TITLE: WATER PRODUCTION FOR AWARE (Parasite)
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:

Regulation 2020/741 Minimum requirements for water reuse. 
Guidelines to support the application of Regulation 2020/741 on minimum requirements for water reuse 
Council Directive 93/88/EEC of 12 October 1993 on the protection of workers from risks related to exposure to biological agents at work. 
Relevant food-borne parasites are not included in any normative. 

[STEPS]
SECTION: Parasite
1. The water production for AWARE main activities includes three stages – disinfection by ultraviolet C radiation (UVC), storage for720 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: Parasite
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 at-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 dose, e.g.,5 mgO3) - MITO3X technology - (G) 
- Effluent of the vacuum UV oxidation (VUV) (H) 
- Effluent of reactive storage / Influent of the recirculation aquaculture system (RAS) (I) 

 

SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2. Parasites: 


Analysis: Detection of parasites.
SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.1. Filtration:

3 Lof water were filtered through 0.45 µm pore size diameter Nitrocellulose / polycarbonate sterile membranes, using a membrane filter holder ( ) apparatus. For every approx.250 mLof water, a new sterile membrane was used to avoid clogging of membranes. Filters corresponding to each litre of water were separated, inserted into well-sealed tubes (50 mL stored a-80 °Cand then transported in dry ice. 
SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.2. Centrifugation:

(1 L)  of water was concentrated by centrifugation at2.500 gfor15 minususing sterilized tubes. Pellets obtained were resuspended using of1 mLof formaldehyde solution and maintained for1440 min. Pellets resuspended in formaldehyde solution were centrifugated at2.500 gfor  , the supernatant was discarded and all pellets were pooled in1 mLof  ethanol.  
SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.3. DNA Isolation, Amplification and sequencing: 

DNA extraction was carried out using a commercially available kit (Powerwater kit, Qiagen) in accordance with the manufacturer's guidelines. The V4 and V9 of the 18S rRNA gene hypervariable regions of the eukaryote 18S rRNA gene were amplified using universal primers. Subsequently, PCR products were purified and subjected to external sequencing using Illumina MiSeq platform 300bp paired-end sequencing.  
SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.4. Bioinformatic analysis: 

Raw sequence data were processed using QIIME2 software 2023.9. Pairedend reads were subjected to quality filtering (FastQC) and denoising (DADA2 plugin). Subsequently, the taxonomic assignment was conducted using the reference database SILVA 18S release 138 with a clustering threshold of similarity. Alpha rarefaction analysis, Shannon diversity index, richness estimation, and relative abundance calculations were performed using the Vegan R package to assess microbial community diversity and composition particularly focusing on the detection of genetic material from potential pathogens. 
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 /100 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.  
SECTION: Methods: Processing of Water samples - The section below summarises the procedures used for analytical control – detailed protocols are annexed to this protocol.
2.5. Observations: Samples were filtered within720 minafter collection the filtering membranes were immediately frozen and stored a-80 °Ctill shipping in dry ice to the respective partner for further analysis.