## 2 Materials and Methods
### Mussel Aclimation and Selected Experimental Treatments  
Sexually mature mussels (_M. galloprovincialis_) were harvested from fishermen of the COPRALMO cooperative in Cesenatico (NW Adriatic Sea, Italy), and acclimatised in laboratory for ten days in artificial seawater (ASW, prepared according to ASTM, [39]) at controlled physico-chemical conditions (16 \({}^{\circ}\)C and at a density of 1 mussel L ASW\({}^{-1}\)). Subsequently, exposures were performed involving early (gametes and embryos) and adult mussel stages to screen the effects of CHR-loaded particles on different phases of the _M. galloprovincialis_ life cycle. Irrespective to the mussel stage, organisms were exposed to virgin MP (MP) and to the same concentration of CHR-loaded MP (hereafter defined as CHR-MP). Notably, these were 25 \(\times\) 10\({}^{3}\) MP/mL FSW for early stages and 5 \(\times\) 10\({}^{3}\) MP/L FSW for adult mussel exposure. These concentrations were selected based on the available knowledge on MP treatments able to induce a ready ingestion by larvae and/or sub-lethal effects in adult marine invertebrates, including bivalves [19; 40]. The stock of CHR-sorbed MP to be used in bioassays or adult mussel exposures was freshly renewed before each administration by co-incubating MP and CHR in Pyrex beakers for 24 h as described previously. At each administration, MP concentrations were checked microscopically using a Burker chamber (HBG, Giessen-Lutzellinden, Germany); based on these measurements, the real tested concentrations were 23.67 \(\pm\) 2.46 \(\times\) 10\({}^{3}\) MP/mL (corresponding to a mass/volume concentration of 0.35 mg MP/L) for early stages tests and 4.57 \(\pm\) 0.30 \(\times\) 10\({}^{3}\) MP/L (i.e., 67.7 mg MP/L) for adult in vivo exposure, indicating consistency with selected nominal concentrations. Further treatments included 0.1 mg/L CHR, chosen as a positive control based on literature information on CHR toxicity to aquatic species (i.e., scallops [41; 42] and shrimps [43]), and a second CHR treatment representing the actual concentration vehiculated by CHR-loaded MP in the CHR-MP condition. The latter was included to comparatively assess the effects of CHR-loaded MP with respect to an identical dosage of freely dissolved CHR and was given by the product of the amount of CHR sorbed per milligram of MP (10.8 \(\upmu\)g) and the amount (in mg) of MP/L ASW\({}^{-1}\) used during early life stages and adult mussel tests (0.35 and 0.0000677, respectively). Notably, these treatments were 3.78 \(\upmu\)g/L CHR for early life stages tests and 0.73 ng/L for adult mussel in vivo exposure. In all performed tests, a control condition represented by ASW-only exposure was performed in parallel to treatments. All experiments were conducted at 16 \(\pm\) 1 \({}^{\circ}\)C, as reported previously [44]. Replicate numbers (\(n\)) adopted in each test are reported in the sections below.  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Sample** & **Incubation Time** & **[CHR]** \\  & **(Hours)** & **mg/L \(\pm\) SD** \\ \hline
0.5 mg/L CHR & & 0.64 \(\pm\) 0.01 \\
0.5 mg/L CHR + MP & 0 & 0.63 \(\pm\) 0.03 \\ \hline
0.5 mg/L CHR & & 0.63 \(\pm\) 0.01 \\
0.5 mg/L CHR + MP & 3 & 0.53 \(\pm\) 0.02 \\ \hline
0.5 mg/L CHR & & 0.64 \(\pm\) 0.03 \\
0.5 mg/L CHR + MP & 6 & 0.52 \(\pm\) 0.08 \\ \hline
0.5 mg/L CHR & & 0.74 \(\pm\) 0.08 \\
0.5 mg/L CHR + MP & 12 & 0.35 \(\pm\) 0.05 \\ \hline
0.5 mg/L CHR & & 0.45 \(\pm\) 0.02 \\
0.5 mg/L CHR + MP & 24 & 0.15 \(\pm\) 0.03 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Chrysene (CHR) concentrations measured using GC-MS in filtered water after incubation of 0.5 mg of 3-\(\upmu\)m MP in 0.5 mg/L CHR solution and in a CHR ‘control’ solution having the same CHR nominal concentration and incubated for the same time intervals in the absence of MP. Data are expressed as mean \(\pm\) SD (\(n\) = 3).