April 30, 2008 Working Draft
The Impact of Sample Holding Time on Nutrient Forms in Agricultural Runoff
Purpose
A study is proposed to determine the impact that storage time and storage conditions have
on the distribution of nutrients between the operational forms of N and P that are measured in the
Pioneer Farm and Discovery Farms runoff (see page 4 for overview of N & P forms). The
results can be used to identify appropriate sample collection and storage requirements.
Methods
1. Runoff will be collected from each study location during a relatively high flow portion of
the hydrograph. Approximately 6 liters will be collected.
2. The sample will immediately be shipped to the laboratory—time the collection so that the
sample arrives in the laboratory the same day that it is collected.
3. Immediately after arrival in the laboratory, the sample will be divided into two three liter
portions by mixing and dividing. Each three liter portion will be mixed in the churn
splitter and used to fill a group of 48 125 ml bottles to approximately 90 mls. This will
mimic a 3:1 water/air ratio in the bottle similar to the ISCO bottles. The 125 ml bottles
have a water volume/bottle wall ratio that is a little less than, although close to, that of the
ISCO bottles (125 ml bottle is approximately 1.0 ml/cm2 versus the ISCO 1.2 ml/cm2).
New, acid-washed polypropylene bottles will be used (the ISCO bottles are
polypropylene). Three suspended sediment analyses will be performed with each churn
splitter batch to confirm that the splitting from 6 liters to two 3 liter batches did not lead
to significant differences in composition during initial splitting.
4. Three of the bottles will be processed and analyzed immediately. Another three will be
processed immediately, then acidified and refrigerated for approximately 7 days before
analysis. Over time, the samples in groups of three bottles will be processed and
ultimately analyzed. Table 1 shows the storage, processing and analysis schedule.
5. Each 125 ml bottle will be processed over time similar to field runoff samples. At each
processing time, a 125 ml bottle will be shaken and a 30 ml aliquot added to a bottle with
sulfuric acid preservative and then refrigerated. This will be used for the digestion and
analysis of TP and TKN. The remainder will be used to fill a 60 ml syringe attached to a
0.45 micron filter. 10 ml of the sample will be used to condition the filter and the
remainder will be filtered into a bottle with sulfuric acid preservative. That sample
portion will be analyzed for soluble reactive P, filtered total P (after digestion), filtered
TKN (after digestion), nitrate+nitrite and ammonium. The bottles will be stored in the
refrigerator until they are analyzed.
April 30, 2008 Working Draft
Table 1. Processing and Analysis Schedule
Process
Time
Storage
Temp
o
Bottles
C
Analysis
Time*
Dissolved
Total
Reactive
Dissolved
P
P
(DRP)
(DTP)
Total
P
(TP)
Total
Kjeldahl
N
(TKN)
Filtered
NO3/
TKN
NO2
(FTKN)
(NO3)
NH4
0 hour
3
1
X
X
X
X
X
X
X
0 hour
3
2
X
X
X
X
X
X
X
3/3/3=9
2
X
X
X
X
X
X
X
3/3/3=9
2
X
X
X
X
X
X
X
3/3/3=9
2
X
X
X
X
X
X
X
3/3/3=9
2
X
X
X
X
X
X
X
1 day
2 days
3 days
5 days
4/10/3
0
4/10/3
0
4/10/3
0
4/10/3
0
10 days
4
3
3
X
X
X
X
X
X
X
21 days
4
3
3
X
X
X
X
X
X
X
Cl
SS
X
X
*Analysis Time: 1) These samples will be analyzed for DRP/NO3/NH4 within 24 hours of processing and TP/TDP/TKN/FTKN within
48 hours (additional time for digestion); 2) These samples will be digested on the approximately the 7th day and analyzed on
approximately the 8 day; and 3) These samples will be digested and analyzed on the 23rd-24th day. All samples will be filtered and
acidified when processed, and then refrigerated until analyzed.
Additional Comments and Supplemental Research Project Testing
The proposal above is narrowly focused. It tries to identify changes that may occur during
storage in bottles after collection and before sample processing. In addition, by comparing results
with an almost immediate analysis, some information on the role that sample storage may play on
nutrient forms will also be obtained. In addition, we have several research projects that would
benefit from some additional information that can be collected in parallel with the study described
above and we would propose to support these additional analysis through those research projects.
a. We would like to learn whether there is a larger unreactive phosphorus
component in the runoff that may be obscured by the acidification and storage
after processing. As we split the sample for the bottles described above, another
three 125 ml bottles will be prepared, filtered and stored in the cold, and then
analyzed without prior acidification for dissolved reactive P within 24 hours (this
will be parallel to the immediate analysis group in Table 1).
b. It would also be useful to know if immediate filtration would affect the
concentration of dissolved P. As another parallel research experiment, we will
prepare another nine 125 ml bottles with runoff that is filtered immediately after
removal from the churn splitter. Those samples will be stored in the cold (4
degree C) and will be processed with the other samples, with three bottles
processed at the 1, 5 and 21 day processing times for dissolved reactive P and
total dissolved P.
April 30, 2008 Working Draft
Some References Related to Sample Storage
Allen-Diaz, B., E. Hammerling, and Chris Campell. 1998. Comparison of standard water quality sampling
with simpler procedures. Journal of Soil and Water Conservation 53:42-45.
Avanzino R.J. and V.C. Kennedy. 1993. Long-term frozen storage of stream water samples for dissolved
orthophosphate, nitrate plus nitrite, and ammonia analysis. Water Resources Research 29: 3357-3362.
Burke, P.M, S. Hill, N. Iricanin, C. Douglas, P. Essex and D. Tharin. Evaluation of preservation methods
for nutrient species collected by automatic samplers. 2002. Environmental Monitoring and Assessment
80:149-173.
Gardolinski, R.C.F.C., G. Hanrahan, E.P. Achterberg, M. Gledhill, A.D. Tappin, W.A. House and P.J.
Worsfold. 2001. Comparison of sample storage protocols for the determination of nutrients in natural
waters. Water Research 35:3670-3678.
Haygarth, P.M., C.D. Ashby and S.C. Jarvis. 1995. Short-term changes in the molybdate reactive
phosphorus of stored soil waters. Journal of Environmental Quality 24:1133-1140.
Klingaman, E.D. and D.W. Nelson. 1976. Evaluation of methods for preserving the levels of soluble
inorganic phosphorus and nitorgen in unfiltered water samples. Journal of Environmental Quality 5:42-46.
Kotlash, A.R. and B.C. Chessman. 1998. Effects of water sample preservation and storage on nitrogen and
phosphorus determinations: implications for the use of automated sampling equipment. Water Research
32:3731-3737.
Lambert, D., W. Maher and I. Hogg. 1992. Changes in phosphorus fractions during storage of lake water.
Water Research 5:645-648.
Nelson, D.W. and M.J.M. Romkens. 1972. Suitability of freezing as a method of preserving runoff
samples for analysis of soluble phosphate. Journal of Environmental Quality 1:323-324.
Thayer, G.W. 1970. Comparison of two storage methods for the analysis of nitrogen and phosphorus
fractions in estuarine water. Chesapeake Science 11:155-158.
April 30, 2008 Working Draft
Overview of Nutrient Analysis Methods
Nitrogen
Measured:
Total Kjeldahl N (TKN)
Filtered TKN (FTKN) (or Dissolved TKN)
Nitrate+Nitrite N (NO3N)
Ammonia-N (NH3N)
Computed:
Total Nitrogen (TN) = Total Kjeldahl-N (TKN) + Nitrate+Nitrite-N (NO3N)
Dissolved Total Nitrogen (DTN) = Dissolved TKN + Nitrate/Nitrite-N (NO3N)
Particulate Total Nitrogen (PTN) = TKN (TKN) – Dissolved TKN (DTKN)
Dissolved Inorganic Nitrogen (DIN) = Ammonia-N (NH3N) + Nitrate/Nitrite-N (NO3N)
Dissolved Organic Nitrogen (DON) = Dissolved TKN – Ammonia-N (NH3N)
Phosphorus
Measured:
Total Phosphorus (TP)
Dissolved Total Phosphorus (DTP) (or Filtered Total P; Soluble Total P)
Dissolved Reactive Phosphorus (DRP) (or Filtered Reactive P; Soluble Reactive P)
Computed:
Particulate Total Phosphorus (PTP) = Total Phosphorus (TP) – Dissolved Total Phosphorus (DTP)
Dissolved Unreactive Phosphorus (DUP) = Dissolved Total P (DTP) – Dissolved Reactive P (DRP)
Solids
Measured:
Suspended Sediment (SS)
Total Suspended Solids (TSS)
Total Dissolved Solids (TDS)
Total Volatile Suspended Solids (TVSS)
Computed:
Total Solids (TS) = Total Suspended Solids (TSS) + Total Dissolved Solids (TDS)
or Total Solids (TS) = Suspended Sediment (SS) + Total Dissolved Solids (TDS)
Total Nonvolatile Solids (TNVS) = Total Suspended Solids (TSS) – Total Volatile Suspended Solids (TVSS)
April 30, 2008 Working Draft
UWSP Nutrients and Solids Analysis Methods
Category
Forms
Solids
Total Suspended Solids
Total Dissolved Solids
Gravimetric, 2540D
Gravimetric, 2540C
Nitrogen
Total Kjeldahl Nitrogen
Block digester, auto salicylate, 4500-NH3 G,
Lachat 8500
0.45  filtration, block digester, auto salicylate,
4500-NH3 G, Lachat 8500
Automated cadmium reduction, 4500 NO3 F,
Lachat 8000
Automated phenolate, 4500-NH3, Lachat 8500
Dissolved Total Kjeldahl
Nitrogen
Nitrate+Nitrite Nitrogen
Ammonia Nitrogen
Phosphorus
Total Phosphorus
Dissolved Total Phosphorus
Dissolved Reactive Phosphorus

Methods*
Method numbers from Standard Methods, 19th Ed.
Block digester, automated 4500 P F, Lachat
8500
0.45  filtration, block digester, automated
4500 P F, Lachat 8500
0.45  filtration, automated colorimetric, 4500
P F, Lachat 8500