Investigating the Effects of Increasing Copper Sulphate Solution Concentrations on the Germination of Cress Seeds

I think that as the copper concentrations in the solution rise above natural levels (0.06mg/l), then the seeds will suffer from copper toxic levels, and germination will be stopped.

Factors affecting the investigation:

* Light availability

* Micronutrients availability: copper, zinc, boron, chlorine

* Macronutrients availability: nitrogen, sulphur, phosphorus, potassium, calcium, iron, magnesium

* Water availability

* Temperature

* Oxygen availability

* Enough time for germination to occur

* Enough space for the seeds to germinate

Method:

Equipment needed (for doing experiment once):

* 500ml of copper sulphate stock solution of 60mg/dm-3

* 500ml of pure distilled water

* Micropipette

* 7 Large Beakers

* Stirring rod

* 168 clean plant pots, diameter 20cm (7×24)

* Cling Film

* Filter paper, diameter 20cm

* 2520 cress seeds (168×15)

* Ruler with millimetre measurements

* Glass screen

* Needle

* Gloves

* Digital Thermometer

* Magnifying glass

Method For Changing Independent Variable:

I will change the independent variable, copper concentration, by using a stock solution of 60mg/l.

With this stock solution I will use ten fold serial dilutions to make 5 new concentrations of copper sulphate and then I will make a solution of pure water. Each of these will be a solution of 240ml.

The final concentrations will be:

* 60 mg/l

* 6 mg/l

* 0.6 mg/l

* 0.06 mg/l

* 0.006 mg/l

* 0.0006 mg/l

* 0 mg/l

I am going to do this because it will give me differing concentrations which will allow me to run the investigation at the different concentrations which show the effects of the copper on germination. The serial dilution will allow me to create the solutions accurately and easily so there will be no errors in measurements. I am making one at concentration 0.06 so that it is at the natural level that plants will germinate at, to compare them with. I will also prepare one of purely water, so that I can compare with a batch that had no copper sulphate whatsoever in the solution.

I will use pure distilled water to add to each solution.

I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

For the first batch solution I will add 240ml of the stock solution of 60mg/l to a beaker this will create my first solution for the experiment: 60 mg/l

This gives me a 60mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly.

Clean out the micropipette.

This is so that the micropipette is clean for each step, so the solutions are not contaminated.

For the second beaker in the serial dilution, will use a micropipette to add 24ml of the first solution to 216ml of distilled water in a test tube. Then I will mix this with a stirring rod. This creates the second solution: 6 mg/l

This gives me a 6mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly. I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

Clean out the micropipette.

This is so that the micropipette is clean for each step, so the solutions are not contaminated.

For the third solution I will add 24ml of the 6 mg/l with a micropipette and 216ml of distilled water to a beaker. I will then mix it to create a solution of 0.6mg/l

This gives me a 0.6mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly. I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

Clean out the micropipette.

This is so that the micropipette is clean for each step, so the solutions are not contaminated.

For the fourth solution I will add 24ml of the 0.6 mg/l solution with a micropipette to a beaker with 216ml of distilled water and mix. This creates a solution of

0.06 mg/l

This gives me a 0.06mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly. This solution is at the natural level of copper sulphate that is found in the soil for cress seeds to germinate properly. Therefore, this can be used as a natural level to compare with. I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

Clean out the micropipette.

This is so that the micropipette is clean for each step, so the solutions are not contaminated.

For the fifth solution I will add 24ml of the 0.06 mg/l solution with a micropipette to a beaker with 216ml of distilled water and mix. This creates a solution of 0.006mg/l

This gives me a 0.006mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly. I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

Clean out the micropipette.

This is so that the micropipette is clean for each step, so the solutions are not contaminated.

For the sixth solution I will add 24ml of the 0.006 mg/l solution with a micropipette to a beaker with 216ml of distilled water and mix. This creates a solution of 0.0006mg/l

This gives me a 0.0006mg/l solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse more quickly. I am using distilled water in the solution because it means I can control the levels of other macronutrients and micronutrients available. If I remove the other nutrients, it will mean that it is only the levels of copper sulphate available that is changing. The seeds are all cress seeds, so will have the same levels of other micronutrients and macronutrients, so this is controlled to be the same for each batch.

For the seventh solution, I will simply put 216ml of distilled water into a beaker for pure water.

This gives me a pure water solution which I will use as one of my 7 investigation solutions. I use a micropipette because it gives me an accurate measurement for each solution, so there will be no mistakes in the strength of each solution. By mixing the solution as it is made, it means that the solutions will not have all the copper sulphate at the top or bottom, the whole thing will diffuse quicker. This batch can be used as a control as it has no copper sulphate in it.

I will then prepare stands for the seeds to germinate on.

Each stand will be prepared as follows:

Method For Preparing Seed Stands.

Take 168 plant pots of diameter 20cm which have been well cleaned out with hot water beforehand.

This is done so that once the solutions have been put into the pots they are not affected by dirt or pathogens which may have been in the pot before the experiment.

In each set of 24 pots put in 10ml of the seven different solutions prepared earlier. E.g., 10ml of solution 1 in 24 pots (batch 1), 10ml of solution 2 in 24 pots (batch 2) etc. I will do this using a micropipette.

So that each experiment is run separately and the solutions affect each batch of seeds separately for statistically viable results. I will use a micropipette because it means that the measurements will be accurate, keeping the test statistically valid.

Place a piece of filter paper of diameter 20cm over each solution. Do this wearing gloves.

I will do this so that the seeds can sit on the filter paper in the pots, well dispersed so that they are not competing for space in germination. By sitting on the filter paper, it means that they will not be drowned by the solution, and so will not suffer from a lack of oxygen. If they are drenched there will not be enough oxygen available to the seeds and so aerobic respiration cannot take place. Instead, through anaerobic respiration, the seeds will produce ethanol, and will be poisoned and killed. Wearing the gloves will stop enzymes from my hands getting onto the filter paper and disturbing the investigation.

On each layer of filter paper, I will place 15 seeds, with enough space between each for germination. The seeds will be fresh from the packs which they have arrived in.

This is so that there is the same number of seeds in each batch, so they are all equal Thus by not sharing resources between different numbers, the investigation is kept statistically viable. Also they have to be dispersed with enough space, so they do not compete with each other for the resources. Also by using 30 seeds, it means that there are enough seeds to make the test statistically viable, especially if some seeds are already dead. I will use fresh seeds, because these will have been given the right amount of time in storage before being sold, and so should be ready for germination.

Over the pots, I will put a layer of cling film, tightly secured around each pot.

This is so that the solutions will not escape from the pots through evaporation.

I will pierce five small holes in the cling film using a needle.

This gives 5 identically sized holes in the cling film, so that air is still circulated, with enough oxygen getting in, and carbon dioxide getting out. I will use the same stirring rod because it means that the holes in each batch will all be the same size, for statistical validity.

I will put the pots all in one corner of the room.

I will put them in a corner of the room because we do not have enough incubators available to us for the investigation, and our class has to do the investigation at the same time. If the temperature changes, then the activity of the enzymes in the seeds will be affected, and so they will not be able to mobilise food sources and so start germination properly. If each batch suffer from the same changes as a result of being in the same environment then the results are still statistically viable.

I will keep them away from the classroom doors and windows.

Putting them in an isolated corner means they will all be under the same temperature conditions, so none of them will be under different temperature fluctuations which might arise from open doors and windows. Although we cannot control temperature of the classroom, it is sufficient for germination at 18oC, and if it changes, then all batches will have the same changes. So results will still be statistically viable.

I will put a large glass screen in front of the pots.

Away from the doors and windows with a glass screen will stop breezes reaching the pots and changing temperature

I will make sure that they are all under the same lights of the classroom.

Keeping the pots under the same light is important, so that any changes will be the same for each pot of seeds, so none will have more, or less. Light may affect the experiment, in that it could provide extra heat for the seeds.

During the time of the experiment I will add 10ml of the solutions to each batch each day. E.g.

Batch 1 add 10ml of solution 1 on the second day, another 10ml on the third day etc

Batch 2 add 10ml of solution 2 on the second day, 10ml on third day etc.

I will do this using a micropipette.

This is because the seeds will absorb the solutions each day, and so more of each solution will have to be added each day. This needs to be added daily and not all at the start because otherwise the solution would drown the seeds. We know this because in the pilot experiment we ran the solution was absorbed 10ml on each day by the 15 seeds.

I am using a micropipette because it means that each measurement will be accurate.

The seed batches will have to be left for four days before I assess whether germination has occurred or not.

They are left for four days because I first did a pilot experiment, with the cress seeds at the natural level of copper sulphate, and most seeds took four days to germinate. The seeds need this time to absorb enough water from the solution to rupture the testa and start the germination process.

Method for judging if germination has occurred

For this experiment I will say that germination has occurred when:

a) The testa has burst

b) The radicle has emerged

c) The radicle is at least 2mm long

This is because the testa rupturing is commonly known as the start of germination. However this will happen if the seed is in any solution, and is just a result of the seed absorbing water. Therefore it will happen whether germination does or does not take place. However the emergence of the radicle shows that the food stores in the seed have been mobilised and that the seed germination is truly underway.

I will measure each radicle on seeds from which one has emerged to see if germination has occurred as by my statement above.

This is so that I can define which seeds have actually germinated

I will then repeat the whole investigation again, making the solutions and seed stands up again.

This is so that the results I take can be compared with the second time to make sure the test was reliable.

Null Hypothesis

There is no significant difference between the number of seeds which germinate in the differing copper sulphate concentrations

Raw Data

Mass of Copper Sulphate Solution (mg/l)

Number of Seeds Germinated

Test

1

Test

2

Test

3

Test

4

Test

5

Test

6

Test

7

Test

8

60

1

3

0

2

0

1

1

0

6

15

12

8

11

8

2

4

4

0.6

21

17

10

14

10

9

7

6

0.06

13

15

13

15

12

10

11

17

0.006

19

19

15

13

11

13

15

12

0.0006

18

16

16

11

9

12

9

8

0

17

11

13

13

5

10

13

11

Mass of Copper Sulphate Solution (mg/l)

Total number of seeds germinated

Average number of seeds germinated (per batch)

Standard Deviation

Standard Error

60

8

1

1

0.4

6

64

8

4.2

1.6

0.6

94

11.8

4.7

1.8

0.06

106

13.3

1.8

0.7

0.006

117

14.6

3.0

1.1

0.0006

99

12.4

3.5

1.3

0

93

11.6

3.3

1.2

Manipulated Data

I will now use the values and the quantities worked out for my manipulated data to work out if the means of the samples are significantly different. As I have worked out the standard errors and the means, I will first use the 95% confidence standard error test. I am using this because it is used to work out if means of different samples are significantly different in normal distribution values.

Analysis of Standard Error Test

This table below interprets the log of concentration graph to show where there were and were not significant differences between the mean number of seeds germinated at each differing cupper sulphate concentration:

Mass copper sulphate solution mg/l

Significant difference in mean number of seeds germinated ( / )

60

6

0.6

0.06

0.006

0.0006

0

60

6

0.6

0.06

0.006

0.0006

0

Having done the 95% confidence standard error test I would reject my null hypothesis. This is because there were significant differences between the means of the number of seeds germinated per concentration at the 5% level of probability. From the standard error test, it shows that there was a 95% chance that the means were significantly different between the concentrations marked above in the table. But because the standard error test is usually suitable for sizes of samples which are at least 30, and unfortunately, due to limitations we could only do 8 repeat samples, this may not be entirely reliable. So I will also do the t-test, which is another test for finding out if there is a significant difference between two means in normally distributed data, but for samples smaller than 25.

I will now use the values and the quantities worked out for my manipulated data to work out if the means of the samples are significantly different. As I have worked out the standard errors and the means, I will first use the 95% confidence standard error test. I am using this because it is used to work out if means of different samples are significantly different in normal distribution values.

T-Test

For the t-test I will compare closely the results from the 60mg/l, 0.06mg/l and 0mg/l tests. This is because the 0.06mg/l is the natural level for cress seeds to germinate in when in their natural environment in the soil. The 60mg/l is the strongest solution of copper sulphate I used, and the 0mg/l is the pure water solution, which shows germination with no excess copper sulphate.

Mass of Copper Sulphate Solution (mg/l)

Average number of seeds germinated (per batch)

Standard Deviation

Standard Error

60

1

1

0.4

0.06

13.3

1.8

0.7

0

11.6

3.3

1.2

Table of t distribution

Decreasing value of p

Degrees of freedom (df)

p Values

0.10

0.05

0.01

0.001

1

6.31

12.71

63.66

636.60

2

2.92

4.30

9.92

31.60

3

2.35

3.18

5.84

12.92

4

2.13

2.78

4.60

8.61

5

2.02

2.57

4.03

6.87

6

1.94

2.45

3.71

5.96

7

1.89

2.36

3.50

5.41

8

1.86

2.31

3.36

5.04

9

1.83

2.26

3.25

4.78

10

1.81

2.23

3.17

4.59

12

1.78

2.18

3.05

4.32

14

1.76

2.15

2.98

4.14

16

1.75

2.12

2.92

4.02

18

1.73

2.10

2.88

3.92

20

1.72

2.09

2.85

3.85

22

1.72

2.08

2.82

3.79

24

1.71

2.06

2.80

3.74

26

1.71

2.06

2.78

3.71

28

1.70

2.05

2.76

3.67

30

1.70

2.04

2.75

3.65

40

1.68

2.02

2.70

3.55

60

1.67

2.00

2.66

3.46

120

1.66

1.98

2.62

3.37

?

1.64

1.96

2.58

3.29

Analysis of t-test results

Test

The t value

Degrees of freedom

Solution 1 (mg/l)

Solution 2 (mg/l)

60

0.06

16.9

14

0.06

0

1.28

14

The 0mg/l and 0.06mg/l test:

The t value was 1.28 and the degree of freedom was 14. This meant that I had to find the probability using the following column of the t distribution table:

Decreasing value of p

Degrees of freedom (df)

p Values

0.10

0.05

0.01

0.001

14

1.76

2.15

2.98

4.14

This shows that p>0.10 and so the results of these two concentrations are not significant, so the null hypothesis is accepted here as the mean number of seeds germinated between the concentrations is not significantly different.

The 60mg/l and 0.06mg/l test:

The t value was 16.9 and the degree of freedom was 14. This meant that I had to find the probability using the following column of the t distribution table:

Decreasing value of p

Degrees of freedom (df)

p Values

0.10

0.05

0.01

0.001

14

1.76

2.15

2.98

4.14

This shows that the p<0.001. This means that the results are very highly significant and that I can reject my null hypothesis and be almost certain that the mean number of seeds germinated in concentrations 60mg/l and 0.06mg/l are significantly different.

So I have rejected my null hypothesis.

From using both the 95% confidence standard error test and the t-test, I now reject my null hypothesis. This is because the tests show that the mean number of seeds germinated at differing concentrations of copper sulphate was significantly different.

From looking at the manipulated and the raw data, I can see that, from moving down the strength of copper sulphate concentrations, (60mg/l-0mg/l) the mean number of seeds germinated increased with each new concentration to a point, 0.006mg/l with a mean of 14.6, and then as the concentrations of copper sulphate in the solutions get smaller, then the mean number of seeds germinated decreases for each new concentration to 0mg/l and a mean of 11.6. Because the number of samples for each concentration was the same, then this increase and decrease of mean coincided with the increase and decrease of the total number of seeds germinated per concentration over the 8 samples.

At first look, there does not appear to be a trend or pattern in the standard deviations for the 7 concentrations of copper sulphate. But if the 2 concentrations with the highest mean and total numbers of seeds germinating are removed (0.06mg/l, mean 13.3 and standard deviation 1.8 and 0.006mg/l mean 14.6 and standard deviation 3.0) then the standard deviations follow the same trend as the means, with an increase to a point and then a decrease again. This is relevant because the 2 concentrations removed are the 2 closest to the normal value of copper sulphate in soil for cress seed germination to occur. They have lower standard deviations than the other closer concentration strengths. The standard errors follow the exact same trend as the standard deviations.

When looking at the standard error 95% confidence test results and particularly the log of concentration graph showing the means with standard error, I can see clearly the increase and decrease in the means as described above. It is also clear from looking at the results of the standard error test, that all the concentrations had significantly different mean number of seeds germinated than the 60mg/l copper sulphate concentration. But the 2 concentrations with the low standard deviations and standard errors, which were closest to the natural copper sulphate levels, also had significantly different means from the 6mg/l tests.

As I only needed to do the t-test with 3 of the concentrations of the investigation, then no real patterns appeared. Although the t-test did support the fact that I rejected my null hypothesis because it showed a significant difference between the mean number of seeds germinated in some of the concentrations.

From the results it can be seen that the favoured concentrations of the cress seeds for germination are 0.06mg/l and 0.006mg/l of copper sulphate solution. The least favoured concentrations are the strongest concentrations of copper sulphate (6mg/l and 60mg/l), although the weakest solutions, such as the pure water, also did not favour cress seed germination as much as the 0.06mg/l and the 0.006mg/l concentrations.

The cress seeds did not germinate well in the strongest concentrations of copper sulphate, because germination is initiated by the seed taking up water rapidly, which causes the seed to swell and thus rupture the testa. The water will then hydrolyse insoluble storage material to soluble substances which can be transported. These are transported to the point at which the embryo is growing. This was prevented from happening in the strongest solutions.

The uptake of water depends on osmosis, which relates to the diffusion of water across a partially permeable membrane from a less negative water potential to a more negative water potential. Seeds normally have a low water potential, it is normally very negative, but require a water potential of around -2Mpa for germination to start. Because they have such a low water potential they normally take in water quite easily. But in the high concentrations of copper sulphate, they difference in water potentials between the solution and the seed is not great, so they do not take in enough water for the testa to rupture and for the hydrolysis of the insoluble storage material or activation of the enzymes for food mobilisation.

The germination also depends on the activation stage which is the mobilisation of foods such as starch proteins and fats. This occurs through enzymes (amylase, maltase, peptidases, lipase) changing the substances. The starch is broken down to maltose, which in turn is broken to glucose and transported as sucrose for cellulose or used for energy. The proteins in the seed are converted to polypeptides and then broken to amino acids, which are used for structural or enzyme proteins. The fats are converted to glycerol which is converted to transportable sugars or broken to fatty acids for energy or for transport. If these enzymes are not mobilised by the initiation of germination then the seed will die, and no radicle will appear.

Also in these high concentrations of copper sulphate, the seed will take in copper sulphate through diffusion down the concentration gradient. Copper acts as an enzyme inhibitor in high concentrations and it is very likely that it prevents the breakdown of stored food products in the seed and also inhibits the formation of chlorophyll. This results in inhibited germination and radicle growth. The copper levels will change the pH of the solution in the seeds, which will move the pH away form the optimum level of the enzymes needed in the food mobilisation. Because the seed has lost it’s carefully maintained optimum levels for the enzyme activity, then the enzymes will be denatured. This is when the pH change causes the tertiary structure of the enzyme to lose its shape, with the ionic bonds breaking up. When the active site loses its shape from this and from a loss of charge in the amino acids, the enzyme is said to be denatured and no longer functions, or forms enzyme substrate complexes.

The cress seeds had the highest mean number of seeds germinating at the concentrations 0.06mg/l and 0.006mg/l of copper sulphate. I researched the optimum conditions for cress seed germination, and found that the natural level of copper in the soil for cress seed germination is 0.064. The two concentrations with highest means are those closest to this value. These concentrations had the most seeds germinating because they had the right concentration to get enough copper through diffusion for chlorophyll formation and for proper enzyme activity. And also these concentrations were low enough to not inhibit enzyme activity or effect osmosis and stop water entering the cell. The concentration 0.06mg/l of copper sulphate, which was closest to the natural level, had the lowest standard deviation other than the 60mg/l of copper sulphate. This shows that in each batch almost the same number of seeds germinated, showing that this concentration was consistently right for seed germination. This emphasizes how the 0.06mg/l was perfect for germination.

The lowest concentrations would not have had as many seeds germinating as the higher concentrations because there would not be enough copper taken in by diffusion to start chlorophyll formation and help with enzyme activites.

Anomalies and Improvements

In the raw data, some of the results stand out. These were in the 6mg/l, 0.6 mg/l, and 0mg/l copper sulphate concentrations, and one test had other problems:

Mass of copper sulphate solution (mg/l)

Sample Number

Result

Average number seeds germinated for this concentration

6

6

2

8

0.6

1

21

11.8

0.06

6

10

13.3

0.06

7

11

13.3

0

5

5

11.6

These anomalies in the results could be because of a number of reasons.

Although I planned my investigation out carefully, there were some things that I was unable to do as I had planned, and some things which I had planned which should have been done better and there are a few things which I would change if I was going to do the investigation over again.

Although I had planned to repeat each copper sulphate concentration 24 times, due to limitations I could not do this. When I planned the experiment, I planned it so that I would be able to carry out the investigation to achieve the most accurate and statistically valid results, so I planned to do 24 repeats. But as the school could not provide 168 pots or pipette dishes, I could only do 8 repeats of the differing concentrations. I could possibly have done the test 3 times over, but then it would have been three different investigations because it would have meant that the tests were done in different conditions for example different temperatures. Also the solutions would not all have been made together, so there could have been some apparently minor differences in the concentrations, which would have had a big effect on the results of the test. So I would not have been able to use all of the results together. This could have affected the accuracy of the results, and of the statistical tests, because there were not many samples to use, particularly to check the significance of the results using the 95% confidence standard error tests. Because I was not doing as many repeats as I had planned, I also had to change the number of seeds which I was going to put in each pot for each concentration form 15 to 30, so that the test was still statistically valid.

Limitations on time, and availability of usage of the classroom lab also meant that I could not go to the lab when I planned to, to put solutions back into the pots so that they did not dry out. So I decided that I would not put any more solution on any of the pots at all after the investigation had started. This led to the sample number 5 in the 0mg/l copper sulphate concentration drying out. This would have meant that the seeds in that particular pot did not have water available to all of them for the whole time, so the results here were questionable. This is supported by the fact that only 5 seeds germinated in this pot, whereas the average for the 0mg/l concentration was 11.6. But it also seems strange that this was the only pot which did dry out in the whole experiment, this may have been because I was at fault and did not put the full amount of solution into the pot at the start, so it would have been quicker to dry out.

This may also have dried out because I was not able to have access to clean needles to pierce the cling film for air to circulate into the seeds, so I had to use a stirring rod to pierce it. This meant that the wholes were very big, and so evaporation could have occurred. But this should not really have affected the investigation, because all of the pots had cling film pierced with the same size stirring rod, the same number of times.

Another thing I had to change from the plan which would not have affected the investigation because it affected all the seed samples the same was that I had to use smaller pots than I wanted; they were only 15cm in diameter. Also, I had to use syringes to measure out each solution because there were not enough micropipettes for the class to all use one. This could not have affected the investigation because it meant all solutions were being measured the same way, but some may have not been measured very accurately, this may be why I might have measured the solution for sample number 5 in the 0mg/l copper sulphate concentration wrongly.

Although the results for the samples 6 and 7 in copper sulphate concentration 0.06mg/l do not appear to be very wrong, because 10 and 11 seeds germinated in each and the mean number was 13.3, the results were affected by the fact that when I put the seeds onto the filter paper in the solution, some seeds moved about in the solution away from where they were supposed to be and some were very close to each other in the experiment. This would have meant that they were affected by intraspecific competition, as the seeds would have competed for the available water, meaning some might not have got the needed amounts of water for germination to be initiated. If the pots were 20cm in diameter instead of 15cm, then the water would have been shallower and more spread out, meaning the seeds would not have floated at the start.

Something else which may have affected the investigation would have been that although I said I would put up glass screens in front of the pots to stop wind or drafts affecting the temperature and increasing the rate of evaporation into the air, there were no glass screens available for me to use. Therefore, in their place, I wrapped large amounts of cling film around the area with the pots to stop this, and put holes in this facing away from doors for air to circulate, it seemed largely ineffective and fell down quite a bit, not really protecting the seeds in the pots at all. This could have meant that drafts may have increased the rate of evaporation in the pots. But all of the seeds in all of the pots were again under the same conditions stopping the test becoming unfair and keeping it statistically viable. Also I kept a thermometer checking the temperature of the area with the seeds at all times, this was linked to a computer, which drew a graph of the temperature, showing any fluctuations over the time of the investigation. This graph is included over the page. It shows there was a very small 2 degree variation in the temperature and this too would have been experienced by all of the pots together. SO none would have been in different conditions to the other plant pots in the investigation Therefore this would not really have had that big an effect on changing the results of the tests.

If I were to do the investigation again there are some things which I would change, these were mentioned above in the anomalies and the sources of error which occurred with the procedure and the apparatus which I used.

I would do the investigation with more repeats of the samples as I had planned, so that the results would be more statistically viable. I would do this by doing the investigation at a time when I would be able to use all of the pots which I needed, this would mean that I would be able to use them all without disturbing other experiments. I would also make the seed numbers 20, as this would keep the test statistically viable as the 30 did for the 8 samples, but counting out 30 seeds for 168 different pots is unreasonable for the experiment to be done in time.

I would also use an incubator for my tests instead of using glass screens as I planned, or cling film as I had to. This would mean I could keep the temperature constant for the whole experiment and no fluctuations would occur. Therefore, the seeds could be kept at a temperature optimal for the germination of cress seeds.

As I had planned I would pierce the cling film with a needle not a stirring rod so that the wholes were small and all the same size. This would be enough for air to get in and out, and for evaporation to be largely prevented form the solution. I would also use a micropipette to take the measurements because this would make them more accurate and so no miss-calculations would be made. These two precautions in procedure, should prevent any pots drying out like they did in the sample number 5 of the 0mg/l of copper sulphate solution in the investigation. As none of the other pots dried out, I would not have to put more solution on each pot to stop it drying out. This would also mean that I could make a lot less of each solution than I would if I was topping up each pot with more solution. I would only need one quarter of what I needed to plan for.

I would use different pots (20cm in diameter as planned) for my investigation, so that they were big enough to stop the seeds floating in too much solution and to keep them far enough apart, and small enough to stop the solution being to spread out and shallow in places. If this were not possible I would make less solution (6ml for each pot) and still use the pots which were 15cm in diameter.