*2.3. Case Studies*

The proposed methodology was used to screen for plastic NPs in two real-life case studies. The first case study consisted of sampling wild *Mya arenaria* clam populations from a clean or reference site in the Saint-Lawrence estuary (Baie du Moulin à Baude, Québec, Canada) and polluted site (harbor) at Baie Sainte Catherine supporting regular boat tourism activities (Québec, Canada). The polluted site was located some 8 km south from the reference site in the Saint-Lawrence River estuary. A total of 30 clams between 4–8 cm longitudinal shell length were collected by hand and frozen at −20 ◦C for transportation to the laboratory. At the laboratory, the clams (*N* = 4) were thawed on ice and the soft tissues homogenized in a tissue grinder apparatus in ice-cold homogenization buffer as described above. The homogenates were analyzed by chromatography in each individual (homogenates were not pooled). The second case study consisted of experimentally caged *Elliptio complanata* mussels collected at a pristine lake (Lac des Écorces, Québec, Canada) and 5 km downstream of a municipal wastewater dispersion plume of a largely populated city (population of 1.8 million, City of Montréal, Québec, Canada). The mussels (mean size of 6 ± 0.5 cm, *N* = 10 per cage) were exposed for 3 months during the summer of 2017 (July to September) in cages consisting of 1 × 0.25 m cylindered nets fixed on cement blocks (5–10 kg) and immersed between 2–3 m depth. At the end of the exposure period, the mussels were processed in the same way as the clams, with the exception that the digestive glands of mussels were used instead of whole tissues and kept at −80 ◦C.

#### *2.4. Data Analysis*

Sample preparation and column optimisation experiments were repeated three times and the mean value with standard deviation was used as the descriptive statistics. The chromatographic elution profiles in absorbance, conductivity, and NR fluorescence data were normalized at 1 to facilitate data visualization and reporting: (sample reading—blank (elution buffer))/(maximal reading—blank). A number of four individuals were collected for *Mya arenaria* clams and *Elliptio complanata* caging experiments for the chromatography analyses with either direct injection or after NaCl/ACN fractionation. Graphical and statistical analysis were performed using the SYSTAT software package (version 13).
