chilling (USEPA)
     *    filtration and chilling, or chilling alone --affect
          stability of nutrient concentrations in natural water
          samples over a 30-day storage period?

2.   Does water type (ground, surface, municipal supply) affect
stability of nutrient concentrations in samples over a 30-day
storage period for any or all of the three field treatment
protocols?

3.   Are particular analytes -- phosphorus and Kjeldahl nitrogen
(total and dissolved); ammonium, orthophosphate, nitrate +
nitrite, nitrite (dissolved only) -- more or less stable over a
30-day storage period following any of the three field treatment
protocols?

4.   Following any of the three field treatment protocols, are
samples with relatively "high" nutrient concentrations more stable
over a 30-day storage period than those with relatively low ones?

5.   Is there a relationship between the amount of suspended
sediment in samples and the stability of nutrient concentrations
in samples over a 30-day storage period? (DELETE, NO DATA)

6.   For each of the three field treatments, how does the 30-day
stability of nutrient concentrations dissolved in a natural matrix
(real samples) compare with that of nutrient concentrations
dissolved in a deionized water matrix (control samples)?

7.   Are there any statistically significant bottle effects?

8.   Is there a clear-cut relationship between the temporal
stability of nutrient concentrations in a particular sample and
the biological activity in that sample for any of the three
treatments?

HOW THIS EXPERIMENT DIFFERED FROM SOME PREVIOUS ONES

Unpolluted surface and ground water samples collected from 15
stations throughout the continental United States were used in the
Office of Water Quality nutrient preservation experiment performed
at the National Water Quality Laboratory by Patton and Truitt
(April - July, 1992), hereinafter referred to as "THIS STUDY."  In
contrast, highly polluted samples collected from 12 sewage
outfalls in the Metropolitan Chicago area were used in the Chicago
U.S. Environmental Protection Agency nutrient preservation
experiments performed by Carter and Jirka (unpublished work, circa
1974 -- hereinafter referred to as "USEPA STUDY") that ultimately
resulted in nutrient sample preservation protocols mandated (40
CFR, paragraph 136.3, Table II) for National Pollution Discharge
Elimination System (NPDES) compliance monitoring.

In THIS STUDY, sample processing (splitting, filtration -- 0.45
micrometer pore size --, addition of preservatives) was performed
in the field immediately following sample collection according to
protocols described below.  Samples arrived at the NWQL within 24
hours of collection and were immediately analyzed in quadruplicate
for dissolved and total nutrients.  The only sample processing
operation performed at the NWQL entailed splitting one of the two,
1 liter samples from each treatment group (FC and RC mercury, FC
and RC sulfuric acid, FC and RC water [control]) into ten 125
milliliter bottles as described below.  In a previous USGS
nutrient preservation experiment performed by Fishman, Schroeder,
and Shockey (J. Environmental Studies, v. 26, p. 231-238, 1986) --
hereinafter referred to as "FISHMAN STUDY", three, 19 liter
surface water samples were collected from the metropolitan Denver
area in 5 gallon, plastic carboys.  These three bulk samples were
returned to the laboratory, processed through 10 micrometer
filters, and then stored in a refrigerator at 4 degrees Celsius.
Additionally, bulk samples of NWQL tap water and deionized water
were also collected.  These two samples along with one of the
three environmental samples were spiked with organic and inorganic
nitrogen and phosphorus compounds.  One to two days elapsed before
each of the 5 bulk samples was spiked (see above) and split into
sixty-two 250 mL bottles.  Sample splits were dosed with various
preservatives (sulfuric acid, mercuric chloride, chloroform),
three of which were immediately analyzed in triplicate for
dissolved and total nutrients.  On each of three other occasions
(days 4, 8, and 16), three splits were selected and again analyzed
for dissolved and total nutrients.

In THIS STUDY, control samples with certified concentration
estimates (USEPA "Nutrient Concentration 1" and "Nutrient
Concentration 2"), which were prepared in deionized water
containing the appropriate preservative, were analyzed in
quadruplicate each time real samples were analyzed.  Daily
concentration variations in these pristine matrix control samples
could be compared with concentration variations in natural water
samples to estimate the contribution of instrumental variability
to the over all variability in the experiment.  Pristine matrix
control samples were not included in the FISHMAN STUDY.  Data from
the EPA Study are no longer in existence.

At the end of THIS STUDY microbiological activity of samples from
each station was assessed by tritiated adenine uptake experiments
and fluorescent staining techniques.  Samples in the Fishman study
were not assessed for microbiological activity.  Data from the EPA
Study are no longer in existence.

EXPERIMENTAL DESIGN

Samples from fifteen (15) stations around the United States
were selected for this experiment.  Eleven stations were
NASQAN sites (surface water), three were wells (ground
water), and one was tap water (municipal supply).
In THIS STUDY the acid preservation protocol used was that
mandated by the U.S. Environmental Protection Agency for
NPDES compliance monitoring as defined in 40 CFR, paragraph
136.3, Table II (Mark Carter and Andrea Jirka, unpublished
work, circa 1974).  The mercury preservation protocol
stemmed from work at the U.S. Geological Survey as described
by Marvin Fishman, LeRoy Schroeder, and Merle Shockey (J.
Environmental Studies, v. 26, p. 231-238, 1986).

Six (6) field treatments were applied to each sample
collected for determination of digested nutrients (total and
dissolved Kjeldahl nitrogen, total and dissolved phosphorus):

1.   Addition of deionized water (ampule effect control),
and chilling.  (whole water, "RC", subsample)

2.   Addition of mercury (II) (approximately 40 mg per liter
of sample), and chilling to 4 degrees Centigrade.
(whole water, "RC", subsample)

3.   Addition of sulfuric acid (sufficient to reduce sample
pH to less than 2), and chilling to 4 degrees
Centigrade.  (whole water, "RC", subsample)

4.   Filtration, addition of deionized water (ampule effect
control), and chilling to 4 degrees Centigrade.
(filtered water, "FC", subsample)

5.   Filtration, addition of mercury (II) (approximately 40
mg per liter of sample), and chilling to 4 degrees
Centigrade.  (filtered water, "FC", subsample)

6.   Filtration, addition of sulfuric acid (sufficient to
reduce sample pH to less than 2), and chilling to 4
degrees Centigrade.  (filtered water, "FC", subsample)

Three (3) field treatments were applied to each sample
collected for determination of dissolved nutrients (ammonia,
orthophosphate, nitrate + nitrite, nitrite):

1.   Filtration, addition of deionized water (ampule effect
control), and chilling to 4 degrees Centigrade.

2.   Filtration, addition of mercury (II) (approximately 40
mg per liter of sample), and chilling to 4 degrees
Centigrade.

3.   Filtration, addition of sulfuric acid (sufficient to
reduce sample pH to less than 2), and chilling to 4
degrees Centigrade.

Samples were shipped to the NWQL for first nutrient
determinations within 24 hours of collection.

FIELD AND LABORATORY SAMPLE PROCESSING PROTOCOLS

At each of 15 stations, approximately 14 liters of water
were composited in a churn splitter.  The composite sample
was processed in the field into twelve, prelabeled, 1 liter
bottles as follows.  With constant churning, six (6) whole
water samples were collected in bottles marked "RC".  Then
churning was discontinued and six (6) filtered water samples
(0.45 micrometer filter) were collected in bottles marked
"FC".  The twelve bottles were then sorted into three
treatment groups according to color coded labels.  Each
group consisted of two (2) "FC" bottles and two (2) "RC"
bottles.  One (1) ampule containing mercuric chloride
preservative solution was added to each of the four bottles
labeled "MERCURY" (green color code).  One (1) ampule
containing sulfuric acid preservative solution was added to
each of the four bottles labeled "ACID" (red color code).
One (1) ampule containing deionized water preservative
solution was added to each of the four bottles labeled
"WATER" (blue color code).  The twelve treated samples were
then packed in ice and shipped to the National water Quality
Laboratory in Arvada by overnight express.  With one
exception, samples were received at the NWQL, processed
further, and analyzed for eight nutrient parameters within
24 hours of their collection, as described below.

Immediately upon receipt at the NWQL, two bottles (one "FC"
and one "RC") from each of the three treatment groups were
shaken, poured into the appropriate ten-port cone splitter,
and collected in ten, prelabeled, 125 mL, brown polyethylene
bottles.  Head space in 125 mL bottles was minimized by
squeezing their sides gently to bring the sample level to
the top of their necks before caps were secured.  A
different cone splitter was used exclusively for each
treatment group throughout the experiment.  Just prior to
use, each cone splitter was rinsed copiously with 5 percent
v/v hydrochloric acid and deionized water.  In each
treatment group, filtered samples ("FC" bottles) were always
split before unfiltered samples ("RC" bottles).  Samples
from each of the 15 stations in the experiment were analyzed
in quadruplicate for eight nutrient parameters -- dissolved
ammonia, dissolved orthophosphate, dissolved nitrate +
nitrite, dissolved nitrite, dissolved Kjeldahl nitrogen,
dissolved phosphorus, total Kjeldahl nitrogen, and total
phosphorus -- the day of arrival at the NWQL, and five
additional times thereafter.

CONCLUSIONS

     1.   Field preservation treatments had little effect on
temporal variation in nutrient concentrations in the 2
to 5 reporting limit regime.  Concentration changes of
nutrients observed in THIS STUDY (with the exception of
nitrite in acidified samples) do not decrease or
increase linearly.  That is, a concentration gain
followed by a loss, or the reverse, was the rule rather
than the exception in THIS STUDY.  NOTE AGAIN THAT
THESE CONCENTRATION CHANGES WERE IN THE 2 TO 5
REPORTING LIMIT REGIME.

     2.   No statistically significant differences in
storage stability between surface and ground water were
observed in THIS STUDY for any of the preservation
treatments.

     3.   With the exception of nitrite in acidified
samples, stability of nutrient concentrations was
statistically equivalent for all preservation protocols.

     4.   Initial concentrations of nutrients in samples
did not appear to exert a major effect on storage stability.

     5.   Data to assess the affect of suspended sediment
concentration on storage stability apparently does not
exist.

     6.   On the basis of BQA regression equations for
standard reference waters, nutrient concentrations (with the
exception of nitrite in acid preserved samples) in samples
from all 15 stations remained within statistical control
limits for 30 days.

     7.   No statistically significant bottle effects were
observed in this study.

     8.   Temporal variation nutrient concentrations cannot
be unambiguously attributed to biological activity in
samples.