## 2 Results
### Monitoring VOC Emissions during Rotifer-Grazing and Freeze-Thaw Experiments
#### 2.2.3. Comparison of Algae Crop Damage Mechanisms  
\(\beta\)-ionone was monitored in both A + R and FTA experiments using extracted ion chromatograms for its base peak, _m/z_ 177 in Figure 2. The rotifier-grazing VOC profiles (Figure 2A) display maximum \(\beta\)-ionone signals of \(\sim\)7 \(\times\) 10\({}^{5}\) at the first timepoint measured after rotifier addition (48 h post-inoculation). The signal decreased to an average of 58% and 27% of the original signal after 24 and 48 h, respectively, with a final peak area averaging \(\sim\)1 \(\times\) 10\({}^{5}\). A time-matched monitoring of healthy algae cultures (Figure 2A) from the rotifier grazing experiments showed no detectable signal of _m/z_ 177, and presumably \(\beta\)-ionone for all timepoints sampled. For FTA, represented in Figure 2B, an average signal of \(\sim\)3 \(\times\) 10\({}^{6}\) was observed at the first timepoint \(\sim\)1 h post-inoculation. The signal rapidly decreased over the subsequent timepoints, averaging 54% and 3% of the original signal at 6- and 24-h post-inoculation, before becoming undetectable at the final timepoint of 48 h post-inoculation. As with the A + R experiments, time-matched health algae controls (A  
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\begin{tabular}{c conly) for FTA experiments also showed no detectable signal of _m/z_ 177, and presumably \(\beta\)-ionone, for all timepoints monitored. Differences in algal crop damage methods were evidenced by unique VOC profiles, summarized in Figure 3. In the A + R cultures, the presence of \(\beta\)-cyclocitral, \(\beta\)-ionone, and 1-heptadecene were detected at three timepoints only in samples that had rotifers present. The relative abundance of these compounds either remained consistent (\(\beta\)-cyclocitral) or gradually decreased with continued rotifer grazing (\(\beta\)-ionone and 1-heptadecene). All three putative biomarkers were still detected at the final A + R timepoint. Conversely, \(\beta\)-ionone and palmitoleic acid were only detected within the first 6 h after the freeze-thawed algal cells were added to the media.  
Figure 2: Example extracted ion chromatograms for _m/z_ 177 for putative biomarker, \(\beta\)-ionone, observed in both rotifer-grazed _M. gaditana_ (A + R, red) and time-matched healthy algae controls (A only, black) in part (**A**) and freeze–thawed _M. gaditana_ (FTA, blue) with time-matched healthy algae controls (A only, black) in part (**B**) throughout the duration of condition-specific timepoints measured during each experiment.  
Figure 3: Peak areas of extracted ion chromatograms for \(\beta\)-cyclodextrin, \(\beta\)-ionone, and 1-heptadecane in each A + R replicate experiment (top nine graphs) and for \(\beta\)-ionone and palmitoleic acid in each FTA replicate experiment (bottom six graphs). Error bars in A + R experiments represent standard deviation of technical replicates for each sample. Semi-quantitative VOC analysis for the same putative biomarker was set relative to the highest intensity across all three replicate experiments. For comparison across A + R and FTA experiments, relative abundance scale for \(\beta\)-ionone was set relative to the observed highest peak area in FTA Experiment 3.