TITLE: WATER PRODUCTION FOR AWARE (Total Bacteria and Antibiotic Resistance Genes):
AUTHORS: Celia Manaia

[DESCRIPTION]
The protocol summarises the procedures used for analytical control. The protocol describes the Standard Operating Procedure (SOP) for the optimization of advanced tertiary treatment of water, based on a comprehensive quality and risk assessment.

[GUIDELINES]
RECOMMENDED/ACCEPTED VALUE:

No legal recommendation available.  
16S rRNA geneAmount<3 log units/ mL  ; other faecal and antibiotic resistance genes < limit of detection (LOD) inAmount2000 mL

[STEPS]
SECTION: Total Bacteria and Antibiotic Resistance Genes):
1. The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage for1440 min-1440 min(according to water load and season) and ozonation. The water quality is monitored at these three stages, for the parameters indicated in Figure 1 below.

 

SECTION: Total Bacteria and Antibiotic Resistance Genes):
1.1. Sampling, Processing, and Analyses 

Water samples are collected (see Figure 2) and processed within a360 mininterval, before being shipped for the partner responsible for the analyses (Table 1). In case no processing is needed, samples are frozen and stored atTemperature-80 °Cwithin180 min.
For each sampling event, the date, day of the week and hour; the temperature and rain. Sampling points, indicated in Figure 2 were designated from A to I:
- Influent of primary treatment (A) 
- Influent of biological treatment (activated sludge) (B) 
- Treated secondary effluent (C) 
- Sand filter effluent (D) 
- UVC effluent (E) 
- Storage for reuse tank effluent (F) 
- Ozonation effluent (1 dos,  O3) - 5 mgITO3X technology - (G) 
- Effluent of the vacuum UV oxidation (VUV) (H) 
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I) 

 

SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2. Total Bacteria and Antibiotic Resistance Genes: 
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.1. Analysis: Culture-independent detection and/or quantification of bacteria and bacterial contaminants.
SECTION: Methods: The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.2. Observations: Samples were filtered within360 minafter collection the filtering membranes were immediately frozen and stored at-8 °Ctill shipping in dry ice to the respective partner who proceeded for DNA extraction. 

SECTION: Parameters framed by Legal and Regulatory Requirements:
3. Using the EU Drinking Water Directive:  

Mesophilic Bacteria in PCA (Plate Count Agar) – 0 CFU/100 mL  

Total coliforms and Escherichia coli –Number /Amount100 mL   (0 MPN/100 mL) 

Fecal enterococci –Number/100 mL (0 MPN/100 mL)  

Viral concentration - There are no legal requirements for viruses. They are not included in any regulation now. 

Parasite  - EU legislation (2020/741)  

Metals - DIRECTIVE 2008/105/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 16 December 2008 on environmental quality standards in the field of water policy 

Organic contaminants - DIRECTIVE 2008/105/EC OF THE EUROPEAN PARLIAMENT AND THE 
COUNCIL of 16 December 2008 on environmental quality standards in the field of water policy.