Hypochlorous acid

The antioxidant barrier and the concentration of Reactive Oxygen Metabolites (ROMs) were measured using the OXY-Adsorbent (2 μL of plasma) and d-ROMs tests (5 μL of plasma, DIACRON INTERNATIONAL, s.r.l, Italy) following the manufacturer’s protocol (for detailed description of these tests, see [39 (link)]). The OXY adsorbent test was used to quantify the ability of the plasma antioxidant barrier to buffer massive oxidation through hypochlorous acid, while the d-ROMs test mostly measures hydroperoxydes as a marker of global early oxidative damage (principally on lipids and proteins). Antioxidant barrier is expressed as mM of HClO neutralised and d-ROMs as mg of H₂O₂ equivalent/dL. All measurements were run in duplicates and intra-individual variation was low (respectively 1.96 ± 0.34% for the OXY test and 2.76 ± 0.86% for the d-ROMs test). Measurements for the same individual before and after reproduction were run within the same laboratory session, and measurements of all the samples were divided in three laboratory sessions. Inter-session variations in the measurement (based on one sample repeated in all the session) were 4.52% for the OXY test and 5.31% for the d-ROMs test. Repeatability, i.e. the proportion of variability explained by the individual, was calculated following [40 (link)]. Both d-ROMs (ANOVA, F_{1, 35} = 26.41, p <0.001, r = 0.585) and Oxy-Adsorbent (ANOVA, F_{1, 35} = 4.90, p <0.034, r = 0.178) tests were shown to be repeatable over the study.

Hypochlorous acid (HOCl) was prepared immediately before the experiment by adjusting the pH of a 10% (v/v) solution of NaOCl to 6.2 with 0.6 M H₂SO₄, and the concentration of HOCl was determined by measuring the absorbance at 235 nm using the molar extinction coefficient of 100 M^-1 cm^-1. The assay was carried out as described by Aruoma and Halliwell [19 (link)] with minor changes. The scavenging activity was evaluated by measuring the decrease in absorbance of catalase at 404 nm. The reaction mixture contained, in a final volume of 1 ml, 50 mM phosphate buffer (pH 6.8), catalase (7.2 μM), HOCl (8.4 mM) and increasing concentrations (0–100 μg/ml) of plant extract. The assay mixture was incubated at 25°C for 20 min and the absorbance was measured against an appropriate blank. All tests were performed six times. Ascorbic acid, a potent HOCl scavenger, was used as a reference [20 (link)].

We performed initial screening of a range of methods that have been suggested in the popular media for home seed disinfestation. The goal was to identify candidate disinfestation methods that significantly reduced fungal contamination of seeds prior to sprouting and that did not adversely affect germination rates. We performed these initial tests on onion seeds because our initial assessments suggested they had consistently high surface contamination with both fungi and bacteria. Seeds were aseptically removed from the source bag, and 0.25 g (58 ± 6 seeds) were placed into sterile 2-mL Eppendorf^TM (Hamburg, Germany) microcentrifuge tubes for each of three replicates of 25 different disinfection treatments. Chemical treatments included (1) sterile deionized water (2 min), (2) sodium hypochlorite (0.6% NaOCl; 10% household Clorox bleach, 2 min), (3) hypochlorous acid (HClO; freshly prepared Force of Nature^TM Multi-purpose cleaner (Westford, MA, USA), ~800 ppm chlorine, 2 min), (4) hydrogen peroxide (3%, 2 min), (5) ethanol (70%, 2 min), and (6) glacial acetic acid (5%; similar to vinegar). Each of these six chemical treatments was performed both as a simple soak with agitation and with a 2-min suspension in a water-bath sonicator. Heat treatments were conducted as (1) boiling in sterile deionized water (1 min), (2) held at 55 °C (5, 10, and 30 min), and (3) held at 70 °C (5, 10, and 30 min). Seeds were treated (50 seeds per treatment) and then placed aseptically in 1.5% water agar in a Petri dish. We recorded the percentage of seeds from which fungi were growing after 3 days and the percentage of seeds that had germination after 7 days. The seed of origin of fungal growth could not be reliably determined after 3 days, and percent germination did not meaningfully increase after 7 days.
We compared the mean outcomes of each treatment to that of the control treatment (sterile deionized water) using a Dunnett test (DescTools package in R). A Wilcoxon paired rank test (wilcox.text, stats package in R) was used to test whether sonication significantly affected the effectiveness of the disinfectant treatments (boiling was excluded from this analysis because there was no sonication for that treatment).

We brought together insights from the initial screening and factorial experiments for a final test of the effects of six disinfection treatments on the bacterial and fungal loads on seedlings after 24 h and on seed germination success. Disinfection treatments were bleach (10% commercial Clorox^TM, 0.6% sodium hypochlorite), hypochlorous acid (HCA; freshly produced Force of Nature^TM Multi-purpose cleaner, ~800 ppm chlorine), vinegar (diluted Heinz^TM (Sharpsburg, PA, USA) white vinegar, 3% acetic acid), H₂O₂ (3%), 55 °C water; Sonication in 23 °C (room temperature) water, and a control treatment of sterile deionized water (23 °C). Note that H₂O₂ was used at 3% rather than the higher concentrations tested earlier because concentrations of food-grade H₂O₂ at higher concentrations are widely restricted for consumer purchase. Each treatment was replicated 5 times. The entire experiment was conducted twice with treatments lasting 5 min and twice for treatments lasting 15 min.
To conduct each experiment, 200 broccoli seeds were placed in sterile test tubes with a slip-on cap (35 tubes total). The disinfecting agent was poured into the tubes to cover the seeds. At the conclusion of the treatment time, the agent was decanted from the tubes, and seeds were rinsed 3 times with sterile deionized water. Sterile deionized water was then added to each tube to cover seeds and allowed to soak for 4 h before draining. At 24 h from the initial treatment, 9 mL of sterile deionized water was added to each tube. Tubes were vortexed for 10 s, then two 100-fold dilutions of the solution were prepared. This created three 100-fold dilutions: 10⁻², 10⁻⁴, and 10⁻⁶; aliquots (100 µL) of each of the three dilutions were individually transferred to 100-mm Petri dishes containing 0.1TSA and spread evenly with a sterile bent glass rod. The same was done for the 10⁻² on MEA medium for quantification of fungi. Seeds from each tube were then placed into a Petri dish lined with sterile filter paper; percent germination was recorded 5 days after initial treatments. Bacterial and fungal densities were expressed as log₁₀(CFU per 200 seeds + 10); the 10 was added to allow for the inclusion of replicates that recovered zero bacteria at the 10⁻² dilution and was chosen as 10-fold below the expected minimum level of detection). Percent seed germination was measured after 5 days.
Differences among treatment means were tested using analysis of variance, treating each experimental repetition as a block (function aov in R). Soaking times of 5 min and 15 min were analyzed separately. Least squares means and 95% confidence intervals for the mean of each treatment were calculated using emmeans function (emmeans package in R). Post-hoc comparison of means was performed using Tukey’s HSD multiple comparisons test (TukeyHSD in R) with 95% confidence.