***WATER QUALITY MONITORING PROTOCOL***

***FOR KETTLE PONDS OF***

***CAPE COD NATIONAL SEASHORE***

Prepared by:

J. Portnoy, K. Lee, J. Oset, E. Gwilliam & J. Budreski

April 2003

**Table of Contents**

Preface [4](#preface)

Acknowledgements [4](#acknowledgements)

Introduction and Objectives [5](#introduction-and-objectives)

Integration with Other CACO Monitoring
[6](#integration-with-other-caco-monitoring)

Background for selection of monitoring variables
[7](#background-for-selection-of-monitoring-variables)

Brief explanation of selected monitoring variables
[9](#brief-explanation-of-selected-monitoring-variables)

Measurement methods [11](#measurement-methods)

Seasonal Sampling Program and Collection Overview
[12](#seasonal-sampling-program-and-collection-overview)

Sample Location and Procedure [14](#sample-location-and-procedure)

Field Logistics and Safety [15](#field-logistics-and-safety)

Data management/Reporting/Staffing
[15](#data-managementreportingstaffing)

Literature Cited [17](#literature-cited)

Appendix A. -- YSI (temperature, conductivity, redox potential,
dissolved oxygen, pH, depth)
[18](#appendix-a.-ysi-temperature-conductivity-redox-potential-dissolved-oxygen-ph-depth)

Appendix B. -- Transparency (Secchi and Li-cor Measurements)
[24](#appendix-b.-transparency-secchi-and-li-cor-measurements)

Appendix C. -- pH and Alkalinity titration (potentiometric, low-level)
[27](#appendix-c.-ph-and-alkalinity-titration-potentiometric-low-level)

Appendix D. -- Dissolved Nutrients Analysis (Ammonium, Nitrate/Nitrite
and Orthophosphate)
[29](#appendix-d.-dissolved-nutrients-analysis-ammonium-nitratenitrite-and-orthophosphate)

Appendix E. - Total Phosphorus and Nitrogen: Acid Persulfate Digestion
[31](#appendix-e.---total-phosphorus-and-nitrogen-acid-persulfate-digestion)

Appendix F -- Cations (Ca, Mg, K, Na)
[37](#appendix-f-cations-ca-mg-k-na)

Appendix G -- Anions (Cl^-^ and SO~4~^-^)
[38](#appendix-g-anions-cl--and-so4-)

Appendix H - Sampling Procedures for the Pond Monitoring Program
[39](#appendix-h---sampling-procedures-for-the-pond-monitoring-program)

Appendix I -- PondStage. [47](#appendix-i-pond-stage.)

Appendix J - Data Forms. [48](#appendix-j---data-forms.)

Appendix K. The Need for Annual Sampling.
[52](#appendix-k.-the-need-for-annual-sampling.)

**Tables and Figures:**

# 

**Table 1. CACO Ponds Included in the Kettle Pond Monitoring Program.
[4](#caco-ponds-included-in-the-kettle-pond-monitoring-program.-see-summer-biweekly-monitoring-p.11.-gps-xy-coordinates-eastingnorthing-of-deepest-point.)**

**Table 2. Variables and Measurement Methods.
[11](#variables-and-measurement-methods.)**

**Table 3. Changes in Secchi Transparency (m) Versus the Seasonal Range
at CACO Kettle Ponds as an Indicator of Necessary Sampling Frequency.
[13](#changes-in-secchi-transparency-m-versus-the-seasonal-range-at-caco-kettle-ponds-as-an-indicator-of-necessary-sampling-frequency.)**

**Table 4. Summary of water quality variables by sampling period - Field
Sampling Information
[14](#summary-of-water-quality-variables-by-sampling-period---field-sampling-information)**

Figure 1. Map of CACO
ponds........................................................................5

Figure 2. Flow diagram of pond water sample
collection..................................13

# 

# 

# 

# Preface

This protocol describes the conceptual underpinnings and field and
laboratory methods employed in the monitoring of 20 kettle ponds within
Cape Cod National Seashore (CACO). Selection of monitoring variables and
methods derived from three prior reviews (Martin et al. 1993; Portnoy
and Farris 1996; Portnoy and Lee 2000) by scientists and resource
managers with expertise and experience in Cape Cod limnology, hydrology
and biogeochemistry.

# Acknowledgements

CACO is indebted to the following individuals for periodic technical
assistance and critical review: Elizabeth Colburn, Harvard Forest, Anne
Giblin, Marine Biological Laboratory, Paul Joseph Godfrey, University of
Massachusetts, Harry Hemond, Massachusetts Institute of Technology,
National Park Service, Charles Roman, U.S. Geological Survey, University
of Rhode Island, Marjorie Winkler, Center for Climatic Research,
University of Wisconsin.

##  CACO Ponds Included in the Kettle Pond Monitoring Program. See Summer Biweekly Monitoring P.11. GPS X/Y Coordinates (Easting/Northing) of deepest point.

  ------------- -- ------------- -- -------------- -- --------- ---- ------------- -- ---------
  **PRIMARY        **Easting**      **Northing**      **Depth        **SECONDARY      **Depth
  Ponds**                                             (ft)**         Ponds**          (ft)**

  Gull             416491           4644967           60             Higgins          20

  Duck             417050           4642801           60             Williams         4

  Ryder            414538           4646672           39             Kinnacum         12

  Spectacle        417457           4644849           24             Northeast        11

  Great (Truro)    414652           4647453           36             Southeast        12

  Great            417193           4643575           52             Turtle           3
  (Wellfleet)                                                                         

  Dyer             416348           4643454           21             Horseleech       13

  Long             416303           4644036           47             Slough           25

  Snow             414867           4646937           23             Round (west)     31

  Herring          415967           4646237           12             Round (east)     24
  ------------- -- ------------- -- -------------- -- --------- ---- ------------- -- ---------

![](media/image1.png){width="6.802777777777778in"
height="4.830555555555556in"}

# Introduction and Objectives

Kettle pond management issues and monitoring objectives are detailed in
Martin et al. (1993). In brief, concerns include:

1\) Increased human use and accompanying nutrient (phosphorus and
nitrogen) loading from a variety of anthropogenic sources including:

a\) Bank and soil erosion;

b\) Shoreline wastewater disposal;

c\) Visitors and their pets;

2\) Atmospheric deposition of metals, sulfate, and nitrate and their
effects on pH, alkalinity and sulfur and phosphorus cycling;

3\) Hydraulic alterations, especially stream channelization, which may
alter hydrology, trophic structure and native species diversity;

4\) Fish stocking, for the enhancement of recreational fishing, and its
possible effects on pond trophic dynamics.

To document current status and to monitor future change in pond water
quality, this monitoring program is designed to:

1\) Characterize the trophic status of the ponds;

2\) Develop repeatable methods of monitoring trophic status and
limnological processes to detect important changes;

3\) Describe pond-specific and seasonal limnological and hydrological
processes affecting water quality;

4\) Identify and develop management actions to mitigate anthropogenic
effects.

The selection of monitoring variables and temporal and spatial sampling
frequencies is guided by expectations (i.e. hypotheses) regarding the
biogeochemical and limnological responses of the ponds to added
nutrients (nitrogen and phosphorus), acids (as from "acid rain") or to
manipulations of hydrography or of the resident fish community.
Monitoring the effects of \"external\" (extra-watershed) phosphorus
loading, for example, on pond trophic status requires data on nutrient
status, transparency, and dissolved oxygen.

##  {#section-4 .unnumbered}

# Integration with Other CACO Monitoring

Besides assessing the effects of nutrient and acid loading on the
limnology and ecology of the ponds themselves, this monitoring program
provides important information for a diverse array of research and
management questions. Pond water level data is critical to hydrologic
monitoring which is relevant to understanding pond water quality changes
that are linked to water level and/or flow direction. Pond chemistry
affects biota and often determines the species composition and abundance
of aquatic macrophytes, benthic and pelagic invertebrates and fish,
including rare species. Pond chemistry has also been shown to reflect
long-term and landscape-scale changes in the surrounding uplands, and
thus to integrate watershed-wide changes in soils and vegetation. Data
from the National Atmospheric Deposition Program (NADP), including the
local CACO-operated Truro site (MA01), have been employed for over two
decades to help interpret pond chemistry and to assess the importance of
atmospheric deposition in eutrophication and acidification.

# Background for selection of monitoring variables

##  {#section-5 .unnumbered}

##### Nutrient loading and eutrophication

Phosphorus (P) is the nutrient most likely limiting primary production
and controlling water clarity in the clearest (oligotrophic) kettle
ponds. Local glacial outwash soils are low in P, probably explaining the
natural clarity of most ponds. \[Nitrogen, the other major nutrient for
plant and algal growth, is supplied by microbial fixation of atmospheric
nitrogen gas in freshwater environments.\] It is hypothesized that
kettle pond water quality deteriorates when humans import P into the
pond basin. P inputs from precipitation are minor compared to other
sources, based upon data collected since 1981 at the NPS-operated NADP
site in South Truro. Wildlife (primarily bird) contributions of P are
probably of importance for Gull Pond (NADP data, Portnoy and Soukup
1990). With increasing shoreline development, cottage occupancy and pond
use, humans provide additional P to pond nutrient budgets from septic
wastes and through shore erosion. Human wastes from shoreline septic
systems are likely the major source of P to the kettle ponds.

Because P has no common gaseous form, once imported into a lake
watershed it tends to remain there. Most P is adsorbed to iron minerals
on or in the sediments, where it remains unless released mechanically or
through a change in the chemical environment. Chemical release of P
generally occurs in summer as a result of thermal stratification and the
loss of oxygen from bottom waters. The process is as follows: In spring
and summer pond surface waters warm. This warm water tends to remain
"floating" at the surface because it is less dense than colder water;
also less wind energy is available for mixing than in fall and winter.
Colder water accumulates at the bottom and, because of its higher
density, is not readily brought to the surface by weak summer winds.
Bottom water thereby stagnates during this thermal stratification.
Because there is little or no light in the bottom waters of the deep
(10-20 m) kettle ponds, oxygen production through photosynthesis is nil,
while oxygen consumption by bacteria which decompose organic matter is
high. Oxygen depletion (anoxia) of the bottom waters results. Once
oxygen gas is no longer available to bacteria for the oxidation
(decomposition) of organic matter, the microbes then use oxidized iron
minerals. This results in the dissolution of the iron minerals and, most
importantly for pond water quality, the release of phosphate that is
normally immobilized by the iron under more aerobic conditions. Fall
brings surface water cooling so that the density difference between
surface and bottom waters decreases and autumn winds can eventually mix
the entire water column, i.e. fall overturn. With renewed mixing,
PO~4~-enriched bottom water is carried to the well-lit surface where it
is assimilated by phytoplankton, thereby stimulating algal blooms.

Pond eutrophication and accompanying algal blooms can cause earlier and
more intense heating of the surface waters because the algal cells
suspended in the water column absorb solar energy and re-radiate it as
heat, unlike clear water which allows much of the energy to pass through
to deeper water. Thus, more eutrophic ponds thermally stratify sooner in
the warm months, resulting in more prolonged and extensive deep-water
anoxia and P release, which can feed back to increase algal growth. The
degree of temperature stratification and oxygen depletion within the
water column can be monitored with submersible temperature, conductivity
and oxygen probes.

Iron is also active in the lake\'s sulfur cycle. Chemically reduced iron
(Fe^++^), which is found in the sediment and in bottom waters during
oxygen depletions, precipitates the sulfides produced by anaerobic
sulfate-reducing bacteria in the sediment. Sulfide precipitation may in
fact limit PO~4~ binding sites on Fe complexes. Thus, the presence of
free sulfides, particularly the foul-smelling hydrogen sulfide (H~2~S)
gas, can indicate that Fe is in short supply for sulfide binding, and
more importantly, for phosphate complexation. The odor of H~2~S in
collected bottom water therefore indicates PO~4~ release from anaerobic
sediment. Acid deposition may increase the competition between PO~4~ and
sulfide for Fe. The Cape's acidic precipitation is acidified primarily
by sulfate contamination. In this way "acid rain" can contribute to pond
eutrophication, as well as acidification (Caraco et al. 1989).

Nutrient additions manifest their effect in the water column; however,
phytoplankton assimilation of a limiting nutrient is so rapid that
little dissolved inorganic nitrogen or phosphorus may be detectable in
pond water even during conditions of high loading. Also, although P
clearly limits algal growth in oligotrophic ponds, once P concentrations
increase above a critical threshold (e.g. 30 ppb,) (Downing and McCauley
1992 ), nitrogen can limit phytoplankton growth. Thus, to assess total
nutrient status, whole water samples must be chemically digested to
measure their total phosphorus (TP) and nitrogen (TN) content. TP and TN
include all dissolved and particulate forms of these elements in the
water column.

Increased algal productivity in kettle ponds should be evident as
reduced clarity, as algal biomass increases. Clarity can be measured
directly using light attenuation and Secchi depth; chlorophyll
concentration serves as a widely used surrogate for phytoplankton
biomass. Chlorophyll measurements were discontinued in 2002 due to poor
data trends and major variability in sampling and analyses.

##### Acidification

The scarcity of natural inorganic buffers in native granitic soils makes
the kettle ponds apparently sensitive to acid deposition. All 20 kettle
ponds are poorly buffered with alkalinities less than 4 mg/L and pH
ranging from 4.3 to 6.5. For the past two decades of record,
precipitation pH has averaged about 4.6 with high sulfate loading of
surface waters. However, pond pH with few exceptions has remained stable
both over the short-term (1981 -- present) and apparently over the past
10 millennia (Winkler 1988). Nevertheless, there is the potential for
long-term effects on base cation leaching and pH due to continued acid
deposition within pond watersheds. Further, nitrate contamination from
shoreline septic systems has the potential to acidify surface waters
once the microbial community within a pond which normally removes
nitrate is saturated (Kelly et al. 1990) .

# Brief explanation of selected monitoring variables

Pond Stage, especially relative to adjacent groundwater levels, affects
both inflow and outflow of water and dissolved species. It also affects
the redox potential and pH of littoral sediments, in turn affecting the
mobility of sedimentary Fe, P and S.

***Temperature*** is measured along with electrical conductivity
(specific conductance) to follow thermal stratification of the water
column. Water masses of different temperature and, thus, density can
stratify with little mixing between layers during summer months leading
to stagnant, and often oxygen-poor, conditions in bottom waters.
Temperature profiling describes the relative thermal resistance to
mixing. Anoxia induces the chemical reduction of iron and the release of
sedimentary phosphorus and sulfides into the water column. Phosphorus
release can trigger algal blooms, if transported to well-lit depths, and
sulfides are toxic to aquatic life. Temperature profiles can also be
useful for evaluating groundwater inputs and outputs and for computing
heat budgets.

***Electrical conductivity*** is a measure of the amount of dissolved
constituents in water. It is temperature-dependent (and readings must be
temperature-compensated) and thus aids in identifying thermal
stratification. More importantly, the loss of oxygen from deep pondwater
in summer causes the dissolution of limnologically important substances
(iron, phosphorus, and sulfides) that show up as increased conductivity.
Lower conductivities are often associated with purer waterbodies.

***Dissolved oxygen*.** Besides being essential for aerobic respiration
(and therefore critical for the survival of aquatic life), oxygen
concentrations influence all major nutrient cycles, including the
storage of phosphorus in lake sediments. Lakes undergoing eutrophication
typically show increases in the duration of deep-water anoxia and the
thickness (i.e. vertical extent) of anoxic bottom waters. By calculating
the anoxic factor for our primary ponds, trends in pond health can be
followed (Nurnberg, 1995).

***Light penetration*.** A light meter is used to measure incident light
(visible range) above the lake surface and at specific depths,
preferably simultaneously, to calculate the percent transmission through
the intervening water column. Photosynthesis and aquatic plant
production are dependent upon available light; thus, photosynthetic
oxygen production is often limited by light transmission to the bottom
waters of turbid lakes. This phenomenon can exacerbate deep-water oxygen
depletions. Light profiles can also show where plankton organisms are
concentrated and thereby explain subsurface peaks in the oxygen profile.

***Secchi depth*.** A single measurement of the maximum depth at which
the black and white 20-cm Secchi disk can be seen is widely used as an
index of clarity. The Secchi disk measures the transparency of the
water. The transparency can be affected by the color of the water,
suspended sediments and algae. Transparency decreases as color,
suspended sediments, or algal abundance increases. Water may be stained
yellow or brown by decaying plant matter; however, Cape Kettle Ponds
have very low dissolved organic material (DOM), therefore, color is
relatively unimportant in affecting water clarity. Suspended sediments
often come from sources such as re-suspension from the lake bottom,
construction sites, agricultural fields, and urban storm runoff. Algae
abundance is related to the amount of plant nutrients, especially
phosphorus and nitrogen. Transparency can therefore be affected by the
amount of plant nutrients coming into the lake from sources such as
sewage treatment plants, septic tanks, and lawn and agricultural
fertilizer (Great North American Secchi Dip-In, 2002).\
\
Transparency is an indicator of the impact of human activity on the land
surrounding the lake. If transparency is measured through the season and
from year to year, trends in transparency may be observed. Transparency
can serve as an early warning that activities on the land are having an
effect on a lake.

***Hydrogen ion concentration*** is represented by **pH**, the negative
logarithm of \[H^+^\], and measures the acid-base balance of water. Pond
water pH is determined by the combination of ions in solution. Ionic
composition is affected by surrounding soils, atmospheric inputs and
in-pond chemical and biological processes. Conversely, pH affects both
chemical reactions and the aquatic biotic community.

***Alkalinity*** measures the acid-neutralizing (i.e. buffering)
capacity of pondwater, and is therefore a measure of the ability to
resist pH change while receiving acidic inputs, as from acid deposition.

***Total Nitrogen and Total Phosphorus (TN and TP)*** are measures of
the total concentration of these elements in the water column, including
dissolved species, which are scarce in nutrient-limited kettle ponds,
and the more abundant N and P contained in organic particles including
plankton. To measure TN and TP colorimetrically, water samples must be
digested to convert all N and P to inorganic forms. Since P is rapidly
cycled by water column biota in P-limited pond systems, soluble reactive
P is often undetectable in the water column and total P is a better
measure of this element\'s availability. Total P is measured by
digestion of unfiltered samples.

***Dissolved Nutrients, (orthophosphate, nitrate and*** ***ammonium)***
are included as potentially sensitive indicators of septic leachate
loading. Because most ponds are likely P- rather than N-limited,
dissolved inorganic N should persist in the water column to provide a
tracer of contamination from shoreline wastewater disposal systems.

***Anions (SO~4~^-^ and Cl^-^)*** are indicators of the atmospheric
deposition and marine influences on the ponds. SO~4~ is the principal
acidifying anion in Cape Cod precipitation and, in addition to
acidification, can contribute to eutrophication by enhancing P release
from sediment. Concurrent chloride monitoring allows SO~4~ from marine
aerosols to be subtracted from the total, yielding a measure of
non-sediment SO~4~.

***Cations*** (**Mg, K, Na, Ca**) are essential both physiologically and
for maintaining a stable acid balance in pond water. Cation depletion
has been observed in freshwater and forest soils in areas of the
Northeast United States receiving acid precipitation.

# Measurement methods

Field and laboratory measurements employ the following equipment and
methods. Staff adheres strictly to these procedures unless analytical
problems or safety concerns arise. Deviation from these protocols for
any reason is reported to the project Technical Coordinator within 24
hours to ensure rapid resolution of the problem and continuance of the
program. Table 2 lists current methods used.

## Variables and Measurement Methods.

  ---------------------------- --------------------------------------------------
  **[Variable]{.underline}**   **[Method]{.underline}**

  Temperature                  YSI (see Appendix A,H)

  Conductivity                 YSI (see Appendix A,H)

  Redox potential (Eh)         YSI (see Appendix A,H)

  Dissolved oxygen             YSI (see Appendix A,H)

  pH for Profiles              YSI (see Appendix A,H)

  pH for Apr, Jul, Oct survey  Beckman meter with glass KCl-filled electrode,
                               calomel junction (see Appendix C,H)

  Transparency                 *Licor light meter* with two underwater sensors
                               (see Appendix B,H)

                               *Secchi disk* (20-cm diameter, black and white
                               quadrants) (see Appendix B,H)

  Alkalinity                   Potentiometric titration using Beckman meter,
                               Dosimat (see Appendix C,H)

  Ammonium                     Phenolate method, flow injection analysis
                               (FIA-Lachat QuickChem Method 10-107-06-1-C; see
                               Dissolved Nutrients Analysis Appendix D)

  Nitrate                      Cadmium reduction (FIA-Lachat QuickChem Method
                               31-107-04-1-C, see Dissolved Nutrients Analysis
                               Appendix D)

  Orthophosphate               Ammonium molybdate and antimony potassium tartrate
                               -ascorbic acid reduction (FIA-Lachat QuickChem
                               Method 31-115-01-1-G, see Dissolved Nutrients
                               Analysis Appendix D)

  Total phosphorus/ Total      FIA- Simultaneous Determination of Total
  nitrogen                     Phosphorus and Total Nitrogen in Natural Waters
                               (alkaline persulfate digestion, see Appendix E)

  Major cations Ca^++^,        Atomic absorption spectrometry (see Appendix F,H)
  Mg^++^, K^+^, Na^+^          

  Major anions *SO~4~ and      Turbidimetric method for sulfate and mercuric
  Cl^-^*                       nitrate for chloride (see Appendix G,H)

  Pond stage                   Siphon well or staff gauge (see Appendix I)
  ---------------------------- --------------------------------------------------

#  

# Seasonal Sampling Program and Collection Overview

Monitoring comprises four sub-programs. See the Field Sampling section
for operational details and Table 4 for a complete summary of variables
by sampling period. Refer to Appendix A-K for further details.

#####  1) Sampling of pH and alkalinity three times per year (April, July and October) for all 20 

##### ponds. This profiling tracks long-term trends in acidity and acid-neutralizing capacity, both of which can vary seasonally. (See Appendix H and C for procedures)

> Note: The original monitoring program, begun in 1984 in cooperation
> with a statewide
>
> survey **(Massachusetts Acid Rain Monitoring Project -ARM)**, included
> January
>
> sampling. This mid-winter sampling was discontinued after January 2000
> because of safety
>
> concerns and because pH and alkalinity data did not differ
> significantly (p\>0.05) between
>
> January and April. Other ARM sites are included with the sampling.
>
> ***2) Spring*** ***Survey*** of all 20 ponds. During the late March --
> early April well-mixed period, all 20 ponds are visited to
> characterize temperature, pH, Eh (redox potential), conductivity,
> dissolved oxygen, Secchi and light, dissolved nutrients (nitrate,
> ammonium and orthophosphate) TP, TN, major anions (SO~4~ and Chloride)
> and major cations (Ca, Mg, K, Na). Field observers position a boat
> above the pond's deepest point and collect depth profiles for field
> variables using a YSI. Samples for laboratory analysis are collected
> from the same anchored position. This spring visit can double as an
> opportunity to collect the spring pH/alkalinity samples (See Appendix
> H)

##### 3) Summer Biweekly (May through Fall overturn) sampling of the ten "primary" ponds for secchi transparency and for YSI profile measurements - temperature, conductivity, pH, redox potential, dissolved oxygen and depth. Ten "primary" ponds were selected to represent trophic groups as follows: oligotrophic (Gull, Dyer, Great-Wellfleet, Great-Truro, Spectacle and Long), mesotrophic (Duck, Snow and Ryder) and eutrophic (Herring). Past monitoring has shown that transparency (e.g. Secchi depth) must be measured at least biweekly to document major temporal change (Table 3). The objective is to conduct field profiling, particularly of dissolved oxygen, to document the depth and duration of oxygen depletion in deep waters (i.e. hypolimnetic anoxia). Thus it is essential that this sub-program completely encompass the period from onset of stratification in spring to fall overturn. 

4)  ***August*** ***Survey*** for all 20 ponds. For most ponds,
    stratification, hypolimnetic anoxia and sediment P mobilization are
    at an annual extreme during late summer; therefore, an annual
    characterization of the degree of eutrophication of all 20 ponds is
    performed during this worst-case period. With the exception of major
    anions, August variables are the same as the spring survey set.

> ***5) Monthly Pond Stage*** Pond water levels are monitored at all
> twenty ponds on a monthly basis to document major seasonal and annual
> hydrologic changes that could affect water chemistry and biota. Siphon
> wells are read at Gull, Duck, Great-Wellfleet, Ryder, Dyer, Long,
> Herring, Snow and Great-Truro using an electric or chalked tape.
> Elsewhere, an auto-level is used to determine pond surface elevation
> by differential leveling from local benchmarks. (see Appendix I)

## Changes in Secchi transparency (m) versus the seasonal range at CACO kettle ponds as an indicator of necessary sampling frequency.

> 1994 1995

[Biweekly Change]{.underline} [Biweekly Change]{.underline}

Seasonal Seasonal

Pond Mean ± SE Maximum Range Mean ± SE Maximum Range

Ryder 1.2 ± 1.2 3.7 8.4 1.1 ± 1.0 3.0 4.5

Duck 0.8 ± 0.9 2.5 2.2 2.2 ± 1.5 4.6 5.7

Great(T) 1.5 ± 0.9 2.4 2.6 0.4 ± 0.5 1.0 1.0

Gull 0.5 ± 0.6 1.8 2.2 0.4 ± 0.2 0.5 1.0

Spectacle 1.7 ± 1.0 2.6 3.7 0.8 ± 0.9 2.3 3.5

###### 

## Summary of water quality variables by sampling period - Field Sampling Information

+-------+-------------------+-----+------------------+---+---+---+---+---+
| *     | **Sample          | **  | # Sample Locati  | * | * | * | * | # |
| *Para | Equipment,        | Num | on and Procedure | * | * | * | * |   |
| meter | Container         | ber |                  | A | A | S | A |   |
| to be | Sizes,**          | of  | (Always the      | p | p | u | u | * |
| M     |                   | s   | deepest point    | r | r | m | g | * |
| onito | **Types and       | amp | except pH/ALK)   | i | i | m | u | M |
| red** | Required Prep**   | les |                  | l | l | e | s | o |
|       |                   | or  |                  | , | S | r | t | n |
|       |                   | r   |                  | J | p | B | S | t |
|       |                   | ead |                  | u | r | i | u | h |
|       |                   | ing |                  | l | i | w | r | l |
|       |                   | s** |                  | y | n | e | v | y |
|       |                   |     |                  | , | g | e | e | * |
|       |                   |     |                  | O | S | k | y | * |
|       |                   |     |                  | c | u | l | * |   |
|       |                   |     |                  | t | r | y | * | * |
|       |                   |     |                  | * | v | * |   | * |
|       |                   |     |                  | * | e | * | * | ( |
|       |                   |     |                  |   | y |   | * | a |
|       |                   |     |                  | * | \ | * | \ | l |
|       |                   |     |                  | * | ( | * | ( | l |
|       |                   |     |                  | ( | a | ( | a | p |
|       |                   |     |                  | a | l | p | l | o |
|       |                   |     |                  | l | l | r | l | n |
|       |                   |     |                  | l | p | i | p | d |
|       |                   |     |                  | p | o | m | o | s |
|       |                   |     |                  | o | n | a | n | ) |
|       |                   |     |                  | n | d | r | d | * |
|       |                   |     |                  | d | s | y | s | * |
|       |                   |     |                  | s | ) | p | ) |   |
|       |                   |     |                  | ) | * | o | \ |   |
|       |                   |     |                  | \ | * | n | \ |   |
|       |                   |     |                  | * |   | d | * |   |
|       |                   |     |                  | * | * | s | * |   |
|       |                   |     |                  |   | * | ) |   |   |
|       |                   |     |                  |   | \ | * |   |   |
|       |                   |     |                  |   | X | * |   |   |
|       |                   |     |                  |   | * |   |   |   |
|       |                   |     |                  |   | * |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***pH | 250-ml HDPE       | 1   | wade beyond      |   |   |   |   |   |
| and   | bottle, cleaned   | per | littoral weed    |   |   |   |   |   |
| alk   | and rinsed with   | p   | beds, rinse the  |   |   |   |   |   |
| alini | distilled water   | ond | bottle with pond |   |   |   |   |   |
| ty*** |                   |     | water and        |   |   |   |   |   |
|       |                   |     | sample/cap about |   |   |   |   |   |
|       |                   |     | 0.5 m below the  |   |   |   |   |   |
|       |                   |     | surface (elbow   |   |   |   |   |   |
|       |                   |     | depth)           |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| **    | YSI unit          | ve  | take readings at |   | * |   |   |   |
| *temp |                   | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| eratu |                   | cal | meters, 1 meter  |   | X |   |   |   |
| re*** |                   | p   | and every meter  |   | * |   |   |   |
|       |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | YSI unit          | ve  | take readings at |   | * |   |   |   |
| pH*** | (calibrate for pH | rti | 0.1 meters, 0.5  |   | * |   |   |   |
|       | monthly)          | cal | meters, 1 meter  |   | X |   |   |   |
|       |                   | p   | and every meter  |   | * |   |   |   |
|       |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | YSI unit          | ve  | take readings at |   | * |   |   |   |
| condu | (calibrate for    | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| ctivi | conductivity      | cal | meters, 1 meter  |   | X |   |   |   |
| ty*** | monthly)          | p   | and every meter  |   | * |   |   |   |
|       |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | YSI unit          | ve  | take readings at |   | * |   |   |   |
| redox | (calibrate for    | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| pote  | redox potential   | cal | meters, 1 meter  |   | X |   |   |   |
| ntial | monthly)          | p   | and every meter  |   | * |   |   |   |
| (E    |                   | rof | thereafter until |   | * |   |   |   |
| h)*** |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| **    | YSI unit          | ve  | take readings at |   | * |   |   |   |
| *diss | (calibrate for DO | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| olved | daily)            | cal | meters, 1 meter  |   | X |   |   |   |
| oxyg  |                   | p   | and every meter  |   | * |   |   |   |
| en*** |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| *     | YSI unit          | ve  | take readings at |   | * |   |   |   |
| **dep | (calibrate for    | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| th*** | depth daily)      | cal | meters, 1 meter  |   | X |   |   |   |
|       |                   | p   | and every meter  |   | * |   |   |   |
|       |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | 0.5m from bottom |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | Licor light meter | ve  | take readings at |   | * |   |   |   |
| trans | with two          | rti | 0.1 meters, 0.5  |   | * |   |   |   |
| paren | submersible light | cal | meters, 1 meter  |   | X |   |   |   |
| cy*** | sensors           | p   | and every meter  |   | * |   |   |   |
|       |                   | rof | thereafter until |   | * |   |   |   |
|       |                   | ile | the probe reads  |   |   |   |   |   |
|       |                   |     | 1%               |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***S  | Secchi disk;      | 1   | take readings at |   | * |   |   |   |
| ecchi | viewing scope     | per | the deepest      |   | * |   |   |   |
| dep   |                   | p   | point in the     |   | X |   |   |   |
| th*** |                   | ond | pond             |   | * |   |   |   |
|       |                   |     |                  |   | * |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| **    | 50mL clear        | 3   | All three        |   | * |   |   |   |
| *Diss | centrifuge tubes  | per | samples are      |   | * |   |   |   |
| olved | pre-acidified     | p   | pumped from      |   | X |   |   |   |
| Nutr  | with 0.2 ml of 2  | ond | about 0.5 meters |   | * |   |   |   |
| ients | N trace metal     |     | through a 0.45m  |   | * |   |   |   |
| (nitr | grade HCl; filter |     | filter; into     |   |   |   |   |   |
| ates, | holders with 0.45 |     | clear            |   |   |   |   |   |
| ammo  | m filter;         |     | pre-acidified    |   |   |   |   |   |
| nium, | peristaltic pump  |     | 50-ml plastic    |   |   |   |   |   |
| or    | with tubing       |     | centrifuge tubes |   |   |   |   |   |
| thoph |                   |     | **(do not rinse  |   |   |   |   |   |
| ospha |                   |     | bottles)**;      |   |   |   |   |   |
| te)** |                   |     | store on ice     |   |   |   |   |   |
| &     |                   |     |                  |   |   |   |   |   |
| **ca  |                   |     |                  |   |   |   |   |   |
| tions |                   |     |                  |   |   |   |   |   |
| (Ca,  |                   |     |                  |   |   |   |   |   |
| Mg,   |                   |     |                  |   |   |   |   |   |
| K,    |                   |     |                  |   |   |   |   |   |
| N     |                   |     |                  |   |   |   |   |   |
| a)*** |                   |     |                  |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | 60-ml brown       | 3   | All three        |   | * |   |   |   |
| major | Nalgene bottles   | per | samples are      |   | * |   |   |   |
| ani   | cleaned by        | p   | pumped from      |   | X |   |   |   |
| ons,\ | rinsing 3 times   | ond | about 0.5 meters |   | * |   |   |   |
| (chl  | with HCl rinse    |     | below surface    |   | * |   |   |   |
| oride | and then 3 times  |     | through a 0.45m  |   |   |   |   |   |
| &     | with distilled    |     | filter into      |   |   |   |   |   |
| SO~4  | water;            |     | brown 60-ml      |   |   |   |   |   |
| ~)*** | peristaltic pump  |     | bottles ; rinse  |   |   |   |   |   |
|       | with tubing       |     | with filtered    |   |   |   |   |   |
|       |                   |     | water prior to   |   |   |   |   |   |
|       |                   |     | filling; store   |   |   |   |   |   |
|       |                   |     | on ice           |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| ***   | 70-ml test tubes  | 3   | All three        |   | * |   |   |   |
| total | pre-washed with   | per | samples are      |   | * |   |   |   |
| nit   | boiling           | p   | pumped from      |   | X |   |   |   |
| rogen | phosphate-free    | ond | about 0.5 meters |   | * |   |   |   |
| and   | soapy water,      |     | below surface    |   | * |   |   |   |
| total | triple washed     |     | (no filtration)  |   |   |   |   |   |
| pho   | with dH~2~0,      |     | into a           |   |   |   |   |   |
| sphor | triple washed     |     | pre-washed test  |   |   |   |   |   |
| us*** | with HCl rinse    |     | tube (etched at  |   |   |   |   |   |
|       | and triple washed |     | the 40ml level); |   |   |   |   |   |
|       | again with        |     | rinse with pond  |   |   |   |   |   |
|       | dH~2~0; 5mL       |     | water before     |   |   |   |   |   |
|       | repipet dispenser |     | sampling; after  |   |   |   |   |   |
|       | with persulfate   |     | sample is taken, |   |   |   |   |   |
|       | oxidizing agent   |     | add 5 ml of      |   |   |   |   |   |
|       | (made up of       |     | oxidizing agent  |   |   |   |   |   |
|       | potassium         |     | and invert       |   |   |   |   |   |
|       | persulfate, boric |     | several times to |   |   |   |   |   |
|       | acid and NaOH);   |     | mix; store in    |   |   |   |   |   |
|       | peristaltic pump  |     | rack             |   |   |   |   |   |
|       | with tubing       |     |                  |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+
| **    | Use siphon well   | 1   | Use electric     |   |   |   |   |   |
| *pond | or staff gauge    | per | tape for siphon  |   |   |   |   |   |
| water |                   | p   | wells. For ponds |   |   |   |   |   |
| leve  |                   | ond | w/o siphon, 2    |   |   |   |   |   |
| ls*** |                   |     | people use       |   |   |   |   |   |
|       |                   |     | autolevel.       |   |   |   |   |   |
+-------+-------------------+-----+------------------+---+---+---+---+---+

***Figure 1. Flow Diagram of Pond Water Sample Collection.*** For all
lab-analyzed samples, triplicate samples are collected.

# 

# 

# 

# 

# 

# 

# 

# 

# 

# 

# 

# 

# Field Logistics and Safety

Fieldwork is conducted from a canoe or a 10-foot skiff transported to
the ponds by truck. CACO kettle ponds are small (most \< 20 ha) and
sheltered so that oars are sufficient propulsion. All personnel are
required to wear Personal Flotation Devices (PFDs), or survival suits
when surface water temperature is \< 18 C. Two technicians are required
for equipment operation and safety; thus, no one goes out on the ponds
alone.

At the beginning of each field season in late March, each pond's deepest
point is determined by fathometer and marked with an anchored buoy.
These "permanent" buoys must be removed, or their floats submerged, in
the fall to avoid being displaced by ice. Technicians should always
carry a park radio or cellular phone for emergency use, water and
sunscreen. If inclement weather approaches, return to shore and seek
shelter. (Cape Cod National Seashore SOP #S0014)

# Data management/Reporting/Staffing

***Data entry***

-   **Pond profile data** (i.e. temperature, conductivity, pH, Eh and
    dissolved oxygen) are recorded by two different means while in the
    field: (1) profile data are saved in the YSI 650 Datalogger and (2)
    the same data are recorded manually on Form 2 (see Appendix J).
    Secchi and Light data are recorded manually also on Form 2.

```{=html}
<!-- -->
```
-   **pH and alkalinity data** are recorded on Form 1 (see Appendix J).

Completed data forms are proofed by the field biotechnician and then by
the Aquatic Ecologist.

Data for all parameters will be stored in two different places on the Y
drive (CACO Server):

1.  As a working document in the folder titled *labusers/Ponds
    Computer/Working Directory/CCNS Kettle
    Ponds/CACO_KettlePonds_97.mdb*. This file is intended to be updated
    **weekly** as data becomes available.

2.  As archived data in the folder titled *dataarchive/Kettle
    Ponds/PONDS ARCHIVE/Synthesis/CACO_KettlePonds_97.mdb.* This file is
    intended to be updated **annually** by saving the same file from the
    directory indicated in part 1. above to the archive database
    annually, once all data has been entered and validated.

Hard copies of data for all parameters will be stored in the Ponds
binder for that year (i.e. 2002)

***Reporting and analysis***

The Aquatic Ecologist is responsible for data reduction and annual
reporting. Annual reports include: 1) both paper and digital compendia
of all collected data; 2) a summary of important trends relevant to
management concerns and monitoring objectives; 3) method developments
and changes to the protocol; and 4) recommendations for improved
monitoring, research and management.

***Budget and staffing***

The pond field-monitoring program requires one full-time biological
technician and a field assistant, typically an SCA or volunteer, from
April through October. Laboratory analysis is conducted by the above
biotechnician plus the Laboratory Chemist. Data analysis and reporting
are the responsibility of the Aquatic Ecologist.

# Literature Cited

> Caraco, N.F., J.J. Cole, AND G.E. Likens. 1989. Evidence for sulphate
> controlled phosphorus release for sediments of aquatic systems.
> Nature. **341:** 316-318.
>
> Downing, J.A., AND E. McCauley. 1992. The nitrogen:phosphorus
> realtionship in lakes. Limnol. Oceanogr. **37:** 936-945.
>
> Great North American Secchi Dip-In, Department of Biological Sciences,
> Kent State University Kent OH 44242 website: http://dipin.kent.edu
>
> Kelly, C.A., J.W.M. Rudd, AND D.W. Schindler. 1990. Acidification by
> nitric acid - future considerations. Water Air Soil Pollut. **50:**
> 49-61.
>
> Martin, L., J.W. Portnoy, AND C. Roman. 1993. Water quality and
> research plans for Cape Cod National Seashore: Report of a workshop,
> March 2-3, 1992. NPS/NRWRD/NRTR-93/15.
>
> Nurnberg, G.K. 1995. Quantifying anoxia in lakes. Limnol. Oceanogr.
> 40(6) 1100-1111.
>
> Portnoy, J.W., AND C.N. Farris. 1996. Revisions to protocol for kettle
> pond monitoring at Cape Cod National Seashore.
>
> Portnoy, J.W., AND M.A. Soukup. 1990. Gull contributions of phosphorus
> and nitrogen to a Cape Cod kettle pond. Hydrobiol. **202:** 61-69.
>
> Schoenherr, A. 2000. Seawater as an alkalinity standard. Cape Cod
> National Seashore. 5 p.
>
> Winkler, M.G. 1988. Paleolimnology of a Cape Cod kettle pond: Diatoms
> and reconstructed pH. Ecol. Monogr. **58:** 197-214.

# Appendix A. -- YSI (temperature, conductivity, redox potential, dissolved oxygen, pH, depth)

The laboratory owns a YSI 6820 Sonde (Serial \# 0H932) and a YSI 650
Display )Serial \# 02B0008A-B). The 650 compensates for barometric
pressure. Dissolved oxygen and depth should be calibrated every day. All
other parameters (DO Membrane, pH, conductivity and redox) should be
calibrated every two weeks. The sonde can be calibrated either right
side up or upside down. The amount of calibration solution necessary for
the process varies between the two methods. See section 2.6 in the YSI
manual for more information regarding the calibration procedure. YSI can
provide annual instrument inspections and factory calibration / cable
checks. Be sure to utilize the YSI calibration worksheet (Appendix J)
and store this with all monthly calibrations in the laboratory.
Additional YSI information is available at YSI.com, YSI Massachusetts at
(508) 748 0366, or the YSI Manual in the laboratory.

**Calibration Procedure:**

-   Connect the probe, 650 and cable.

-   Remove the probe guard (if performing pH, Eh, conductivity
    calibrations) and use the calibration cup for the calibration
    procedure.

-   Turn on the YSI.

-   Press "Escape" to get to the main menu.

-   Select "Calibration Mode" and press "Enter"

-   A screen allowing you to choose which parameter to calibrate will
    appear.

-   Fill a bucket with room temperature tap water to rinse the sonde
    between calibration solutions. Rinse the probe being calibrated with
    deionized (DI) water between calibrations.

-   Utilize Calibration Worksheet (Appendix J.) during process

# 

**[Conductivity]{.underline}**

1.  Conductivity should be calibrated first. Avoid contamination of
    conductivity standard. Never use less than 1.0mS/cm Conductivity
    Standard.

2.  Rinse the probe and calibration cup with distilled water before
    starting the calibration.

3.  For freshwater pond sampling use the 1,000 uS/cm (microsiemens/cm)
    conductivity standard solution. This is equal to 1 mS/cm
    (millisiemens/cm). The Sonde requires input in millisiemens.

4.  Choose "Conductivity" and then "SpCond" in order to calibrate for
    specific conductivity.

5.  Add a small amount of calibration solution to the calibration cup to
    rinse the probe sensors and cup, then discard.

6.  Fill the calibration cup with the conductivity standard (\~ about
    200 ml) making sure that the standard is covering the entire
    conductivity bridge (ensure no air bubbles are present in
    conductivity hole) and screw the cup onto the probe.

7.  Enter the conductivity value of the calibrator when prompted.

8.  Allow the readings to stabilize for about 30 seconds and then press
    "Enter" to accept the calibration.

9.  The YSI will indicate if the calibration was successful. Check the
    Conductivity Cell Constant (Advanced Menu) under Cal Constants. The
    acceptable range is 5.0 +/-0.45. Contamination is likely if it is
    out of this range. If the sonde reports "Out Of Range", investigate.
    Never override a calibration without understanding the cause. A
    typical error is entering 1000 microsiemens instead of 1.0
    millisiemens.

10. Press any key as prompted, hit "Escape" twice to get back to the
    calibration menu.

**[pH]{.underline}**

1.  Go to the sonde's report menu and turn on the pH mv output. This
    will allow the sonde to display the millivolts (probes raw output),
    in addition to the pH units during the calibration process.

2.  Recondition the probe if a slow response in the field has been
    reported. The procedure can be found in the manual under the "Sonde
    Care and Maintenance Section".

3.  First, rinse the calibration cup with DI water.

4.  Choose "ISE1 pH" from the menu and then "2 Point" calibration.

5.  Use pH 7 standard to calibrate for the first point.

6.  Ensure that the temperature probe is in solution with the standard,
    record pH millivolts at each calibration point.

7.  Add a small amount of calibration solution to the calibration cup
    and rinse the probe sensors and calibration cup.

8.  Discard the solution and refill the cup with the standard. You will
    need about 100 mL to completely immerse the pH sensor when the probe
    is held upright.

9.  Screw the cup onto the end of the probe.

10. Input the calibration value, press "Enter".

11. Allow the readings to stabilize for about 30 seconds and then press
    "Enter" to accept the calibration.

12. The YSI will indicate if the calibration was successful. Press any
    key as prompted.

13. Press "Escape" to get to the second point of the pH calibration.

14. Repeat the same process using pH 4 standard.

15. After successfully calibrating for pH 4 standard, press any key as
    prompted.

> The millivolts indicates the present status of the probe. A good set
> of numbers to use are as follows.

BUFFER 4 = +180 +/- 50mv

BUFFER 7 = 0 +/- 50mv

BUFFER 10 = -180 +/- 50mv

> The ideal numbers when a probe is new are close to the 0 and 180
> numbers. The numbers will shift to the higher side of the tolerance as
> the probe ages.

16. After recording the pH millivolts for the calibration points, you
    must determine the slope of the sensor. This is the difference
    between the two calibration points that were used. For example, if
    you record +3mv for buffer 7 and --177 for buffer 10, then the slope
    is 180. The acceptable slope range is 165 to 180. Once the slope
    drops below span of 165, the sensor should be taken out of service.

17. Press "Escape" twice to return to the calibration menu.

18. Never override a calibration without understanding the cause. Proper
    storage of the sensor will greatly extend the probe life.

# 

**[Redox (ORP or Eh)]{.underline}**

1.  Rinse the probe and calibration cup with distilled water before
    starting calibration. Dry the calibration cup and probe sensors as
    much as possible before adding the calibration standard.

2.  Use the YSI Zobell Solution **(Toxic, Handle with caution! Use and
    discard per MSDS)** to calibrate for redox. Once reconstituted, the
    solution has only a six-month shelf life.

3.  Fill the calibration cup (\~ 250 ml) with the solution and screw the
    cup onto the YSI. The Redox (ORP or Eh) sensor shares the pH sensor
    reference so it must be in solution when the 6000 is being
    calibrated.

4.  To determine the redox value of the standard, use the **Ag/AgCl**
    formula given on the sheet included with the Zobell Solution, or see
    "Principles of Operation" section of the manual. Air temperature is
    needed to calculate the redox value. Enter the number in millivolts
    that is closest to the measured solution temperature of the sonde.

5.  Choose "ISE2 Orp" from the calibration menu

6.  Enter the redox value of the standard when prompted and press
    "Enter."

7.  Allow the readings to stabilize for about 30 seconds and then press
    "Enter" to accept the calibration.

8.  The YSI will indicate if the calibration was successful.

9.  Press any key as prompted; hit "Escape" once to get back to the
    calibration menu.

**[Dissolved Oxygen]{.underline}**

A.  DAILY CALIBRATION:

```{=html}
<!-- -->
```
1.  Rinse the probe and calibration cup with water.

2.  Use a kimwipe to remove all liquid from the dissolved oxygen
    membrane.

3.  Add enough water to cover the bottom of the calibration cup. Make
    sure the probe membrane does not touch the water.

4.  Put the probe into the calibration cup, but do not screw the cup
    onto the probe.

5.  Allow the air within the cup to become fully saturated by letting
    the probe sit in the calibration cup for ten minutes.

6.  Select "Dissolved Oxy" from the calibration menu and then "DO%" from
    the next menu.

7.  Allow the readings to stabilize for about 30 seconds and then press
    "Enter" to accept the calibration.

8.  The YSI will indicate if the calibration was successful.

9.  When calibration is complete, go to the sondes "Advanced Menu" and
    then to the "Cal Constants" and record the "DO Gain". The Gain
    should be 1.0 with a range of --0.3 to +0.5. The probe should now be
    successfully calibrated and ready to go. Investigate any warning
    messages.

10. Press any key as prompted, hit "Escape" twice to get back to the
    calibration menu.

```{=html}
<!-- -->
```
B.  *3 WEEK CALIBRATION* *AND MEMBRANE CHANGE:*

```{=html}
<!-- -->
```
1.  Replace the DO Membrane using the 6035 Probe Reconditioning Kit and
    Directions.

2.  DO membranes will be slightly unstable for 3-6 hours after
    installation. It is recommended that final calibration of DO sensor
    take place after replacing the membrane. (The Sonde should be in a
    saturated evironment).

3.  Go to the sonde's "Report" menu and enable the "DO Charge"

4.  Now go to the "Run" menu and start the sonde in "Discrete Run" mode
    at a 4 second rate. Allow the sonde to run for 10 minutes.

5.  Record the DO Charge after 5 minutes. It should be 50 +/- 25.

6.  Once again, start the probe in "Discrete Run" mode at a 4 second
    rate and record the first 10 DO% numbers on paper. The numbers
    should start high and drop with each 4 second reading (eg) 110, 105,
    102, 101.5, 101.1, 101.0...etc..It does not matter that exactly 100%
    is not reached, only that the high to low trend exists.

> **Note**: Initial power-up can corrupt the first two DO % samples;
> disregard. Now the probe can be calibrated as explained above for
> daily calibration.

**[Depth]{.underline}**

1.  Select "Pressure-Abs" from the calibration menu.

2.  Enter "0.0" at the prompt and then press "Enter".

3.  Allow the readings to stabilize for about 30 seconds and then press
    "Enter" to accept the calibration.

4.  The YSI will indicate if the calibration was successful.

5.  Hit "escape" until the prompt has returned to the main menu.

YSI Calibration information adapted from M. Lizotte, YSI MA, May 2002.

# Appendix B. -- Transparency (Secchi and Li-cor Measurements)

# 

1.  **Secchi Measurements**

The laboratory owns numerous secchi disks. It is vital to examine the
ponds secchi disk to ensure the line is correctly marked in meter
increments. Secchi depth (SD) should be measured first so as to
ascertain the depth for subsequent YSI measurements and minimizing the
chance of the YSI sonde disturbing the bottom sediments which will
adversely affect readings.

2.  **Li-cor Measurements for Light Attenuation**

The laboratory owns two Li-cor underwater sensors : (serial number --
UWQ4539 and serial number UWQ6339). These sensors must be calibrated
every two years. In the past the light sensors have been calibrated
every other spring before the April sampling season has begun. It will
take at least a full month for the calibration process to be completed,
so be sure to plan ahead. If there are any questions, consult the
aquatic ecologist. Also, all calibration information should be given to
the aquatic ecologist. The calibration needs to be scheduled with
Li-cor. Call the company at (402) 467-3576 to receive an RMA number.
Then box up the sensors and send them to the company.

The datalogger does not need to be calibrated. However, before the
season begins it is a good idea to check that the Datalogger has been
programmed with the correct multipliers. Each light sensor has a unique
multiplier that is stored with Li-cor information in the lab.

> **Li-cor setup:**

1.  Connect a sensor to an underwater cable by pushing the two-pronged
    connector of the sensor itself into the cable, aligning the marks on
    the cable and sensor. If the marks are opposite you will get
    negative (but accurate) readings. The cable can then be connected to
    the logger at one of the bayonet mount terminals on the top of the
    logger. The terminal to the left is for \"Channel 1\" or "surface"
    and the one on the right is for \"Channel 2\" or deep profiling
    sensor.

2.  Turn on the LI-1000 by pushing the \"FCT ON\" button firmly.
    Instantaneous mode is the correct mode for CACO operations. Before
    taking readings channels MUST be configured. Once started you must
    complete the configuration procedure before doing anything else; the
    logger will not let you do otherwise.

3.  Configurations are done by channel, starting with Channel 1. Push
    the CFG button and the display will read \"MODE IS INST\", meaning
    that instantaneous mode has been selected. Press ENTER to accept (or
    up or down arrow to toggle to LOG). A message saying \"ch1 is OFF\"
    (or LIGHT or GEN or THERM) appears. Using the up or down arrows to
    toggle to LIGHT and press ENTER. The next display says \"range =
    A\". This means the machine\'s sensitivity range will be
    automatically adjusted for different light intensities. Press ENTER
    to accept, and don\'t be concerned with other choices. The next
    display is \"mult = xxx.x\" where the number is a calibration
    coefficient. Each sensor has a small plastic card listing its
    factory-calibrated coefficient. This number differs for measurements
    of light flux in air or water. Choose the proper one and enter the
    number, including the minus sign first, from the keypad, then press
    ENTER. The next display is \"label=XX\" where XX is simply 2
    characters chosen to denote the particular channel or type of
    measurement in progress. The 2 characters you choose will be
    displayed in data readouts. Note that letters must be selected by
    using the up or down arrow then pressing the appropriate button,
    e.g., up arrow-9 gets you \"Q\". Press ENTER to accept the
    characters chosen. The next display is \"ave=X\" where X is some
    number. Any number can be entered; this denotes the period in
    seconds over which the logger will take a moving average of light
    flux. This feature allows the LI-1000 to average short scale
    fluctuations in light intensity, such as from moving clouds, but
    requires patience to get an accurate reading. Ten seems to work
    well. Press ENTER to accept the number chosen. This completes
    configuration of channel 1. This process may be repeated for channel
    2 if two sensors are in use (and likewise for additional sensors
    hard-wired in on the pin connectors), or simply toggle each channel
    OFF and press ENTER until past channel channels 8 and M (the latter
    used for math functions). The next display will be 1X and a decimal
    number, which gives readings in microeinsteins (=micromoles of
    photons) m-2 sec-1. Configuration is now complete. The logger can be
    turned off by pushing OFF; it also will turn itself off after a 15
    minutes with no key activity to save battery life.

4.  Measurements of light flux can now be taken by reading the display
    after a suitable time has elapsed (bearing in mind the averaging
    time) and/or by hitting ENTER whenever storing a reading in memory.

5.  Use of the memory to store data requires that time and date be set
    before beginning (also important for use of log mode). To do this,
    push TIME. The display will read a preset date. If it is incorrect,
    simply enter two numbers for year (YY), two for month (MM) and two
    for day (DD), then press ENTER. If a mistake is made, push the back
    arrow to delete the wrong character and enter the right one. Push
    ENTER to accept the date. The next display will give military time
    in hours and minutes. If this is not right, enter the correct
    numbers as above, then push ENTER, which will return to the main
    display.

# Notes & Troubleshooting

-   On sunny, windy days the light readings will fluctuate. There are a
    few things you can do to reduce the amount of fluctuation. First,
    adjust the range of the submerged probe as it's moved through the
    water column. Pages 4-1 and 4-2 in the LI-1000 Datalogger
    Instruction Manual explain the range settings in the instantaneous
    mode (the mode which should be used to collect data). The
    autorange (A) setting covers the widest input signal range but tends
    to fluctuate rapidly when underwater radiation sensors are used.
    Therefore, adjusting the range as the probe is lowered through the
    water column can decrease the amount of fluctuation.

```{=html}
<!-- -->
```
-   To change the range settings:

```{=html}
<!-- -->
```
-   Press the "CFG" button.

-   Use the "ENTER" button to scroll down to the channel, typically
    channel 2 (the submerged probe), which will be changed.

-   Press any button to scroll through the range values.

-   Choose a range value by hitting "CFG".

```{=html}
<!-- -->
```
-   Another way to stabilize readings is to adjust the average value;
    the time interval over which values are averaged ("ave =").

Adapted from:

<http://people.uncw.edu/cahoon/LiCor.htm>

# Appendix C. -- pH and Alkalinity titration (potentiometric, low-level)

Surface samples (0.5 m depth) are collected April, July and October by
wading out beyond weed beds, rinsing a 250 mL HDPE bottle with pond
water, and filling the bottle to the top, i.e. no air in head space.
Samples are kept cold in a cooler, or refrigerated, and titrated within
24 hours of collection.

[Stock seawater standard]{.underline}:

Collect and filter (0.45 m) seawater from Coast Guard Beach, Truro.

[Dilute standard]{.underline}:

Pipet exactly 10.0 ml of the above stock into a 1-L volumetric flask and
fill to the mark with distilled water. The alkalinity of this solution
should be about 0.0232 mEq/L = 1.16 mg CaCO~3~/L (Schoenherr 2000).

[pH & titration procedure]{.underline} (All solutions at room
temperature):

1\. Calibrate pH meter and electrode pair in pH 7 and pH 4 buffers
**with stirring**.(medium speed)

2\. Measure 100 ml of the standard into a 150-ml beaker. Add a small
stir bar.

3\. Turn stirrer speed to medium.

4\. Place pH and reference electrodes in the standard and record pH
after 3 min or sooner if pH is stable, i.e. no change \> 0.05 pH units
for 30 sec.

5\. Submerse the tip of the Dosimat dispenser, containing 0.1*N* HCl
(certified 1*N* diluted 1:10), into the solution. Set mode to DIS C and
delta volume to 0.005 ml.

6\. Add titrant slowly to reduce pH to **4.50** (see notes below).

7\. Record titrant volume to pH 4.5 (quantity **A**).

8\. Again add titrant until pH reads exactly **4.20**.

9\. Record **total** titrant volume (quantity **B**).

10\. Alkalinity (mg CaCO~3~ L^-1^) is equal to:

**(2A-B) X 0.1 X 50,000/100**

11\. Compare your results with previous calibration data for the
standard. If they differ more than

0.5 pH units and/or more than 0.3 alkalinity units, probe (or, less
likely, meter) maintenance is

indicated.

Notes:

If you overshoot the 4.50 pH by a few hundredths (**no more**!) don\'t
abandon the titration. Simply record the pH (e.g. 4.48) and the titrant
volume used and then titrate **exactly** **0.3 pH units lower** (e.g.
4.18) and record total titrant volume. The formula still works.

For ponds with pH below 4.50, record the ambient pH as usual and then
titrate to exactly 0.3 pH units lower. Alkalinity is computed as usual
although quantity **A** is zero.

# Appendix D. -- Dissolved Nutrients Analysis (Ammonium, Nitrate/Nitrite and Orthophosphate)

***Sample Handling for Dissolved Nutrients***

Samples taken for dissolved nutrient analysis are also used for cation
analysis.

> For cation and dissolved nutrient samples, direct the GeoPump tube
> discharge through a 47-mm filter holder with 0.45-m membrane filter
> (Millipore HA). Allow the first few milliliters to discharge to waste
> and then direct the flow into a pre-acidified (0.2 ml of 2*N* trace
> metal grade HCl) 50-ml clear freestanding HDPE centrifuge tube.
> Labeled samples are stored on ice or refrigerated.

1.  **Ammonium (Phenolate method*)***

(Adapted from Lachat QuickChem Method 10-107-06-1-C)

Note: EDTA is added to the sample in-line in order to prevent
precipitation of calcium and magnesium ions.

***Principle***

This method is based on the Berthelot reaction. Ammonia reacts with
alkaline phenol, then with sodium hypochlorite to form indophenol blue.
Sodium nitroprusside is added to enhance sensitivity. The absorbance of
the reaction product is measured at 630 nm and is directly proportional
to the original ammonia concentration.

See Lachat QuickChem Method 10-107-06-1-C for preparation of reagents,
standards, timing specifications, manifold diagram, and system notes.

2.  **Nitrate/Nitrite (Cadmium reduction method*)***

(Adapted from Lachat QuickChem method #31-107-04-1-C)

Note: Samples containing high concentrations of iron, copper or other
metals may give low results. EDTA is added to the buffer to reduce this
interference.

***Principle***

> Nitrate is quantitatively reduced to nitrite by passage of the sample
> through a copperized cadmium column. The nitrite (reduced nitrate plus
> original nitrite) is then determined by diazotizing with sulfanilamide
> followed by coupling with N-(1-naphthyl)ethylenediamine
> dihydrochloride. The resulting solution has a magenta color, which is
> detectable at 520 nm.

See Lachat QuickChem Method 31-107-04-1-C for preparation of standards,
reagents, packing of cadmium column, timing specifications, manifold
diagram, and system notes.

3.  **OrthoPhosphates**

(Adapted from Lachat QuickChem method #31-115-01-1-G)

***Principle***

Ammonium molybdate and antimony potassium tartrate react in an acid
medium with 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
in the sample. Though there is a density difference between seawater and
reagent water the bias is less than 2%.

Though the method is written for seawater and brackish water it is also
applicable to non-saline sample matrixes. The method is calibrated using
standards prepared in deionized water. Once calibrated, samples of
varying salinities (0 to 35 ppt) may be analyzed. The determination of
background absorbance is necessary only for samples, which have color
absorbing at 880 nm.

See Lachat QuickChem Method 31-115-01-1-G for preparation of standards,
reagents, timing specifications, manifold diagram, and system notes.

##### Special Apparatus

Heater Module (175 cm of 0.8 mm i.d. Teflon tubing).

# Appendix E. - Total Phosphorus and Nitrogen: Acid Persulfate Digestion

(Adapted by K. Lee from Lachat QuickChem method #'s 31-115-01-1-F and
31-107-04-1-B or C)

Note: This method is extremely sensitive to pH in the sample. The higher
the acidity, the less sensitive the method.

***Principle***

All forms of phosphorus are converted to orthophosphate. The
orthophosphate ion (PO~4~^-^) reacts with ammonium molybdate and
antimony potassium tartrate under acidic conditions to form a blue
complex which absorbs light at 880nm.

##### Sample Handling

Wash all sample test tubes (70 mL etched glass tubes) with HOT phosphate
free soapy water and triplicate flush with dH~2~O. Acid wash the tubes
and triplicate rinse with DI H~2~O. After drying, store sample tubes
capped until day of sample collection. Collect triplicate 40 mL samples
and immediately treat with 5ml of persulfate oxidizing reagent (50 g
Aldrich low nitrogen Potassium Persulfate, recrystallized; 30g Fisher
Boric Acid and 350 ml of Fisher1M NaOH \[40g/L\]). Cap tubes securely.
Invert twice to mix. Record reagent date on each tube and run a blank.

***Sample Digestion***

> Place samples in pressure cooker and process at 15 psi for
> approximately 50 minutes on the day of collection. Allow sample tubes
> to cool and store upright at room temperature until analyzed. Holding
> time for samples should be limited to one month (28 days) if at all
> possible. (Ameel, 1993 and Lachat Instruments method 10-115-01-1-F)

##### Method

> **[Introduction]{.underline}**
>
> The method described here is an adaptation of the *Total Dissolved and
> Total Particulate Nitrogen and Phosphorus* method by Barbara L.
> Nowicki, published in the [Manual of Biological and Geochemical
> Techniques in Coastal Areas]{.underline}, MERL Series, Report No. 1,
> 3^rd^ edition. This method is performed on a whole water sample
> utilizing an alkaline persulfate digestion in order to oxidize both
> organic nitrogen and ammonia to nitrate. Total phosphorus can also be
> determined from the same digestion. The samples are analyzed on a
> multi-channel Quikchem FIA + 8000 Series autoanalyzer (Lachat
> Instruments, Inc., Milwaukee, WI).
>
> For simultaneous oxidation of total nitrogen (TN) and total phosphorus
> (TP) it is necessary to utilize an alkaline medium for nitrate
> production and an acidic medium for the oxidation of phosphorus. This
> simultaneous oxidation method starts at a pH of approximately 10 and
> ends at a pH of approximately 3-4. These conditions may be obtained
> from a sodium hydroxide-boric acid system. The digestion time is a
> function of the autodecomposition of peroxodisulfate and temperatures
> must reach at least 110C in the pressure cooker to achieve this
> decomposition (Valderrama, 1981).
>
> **[Equipment]{.underline}**

-   Digestion tubes are 70 mL Pyrex #9825 borosilicate glass screw-top
    tubes with autoclavable rubber-lined screw caps (Fisher Scientific,
    catalog #15-327). **Sample caps and tubes are washed thoroughly
    with** HOT soapy water (Citranox; phosphate free acidic detergent),
    distilled water rinse to remove all soapy residue triplicate acid
    rinse with 1:4 HCl solution triplicate distilled water rinse, air
    dried and capped until used.

```{=html}
<!-- -->
```
-   Hot plate

-   Pressure cooker (maximum pressure \~20psi)

-   Orion pH meter

-   Quikchem FIA + 8000 Series Lachat autoanalyzer with nitrate/nitrite
    and orthophosphate channels

-   Eppendorf pipettes and tips

> **[Reagent Preparation]{.underline}**
>
> Note: Use deionized water for all solutions.
>
> [**Reagent 1: Stock Ammonium Molybdate Solution**:]{.underline}
> Dissolve 40.0 g. ammonium molybdate tetrahydrate in approximately 800
> ml of dH~2~O. Dilute to the mark in a 1L volumetric flask and invert
> to mix. Store in plastic and refrigerate.
>
> [**Reagent 2: Stock Antimony Potassium Tartrate
> Solution**:]{.underline} Dissolve 3.0 g. antimony potassium tartrate
> hemihydrate (K(SbO)C~4~H~4~O~6~·1/2H~2~O) in approximately 800 ml of
> dH~2~O. Dilute to the mark in a 1L volumetric flask and invert to mix.
> Store in a dark bottle and refrigerate.
>
> [**Reagent 3: Molybdate Color Reagent**:]{.underline} Slowly add 35.0
> ml concentrated TMG sulfuric acid to about 500 ml dH~2~O. Mix and
> allow to cool. Add 72.0 ml of Reagent 2 and 213.0 ml of Reagent 1.
> Dilute to the mark in a one-liter volumetric flask and invert to mix.
>
> [**Reagent 4: Ascorbic Acid Reducing Solution**:]{.underline} Dissolve
> 60.0 g. ascorbic acid in about 700 ml dH~2~O. Dilute to the mark in a
> 1L volumetric flask and invert to mix. Prepare this reagent weekly.
>
> [**Reagent 5: Sodium Hydroxide-EDTA Rinse**:]{.underline} Dissolve 65
> g. sodium hydroxide and 6 g. tetrasodium ethylene diamine tetraacetic
> acid in 1L of dH~2~O.
>
> [**Reagent 6: Carrier Solution**:]{.underline} The carrier solution is
> distilled water.

# 

> **[Standard Preparation]{.underline}**
>
> The linear range of standards found to be appropriate for analysis of
> natural water samples by this method are 1-100M NO~3~ and 0-10M PO~4.~
> Inorganic standards (the same standards used in the dissolved
> nutrients method) are used for the standard calibration. An organic
> standard (B) is prepared as follows and digested exactly as
> sample/standard procedure outlined in the next section and is analyzed
> as an unknown.
>
> NOTE: Seashore vernal pool samples will likely exceed the range for
> NO~3~ and should be diluted accordingly. All other Cape Cod National
> Seashore natural water samples are expected to fall within these
> standard ranges for TP and TN.

**Preparation of Inorganic Standards:**

The standard used for Total Phosphorus/Total Nitrogen is the same
standard used in the Dissolved Nutrients Analysis. It consists of one
standard (Standard A). The Lachat Total Phosphorus/Total Nitrogen
calibration method should be programmed to dilute this Standard A for
determining the standard curve. This program is set so that the first
standard uses no dilution and the last standard is the blank. Make sure
at least 4 test tubes in the first column, (first positions of Tray 4)
are ready for dilution. The total points for the standard curve will be
6.

To make Standard A, a stock standard solution is prepared for each
nutrient and a given amount of each of the three stock solutions are
combined in distilled water for the desired resulting concentration.
Below is the procedure for preparing each of the stock solutions along
with Standard A.

> **Stock Standard Solution for NH~3~-H^+^ Stock Solution, 10mM**

In a 1 L volumetric flask dissolve 0.535 g of ammonium chloride
(NH~4~Cl) that has been

dried for two hours at 110C in about 800 mL DI water. Dilute to the mark
with DI water

and invert to mix. Store refrigerated in a properly labeled bottle for
up to six months

.

> **Stock Standard Solution for PO~4~** **Stock Solution, 10 mM**
>
> In a 1 L volumetric flask dissolve 1.361g primary standard grade
> anhydrous potassium dihydrogen phosphate (KH~2~PO~4~) that has been
> dried for two hours at 110C in about 800 mL DI water. Dilute to the
> mark with DI water and invert to mix. Store refrigerated in a properly
> labeled bottle for up to six months.
>
> **Stock Standard Solution for NO~3~ Stock Solution, 10 mM**
>
> In a 1 L volumetric flask dissolve 0.850g of sodium nitrate (NaNO~3~)
> that has been dried for one hour at 60C in about 800 mL water. Dilute
> to the mark with DI water and invert to mix. Store refrigerated in a
> properly labeled bottle for up to six months.
>
> **STANDARD A**
>
> In a 100 mL volumetric flask, add the following to approximately 80 mL
> of distilled water:

  ----------------------------------------- -----------------------------
  **To 80 mL of DI H~2~0 in a volumetric    **Resulting Concentration**
  flask add:**                              

  1000 μL NH4 Stock Standard                1400 ppb

  100 μL PO4 Stock Standard                 310 ppb

  500 μL NO3 Stock Standard                 700 ppb
  ----------------------------------------- -----------------------------

> **Organic Standard for analyses as an unknown**:
>
> **Nitrogen Organic Standard**: Urea (NH~2~CONH~2~) M.W. = 60.06
> (Fisher certified A.C.S.)

10mM stock solution: 0.060g/100mL dH~2~O

> [Standard]{.underline} [M]{.underline} [mL of 10mM
> stock/100mL]{.underline} [mg/L N]{.underline}

B 40 0.4 0.56

> **Phosphorus Organic Standard**: D-fructose 1,6-diphosphate
> (C~6~H~11~O~12~P~2~Na~3~)
>
> M.W. = 406.1 (Sigma grade)

1mM stock solution: 0.0413 g/100mL dH~2~O

[Standard]{.underline} [M]{.underline} [mL of 1mM
stock/100mL]{.underline} [mg/L P]{.underline}

B 5 0.5 0.155

> Percent recovery of organic standards is checked against the recovery
> of inorganic nitrate and phosphate standards.
>
> **[Sample/Standard Procedure and Storage]{.underline}**
>
> **Recrystallization of Potassium Persulfate**:
>
> Acid clean all glassware and dry in oven. Dissolve 48 g. of potassium
> persulfate (Aldrich, low nitrogen) in 300 mL of distilled water in a
> 1000 mL Erlenmeyer flask. Heat the solution with stirring and swirling
> to 65C at which point the persulfate dissolves. Continue heating to
> 75C. Place immediately in ice-water bath for approximately 45 minutes
> while crystals form. Pour through acid-cleaned, Distilled water-rinsed
> Buchner funnel with No. 40 Whatman filter paper. Apply 5 psi vacuum.
> Rinse and scrape remaining crystals from flask into funnel. Dry and
> fluff crystals in funnel for 5 minutes. Put in petri dish and dry in
> dessicator.
>
> **Persulfate Oxidizing Reagent**:
>
> 50g Potassium persulfate (low nitrogen) (Aldrich)
>
> 30g Boric Acid (Fisher)
>
> 350mL 1M NaOH (40g/L) (Fisher)
>
> Dissolve above reagents in a 1-liter volumetric flask (acid and dH~2~O
> rinsed!) and bring to volume. Transfer to amber 5mL-repipet dispenser.
> Holding time of oxidizing reagent not to exceed one week, although it
> is recommended that new reagent be made daily.
>
> **[Sample/Standard Digestion:]{.underline}**
>
> Forty milliliters (40mL) of organic standard B or whole water sample
> is collected directly into the 70 mL glass tubes. Five milliliters
> (5mL) of persulfate oxidizing reagent is added immediately following
> sample collection to each tube and tubes are capped. Sample/standard
> tubes are cooked for approximately 50 minutes at 15psi in pressure
> cooker. Tubes are allowed to cool to room temperature and stored
> upright until analysis is performed. Samples should be analyzed as
> soon as possible, with a maximum holding time not longer than 28 days
> following digestion (Ameel, 1993 and Lachat Instruments method
> 10-115-01-1-F).
>
> **[Sample analysis]{.underline}**
>
> Simultaneous analysis of TP and TN is performed on a Quickchem 8000+
> Series autoanalyzer (Lachat Instruments, Inc., Milwaukee, WI). TN is
> determined by cadmium reduction using Lachat Instruments method
> 31-107-04-1-B or C, based on U.S. EPA Method 353.2. TP is measured as
> orthophosphate by automated ascorbic acid method on the Quickchem IV
> autoanalyzer (Lachat Instruments method 31-115-01-1-F, based on U.S.
> EPA Method 365.3).
>
> NOTE: Carrier solution should be prepared to match sample/standard
> matrix (i.e. add an appropriate amount of TMG H~2~SO~4~ to match pH of
> samples).

***REFERENCES***

> Nowicki, B.L. 1994. Total Dissolved and Total Particulate Nitrogen and
> Phosphorus. MERL Series, Report No. 1, 3^rd^ Edition, pages 88-91,
> 1994.
>
> Ameel, J.J., R.P Axler, and C.J. Owen, 1993. Persulfate digestion for
> determination of total nitrogen and phosphorus in low-nutrient waters.
> *American Environmental Laboratory*. 1993.
>
> Valderrama, J.C. 1981. The simultaneous analysis of total nitrogen and
> total phosphorus in natural waters. *Marine Chemistry*, 10:109-122.
>
> American Public Health Association. Standard methods for the
> examination of water and wastewater, 19^th^ edition. Eaton, AD, ed.
> Washington DC, 1995.

# Appendix F -- Cations (Ca, Mg, K, Na)

***Sample Handling for Cations***

Samples taken for cation analysis are also used for dissolved nutrient
analysis.

For cation and dissolved nutrient samples, direct the tube discharge
through a 47-mm filter holder with 0.45-m membrane filter (Millipore
HA). Allow the first few milliliters to discharge to waste and then
direct the flow into a pre-acidified (0.2 ml of 2*N* trace metal grade
HCl) 50-ml clear HDPE centrifuge tube. Samples are stored on ice or
refrigerated.

**Direct Air-Acetylene Flame Atomic Absorption Method**

***Principle***

In flame atomic absorption spectroscopy a liquid sample is aspirated and
mixed as an aerosol with combustible gases (acetylene and air). The
mixture is ignited in a flame of temperature ranging from 2100 c to
2800 c (depending on the fuel gas used.) During combustion, atoms of the
element of interest in the sample are reduced to the atomic state. A
light beam from a lamp whose cathode is made of the element of interest
is passed through the flame into a monochronometer and detector. Free,
unexcited ground state atoms of the element absorb light at
characteristic wavelengths; this reduction of the light energy at the
analytical wavelength is a measure of the amount of the element in the
sample.

(Follow procedures outlined in the Cape Cod National Seashore, North
Atlantic Coastal Lab Method A-001 )

# Appendix G -- Anions (Cl^-^ and SO~4~^-^)

# 

***Sample Handling for Anions***

Filter samples (Millipore type HA 0.45-M filters) directly into brown,
60-ml polyethylene, distilled water-rinsed bottles. Refrigerate samples
until analyzed.

***Principle\
*Sulfate**

Sulfate ion (SO~4~^-2^) is precipitated in an acetic acid medium with
barium chloride so as to form barium sulfate crystals of uniform size.
Light absorbance of the barium sulfate suspension is measured by a
photometer and the SO~4~^-2^ concentration is determined by comparison
of the reading with a standard curve.

Refer to method E Turbidimetric Method 4500-SO~4~^-2^. in **Standard
Methods for the Examination of Water and Wastewater, 20^th^ Edition**

**Chloride**

Chloride can be titrated with mercuric nitrate because of the formation
of soluble, slightly dissociated mercuric chloride. In the pH range 2.3
to 2.8, diphenylcarbazone indicates the titration end point by formation
of a purple complex with the excess mercuric ions. Xylene cyanol FF
serves as a pH indicator and end-point enhancer. Increasing the strength
of the titrant and modifying the indicator mixtures extend the range of
measurable chloride concentrations.

Refer to method C. Mercuric Nitrate Method 4500-Cl^-^ in **Standard
Methods for the Examination of Water and Wastewater, 20^th^ Edition**

.

# Appendix H - Sampling Procedures for the Pond Monitoring Program

######### 

1.  [Biweekly Pond Sampling Procedure]{.underline}

Equipment List for Biweekly Sampling

*Check here*

  ---------------------------------------------------------------- ------
  Truck                                                            

  Boat                                                             

  Oars/Paddles                                                     

  Radio                                                            

  2 Anchors                                                        

  Fathometer                                                       

  Clipboard                                                        

  Pencils                                                          

  Pond Field sheets copied onto "Write in the Rain" paper          
  (Profile)                                                        

  YSI (Four parts -- 650, cable, probe and sensor guard)           

  Secchi disk                                                      

  Viewing Scope                                                    

  Light meter (Three parts -- Datalogger, long cable with attached 
  light sensor for variable depth readings and short cable with    
  attached sensor for 10 cm readings)                              
  ---------------------------------------------------------------- ------

> ***[Before Sampling]{.underline}***

-   Before sampling, the YSI should be calibrated. DO and depth must be
    calibrated

> each day. See Appendix A. for more information.

-   Make sure the YSI is properly charged. Once fully charged the
    battery will run for 6-8

> hours. When recharging make sure that the charger is on the positive
> setting.

-   Copy enough pond field sheets onto "write in the rain paper".

> Put the YSI together in the laboratory before going out into the
> field. Replace the calibration cup with the probe guard. Be careful
> when doing this because the DO
>
> membrane is not protected and can easily be nicked. To keep the DO
> membrane wet during transport, wrap a wet towel around the probe and
> place the entire probe upright into an empty bucket.

-   Load equipment and boat into the truck. Check twice to make sure
    nothing has been forgotten.

```{=html}
<!-- -->
```
-   Some sampling sites require you to walk across private property. Be
    aware of the private property owners and their personalities
    especially during July and August. Shoreline residents at Duck,
    Ryder, Herring and Southeast, Great (W) ponds support NPS monitoring
    but are especially sensitive to trespass.

```{=html}
<!-- -->
```
-   Paddle out to the marker buoy or find the deepest point in the pond.
    Use the anchor in conjunction with the buoy line to situate the
    canoe over the deep point. If windy, do not tie the boat to the
    buoy. Instead paddle up-wind of the buoy about 20 m, drop the bow
    anchor (the heaviest anchor) and allow the boat to float back to the
    buoy. Cleat off the front anchor and drop the back anchor. This
    should keep the boat from dragging anchor or drifting with the wind.
    In high winds let out more anchor line (i.e. increase scope) to hold
    the bottom.

***[Data Collection -- Transparency]{.underline}***

**Note: Data collection should take place as close to the deepest point
as possible**.

##### 

> **[Secchi Disk]{.underline}**

-   Lower the Secchi disk over the sunny side of the boat. Using the
    viewing scope, record the depth at which the disk cannot be seen by
    the naked eye. The disk usually has to be raised and lowered a
    couple of times close to the point where the disk disappears in
    order to determine the Secchi depth. Remove sunglasses when testing
    the Secchi depth. Have helper repeat this procedure and record the
    average of the two readings.

> **[Light Meter]{.underline}**

-   Set up the light meter. The cords connecting the cable to the
    datalogger are color-coded. The blue cord is the surface sensor.
    Take the protective red covers off of the two light sensors (cells).
    Try to keep the datalogger out of direct sunlight.

-   Turn the light meter on (press the "FCT ON" button) and switch to
    "MI" by pressing the "Chan" button twice. "MI" is the math channel.
    It calculates the ratio between values received from the two light
    cells. Channel 1 displays the light readings from the water surface,
    while the second channel 2 displays the values from the profiling
    cell.

-   Position the two light probes next to each other in the boat and
    record this measurement, so that their relative sensitivity can be
    assessed.

-   Next, lower the surface cell and the profiling cell to 0.1m and take
    a reading. Avoid shadows (especially from the boat as the probe is
    lowered through the water column) which will result in incorrect
    light measurements.

-   Take readings at 0.5 meters, 1 meter and every meter thereafter
    until the probe reads one percent.

######## ***Data Collection --YSI (temperature, pH, conductivity, redox potential, dissolved oxygen, depth)***

-   Record DO/Temperature in saturated towel conditions before deploying
    sonde.

```{=html}
<!-- -->
```
-   Unwrap the YSI probe from the towel and turn it on. Try to keep the
    650 Display out of direct sunlight.

-   Place the probe in the water and allow it to equilibrate to the pond
    conditions. This usually takes 5 mins.

-   Take readings while out of the water and then all profile data at
    0.1 meters, 0.5 meters, 1 meter and every meter thereafter until
    about 0.5 m off of the sediment avoiding contact with the sediment
    if possible.

-   Before taking each reading, make sure the values have stabilized.
    Dissolved oxygen is usually the one parameter that takes the longest
    to stabilize, especially when passing through the thermocline. Allow
    60 seconds at each depth interval for meter stabilization.

-   Record readings in the YSI datalogger and on the field sheets (see
    Appendix I, Form 2)

######## ***After Sampling***

-   Upon returning to the lab, properly store all equipment.

```{=html}
<!-- -->
```
-   Disconnect the YSI Sonde from the cable and 650 datalogger. Fill the
    calibration cup with a small amount of water and replace the cup
    onto the end of the probe. Replace the plastic protectors on the
    cable ends and the top of the Sonde.

> Download the YSI 650 data file to the ECOwin program at the ponds
> computer.

-   Enter the data collected that day into the working directory.
    *CACO_KettlePonds_97.mdb* file.

***[Division of labor]{.underline}***

> CACO found the most efficient division of labor was to have one person
> working with the YSI/ Light Meter in combination. The second person
> was in charge of recording the YSI profile values as they are read
> aloud by the first person.

2.  [April and August Sampling Procedures]{.underline}

**NOTE: Major Anions (Cl^-^ and SO~4~^-^) samples are only collected in
April.**

#### 

#### *[Equipment List for April and August Sampling]{.underline}* 

####  *Check here*

  ---------------------------------------------------------------- ------
  Truck                                                            

  Boat                                                             

  Oars/Paddles                                                     

  Survival suits; if water 40 c                                    

  2 Anchors                                                        

  Fathometer                                                       

  Clipboard                                                        

  Pencils                                                          

  Marker                                                           

  Labeling tape                                                    

  Pond Field sheets copied onto "Write in the Rain" paper          
  (Profile)                                                        

  Cooler                                                           

  Icepacks                                                         

  Peristaltic pump with tubing                                     

  YSI (Four parts -- 650 datalogger, cable, probe and sensor       
  guard)                                                           

  Secchi disk                                                      

  Viewing Scope                                                    

  Light meter (Three parts -- datalogger, long cable with attached 
  light sensor for variable depth readings and short cable with    
  attached sensor for 10 cm readings)                              

  60-mL plastic syringe                                            

  Clean beaker                                                     

  Filter holders                                                   

  Luer-lock/threaded stainless steel adapter                       

  Millipore filters (type HA 0.45-m)                               

  50-ml pre-acidified clear freestanding HDPE centrifuge tubes (3  
  per pond)                                                        

  60-ml amber HDPE bottles (3 per pond) without acid **(FOR APRIL  
  SAMPLING ONLY)**                                                 

  70-ml glass test tubes (3 per pond)                              

  Potassium persulfate oxidizing reagent (optional -- this may be  
  added immediately upon returning to lab)                         

  Bottle of distilled water                                        

  250-mL plastic bottles (1 per pond), *if doing ARM pH and        
  alkalinity on same visit*                                        
  ---------------------------------------------------------------- ------

*[Laboratory Preparation Before Sampling]{.underline}*

-   Collect all equipment

-   Use a short length (2 m) of tubing on the pump. Attach a float to
    the tubing in order to suspend the submerged end of the tubing 0.5
    meters under the water's surface. Clean out tubing by pumping 50 ml
    1N HCl and then rinsing with distilled water until pH returns to
    about 5.6.

```{=html}
<!-- -->
```
-   Clean 70-ml glass test tubes.

> Pre-rinse tubes with tap water. Fill the Branson ultrasonic cleaner
> with tap water. Add a dash of Citranox. Heat water in ultrasonic unit
> to maximum heat and then turn on ultrasound for minimum 90 minutes.
> Check all caps to make sure the seals are still tight. Once cleaned in
> the ultrasonic bath, flush the tubes with DI water to eliminate soapy
> residue. Rinse three times or soak over night in HCl rinse (1:4
> HCl:water). Caution: When making this acid rinse, remember to add the
> acid to water. Rinse three times with distilled water. Dry tubes
> inverted in test tube rack and once dry, cap test tubes to keep clean.

-   Clean the 50-ml centrifuge bottles, scintillation vials and 250-mL
    plastic bottles by rinsing three times with HCl rinse (1:4 HCl to
    water) and then rinse three times with distilled water.

-   Pre-acidify 50-ml clear, HDPE centrifuge bottles by adding 0.2 ml of
    2 *N* trace metal grade HCl to each bottle.

-   Label all bottles and glassware according to the pond name, date and
    method (if applicable).

-   Calibrate the YSI (see Appendix A).

-   Copy field sheets onto "Write on the Rain" paper.

-   Make reagents:

> [Potassium persulfate oxidizing reagent (for Total Nitrogen/Total
> Phosphorus)]{.underline}
>
> 50 g Potassium persulfate (low nitrogen-- see page 33 for procedure)
>
> 30 g Boric Acid
>
> 350 ml 1M NaOH (40 g/L)
>
> Dissolve above reagents in a 1-L volumetric flask (acid and DI water
> rinsed) and bring to volume. Transfer to amber 5-mL repipet dispenser.
> Holding time of oxidizing reagent is one week although it is
> recommended that new reagent be made daily.

*[Data Collection - Profile and Transparency]{.underline}*

-   **Follow the directions for biweekly pond sampling.**

######### *[Sample Collection - Alkalinity and pH]{.underline}*

-   Rinse the 250-ml plastic bottles with pond water.

-   Fill the bottle with pond water. Eliminate all air pockets from the
    top of the bottle by holding the bottle and bottle top under water
    while screwing on the cover.

-   Store in the cooler.

**[*Sample Collection and Preservation for Dissolved Nutrients
(orthophosphate, NH~4~, NO~3~ and major cations (Ca, Mg, K, Na)* (in
triplicate*)*]{.underline}**

**Note**: Water samples for nutrients, metals, cations and anions are
collected in triplicate using the Geotech peristaltic pump.

-   Connect the battery to the pump.

-   Lower the intake tubing to 0.5 meters below the pond surface.

-   Rinse and condition the tubing by pumping pond water though the
    hose. To make sure the tubing has been thoroughly rinsed, pump about
    250 ml of waste before sampling.

-   Attach a 47mm Millipore filter holder with a 0.45 millipore filter
    to the discharge end of the tubing.

-   Turn on the pump. Allow the first few milliliters to discharge to
    waste and then direct the flow into a pre-acidified (0.2 ml of 2*N*
    trace metal grade HCl) 50-ml clear HDPE centrifuge tube. Cap each
    sample, shake and place in the cooler. Samples are stored on ice or
    refrigerated. Replace filter when pump begins laboring

-   See Appendix D for Dissolved Nutrients laboratory methods and
    Appendix F for cations laboratory methods..

###### 

*[**Sample Collection - Anions** **(Cl^-^ and SO~4~^-^)**]{.underline}*
**(collected in triplicate only during the April survey)**

-   This procedure is most efficiently done before or after the
    Dissolved Nutrient procedure since the equipment needs for both are
    basically the same.

-   You may use the same filter holder and filter (change the Millipore
    filter when it becomes clogged and the pump starts to labor).

-   Rinse each vial with filtered pond water before filling.

-   Pond water is passed from the pump tube through a 0.45-m membrane
    filter and into an acid-cleaned, distilled-water rinsed 60-ml brown
    polyethylene bottle. **Note that this bottle must be thoroughly
    rinsed as any acid residue will contaminate samples with acid
    anions.** Samples are stored on ice or refrigerated.

See Appendix G for anions laboratory methods.

***[Sample Collection - Total Phosphorus and Total Nitrogen
(triplicates)]{.underline}***

-   Pump 40 mL unfiltered pond water directly into the clean
    borosilicate test tube.

-   Add 5 mL of persulfate oxidizing reagent. *Alternatively, cap and
    store samples in cooler and add the persulfate oxidizing reagent
    immediately upon returning to the laboratory.*

-   Cap and invert numerous times to mix.

-   Samples are digested the same day of collection by pressure cooking
    at approximately15 psi for 50 min and then stored at room
    temperature for no more than 28 days.

See Appendix E for the total phosphorus and total nitrogen laboratory
method.

***[After Sampling]{.underline}***

-   Refrigerate the dissolved nutrients and anion samples (record
    information on the log-in sheet on the refrigerator).

###### If TP/TN samples were not treated with oxidizing reagent in the field, add 5 mL of persulfate oxidizing reagent to the TP/TN samples. Place the samples in a pressure cooker and cook at 15 psi for 50 minutes. Label the reagent date on tubes.

-   Clean all used filter holders with distilled water and fill with new
    filters.

-   Recharge pump battery if needed.

-   Put ice packs back into the freezer.

-   Discard used Millipore filters.

-   Put the equipment away. Disconnect the YSI Sonde from the cable and
    650 datalogger. Fill the calibration cup with a small amount of
    water and replace the cup onto the end of the probe. Replace the
    plastic protectors on the cable ends and the top of the Sonde.

-   Allow the alkalinity and pH samples to warm up to room temperature.
    **These samples must be analyzed the day they are collected.** The
    procedure for this analysis is described in Appendix C.

####  

# Appendix I -- Pond Stage. 

# 

> **[Siphon Measurements]{.underline}**
>
> Ryder, Duck, Great Wellfleet, Great Truro, Herring, Snow, Dyer and
> Long Pond all have siphon wells.
>
> Using an electric well tape, lower the sensor until an audible beep
> occurs. Record the position where the tape intersects the well casing
> and record to the nearest 0.01ft. The casing will be marked with ink
> at the measurement point. Refer to the Long Term Hydrologic Monitoring
> Protocol for Coastal Ecosystems, 2003.

# Appendix J - Data Forms. 

***Form 1 - pH and Alkalinity Form***

  ----------------- --------- ------------ ---------- ---- --------- ------- ------------ ------
  Alkalinity Form                                                                         

                                                                                          

  (P=Provincetown                                                                         
  T=Truro                                                                                 
  W=Wellfleet)                                                                            

                              date         date            ml                             
                                                           titrant                        
                                                           to:                            

  POND              ID#       Collection   Analysis   pH   pH4.5     pH4.2   Alkalinity   

  Seawater                                                                                

  DUCK (P)          96866                                                    0            

  DUCK (W)          96868                                                    0            

  DYER (W)          96070                                                    0            

  FRESHBROOK (W)    9661575                                                  0            

  GREAT (T)         96114                                                    0            

  GREAT (W)         96117                                                    0            

  GULL (W)          96123                                                    0            

  HERRING POND (W)  96134                                                    0            

  HERRING RIVER (T) 9661650                                                  0            

  HIGGINS (W)       96137                                                    0            

  HORSELEECH (W)    96144                                                    0            

  KINNACUM (W)      96163                                                    0            

  LIT.BENNET (P)    96347                                                    0            

  LONG1 (W)         96179                                                    0            

  LONG2 (W)                                                                  0            

  NORTHEAST(W)      96226                                                    0            

  PAMET RIVER (T)   9661725                                                  0            

  ROUND EAST (T)    96261                                                    0            

  ROUND WEST (T)    96260                                                    0            

  RYDER (T)         96268                                                    0            

  SLOUGH (T)        96298                                                    0            

  SNOW (T)          96303                                                    0            

  SOUTHEAST (W)     96305                                                    0            

  SPECTACLE (W)     96306                                                    0            

  TURTLE (W)                                                                 0            

  WILLIAMS (W)      96341                                                    0            

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

***Note: The non-kettle ponds listed are part of the A.R.M. study.***

***Form 2. Field form for kettle pond water quality monitoring***.

  ---------- --------- --------- ------------- -------- -------- -------------- --------
  **Kettle                       Fathometer:            (M or                   
  Pond Field                                            ft)                     
  Form**                                                                        

  Pond                           Date                            Pre-profile    
                                                                 %DO:           

                                                                                

  Weather                        Secchi                          Post-profile   
                                                                 %DO:           

                                                                                

  Depth M    F/CTemp   Conduct   %DO           DO ppm   pH       Eh             %Trans

  Air                                                                           

  0.1                                                                           

  0.5                                                                           

  1                                                                             

  2                                                                             

  3                                                                             

  4                                                                             

  5                                                                             

  6                                                                             

  7                                                                             

  8                                                                             

  9                                                                             

  10                                                                            

  11                                                                            

  12                                                                            

  13                                                                            

  14                                                                            

  15                                                                            

  16                                                                            

  17                                                                            

  18                                                                            

  19                                                                            

  20                                                                            
  ---------- --------- --------- ------------- -------- -------- -------------- --------

# ![](media/image2.wmf)  {#section-32 .unnumbered}

# Appendix K. The Need for Annual Sampling.

Analysis of transparency data collected through year 2000 indicates that
water quality monitoring should continue on an annual basis. Reasons are
as follows:

The most direct measure of clarity is Secchi depth. We compare
inter-annual coefficients of variation (CV) for Secchi given 1, 2, 3, 4
or 5 yr. monitoring intervals for the six ponds with the most data
(Fig.1). Inter-annual variation is very high even with annual sampling.
Low CVs for the 4- or 5-yr interval are attributed to chance given the
small sample size, in most cases only 7-15 years of fairly continuous
annual data (Fig 2).

We also compare CV in Secchi within years to inter-annual CV at annual,
biennial, etc. sampling intervals for representative Duck (Fig. 3) and
Ryder (Fig. 4) ponds. Given the large annual CVs, there is simply not
enough data, i.e. years of observation, to say that less frequent
sampling can discern trends. We have already determined that biweekly
observations are necessary to characterize Secchi depth, because month
to month changes can equal the full range of Secchi over the entire
April-October season of stratification (see Table 3, above).

Other monitoring variables (TN, TP, DO and light profiles) do not
directly indicate the water's clarity, but are essential in
understanding the reasons for water quality changes, though they do take
time and considerable expertise to do well. A recent review of data from
throughout the world in "Limnology & Oceanography" (Nurnberg, 1995,
40:1100-1111) showed that at least monthly profiles of DO are needed to
track changes in hypolimnetic anoxia, a variable that reviewers of this
protocol have consistently stressed as critical to assess
eutrophication.

![](media/image3.wmf){width="5.1in" height="3.4875in"}

![](media/image4.wmf){width="5.794444444444444in"
height="3.9618055555555554in"}

![](media/image6.wmf){width="5.9in" height="4.034722222222222in"}