Before starting work in the lab, members who carried out wet lab activities validated the NEO training regarding biological and chemical hazards. NEO is an educational platform designed to provide safety training to newcomers in CNRS units. Before beginning any laboratory work, every member read the entire laboratory safety manual in order to understand the best practices to adopt in case of unexpected or hazardous situations. Because we worked with PFAS, which are toxic compounds, we first had to determine the most appropriate way to handle and dispose of them. We studied each PFAS we planned to use by carefully reviewing their Safety Data Sheets. Based on their characteristics, we worked closely with the person in charge of chemical hazards and waste treatment.
After two months of work, we obtained validation from all relevant parties, and a treatment method was established. In addition, before every experiment involving PFAS, we sent a detailed protocol to the chemical safety officer (example: click here). Only after it was validated did we proceed with the experiments. While working in the lab, all members wore the appropriate PPE and complied with standard dress code requirements (long hair tied back, long pants, closed-toed shoes, etc.). Members were supervised by experienced researchers, who provided instructions on how to conduct experiments safely and remained available for guidance at all times. Members also followed good laboratory practices, such as washing hands before entering the lab, disinfecting the bench before and after use, organizing and cataloguing all chemicals and reagents, and complying with storage instructions.
1) Biological Safety
Bacterial strains: We mainly worked with E. coli DH5α, E. coli K16MG55, and L. portucalensis F11, which belongs to biosafety risk group 1.
During experiments, members wore appropriate PPE (lab coat and disposable gloves) and worked in designated areas. Appropriate reagents and disinfectants were used to sterilize lab equipment, and all materials that came into contact with bacteria were autoclaved. Experiments involving living bacteria were exclusively performed in a Biological Safety Cabinet (BSC) to limit potential spread and contamination. The BSC was disinfected with 70% ethanol before and after use. Culture media and all materials that came into contact with bacteria were autoclaved to ensure sterile working conditions. Regarding waste management, all biohazardous materials were handled according to laboratory instructions. Any item that came into contact with bacteria was treated following the DASRI (infectious clinical waste) disposal pathway.
2) Chemical Safety
Chemical hazards played a major role in our project. Since we worked with PFAS, it was essential to test our mutant enzymes and bacterial responses on the actual molecules of interest. More precisely, we worked with PFOA and TFA. Before ordering these two compounds, we thoroughly documented them using their CAS numbers and Safety Data Sheets (SDS). From these documents and the scientific literature, we designed an internal reference file compiling the following information (example attached):
- → General characteristics, physical and chemical properties
- → Required PPE for safe handling
- → Toxicity and effects on human health
- → Storage conditions
- → Environmental risks
- → Classification and labeling (according to GHS/CLP)
- → Procedures in case of incidents (spill, skin or eye contact, accidental injection, etc.)
- → Emergency response measures and incident management protocols
- → Approved treatment and disposal methods
- → Photographs of laboratory spaces and equipment dedicated to PFAS storage and treatment
Because experiments with these compounds involve significant risks, every step of each protocol was meticulously organized to minimize hazards. We were supported in this task by Mr. Chabot, Risk Prevention Technician at Université Lyon 1, and Ms. Provost, Safety Officer at the CIRI laboratory, where a large part of our experiments took place. With their guidance, we implemented a dedicated PFAS treatment workflow in accordance with French regulations governing chemical safety in laboratories.
In order to reduce the risks associated with handling PFOA salt and concentrated TFA solutions, one of our supervisors prepared dilutions of each compound to make them less hazardous.
Before each experiment involving PFAS, a detailed protocol was drafted collaboratively with the team leader and validated by Ms. Provost.
Each protocol was systematically structured as follows (example: click here) ):
- Title of the experiment
- Objectives
- Laboratory room(s) and equipment used
- Materials and reagents required
- Contact persons in case of emergency
- People present in adjacent rooms during the experiment
- Detailed experimental procedure, with diagrams where needed
- Assignment of responsibilities (who performs which step)
- Summary of manipulations by location/equipment, including schematic overview
- Chemical waste management instructions (where and how to dispose of contaminated material)
- List of preventive measures and emergency procedures (who to contact, steps to follow in case of unexpected incidents)
