## 5 Conclusions  
Glyphosate-based pesticides, particularly at high concentrations, have a clear effect on several metabolic pathways in marine diatoms (changes in pigment profile, photosynthetic impairment, and decreased antioxidant capacity). Even though photoprotective mechanisms were induced, membrane damage still occurred. These facts highlight the application of pulse amplitude modulated chlorophyll fluorescence bio-optical methods as a promising tool for inclusion in ecotoxicological studies with several advantages relative to the classical biochemical approaches, namely fast data acquisition, lower monetary costs, and a high number of variables obtained while ensuring a high degree of accuracy. Thus, bio-optical tools appear as a fast, inexpensive, and reliable method for toxicophemomic assessment of the impacts of this widespread pesticide in marine diatoms, with a clear dose-response pattern. Moreover, the high volume of data produced by these bio-optical techniques can be efficiently applied in multivariate analysis, providing a classification with a high degree of accuracy. With this in mind, PAM fluorometry appears to be a promising tool for inclusion in ecotoxicological studies aiming towards a possible use in long-range detection through satellite imagery in a global chemical pollution-monitoring network.  
**Supplementary Materials**: The following are available online at [[http://www.mdpi.com/2076-3417/10/21/7391/s1](http://www.mdpi.com/2076-3417/10/21/7391/s1)]([http://www.mdpi.com/2076-3417/10/21/7391/s1](http://www.mdpi.com/2076-3417/10/21/7391/s1)), Figure S1: Photosystem II and ETC related photochemical traits, Figure S2: Rapid light curves and derived parameters, Figure S3: Major fatty acids classes, Figure S4: Fatty acid ratios, Figure S5: Omega 6 to omega 3 fatty acid ratio, Table S1: Fluorometric analysis parameters and their description.  
**Author Contributions**: Conceptualization, B.D., V.F.F. and P.R.-S.; methodology, B.D.; formal analysis, R.C.d.C. and B.D.; investigation, R.C.d.C., E.F. and S.C.N.; data curation, R.C.d.C.; writing--original draft preparation, R.C.d.C.; writing--review and editing, E.F., A.R.M., M.T.C., S.C.N., M.F.L.L., I.C., J.C.M., P.R.-S., V.F.F., B.D.; supervision, B.D.; project administration, B.D.; funding acquisition, B.D. All authors have read and agreed to the published version of the manuscript.  
**Funding**: This research was funded by Fundacao para a Ciencia e a Tecnologia (FCT) via project grants PTDC/CTA-AMB/30056/2017 (OPTOX), UID/MAR/04292/2019, UID/MULTI/04046/2019. Work was also funded by the Integrated Programme of SR&TD SmartBioR (reference Centro-01-0145-FEDER-000018), co-funded by Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund. [PERSON] and [PERSON] were supported by investigation contracts (CEECIND/00511/2017 and DL57/2016/CP1479/CT0024). [PERSON] was supported by FCT through a postdoctoral grant (SFRH/BPD/95784/2013).  
**Acknowledgments**: The authors would like to thank [PERSON] and [PERSON] for revising the English language, grammar, punctuation, and spelling of the manuscript.  
**Conflicts of Interest**: The authors declare no conflict of interest.