## 2 Materials and Methods
### Suspension Test  
Each _Clostridium_ spp. was used as a single strain to establish individual inactivation profiles for both _C. estertheticum_ and _C. gasigenes_. The suspension testing procedure was based on BS EN 13704:2018 \"Chemical disinfectants-Quantitative suspension test for evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas-Test method and requirements (phase 2, step 1)\" which describes the use of suspension tests when evaluating the efficacy of chemical agents against bacterial spores and recommends the use of one test organism per suspension with a well-defined sporulation behavior.  
Sporicidal solutions were prepared in 30 mL polystyrene tubes the day before conducting the experiments by adding double strength 10% \(v/v\) treatment 1, % \(v/v\) treatment 2, 4% \(w/v\) treatment 3 and 2% \(v/v\) treatment 4 to SDW to make up a final volume of 10 mL. The sporicidal products were then placed in a 25 \({}^{\circ}\)C incubator to equilibrate overnight. Depending on the final concentration of spores, approximately 10\({}^{5}\) CFU/mL spore suspensions were prepared by diluting in sterile distilled water (SDW). If the final spore concentration was lower than 10\({}^{5}\) CFU/mL, aliquots of spore suspensions were added together and centrifuged at 7500\(\times\)\(g\) for 10 min. The resultant pellet was resuspended in SDW and made up to 10 mL suspensions of approximately 10\({}^{5}\) CFU/mL. Spore suspensions were thoroughly vortexed before use to ensure homogeneity and a small volume (10 mL) was transferred to tubes containing each sporicidal treatment (10 mL) making a final volume of 20 mL. Based on the preliminary data for these treatments the following contact times (t) 0, 10, 20, 30, 40, 50, and 60 min were used to calculate D-values. At each sampling time, 10 mL Dey-Engley's neutralising broth (D3435; [PERSON], Arklow, Co. Wicklow, Ireland) was added to each tube for 15 min to stop further reaction between spores and the respective sporicidal agents. This was followed by centrifugation at 7500\(\times\)\(g\) 10 min. The supernatant was removed and the pellet was resuspended in 3 mL SDW.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Disinfectant/** & **Chemical Composition** & **Recommended** \\
**Treatment** & **Chemical Composition** & **Concentration** \\ \hline \hline
1 & Hydrogen Peroxide (10–30\%), Acetic acid (1–10\%), & \\  & Peracetic acid (1–10\%) & 10\% \\ \hline
2 & Sodium hypochlorite (5.2–10\%), Sodium hydroxide & \\  & (5–10\%), Alkylamine oxide (3–5\%) & 5\% \\ \hline
3 & Peroxymonosulphate (30–50\%), Sulphamic Acid (1–10\%), & 4\% \\  & Trolosene Sodium (1–10\%) & \\ \hline
4 & Alcohols, C9–11, Ethoxylate (10–30\%), Orthophosphoric & \\  & acid (10–30\%), Sulphuric acid (1–10\%), Iodine (1–10\%) & 2\% \\ \hline \hline \end{tabular}
\end{table}
Table 1: The sporicidal product formulations and concentrations used in this study.  
This procedure was then repeated in a bovine serum albumin (BSA) solution to mimic \"dirty conditions\" as recommended by the British Standards Institution (BSI) (EN 13704:2018). Exactly, 3 g/L of BSA was made up in SDW and sterilized by membrane filtration (0.22 \(\upmu\)m pore size) using vacuum (Millipore, Merck, Cork, Ireland). The contact times (t) used were also as for SDW.  
Surviving spore suspensions were serially diluted in maximum recovery diluent (MRD; Sigma Aldrich 07233) and plated out in duplicate on Columbia blood agar (CBA; Sigma Aldrich 27688) and incubated anaerobically at 4 \({}^{\circ}\)C for three weeks. Thereafter, presumptive surviving C. _estertheticum_ and _C. gasigenes_ were counted and confirmed by performing quantitative real time PCR (qPCR) as described by [36].