ERKEN LABORATORY 2004
Characterizing
Phosphorus
Concentration in the
outflow of Lake Erken
(River Järsöströmmen)
Authors:
Martin Kask – Estonia
Zanda Ceirane – Latvia
Supervisor:
Karen Moore
39
INTRODUCTION
This research is concentrating on the outflow of Lake Erken. The lake has only
one major outflow – River Järsöströmmen (Figure 1.). In October 2003, a new sampling site
at Norr Järsö was established. Between this site and the traditional sampling site (Utloppet) a
difference in phosphorus has been noticed. These differences might be smaller than the
differences within a site. We decided to test for differences within and between stream
outflow sites. In addition the current monitoring sites, we looked at other locations between
and downstream near a main road. A dam located between the established monitoring sites
might also influence water chemistry, so it might be one of the factors that account the change
between upstream and downstream phosphorus concentration. Samples from both places and
between them will be taken and compared.
There is a road and runoff (street sweepings of crud that accumulates between
rainfalls) from it may increase the phosphorus budget though the road is not between the two
major sites.
In the past, samples were collected from one location from the stream channel.
This may not be representative across the whole stream cross section.
Our study examines the variability in total phosphorus (TP) and dissolved
inorganic phosphorus (PO4) for a two week period in June 2004. Water samples were also
analysed for turbidity (cloudiness) to get an idea of how much sediment was suspended in the
water. Phosphorus is often sorbed onto sediment and soils particles that may wash in from the
surrounding landscape.
Hypothesis:
¤ There is no significant different in phosphorus concentration between sites at
the outflow of Lake Erken.
Research questions:
¤ Is there any significant different between two outflow monitoring sites for
Lake Erken?
¤ Does the dam and road have effect on phosphorus budget?
¤ Where is the optimal place to take the samples from?
40
MATERIALS & METHODS
Samples from different parts of river Järsöströmmen were taken in two dates
(22/06/04 9:15 - 11.00 and 29/06/04 9:15 - 10:45). Altogether there was four samples (Figure
2.) from five different places (Figure 3), including the sampling stations. A fibreglass pole
with a bottle attached to it and rubber boots (Figure 4 - 9) were used to obtain 20 different
samples all of the volume 250 ml into plastic bottles, with very little space for air.
Fifty ml of water from all the samples was filtered (Figure 12) into empty 250
ml plastic bottles. Bottles were stored in a dark fridge (+4 °C) for ~ 9h. Then (22/06/04 20:12
and 29/06/04 14:33) the Murphy & Riley’s method, was used on the filtered samples to
determine the concentration of PO4. After 24h the turbidity was measured with turbidity meter
and samples were put back to the fridge. In 26/06/04 22:44 and 30/06/04 15:52 the total
phosphorous was measured.
To compare the results of tests we used programs Microsoft Excel and Statistica
(ANOVA).
Overview of ANOVA (references 2 and 4):
Analysis of variance (ANOVA) is used to test for significant differences
between means of groups called factors or effects. Variances are divided into two parts: the
factors or effects used to explain variation in a measured variable (such as phosphorus
concentration) and the error variance due to variation within a group. The sum of squares (SS)
for each effect is the variance we can explain due to differences in means between groups.
The error SS is the amount of variance that cannot be accounted for by the effects used in an
ANOVA model (Statistica 2004).
In the case of our outflow data, the groups or effects in our ANOVA model are
the 5 locations along the stream from the lake to the downstream site at the road (called
“Place”) and the sampling points within the stream channel at each Place (called “In Site”).
We wanted to know how much variance these effects accounted for in three dependent
variables: total phosphorus, orthophosphate, and turbidity. We used this design to ask whether
there were statistically significant differences in phosphorus concentrations or turbidity
between “place” and between “In site.” Our null hypothesis is that there are no differences in
mean concentrations between these groups. The alternative hypothesis is that the group means
41
are different from each other at an accepted level of statistical significance such as 0.05 or
less, expressed as a p-value in the ANOVA table.
The F-test is used to evaluate whether the variance between groups (Place and In
Site) is the same as the variance within a group. The test is based on a comparison of the
mean squares (MS, obtained by dividing the SS by df, the degrees of freedom) for the effects
(model MS, further broken down into MS for each effect) and the variance within groups
(error MS). If the ratio of these variances is significantly greater than 1 using the Fdistribution, then the null hypothesis of no difference between groups is rejected (Statistica
2004). If the F-ratio is near 1, then it is assumed that differences in group means are due to
random variation and the null hypothesis (no difference) is accepted (Helsel and Hirsch
2002).
A summary of laboratory methods follows (After Broberg 1998):
Test 1 (MRP: PO4 µg/l):
1. Making the reagent: Mix together 250 ml Sulphuric acid (2.5 M), 75 ml
Ammonium molybdate and 25 ml of Potassium antimony(III)oxide tartrate (1 mg Sb/ml);
right before use mix with 150 ml Ascorbic acid (0.1 M)
2. Pipetting: 10 ml of filtered water from every sample to different 20 ml glass
scintillation vial (Figure 13.).
3. Adding reagent: 2 ml of reagent to every glass vial and then waiting for at
least 30 minutes (~ 45 min).
4. Measuring: Measure the absorbance of the solution in 5 cm-cuvette at 882 nm
(with Spectrophotometer (Hitachi U-2000)) (Figure 14.).
5. Reading the concentration of PO4 (equation based on the calibration curve
that the machine uses to calculate the concentration of phosphorous: concentration = 339.1 *
absorbance + 0.623)
Test 2 (Turbidity):
1. Turning the bottle gently to mix the sediment.
2. Pouring about 20 ml of unfiltered samples into glass vial.
3. Putting the vial into turbidity meter.
42
4. Reading the results from the screen.
Test 3 (Total phosphorus):
1. Pipetting 10 ml of unfiltered sample and 2 ml of potassium persulphate
solution into closed glass vials.
2. Autoclaving vials in 120 ºC for 60 min.
3. Prepare reagent mixture (250 ml Sulphuric acid (2.5 M), 75 ml Ammonium
molybdate and 25 ml of Potassium antimony(III)oxide tartrate (1 mg Sb/ml); right before use
mix with 150 ml Ascorbic acid (0.1 M))
4. Let the samples to cool off.
5. Add 2 ml of reagent mixture to all of the samples and mix thoroughly
6. Wait at least 30 minutes (~ 45 min) and measure the absorbance of the
solution at 882 nm in 5 cm-cuvette (with Spectrophotometer (Hitachi U-2000)).
.
7. Reading the concentration of PO4 (equation based on the calibration curve
that the machine uses to calculate the concentration of total phosphorous: concentration =
394.1 * absorbance + 0.039).
RESULTS
Looking at the historical data (Table 1) revealed that there are some differences
between Utloppet and Norr Järsö phosphorus levels, but they don’t have any consistent
pattern, as the numbers vary from negative to positive values. Based on this we made our first
hypothesis: there is no significant different in phosphorus concentration between sites at the
outflow of Lake Erken.
Table 1 Historical data
Place
Date
PO4 (μg/l)
Total
phosphorus
(μg/l)
Utloppet
Norr Järsö
Utloppet
Norr Järsö
Utloppet
13/10/2003
13/10/2003
18/11/2003
18/11/2003
09/12/2003
23
29
27
26
26
44
50
40
37
43
Differences
in PO4
Differences
in total
phosphorus
6
6
-1
-3
0
-7
43
09/12/2003
13/01/2004
13/01/2004
17/02/2004
17/02/2004
16/03/2004
16/03/2004
13/04/2004
13/04/2004
12/05/2004
12/05/2004
Norr Järsö
Utloppet
Norr Järsö
Utloppet
Norr Järsö
Utloppet
Norr Järsö
Utloppet
Norr Järsö
Utloppet
Norr Järsö
26
30
35
41
40
37
35
1
2
5
5
36
46
47
46
46
49
45
33
32
20
21
5
1
-1
0
-2
-4
1
-1
0
1
Samples A5 (22/06/2004) and D1 (29/06/2004) (Highlighted in Table 2) were
excluded from statistical tests which were made for total phosphorus and turbidity, because
they were contaminated with sediment and did not therefore represent the average water from
those sites. These samples were used in orthophosphate tests because the water was filtered
before measuring it.
Table 2 All results from test
22/06/2004
Behind road
Utloppet
Before dam
After dam
Norr Järsö
No.
PO4 µg/l (22/06/04)
Turbidity (FTU)
(23/06/04)
Total phosphorous
(26/06/04)
A1
B1
C1
D1
A2
B2
C2
D2
A3
B3
C3
D3
A4
B4
C4
D4
A5
B5
C5
D5
11.47
9.78
6.73
7.07
7.41
6.39
6.39
6.39
6.05
5.71
5.03
5.71
5.37
7.07
7.07
6.05
6.73
5.03
5.03
4.69
1.13
1.61
1.33
1.85
1.84
1.43
1.31
1.32
2.50
1.48
1.28
1.47
1.53
1.47
1.37
1.25
24.00
0.84
1.03
1.24
14.82
19.92
21.95
11.45
15.21
10.49
16.75
18.00
14.63
22.04
15.79
20.02
12.99
11.36
10.97
10.78
117.10
12.61
7.22
9.91
44
29/06/2004
Behind road
Utloppet
Before dam
After dam
Norr Järsö
No.
PO4 µg/l (06/29/04)
Turbidity (FTU)
(01/07/04)
total phosphorous
(30/06/04)
A1
B1
C1
D1
A2
B2
C2
D2
A3
B3
C3
D3
A4
B4
C4
D4
A5
B5
C5
D5
5.09
5.77
5.09
5.77
5.09
4.41
4.75
6.78
4.41
4.07
5.09
4.07
5.77
7.12
5.43
4.41
4.07
1.70
1.70
1.36
1.94
1.50
1.73
9.00
2.40
1.33
1.65
1.37
1.91
1.85
1.51
1.66
1.23
1.07
1.18
1.51
1.83
1.43
0.80
3.50
14.44
19.92
22.00
69.69
13.96
13.38
25.00
15.30
10.13
13.28
23.00
13.96
12.18
12.51
24.00
13.76
21.66
11.45
20.00
28.68
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Sites have been put into order from (Graphs 1.-3.) downstream to upstream to
get a better picture of changes.
Ortophosphate (µg/l)
Current effect: F(4, 32)=5.0217, p=.00295
Vertical bars denote 0.95 confidence intervals
9
8
PO4 (µg/l)
7
6
5
4
3
2
Norr Järsö
Before dam
After dam
Utloppet
Behind road
Place
Graphic 1 Average orthophosphate
Average total phosphorus
Place effect: F(4, 30)=.71962, p=.58528
Vertical bars denote 0.95 confidence intervals
24
22
Total phosphorus (µg/l)
20
18
16
14
12
10
8
Norr Järsö
Before dam
After dam
Utloppet
Behind road
Place
Graphic 2 Average total phosphorus
46
Turbidity
Current effect: F(4, 30)=.72205, p=.58371
Effective hypothesis decomposition
Vertical bars denote 0.95 confidence intervals
2.2
2.0
Turbidity (FTU)
1.8
1.6
1.4
1.2
1.0
0.8
Norr Järsö
Before dam
After dam
Utloppet
Behind road
Place
Graphic 3 Average turbidity
Results that have significant value have been highlighted in the tables of F-test.
(Table 3 -5)
Table 3 PO4 ANOVA results
Univariate Test of Significance for PO4
Sigma-restricted parameterization
Effective hypothesis decomposition
SS
Effect
Intercept 1234.824
Place
49.331
In site
5.805
Error
78.588
Degr. Of
freedom
1
4
3
32
MS
F
p
1243.824
12.333
1.935
2.456
506.4677
5.0217
0.7879
0.000000
0.002953
0.509565
This model explained 42% of the variance in PO4 concentration (R2 = 0.41).
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Table 4 Total phosphorus ANOVA results
Univariate Test of Significance for Total
phosphorus Sigma-restricted parameterization
Effective hypothesis decomposition
SS
Effect
Intercept 9580.864
Place
72.489
In site
110.311
Error
755.501
Degr. Of
freedom
MS
F
p
1
4
3
30
9580.864
18.122
36.770
25.183
380.4443
0.7196
1.4601
0.000000
0.585282
0.245201
Table 5 Turbidity ANOVA results
Univariate Test of Significance for Turbidity
Sigma-restricted parameterization
Effective hypothesis decomposition
SS
Effect
Intercept 91.30075
Degr. Of
freedom
MS
F
p
1
91.30075
420.7398
0.000000
Place
0.62674
4
0.15669
0.7221
0.583706
In site
1.54695
3
0.51565
2.3763
0.089697
Error
6.51001
30
0.21700
DISCUSSION
After receiving statistical results and analysing them we came to a conclusion
that turbidity and total phosphorous doesn’t have any significant changes compared to
differences in a site and between sites (Table 4 and 5; Graphic 2 and 3), that means that
turbidity and total phosphorus values are about the same in everywhere or if they are not, than
the variation of phosphorus level is not bigger than between places compared to inside
variation. Though from these graphics we can draw an conclusion, that the total phosphorus is
largely dependent from the amount of sediment that the samples contain.
But results concerning orthophosphate indicated the opposite; the differences
between sites were more significant than within a site. And the graphic (No. 1) indicates that
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the PO4 level, along the river, is growing. That means that river gets more phosphate from
other sources than the lake such as farms, road, ditches, streams, groundwater and so on.
Therefore our hypothesis that was based on historical data was proven wrong,
but that can be said only for this time of year, the river may act differently in other seasons.
That is why more study is needed to find the best sampling place. But with the data available
to us the optimal site with the water most representative to the lakes outflow is Norr Järsö.
CONCLUSION
Hypothesis (corrected): There is significant different in phosphorous
concentration between sites at the outflow of Lake Erken.
Answer 1: There is significant difference between two outflow monitoring sites
for Lake Erken.
Answer 2: The dam and the road has visible effect on the phosphorus budget.
Answer 3: The optimal place to take samples from is Norr Järsö.
Possible sources of errors:
¤ Limits of Detection (LOD) for total phosphorus and orthophosphate:
LOD (cons.≤100µg/l)=1µg/l
LOD (cons.>100µg/l)=5µg/l
For turbidity: LOD (turb.≤10 NTU)=0.01 NTU
LOD (turb.>10 NTU)=0.1 NTU
LOD (turb.>100 NTU)=1 NTU
Error of the estimate or laboratory results:
Total phosphorus:
cons.≤20 µg/l = ±26%; cons. >20µg/l = ±10%
Orthophosphate:
cons.≤20 µg/l = ±24%; cons. >20µg/l = ±10%
Turbidity:
all = ±10%
¤ Sampling with rubber boots might have caused sediment to rise from the bottom and
contaminate the samples, changing the average values of turbidity and total phosphorus in that
site.
¤ While sampling with an fibreglass pole from the bottom of the river, and bringing the bottle
from the bottom to the surface there might have been an slight inflow to the bottle from other
depths of river.
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ACKNOWLEDGEMENT
We would like to thank our supervisor Karen Moore for the time and effort she
used to aid us. We also thank Mia Arvidsson for her help with fieldwork and Irina Persson for
assistance in the laboratory.
REFERENCES
1. Broberg A., Uppsala 2002, Water and sediment analyses, 3rd edition, pg. 2629.
2. Helsel, D.R. and R.M. Hirsch. 2002. Statistical Methods in Water Resources.
Techniques of Water-Resources Investigations of the United States Geological Survey, Book
4, Chapter A-3.
3. Phosphorus budget: “http://lakeaccess.org/lakedata/lawnfertilizer/pdiagram.htm”
4. ANOVA: “http://www2.chass.ncsu.edu/garson/pa765/anova.htm” and
“http://www.statsoft.com/textbook/stathome.html”
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APPENDIX
Figure 1 Aerofoto of River Järsöströmmen (Copyright © Lantmäteriet)
51
Figure 2 Stream cross section
Figure 3 Sampling sites (Copyright © Lantmäteriet)
52
Figure 4 Behind road
Figure 5 Norr Järsö
Figure 6 Dam
Figure 7 Before dam
Figure 8 After dam
Figure 9 Utloppet
53
Figure 10 Fridge +4 °C
Figure 11 Filtering
Figure 12 Pipetting.
Figure 13 Spectrophotometer (Hitachi U2000)
Figure 14 Turbidity meter
54