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WorldSkills Laboratory Technician Competition – NEC Birmingham 2017 LIST OF TASKS: DAY 1

TASK A: Electrical conductivity of water samples (90min) (points: 10) Skills tested: following SOP, producing a standard curve, technical skill in using lab equipment, accuracy, trouble-shooting and interpreting results

TASK B: Preparation of solutions and dilutions (60 min) (points: 10) Skills tested: accuracy in weighing and measuring, following SOP, preparing for lab tasks, using instrumentation

TASK C: Serial dilution (30min) (points: 5)

Skills tested: aseptic technique, following SOP, using instrumentation, accuracy and precision

TASK D: Determining the Concentration of Aspirin Using UV/Vis Spectrometry (150min) (points: 25)

Skills tested: following SOP, producing a standard curve, technical skills in using lab equipment (spec), accuracy, basic calculations, analysing and interpreting data

DAY 2

TASK E: DNA extraction from plant tissue (120min) (points: 20) Skills tested: following SOP, correct labelling of samples, using lab equipment

TASK F: DNA Visualisation (90min) (points: 25) Skills tested: following SOP, using lab equipment, analysing and evaluating results

TASK G: Preparing a risk assessment (45min) (points: 5)

Skills tested: Risk assessment, COSHH regulations, laboratory safety, ability to recognise risks and apply controls.

DAY 3

Feedback, discussing results

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 1 TASK A: Electrical conductivity of water samples Weighting (points out of total): 10/100 Maximum time allowed: 90 minutes Task: Electrical conductivity is a measurement of the mineral salts dissolved in the water. The unit of electrical conductivity is microsiemens per cm (µS/cm). You will use a conductivity meter to measure the electrical conductivity of sodium chloride solutions of different concentrations to draw a standard curve. You will then measure the conductivity of two unknown samples containing sodium chloride, and infer the sodium chloride concentration of the unknowns by comparing your conductivity measurement to the standard curve. NOTE: the probe is very sensitive to any solutes. Make sure it is thoroughly rinsed inside and out before measuring conductivity. Materials

• portable conductivity meter

• conductivity meter manual

• glass beakers

• wash bottle with ultra-pure water for rinsing the probe

• sodium chloride solutions of five different concentrations for standard curve

• two sodium chloride solutions of unknown concentrations

• graph paper Method

1. Use the manual to familiarise yourself with the conductivity meter.

2. Thoroughly rinse the probe with ultra-pure water straight from the wash bottle before you start and between measurements.

3. Measure the conductivity of the five sodium chloride solutions of known concentrations starting with the lowest concentration, followed by the two unknown solutions. Remember to rinse the probe between measurements

4. Fill the sample to be tested into a clean glass beaker.

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5. Measure the conductivity of the sample by dipping the probe into the sample; wait until the reading has stabilised. Record your result in Table A1 below.

6. Measure each sample three times with a rinse of the probe in-between measurements. Calculate the average of these repeated measurements.

7. Repeat the above steps 3 to 5 to measure all samples provided.

8. Use the values obtained from the standard solutions to draw up a reference standard curve (using the graph paper supplied). Put the dependent variable on the Y-axis.

9. Use the reference standard curve to read the corresponding sodium chloride concentration of the unknown solutions. Write the value obtained in Table A2 below.

Results Table A1. Electrical conductivity measurements (in µS/cm).

Sample Measurement 1 Measurement 2 Measurement 3 Average

50mg/L

200mg/L

400mg/L

600mg/L

800mg/L

Unknown 1

Unknown 2

Use graph paper to draw up a standard reference curve using the average values calculated above. Put the dependent variable on the Y-axis. Use the graph to infer the sodium chloride concentration of the unknowns and enter your result in Table 2 below.

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Table A2. Sodium chloride concentration of the unknown samples:

Sample Sodium chloride (mg/L)

Unknown 1

Unknown 2

Analysis

• Table A3 (below) shows the conductivity of different types of water. According to the table, which water sample could your two unknown samples be? Write your answer below:

• The standard for drinking water in the UK is an electrical conductivity < 2500 μS/cm @ 20°C (EU standards). Do your samples comply with these standards? Write your answer below:

Table A3. Typical conductivity measurements of different types of water.

Water source Electrical conductivity (µS/cm)

Sea water 55 000

Domestic tap water 500-800

Rain or snow 2-100

Distilled water 0.5-3.0

Pure water 0.05

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 1 TASK B: Preparation of solutions and dilutions Weighting (points out of total): 10/100 Maximum time allowed: 60 minutes Task: Solutions are homogeneous mixtures of at least two substances, the solvent and the solute. The solvent is usually the component present in the largest amount and is the medium into which the solutes are dissolved. A solute is any substance dissolved in the solvent and can be gas, liquid, or solid. The concentration of a solution is used to describe its composition and is often referred to as molarity or molar concentration. Molarity is abbreviated to M. A 1 molar (1M) solution of any substance is prepared by dissolving a molar mass (1 mole) amount of solute (in grams) in 1L of solvent. You can calculate the molar mass of a compound by using the chemical formula of the compound and referring to the molar masses of its components according to the periodic table of elements. The equation that defines molar concentration is:

c =n(mol)

V (L)c =n(mol)

V (L)

with: c – concentration of solute, n – substance of solute, V – volume of solution The objectives of this task are: 1) to prepare a stock solution of copper sulphate pentahydrate and 2) to perform a series of dilutions from this stock solution. Materials

• CuSO4.5H2O

• deionised water in wash bottle

• 2 x 100 mL volumetric flasks

• 6 x 50 mL volumetric flasks

• 2 weighing boats (one large, one small)

• pipettes

• marker pen

• funnel

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Method Stock solution concentration:

1. Calculate how many grams you will need to prepare 100 mL of a 1 M solution of CuSO4.5H2O

(using the periodic table of elements provided). Show your work in the space provided below:

2. Use the scale to weigh out the mass of CuSO4.5H2O that you calculated in step 1, onto a large

weighing boat. 3. Carefully add your CuSO4 to the 100 mL volumetric flask using a funnel and use the wash bottle

to get all of the particles from the weighing boat and funnel into the flask. 4. Fill the volumetric flask with deionized water half-way and gently shake until all solid dissolves. 5. Fill the flask to the line with deionized water using the wash bottle. 6. Label the flask with solution name and concentration.

Dilutions: You will now use your stock solution to prepare a series of dilutions. A useful equation to do this is the following: C1V1=C2V2

Where C1 = initial concentration V1 = initial volume C2 = target concentration V2 = target volume

7. Using the equation C1V1=C2V2 calculate the volume of the initial solution (V1) required to

prepare 50 mL of 0.2 M CuSO4 solution. C1 is the concentration of your initial (stock) solution. Show your work below.

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8. Using a pipette, transfer the volume you calculated in step 7 (V1) into a 50 mL volumetric flask. 9. Fill in the flask to the line with deionized water using the wash bottle. 10. Label the flask with the solution name and concentration. 11. Repeat steps 7-10 to obtain a 0.1 M solution to a final volume of 50 mL, from the stock

solution. Show your work below.

12. Repeat steps 7-10 to obtain a 0.02 M solution to a final volume of 50 mL, from the stock solution. Show your work below.

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 1 TASK C: Serial Dilution Weighting (points out of total): 5/100 Maximum time allowed: 30 minutes

Task: In order to determine how many bacterial cells are in the original culture broth a serial dilution is carried out.

A serial dilution is a microbiological technique whereby sequential dilutions are carried out to reduce the density of billions of bacterial cells in a culture broth. Each dilution will reduce the concentration of bacteria by a specific amount and by calculating the dilutions we can determine the number of bacteria in the original culture broth.

For this task you will be carrying out a serial dilution of a bacterial culture broth. You will be assessed on your aseptic technique and accuracy of pipetting.

Materials

• 5x 2ml micro-centrifuge tubes

• 100µl -1000µl micro-pipette

• Sterile pipette tips

• Culture broth

• Sterile water

Method

1. Label tubes 10-1, 10-2, 10-3, 10-4 and 10-5. 2. Pipette 900µl of sterile water into each tube. 3. Using sterile pipette tip transfer 100µl from culture broth to tube labelled 10-1. Discard

pipette tip, close cap and invert until thoroughly mixed.

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4. Use a sterile pipette tip transfer 100µl from tube labelled 10-1 to tube labelled 10-2. Discard pipette tip, close cap and invert until thoroughly mixed.

5. Repeat for 10-3, 10-4, 10-5 as seen in diagram below.

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 1 TASK D: Determining the Concentration of Aspirin Using UV/Vis Spectrometry Weighting (points out of total): 25/100 Maximum time allowed: 150 minutes Task: Acetylsalicylic acid (commonly known as aspirin) has been used to treat pain, inflammation and fever since 1899. The aspirin content of commercial tablets can be determined quantitatively using a variety of techniques. One way involves the usage of a spectrophotometer. This instrument measures the amount of light that is absorbed by a solution. According to the Beer-Lambert Law, the amount of light absorbed by a solution is directly equivalent to the amount of solute in that solution. You will be quantifying the amount of aspirin in a commercial tablet using a spectrophotometric technique. First you will prepare a series of known aspirin concentrations (a standard solution series), draw up a graph with your standard solutions, and then measure the absorbance of the commercial tablet. The aspirin content of the commercial tablet will be calculated using the standard solution graph. Materials

• Hydrochloric acid (HCl) 0.2M

• Sodium hydroxide (NaOH) 0.2M

• Ethanol

• Salicylic Acid

• Citric acid

• Volumetric flasks (5x100mL and 1x50mL)

• 15mL tube

• Mortar and pestle

• Commercial aspirin tablets

• Spectrophotometer

• Pipettes

• Graph paper

• Funnels

• Spatula

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Method Standard solution preparation

1. Dissolve 19.2 mg of salicylic acid and 100mg of citric acid in a small amount of purified water in a 100mL volumetric flask (use the minimum amount that allows the chemicals to dissolve).

2. Add ethanol to dilute the solution to 100ml and obtain a stock standard equivalent to 25mg aspirin/100ml. Label the flask with the content and concentration.

3. Transfer 5,10 and 20ml aliquots of the stock standard solution into separate 100ml volumetric flasks. Label the flasks appropriately.

4. Add 25ml of 0.2M NaOH to each flask. 5. Dilute each sample to volume in the volumetric flask with 0.2M HCl.

Commercial tablet preparation

1. Weigh a single aspirin tablet and record its weight in the results section below (Table D1). 2. Crush the tablet with a pestle in a mortar. 3. Weigh an amount equivalent to 25mg of aspirin from the crushed tablet. To calculate this,

refer to the aspirin content of each tablet as reported on the packaging. Record how much of the crushed tablet you’ve weighed out in the results section (Table D1, “sample weight”).

4. Transfer the weighed, crushed sample into a 50ml volumetric flask. Rinse any excess sample from the boat with ethanol if necessary.

5. Add ethanol to the 50mL mark of the flask and label it appropriately. Shake the flask for 2 min. 6. Pipette a 5mL aliquot from the prepared tablet solution into a 100mL volumetric flask. 7. Add 25mL of 0.2M NaOH to the flask. 8. Let the flask stand for 30 min. 9. Dilute the flask to volume with 0.2M HCl.

Spectrophotometric measurement

1. Prepare a blank sample by adding 0.5mL ethanol, 2.5mL 0.2M NaOH, and 7mL 0.2M HCl to a 15mL tube.

2. Set the spectrophotometer to measure at λ=302nm. 3. Transfer 1mL of blank sample to a cuvette and zero the spectrophotometer. 4. Measure the absorption of 1mL of each standard solution using a separate cuvette for each

standard. Record your readings in the results section (Table D2). 5. Measure the absorption of 1mL of the tablet sample in a clean cuvette and record your

reading (Table D1).

Standard curve preparation 1. Calculate the concentrations of the standard solutions in the Calculations section below. 2. Draw a standard curve on the supplied graph paper using the concentration of the standard

solutions and the corresponding absorption of the standards. The standard curve should represent a line of best fit through your data points.

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3. Use the standard curve and the absorbance of your sample at 302nm to determine the

concentration of aspirin in your sample. Write your result in Table D1. 4. Calculate the aspirin content in the whole commercial tablet. Show your calculations in the

Calculations section below. 5. Compare your calculated aspirin content to the stated aspirin content on the packet. Describe

your results in the Analysis section below. Results Table D1. Tablet measurements

Tablet weight (g) Sample weight

(mg) Sample absorbance

(302nm) Aspirin concentration

in sample (mg/mL)

Table D2. Absorbance values measured in standard solutions

Stock concentration (mg/100mL)

Volume used (mL) Absorbance (302nm)

Aspirin final concentration

(mg/mL)

25 5

25 10

25 20

Calculations In order to draw a standard curve, you will have to calculate the concentration of aspirin (mg/mL) in your prepared standard solutions. Use the concentration of the stock solution (25mg/100mL), along with the volumes you used to complete the following calculations. Use the equation C1V1=C2V2

Where C1 = initial concentration V1 = initial volume C2 = target concentration V2 = target volume Show your calculations below and write the calculated aspirin values (mg/mL) in Table D2.

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Calculate the weight of aspirin in the original tablet here:

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Analysis Compare the calculated value to the value declared on the box (is it the same? By how much does it differ?). Write your answer below:

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Task Information

WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 2 TASK E: DNA extraction from plant tissues Weighting (points out of total): 20/100 Maximum time allowed: 120 minutes Task: Genomic DNA extraction is a key technique in molecular biology and is an essential step in many downstream procedures such as cloning, polymerase chain reaction (PCR), and restriction enzyme digestion. These applications require high quality genomic DNA. In plant (and other eukaryotic) cells, genomic DNA is closely associated with DNA binding proteins. To extract genomic DNA, you will have to separate the DNA from proteins and other components that are also found inside the cell. In addition, the DNA must be extracted carefully to ensure that it remains intact during the purification process. You can assess the quality of your extracted DNA in different ways, including visualising it through gel electrophoresis. The basic steps in DNA extraction from plants are as follows:

1. Grow/collect plant tissue 2. Grind tissue to disrupt cell walls 3. Break open (lyse) cells with lysis buffer 4. Remove cellular debris 5. Purify DNA by removing remaining proteins and RNA 6. Concentrate DNA if necessary 7. Determine purity and concentration of DNA

Materials

• 4 2ml micro-centrifuge tubes

• 2 scalpels

• 2 micropestles

• 2 capless collection tubes

• Sterile water

• Lysis buffer

• Wash buffer

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• 2 purple mini columns

• 100-1000µl micro-pipette

• 10-100µl micro-pipette

• Micro-pipette tips

• Marking pen

• 70% ethanol

• Plant samples

• Sand

• Timer Method

1. Label each 1.5 ml microcentrifuge tube with your initials and plant name.

2. Pipet 200 μl of lysis buffer into each 1.5 ml microcentrifuge tube. 3. Weigh 50–100 mg of each plant material. Record the weight of each plant material.

Name of plant Weight (mg)

4. For each plant, use a razor blade or scalpel to cut the material into small pieces (less than 1–2

mm in diameter). Use a new razor blade or scalpel for each plant type used to avoid

contaminating samples.

5. Add the chopped plant material into a microcentrifuge tube containing 200 μl of lysis buffer.

Add a small amount of sand, just enough to help with homogenizing of samples.

6. For each plant, use a clean micropestle to grind the plant material for at least 3 minutes. Be

careful not to let lysis buffer spill over the side of the tube, which would result in loss of

sample.

7. Once a homogeneous lysate has been generated, add an additional 500 μl of lysis buffer.

Continue grinding using the micropestle until the lysate is homogeneous.

8. Cap (close) the microcentrifuge tube and place it in a microcentrifuge. Centrifuge at full speed

for 5 minutes at room temperature.

9. While the tubes are centrifuging, add 500 μl of 70% ethanol into one labeled colored

microcentrifuge tube for each plant extract.

10. Retrieve the samples from microcentrifuge. For each sample, carefully remove 400 μl of

supernatant (taking care not to disturb the pellet) and add it to the 500 μl of 70% ethanol in

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the appropriately labeled tube. Avoid transferring any solid plant material to the ethanol; if

necessary, recentrifuge the lysate. Pipet up and down to thoroughly mix the lysate and

ethanol into a homogeneous solution. Cap tubes.

11. Label the top edge of two purple mini DNA extraction columns with your initials and plant

names. Place each column into a 2 ml capless collection tube.

12. For each sample, transfer 800 μl of cleared lysate and ethanol mixture to each column. This

step binds DNA to the column.

13. Place the capless collection tube containing the column into the microcentrifuge. Make sure

that the microcentrifuge is balanced. Centrifuge for 1 minute at full speed at room

temperature. Discard the flow-through from the collection tubes.

14. Add 700 μl of wash buffer to each column. Centrifuge at full speed at room temperature for 1

minute. Discard the flow-through. Repeat the wash step two more times for a total of 3

washes. Check the appropriate box after completing each wash step:

❏ 1st Wash

❏2nd Wash

❏3rd Wash

15. After the final wash step, discard the flow-through and place each DNA extraction column

back in its capless collection tube. Dry columns by centrifuging for 2 minutes at full speed at

room temperature. This step is vital to ensure that none of the wash buffer contaminates the

DNA sample.

16. Transfer each DNA extraction column to a clean, appropriately labeled, colored

microcentrifuge tube.

17. Obtain the sterile water from the 70°C water bath. Immediately pipet 80 μl of the warmed

sterile water onto the membrane at the bottom of each column, making sure that the water

wets the column bed. Leave for 1 minute at room temperature to allow the water to saturate

the membranes in the column.

18. Place the column, still in the microcentrifuge tube, into the microcentrifuge. Orient the loose

cap of the microcentrifuge tube downwards, towards the center of the rotor, to minimize

friction and damage to the cap during centrifugation. Centrifuge at full speed at room

temperature for 2 minutes. This step elutes DNA from the column.

19. Remove the column from the microcentrifuge. Cap microcentrifuge tube containing the gDNA

and store at –20°C. Be sure that your tubes are labeled as gDNA with your initial, plant name,

and date.

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 2 TASK F: DNA visualisation Weighting (points out of total): 25/100 Maximum time allowed: 90 minutes Task: Materials Agarose gel preparation

• Agarose

• Spatula

• Weighing boat

• 100ml bottle

• 1x TAE Buffer

• Electrophoresis tank

• Power pack

• SYBR Safe DNA gel stain

• 50ml Measuring cylinder Sample preparation

• Eluted DNA samples

• Loading dye

• 0.1-10 μl micro-pipette

• 10-100 μl micropipette

• Para-film

• Scissors

Method Agarose gel

1. To prepare a 0.8 % agarose gel, weigh out 0.4g of agarose and pour into 100ml bottle 2. Measure out 50ml of 1x TAE buffer using measuring cylinder. 3. Add the TAE buffer to the 100ml bottle containing the 0.4g of agarose, mix by inverting.

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4. Open the bottle slightly to allow steam to escape, place in the microwave for 2 minutes at high power. DO NOT LEAVE THE MICROWAVE UNATTENDED- it’s easy for the gel to bubble over and spill.

5. Once the gel is clear and no beads can be observed, remove from the microwave using the heat resistant gloves provided.

6. Add 5μl of SYBR Safe DNA stain to the gel and mix by gently shaking bottle. 7. While the gel cools, assemble your gel plate. Place the comb into the gel plate and place

the plate into the electrophoresis tank to form a tight seal with the tank wall. 8. Slowly pour your cooled gel into the gel plate taking care not to form bubbles. 9. Allow the gel to cool; this should take approximately 20 minutes. 10. While the gel cools, begin preparation of your samples for loading, instructions can be

found in the Sample preparation section below. 11. When the gel has cooled it will have a cloudy appearance and be cool to the touch,

remove the comb. Adjust the gel plate so the electric current from the power packs will allow the DNA bands to migrate up the gel.

12. Pour in the 1X TAE buffer to the max line on the gel electrophoresis tank, ensuring the agarose gel is completely immersed in the TAE buffer.

Sample preparation Run gel electrophoresis with 10 μl and 20 μl of each of your samples.

1. Cut a piece of 5cm x 5cm para-film- you will be working on the para-film itself to mix your samples with the loading dye. Remove the paper and place the para-film on your work bench sterile part upwards.

2. Pipette 2 μl and 4 μl of loading dye directly on to the para-film and discard the pipette tip. 3. Take 10 μl of your first sample of DNA and mix with the 2 μl of loading dye on the para-

film. Mix by gently pipetting up and down, taking care to not allow bubbles to form. 4. Repeat step 3 with 20 μl of your first sample and mix with the 4 μl of loading dye. 5. Repeat steps 1-4 for your second sample and with 20 μl of the DNA ladder. 6. Using the 10-100 μl pipette, take up your DNA samples, bearing in mind you need to

adjust the pipette to accommodate for the loading dye that has been mixed with your sample.

7. Pipette 10 μl of the ladder into the first well. 8. Pipette your samples into the remaining wells. 9. Cover the electrophoresis tank and attach the electrodes to the power pack. 10. Set the gel to run for 30 minutes at 100V. 11. Tidy your station.

Visualising DNA 1. Turn off the power pack and remove the lid from the electrophoresis tank. 2. Remove the gel plate containing the agarose gel and place on to paper towel. 3. Take your gel to the Dark Reader and note down the location of the bands from your DNA

samples in the results section.

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4. Using the table and diagram below, estimate the size and mass of the DNA you extracted from each sample.

Fragment Size Mass (ng/0.5μg)

1 23130 238.4

2 9416 97.1

3 6557 67.6

4 4361 45.0

5 2322 23.9

6 2027 20.9

7 564 5.8

8 125 1.3

Results

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Task Information WorldSkills UK – Lab Technician – ADVANCED – National Final 2017 DAY 2 TASK G: Preparing a risk assessment Weighting (points out of total): 5/100 Maximum time allowed: 45 minutes Task: Laboratory risk assessments are used to evaluate hazards and potential risks that are associated with working in a laboratory environment. The responsibility of completing a risk assessment is shared between the experimenter and the laboratory technician in charge. Before any laboratory work is carried out, they should consider chemical, biological, equipment and work environment hazards. Who they affect and how? What control measures can be put in place to minimise risks or if the activity is deemed unsafe to take place after control measures have been applied. Using the documentation supplied, you are tasked with completing a risk assessment for the DNA extraction and visualisation you carried out in Tasks D and E. Materials Material Safety Data Sheets (MSDS) Control of Substances Hazardous to Health (COSHH) symbols Risk Assessment template Equipment operating manuals

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Task G: Risk assessment for DNA extraction

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Hazard Persons affected

Staff/ competitors/

general public

Risk Controls

(outline the controls in place)

Risk Rating Future Requirements

(with timescales)

Se

ve

rity

Lik

elih

oo

d

Ris

k Acc

ep

t

Ris

k?

Y/N

Chemical hazards

Biological Hazards

Equipment Hazards – Including SAFETY CRITICAL Equipment (ie equipment whose failure to operate correctly would result in unsafe conditions)

Other Hazards

Task G: Risk assessment for DNA extraction

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Risk Calculation and Interpretation

Severity

Likelihood

High – Death or major injury (as defined by RIDDOR) or illness causing long term disability

High – where it is certain or near certain that harm will occur

Medium – Injuries or illness causing short tem disability Medium – where harm will often occur

Low – All other injuries or illness Low – where harm will seldom occur

Risk Rating

Low likelihood 1

Medium likelihood 2

High likelihood 3

Persons affected

Low severity 1 Trivial Risk

1 Tolerable Risk

2 Moderate Risk

3 e – employees s – students v – visitors c – contractors

p – public

Medium severity 2 Tolerable Risk

2 Moderate Risk

4 Substantial Risk

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High severity 3 Moderate Risk

3 Substantial Risk

6 Intolerable Risk

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A guide to a risk control plan

Risk Level Action and Timescale (Timescales where required shall be determined by the appropriate school/service or management unit)

Trivial No action required and no documentation necessary

Tolerable No additional controls are required. Monitoring is required to ensure that the controls are maintained.

Moderate Efforts should be made to reduce the risk further where reasonable and practicable. Risk reduction measures (where identified) should be implemented within a defined period.

Substantial Work should not be started until the risk has been reduced. If work is already in progress then urgent action should be taken.

Intolerable Work should NOT be started or continued until the risk has been reduced. If it is not possible to reduce the risk then work has to remain prohibited