Figure july-27-2004-f.gif Moore Park colonies with barely straw extract and
H2O2, reptisun8
mean with low and high range shown (not error bars)
http://web.pdx.edu/~rueterj/ukl/july04-trip-summary.html
John Rueter
August 4, 2004
My purpose on this trip was to develop a method for assay for the short term effect of marsh water or barley extract on AFA. I used Pulse Amplitude Modulated Fluorometery, PAM F, to examine the change in fluorescence characteristics that are associated with photosynthetic efficiency, regulation and photoinhibition.
I collected samples at five sites.
I have GPS coordinates for each of these locations.
Many different versions of the following protocol were used. The most common protocol was to take a fresh sample of AFA colonies and mixing these 50/50 with different sources of water. These samples were followed for F0, Fm and light curves. During this time period, live samples were taken from Moore Park Marina. The cells at Running Y - apple tree site were very unhealthy looking. Ten to twenty mL samples were incubated in ice cube tray compartments under different light sources; a fluorescent lamp, a UV enhanced fluorescent lamp (Reptisun 8 which is supposed to have 8% more UV radiation) and GE germicidal lamp. The results reported here were incubated with short exposures to the germicidal lamp that would give a large dose of UV.
Some incubations were treated with a dilution of a commercial barley straw extract. The supplier suggests an applying a dose of 1 oz per 100 gallons (6 x 10^-4 dilution). In my experiments I used a 10^-2 dilution (100 uL into 10 mL). The barley straw extract is:
Microbe-lift CSBE
Barley Straw Concentrated Extract
Ecological Laboratories Inc.
www.microbelift.com
On the first day of this trip I compared different ways of measuring the fluorescence in the samples with colonies. Individual readings on samples with intact colonies are highly variable, I assume from the colonies floating around in the cuvette. If I shake the sample vigorously just before reading, I get a reading that is in the range of the colony readings but the individual readings are much less variable.
D1. Incubation of samples for about one hour
Incubation of fresh colonies from Moore Park Marina with and without addition of a commercial barley straw extract showed a pattern in the photosynthetic efficiency. There was an immediate and dramatic decrease in the Fv/Fm ratio in the barely straw extract treatment.
There is an effect of H2O2 on its own that complicates the interpretation of these results.
The initial dip and recovery is a common pattern that was seen in other samples in other incubations. This seems to indicate that the short term effect is reversible with a longer incubation.That is not a good feature of using short term (acute) assays, such as this, to indicate long term (chronic) toxicity.
Figure july-27-2004-f.gif Moore Park colonies with barely straw extract and H2O2, reptisun8
mean with low and high range shown (not error bars)
Fresh colonies from Moore Park Marina were diluted with pure drinking water or filtrate from the Running Y golf course channel. These samples were incubated under the Reptisun8, enhanced UV lamp and measured for photosynthetic characteristics. The marsh water extract inhibited the photosynthetic efficiency of the cells (Fv/Fm) more at the beginning of the incubation than after one hour.
Figure "july-27-2004-g.gif". Moore Park colonies mixed with RunningY brown water.
Reptisun8 light source
each point represent 5 readings on one sample, the point is the mean and the bars show the range (not the standard error)
D2. Short term screening of water samples
There is a lot of detail in the data that was collected that may help understand the mechanism of the inhibition, however for the purposes of a short term incubation as an indicator of inhibition, I am reporting the ratios of the Fv/Fm of treatment/control. A number less than one indicated inhibition.
There were duplicate samples for each treatment. Each value below represents 5 readings on each of two control and two treatment conditions. The samples were incubated under a GE germicidal lamp that provides an intense dose of a limited spectrum in the UV.
The data that I collected on the light curves showed many differences in the responses of these cells to short term UV inhibition including changes in the rates and the amount of quenching. The Fv/Fm ratio, which is related to the photosynthetic efficiency, is usually the most robust measurement. Comparing the Fv/Fm between the controls and the treatments shows that there were both inhibition and stimulation.
Wood River 0.36
Canoe Trail 1 Running Y 1.39
D3. Absorbance spectra
Filtrates of the water samples show strong absorption in the near UV. Canoe trail samples show same pattern but just lower values.
Figure "absorbance.gif". Absorbance of filtrates of Wood River Marsh and Running Y golf course channel.
As part of an attempt to chemically characterize the humic content of these samples, I used a rapid screening fluorescent method developed by Marhaba et al. 2000. This method looks for relative peaks in fluorescence that are supposed to correlate to six fractions of acid, neutral, base with hydrophobic and hydrophilic conditions for each.
The results that I obtained from this screening method show that the samples have higher fluorescence in the basic fractions and that acidic hydrophobic fraction does not show a relative peak. This is in contrast to Barber et al.1999 work on the humic fractions in UKL, which all contained a prominent acidic hydrophobic component.
In the tables below, a "+" means a relatively high fluorescence signal and the "-" is the lowest. The absolute fluorescence signals on the Running Y samples were lower than the Wood River Marsh even though the absorbance (see above figure) was higher.
Wood River Marsh
hydro-
phobichydro-
philicAcid Base Neutral
Canoe Trail 1
hydro-
phobichydro-
philicAcid Base Neutral
Running Y golf course channel
hydro-
phobichydro-
philicAcid Base Neutral
These results are promising but very mixed. The simplest and usually most robust measurement of photosynthetic efficiency (Fv/Fm) did not provide consistent results in these incubations. More detailed examinations of photosynthetic parameters show that the marsh water and UV treatments usually had an effect on non-photosynthetic quenching. The parameters that describe the non-photosynthetic quenching have to be determined in samples that are exposed to ambient light, making this a much more complicated and difficult measurement to make. It would be very interesting if the marsh water effect is to reduce the effectiveness of the cells to deal with photoinhibition. If this is the case, it could have maximum impact during the spring when the water is clearer and colder.
The commercial barley straw extract served as a nice comparison treatment. We should consider using this as one of the treatments that we employ in the limno-corrals.
We need to understand how we might modify the method of Marhaba to apply to correlate to the study of Barber. This might require a more sophisticated spectro-fluorometer that can perform an entire matrix scan. The new diode array spectrofluorometers can capture all the emissions as it scans through a range of excitation wavelengths.
