The DDA is indeed quite simple in principle: take a water sample in a bottle or jar. Shake it so that it becomes evenly mixed. Pour it through a coars sieve (strainer) into containers (vials) that can be sealed, and fill these as full as is possible. Screw or press the lids back on and store the vials in a perfectly dark place at a constant temperature. About once a day, open each container and measure its acidity (pH) using an accurate three-digit pH meter. Plot the results on graph paper or on computer. After one week at elevated temperature which accelerates the decomposition, all biological matter has been decomposed. Wait another week to be sure.

What happens inside the vials? Because plants cannot survive in the dark, the plant plankton immediately stops working and eventually dies, which happens surprisingly quickly, already after two days in the dark. The decomposers which consist of single-celled bacteria, fungi and viruses, will decompose the plant plankton and eventually also one another. Because they break apart the biochemicals of life which consist of carbon chains with many hydrogen bonds, hydrogen ions (H+) are returned to the water which makes the water more acidic. Because the lids are air tight, these hydrogen ions cannot escape in the form of gases, and they are measured by the pH meter. Thus the pH is an indication of the amount of biomass decomposed, and this can be plotted as curves on a graph.

The decomposition happens more quickly at higher temperatures, which means that the shapes of the curves depend on temperature. If it gets colder, the curve flattens. If it becomes warmer, the curve steepens. So it is important to stabilise the temperature for the following reasons:

• curves taken one day in one place can be compared with those taken on other days in other places.
• measurements such as the bacterial Rate of Attack can be read from the shapes of the curves.
• by elevating the temperature (making it warmer), decomposition happens more quickly and this saves time.

Your minimum DDA laboratory set contains the following items: a precision 3-digit pH meter which is battery-operated and portable vials containing calibration buffers for 4.01, 7.00 and 10.01 pH spare button cell batteries, 4 at a time two collar lids with holes for the pH probe. a simple incubator which comes with a mains voltage converter, cigarette-lighter cable and battery clamps two aluminium trays with 48 vials a precise digital thermometer to monitor the incubator's temparature

• a dark beaker in which the measurement is done, shielded from the surrounding light
• a digital timer with spare battery
• data cards for writing the measurements on to
• plotting cards for plotting the curves
• a 4-colour ballpen and white-out tape for erasing errors and a black marker pen
• topographical maps of your area to read co-ordinates from
• a calculator with LOG and ANTILOG function
• a copy of the DDA manual

• a 50 metre measuring tape, rust-proofed, complete with sinker to measure visibility with.
• a salinity meter also capable of measuring temperature, needed when working in brackish water of estuaries.
• a portable battery-powered GPS for recording actual position, needed when working on large lakes or the sea where position cannot easily be determined from a topographical map or chart.

The DDA method works only if all vials are kept in the dark at all times. The incubator should therefore always be securely closed as it also functions as a dark box. But when samples are measured, they are also exposed to the light, which is to be minimised:

• Don't test in very bright surroundings like in bright daylight.
• Don't remove samples from the incubator until ready for testing.
• Once the pH probe has been inserted, place the sample in a dark beaker

• fill each container as full as is possible but a small bubble is unavoidable.
• don't waste liquid while measuring, but some is spilled when shaking the pH probe.
• keep measuring times as short as possible to just allow the meter to settle. Only at a few instances is precision (longer settling time) required:
• at the very beginning, the initial pH, day zero.
• about 48 hours later, where the Rate of Attack is measured at day 2.
• at the very end, after 1 and 2 weeks, on days 9 and 13 when total biodensity is measured.
• screw or press the lids back on tight and verify this.
• minimise the number of measurements by following the recommended time schedule.
• remember that after a few days when the hydrogen ions have accrued (and the pH has dropped), they are more keen to escape (due to a high partial pressure inside the liquid).
• group your tests for similarity as the pH meter then settles faster. Do not alternate salt and fresh water samples for instance.

As part of the measuring routine, it pays to shake each container a few times in order to stir its contents, but we have not been able to demonstrate that this actually matters.
 

The photo shows the collar lid pushed around the pH probe, just before it is pressed home onto the vial for measuring. The collar has a dip that allows the liquid to rise up and outside the vial without spilling it.

The measurement takes place in a dark beaker, in this case a thermos coffee mug which could also be used to scoop a sample from the sea (or to drink hot coffee with).

Although the water looks clearer than drinking water (which it is most times) you may forget that inside lives an ecosystem containing millions of individuals of thousands of species. It is a true mini world to such an extent that each vial decomposes in a different way compared to every other vial. Thus even when samples are taken from a well mixed jar, differences between them will develop. So it is a good idea to take more than one sample from an evenly mixed jar. For non-scientific work about two samples are adequate, but for more precision and to satisfy scientific criticism, more samples are required from a jar taken at one place at a given moment in time. This of course can easily double the work later. One can often limit the number of duplicates when a batch consists of mostly similar samples taken from similar bodies of water, following a coastline for instance.

Having many vials with many different microbes inside them, raises the possibility of cross-contamination. In its worst case, you will end up with each vial containing the same stuff as all the others. Thus minimising cross-contamination should also be a priority. Ideally one would like to sterilise the pH probe each time before inserting it into the next vial but this raises other problems. Fortunately for us, our samples behave more or less like fully populated communities where introduced strangers are not noticeable and cannot multiply unless there is vacant space available. Thus cross-contamination is fortunately not very serious.

But there's more to it. Where we measure the Rate of Attack in the first 48 hours, cross-contamination is still so minimal that it does not play a role. The beginning of each decay curve is truly due to the decomposers already in the sample and to those that are already numerous. What happens later, is less important. But we noticed that complete decomposition is often not happening unless a small amount of cross-contamination has occurred. It seems as if all the bacterium species necessary to decompose other bacteria, must somehow be present and if not, must be introduced in very small numbers.

To minimise cross-contamination, always proceed as follows:

• shake the probe and collar lid thoroughly before inserting it in a sample from a different place and time. The proper shake happens from the wrist in a lashing movement while holding the pH meter by its far (dry) end. Several shakes are necessary. Make sure never to hit the probe to an object because it is a very sensitive instrument and a replacement probe is rather expensive and takes a long time to become stable.
• do the samples in the order of most similarity. Thus it is not a good idea to switch between estuarine and open sea samples and back again. Do all estuarine samples together. Likewise group freshwater samples together, and so on.

In order to be successful, you must commit yourself to your experiment such that the measurements are done timely and accurately, a spying eye is kept on things that could possibly go wrong, and things are kept tidy. One of the most important tidiness should be the way in which you record your results. Write clearly such that there can never be doubt about the figures. In case of mistakes, use a typist's white-out method so that in the end, the data cards look tidy and anyone can clearly read and transcribe them. Remember these cards carry the results of all your work. Make it a good habit to do backups now and then by photocopying all cards that are ready.

All items used have a name like A1, A2, B1 or ALPHA, BRAVO and so on. Where more than one pH meter is used, they also have unique names or numbers. All this is necessary in order to be able to identify the sets used, without confusion so that irregularities can be tracked down. For instance, a certain vial may always be out of line, possibly caused by a crack in the material. This can be tracked back because all results have been labelled.

Once an experiment has completed its course, the data must be plotted to make sense. Plotting cards are provided and a four-colour ballpen. This will need some experience.

Although a new pH meter is delivered completely dry, it is not advisable to let your pH meter's electrodes dry up. It would eventually build a crust of minerals that could affect its precision. A new pH meter must be soaked first in a saline solution, for which a vial of potassium chloride (KCl) is provided. After an hour the pH meter can be calibrated. How this is done precisely, is found in the meter's manual. But it goes in principle like this:

You have been provided buffer solutions with known acidity or alkalinity: pH=4.01, 7.00 and 10.01. Chemical buffers have the property that their pH values are not easily disturbed by contamination, but we will attempt to minimise this. Your pH meter assumes that when it is presented a solution close to pH=4, and the calibration button is pressed, that the solution is exactly pH=4.01, and it will make an internal adjustment accordingly. Begin with pH=4 because it has the most hydrogen ions.
Rinse the pH probe with tap water, shake it dry and present the pH=7.00 solution. Once the reading has stabilised, which can take ten minutes, press the necessary buttons to calibrate. Finally the pH=10.01 solution is done.

Because our measurements range from pH=6.2 to 8.2, the middle value pH=7.00 is very important whereas the furthest away, pH=4.0 and pH=10 are least important. Thus the pH=7.00 calibration is done most often and with most accuracy. That is why you keep three vials with pH=7.00 buffers. One is labelled FIRST to take any contamination first. The other is least contaminated and most precise. Then there is also a spare just in case. You can get new pH buffers from a friendly laboratory or from a laboratory supplier. They recommend that buffers be replaced every year, as they have an expiry date. You can extend the life of buffers by storing them in a dark, cool place.

When a pH meter is new, it drifts so much that it needs to be recalibrated at least twice daily. After a few days it becomes more stable and after a few months it may need calibration twice weekly. Make a note of the old pH=7 values before pressing the calibration buttons. When a pH probe is faulty, it can be replaced without replacing the body holding the electronics. Every time a probe is recalibrated, it is registered in your calibration log. This allows you to detect problems at an early stage.

The lids of these canisters are shaped such that a bubble can be formed right in the top of the lid, allowing the lid to be pressed close. With a cork-borer tool, a hole can be cut in a lid such that the pH probe fits securely in the hole of the collar, and then onto the vial. It fits sufficiently neatly that the container can safely be lifted with the probe. The probe needs to protrude only a little, as the liquid pushes up around it on the outside of the collar. This kind of lid allows for the smallest possible bubble in the container, as well as for the smallest amount of liquid lost to measuring. In this photo the green liquid is the calibration buffer of pH=7.00. The probe's cap is also shown but not visible is a small moist sponge deep down.

Upon completion of each test, the vials are soaked and rinsed in clean tap water or sea water. Note that sea water is less effective as it is less solvent than fresh water, but it has no chlorine bleach which could affect your results. It would be preferable to sterilise the canisters with ultraviolet light, but letting them drip-dry upside down, then placed on tissue paper, drying in bright sunlight achieves enough. Remember that a small amount of contamination appears to be necessary to achieve complete decomposition.
Do not use soap or other detergents, or bleach or any chemical, as this may upset the next test(s).

Using other containers? We have tested many types of container, small and large and came across some inexplicable differences. For some reason the plankton ecosystem is easily inhibited by PVC containers and even glass! So don't be dishearted if you don't get any results with different containers. First work with the FUJI HDPE film can before trying others.

In the scientific literature the Secchi disc method is described and used. This is a 20cm flat disc with 2 opposite quarters painted white and two black. It is lowered in a horizontal position off the measuring tape. Where the black and white pattern disappears, the depth is read from the tape. This method has several problems.

• the disc sinks only slowly as it flutters downward like a falling leaf. When being pulled up, it offers high resistance and is difficult to reel in. It does not have a hydrodynamic shape.
• when employing the disc from a boat, one finds that the boat drifts so much that the tape does not go down in a straight line but in a curve, seriously affecting precision of measurement.
• the disc has a fixed size of 20cm which is right for shallow waters from 6 to 15m but it is too small for deeper deployment and too large for more shallow measurements.
• the measurement depends largely on surface and weather conditions. In a calm sea with a blue sky and bright sunlight, one can see the disc deepest. But on an overcast day in rough conditions the disc is quickly lost out of sight.

The most accurate measurements are done by floating in the sea in a wetsuit and observing the bag through a dark dive mask, for the following reasons:

• by floating in the sea, one does not drift like a boat in the wind and the sinker descends straight down.
• a dark dive mask removes the reflection of the water's surface so that the same accuracy is obtained in rough and calm seas, blue and overcast skies.
• the tape can easily be read without making adjustments for the height from the railing to the water.

When measuring inside estuaries where currents run, a long pole with a white object attached to its end is more suitable. See the chapter on equipment for suggestions.

• a mechanical thermostat has been fitted to regulate temperature. Its bulb (sensor) makes thermal contact with the metal inner box such that it reacts swiftly. As a result, the mechanism switches on and off more frequently than strictly necessary.
• inside the box, making contact with the element, a heat absorbing freezer pack is placed to even out temperature fluctuations.
• an emergency rechargeable battery of 1.3Ah has been placed inside the mechanism. This battery is inadequate for continuous operation as it would only last for 15 minutes at full power. But it protects against power failures and it keeps the incubator running while disconnected, such as for measuring and leaving the car.
• a precision digital thermometer with external sensor reaches down into the box, making internal temperature visible outside.
• inside the box two aluminium trays are placed, each holding 24 vials.
• the box closes tightly so that it also functions as a dark box.

• place the digital thermometer probe between trays inside the incubator
• frequently monitor the displayed temperature because it varies somewhat when the outside temperature changes drastically.
• you will notice that the temperature is not constant, but swings between extremes of about 1.5 degrees up or down. Make sure the average temperature is as desired (e.g. 27ºC)
• swap the trays daily such that the top tray becomes the bottom one and vice versa.
• make sure that the incubator is connected to an external power source because its internal battery soon runs out.
• when you have reached the ideal setting, tape the dial over with wide duct-tape so it cannot be altered accidentally. This is particularly important when travelling in cars and boats. Note that any irregularity in temperature may void your whole experiment!

• the DDA measures the accumulation of hydrogen ions which are easily leaked away, affecting the accuracy of the method. However, the most important use of the DDA is in comparative studies where one time and place is compared with another. So even if not 100% ideal, its most important aim can be met by standardising how it is done.
• to minimise ion escape, the number of measurements is minimised, and this minimises also the amount of work.
• in the beginning, the sample still behaves very much as if it were in situ (in its natural place) but as hydrogen ions accrue and oxygen is used up, it becomes rather artificial and its main purpose is now to decompose all biomatter completely, including the bacteria themselves. The very first four measurements must therefore be done accurately. Note that the pressure for ions to escape is then still minimal.
• On day 1 two measurements are done, as if the first three were spaced 12 hours apart. This is needed to capture the powerplankton which shows a flat shoulder followed by a steep decline after 36 hours.
• On days 3 - 5 the measurements need little accuracy as they change rapidly. One would say that measurement is not required, but the DDA has shown to proceed better, perhaps due to a small amount of contamination needed.
• After the measurement on day 5, three drops of 20% alcohol (one drop per 10ml) are added to the second set, in order to provide the bacteria with the missing energy lost to biochemical conversions. Note that the added alcohol does not speed up the decay but helps it across a threshold.
• The temperature was chosen such that the whole experiment finishes on day 9 when an accurate measurement is done. Day 13 is just there to check. Sometimes the pH begins to increase, a sign that hydrogen ions are being lost. The experiment finishes just in time before the next one in a fortnightly cycle.

Please note that improvements to the DDA technique are made from time to time and that this time schedule may change accordingly. Check this page from time to time.

Here is what a DDA card could look like on front and reverse side:

The hion as unit of biodensity assumes that for most practical situations, the pH does not rise above 9.0, and if it does, the number of hions involved are so small (less than one) that they can be neglected, but fractions of a hion are still technically possible, even if they are not measurable with a three-digit pH meter.

The biodensity decomposed is calculated by subtracting the beginning value from the ending value, with proper ANTILOG (ALOG) conversion. It is not necessary to understand logarithms to do this correctly. Here is the formula where fpH= final pH  and ipH= initial pH:

biodensity (hion) = ALOG( - fpH) - ALOG ( -ipH) in parts per billion (10^-9)

On your calculator, use the memory function to store the initial value and press the following buttons:
 

If you are able to collect rain water from your roof, this is a real asset. If you have access to distilled water, it comes in handy. But you can achieve satisfactory results by simply rinsing the vials in warm tap water. Don't get tempted to use soap but wash your hands with soap first because a smudge of skin grease on the inside of a vial can upset measurements.

The rule for washing or rinsing is successive dilution. If your first wash is effective only by 90%, leaving 10% 'dirt', the next wash will leave 10% of 10% or 1%, and the next one after that 0.1% 'dirt'. So the secret is to wash at least twice.
The third wash can be done with distilled water but this is not strictly necessary. It is more important to dry the containers completely such that pollutants like chlorine and fluoride evaporate. Shake the moisture off and place containers on a towel in the sunlight before a window. This provides enough ultraviolet light to reach the cleanliness desired for the next test. Once the containers and their caps are dry, cap them and store them away in the dark.

From the local chemist you can purchase 90% pure ethyl alcohol (normal alcohol) which needs to be diluted 4.5 times for 20% final strength. You can do this with either a sight glass or with scales. You won't need more than 50ml of it.
Fill the sight glass to 10ml with 90% alchohol, then add distilled water to 45ml (35ml added). With nearly 30 drops per ml, this suffices for 1350 drops or at most 450 samples. Make a new solution when less than 20% or 10ml remain in the dispenser.