The experiment you cannot do
Three of the four factors have been proved by taking them away. The fourth cannot be, and the reason is worth more than the experiment would have been.
The problem
Although water is necessary for photosynthesis, a laboratory experiment cannot be designed to demonstrate that. The reason is that when water is not supplied for the experiment, the plant dies.
Why that ruins the experiment
Every experiment in this chapter works the same way: remove one thing, leave everything else alone, and see whether starch still appears. Remove the water and you have not removed one thing — you have killed the plant, and a dead plant does not photosynthesise for reasons that have nothing to do with water being a raw material. The result would be the same whichever explanation were true, so it would settle nothing.
This is a real limit, not a gap in the syllabus
A well-designed experiment changes one variable. Sometimes the variable you want to change cannot be changed on its own, because changing it destroys the thing you are measuring. Recognising when you are in that position — and saying so, rather than running the experiment anyway and over-reading the result — is part of doing science properly.
How it was done instead
Scientists demonstrated that water is necessary for photosynthesis by using water containing the oxygen isotope 18O. Because that oxygen is heavier than ordinary oxygen it can be told apart afterwards, so the water could be followed through the plant. The labelled oxygen turned up in the oxygen gas the plant released — which shows the water had taken part in the reaction rather than merely being present.
When you cannot remove something, label it
Subtraction was not available, so a different method was needed: instead of taking the water away, mark it and see where it ends up. That is a tracer experiment, and it answers a question subtraction cannot — not just whether water is needed, but what actually becomes of it.
Three by subtraction, one by tracing
Light, carbon dioxide and chlorophyll were each proved necessary by removing them and testing for starch. Water could not be, because a plant without water dies. It was shown instead with water containing the 18O isotope.