UNIVERSITY OF EAST ANGLIA

SCHOOL OF CHEMISTRY

COSHH ASSESSMENT FOR USE OF HAZARDOUS SUBSTANCES IN LABORATORIES

A written assessment of the risks must be prepared for all categories of work with hazardous chemical substances
which are defined in the UEA Code of Practice for Written Assessments under the COSHH Regulations.

1. TITLE OF EXPERIMENT/PROCEDURE: Microbial azo dye reduction assays using Shewanella oneidensis

2. NAME OF RESEARCH SUPERVISOR: Tom Clarke

3. NAME OF ALL PARTICIPANTS Alejandro Morales Florez

4. LOCATION OF EXPERIMENTAL WORK: BIO 2.30

5. HAZARDOUS SUBSTANCES TO BE USED:

Name of substances Principal hazard Quantity OEL*1 Risk
14 M HCl for buffer pH H290 – may be corrosive to metals < 50 mL LD50 277
adjustment H314 - causes severe burns and eye damage mg/kg (rat)
H335 – may cause respiratory irritation
10 M NaOH for buffer pH H290 – may be corrosive to metals < 50 mL LD50
adjustment H314 - causes severe burns and eye damage (dermal)
1350
mg/kg
(rabbit)
Liquid nitrogen for flash Cold burns. Also note potential for small storage
freezing samples; storage of vials rupturing
protein samples
Methyl orange H301 – toxic if swallowed < 100 mg LD50
60 mg/kg
(oral, rat)

OTHER SUBSTANCES TO BE USED:

100 mM NaCl No known hazards <6g LD50 3000
mg/kg (rat)
Ethanol for cleaning glassware H225 – highly flammable liquid and vapour < 10 mL LD50
H319 – causes serious eye irritation 10,470
mg/kg (rat)
Glycerol No known hazards < 10 mL LD50 4090
mg/kg
(oral,
mouse)
HEPES No known hazards < 20 g LD50 316
mg/kg
(bird)
Reactive Black 5 H317 – may cause an allergic skin reaction < 100 mg LD50 5000
H334 – May cause allergy or asthma symptoms or mg/kg
breathing difficulties if inhaled (oral, rat)
Amaranth H319 – causes serious eye irritation < 100 mg LD50 2000
mg/kg
(oral, rat)

*1
OEL=occupational exposure limit for volatile substances in ppm or mg/m3
*2
Based on Risk Matrix suggested by R.S.C.
1= open bench 2=Fumehood should be used. 3=Special facility recommended.
6. EXPERIMENTAL PROCEDURES: SUMMARY

Work will only be carried out during normal working hours in semester. During training, and the initial weeks of the
project, a postgraduate student or a laboratory worker of more seniority will be present in the laboratory to assist and
ensure best practice in safe laboratory working. Once I’m confident in the methods to be used I can work alone in the
laboratory provided a more experienced researcher is available within the vicinity (typically on the same floor of the
building) in case of an incident and to answer questions. I will never carry out potentially hazardous laboratory work
without a more experienced researcher present in the lab.
After experiments, chemicals will be disposed of in appropriate waste bottles and as instructed.

When dispensing the base (acid) stock solution for pH adjustment care will be taken to avoid any contact with
your skin, eyes or clothes and wear gloves and a laboratory coat. Solutions containing the appropriate buffer
components will be brought to the desired pH using aliquots of NaOH or HCl, then increased to the desired
volume with water and the final pH of the solution measured. Safety glasses will be worn but appropriate
handling will not result in contact with eyes. When using liquid nitrogen to freeze samples, safety glasses and
heavy duty cold protective gloves will be used.

Electrical equipment such as spectrophotometers, fluorimeter, computers and centrifuges will be used. These
cause no hazard if used in the manner shown during training on these items of equipment. Centrifuge tubes
will be balanced BEFORE operation.

Stock solutions of azo dyes will be made up and manipulated wearing gloves. Bacterial cultures will be
manipulated using gloves, and the azo dye will be added to reduced Shewanella oneidensis cells, acting as
terminal electron acceptors. The decolourisation by reduction of azo bonds will be monitored using a
spectrophotomer.

Anaerobic samples will be prepared in the glovebox. Nitrogen chambers will be used appropriately as
demonstrated by a supervisor and pose no risk if used correctly. The chamber will be checked for
leaks/unexpected outgassing and if there are any concerns, a supervisor will be consulted to avoid any
possibility of N2 asphyxiation.

If using liquid nitrogen to freeze samples, safety glasses will be worn as well as the heavy duty gloves
provided for working with liquid cryogens (gloves in BIO 2.30). When removing samples from the liquid
nitrogen storage dewar, the appropriate gloves and safety glasses will be worn and there will be awareness of
the possibility of small sample vials rupturing as they warm to room temperature.

There is a risk of electrical shock associated with the operation of a UV-visible absorption spectrometer and
the computers that control their operation. Only dismantling the equipment will expose the user to this risk. If
normal procedures for operation of the equipment are followed then there is no risk associated with the work.

7. PROTECTIVE CLOTHING REQUIRED: (for any part of the work - specify if necessary)
X EYE PROTECTION
(safety glasses, goggles or full face visor) : When weighing chemical and using NaOH and acid in buffer prep
X GLOVES
(specify material if important) : Wear gloves at all times when using solutions containing viologens
RESPIRATORY PROTECTION
(consult safety service first) :

8. CONTAINMENT REQUIRED:
FUMEHOOD : CLASS 1 CLASS 2 CLASS 3 GLOVEBOX X

DUST EXTRACTION: OTHER:

9. RESTRICTIONS ON USE/ACCESS: (whole or part of work may be specified)

X WORK NOT TO BE DONE OUTSIDE NORMALS HOURS :
 WORK NOT TO BE LEFT UNATTENDED :

WORK TO BE DONE IN SPECIAL LABORATORY FACILITIES :

OTHER:

10. IS SPECIAL MONITORING OR HEALTH SURVEILLANCE likely to

(e.g. air sampling) be appropriate?

11. SPECIAL PRECAUTIONS FOR SPILLAGES: (specify)

Any spillage spillages will be mopped up with paper towel and the area rinsed with water. Contaminated tissue will be
contained and a waste disposal technician contacted.
In cases of contamination of clothing the cloths will be removed.
If spillages result in contact with the skin or eyes the contaminated area should be washed with copious amounts of
water. Seek medical help if feeling unwell. If in contact with eyes use an emergency eye within the lab.

Reactive Black 5, methyl orange, and amaranth will NOT be disposed of in the sink. The product is not
allowed to enter drains. Keep all solutions in suitable, closed containers prior to making arrangements for
safe disposal. For any spills of the solid, liaise with a senior researcher to pick up and collect without
creating dust (sweep up and shovel), then arrange for safe disposal.

12. SPECIAL FIRST AID PROCEDURES IN THE EVENT OF AN ACCIDENT: (specify)

In the event of accidental spillage on body and/or clothing ensure all affected areas are washed with large quantities
of water. Seek advice from colleagues for additional procedures.

13. SPECIAL WASTE DISPOSAL ARRANGEMENTS (specify)

For Reactive Black 5, methyl orange, and amaranth, offer surplus and non-recyclable solutions to a licensed disposal
company.

14. REFERENCE BOOKS CONSULTED:

Merck – Sigma-Aldrich Safety Data Sheet
Fisher Scientific Material Safety Data Sheet
(online resources consulted 11/10/2023)

15. DECLARATION:
The above information is, to the best of my knowledge, an accurate statement of the known or foreseeable hazards
and of the precautions which are to be taken. It has been discussed with and shown to all persons who will participate
in the work.

Signed: Date:

Copies of this form must be kept centrally in the school and in the laboratory where the work is to be done.

TO BE COMPLETED BY STUDENT BEFORE ANY PRACTICAL WORK IS STARTED

Student name(s): Alejandro Morales Florez Student Registration number: 100314456

Course: PhD in Biochemistry

I / We have read and understood the above hazards associated with this project and will take the precautions listed
above

Signature(s) ......Alejandro Morales Florez............ Date ...............11 October 2023....................