*3.1. PAHs Analysis*

Samples were analyzed to verify NAP, PHE, FLT, and BkF nominal concentrations ranging from 0.025 to 10 mg/L, 0.36 to 2.3 × 102 mg/L, 0.41 to 3.9 × 102 mg/L, and 0.025 to 94 mg/L, respectively (shown in Table 1). The gas chromatography-mass spectrometry (GC-MS) (2010plus-TQ8030, Shimadzu, Japan) determinations showed a good agreement between nominal vs. analytical concentrations, whose ratios were less than 1.5 in most cases (Table 1).

#### *3.2. Naphthalene, Phenanthrene, Fluoranthene, and Benzo(k)fluoranthene Toxicity on Nauplii*

After 24 h of exposure to NAP, a statistically significant increase in toxicity (*p* < 0.0001; see Supplementary Table S2) was observed only at the two highest tested concentrations of 4.23 mg/L (38 % mortality) and 10.1 mg/L (43% mortality) (Figure 1A).

After 48 h, mortality became statistically significant (*p* < 0.01) already at 0.11 and 0.26 mg/L and reached 80% (*p* < 0.0001) at 4.23 and 10.1 mg/L (Figure 1A and Supplementary Table S2).

After 24 h of exposure (Figure 1B) PHE induced an increase of the percentage of dead (about 26%) with respect to control already from 3.45, 4.23, and 7.48 mg/L. The data reported at these concentrations were statistically significant as compared to the two lowest (0 and 0.21 mg/L; *p* < 0.01) and highest (98.6 and 223.4 mg/L; *p* < 0.01; see Supplementary Table S2) concentrations. At 48.7, 98.6, and 223.4 mg/L, significant increase of toxicity (about 43.3%, 50%, and 50%, respectively) respect lower tested concentrations (0, 0.21, 0.71, 1.15, and 2.26 mg/L; *p* < 0.0001; see also Supplementary Table S2) has been shown. The scenario after 48 h of exposure was similar to the one described after 24 h. About 26% of dead nauplii was registered at 1.15 mg/L (*p* < 0.05; Supplementary Table S2). At 2.26 and 3.45 mg/L, the toxicity (about 34%) was statistically significant compared to 0, 0.21, 7.48, 48.7, 98.6, and 223.4 mg/L (*p* < 0.0001; Supplementary Table S2). At 7.48, 48.7, and 98.6 mg/L, about 70% of mortality was registered, whereas a percentage of about 90% of dead was displayed at 223.4 mg/L.


**Table 1.** Comparisons of nominal vs. analytical concentrations of naphthalene (NAP), phenanthrene (PHE), fluoranthene (FLT), and benzo(k)fluoranthene (BkF) in seawater. The data were reported in mg/L.

**Figure 1.** After 24 h and 48 h, the percentage of dead nauplii in control (0 mg/L) and treated samples with (**A**) NAP at the concentrations of 0.025, 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2.5, 5, and 10 mg/L; (**B**) PHE at the concentrations of 0.36, 1, 2, 3, 4, 5, 10, 57.5, 115, and 230 mg/L; (**C**) FLT at the concentrations of 0.41, 1, 2.5, 5, 12.5, 25, 50, 97.5, 195, and 390 mg/L; and (**D**) BkF at the concentrations of 0.025, 0.5, 1, 1.5, 3, 6, 12, 23.5, 47, and 94 mg/L was regarded. Data are reported as mean ± standard deviation two-way ANOVA by Tukey's test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001).

Taking into the consideration FLT exposure (Figure 1C), an increase of the percentage of dead (about 18%) with respect to control was already recorded from 9.91, 20.4, 45.6, and 91.6 mg/L. The data reported at these concentrations were statistically significant compared to the four lowest (0, 0.29, 0.81, and 2.14 mg/L; *p* < 0.01) and the two highest (179 and 325 mg/L; *p* < 0.0001; see Supplementary Table S2) concentrations. At 179 and 325 mg/L, significant increase of toxicity (about 35% and 70%, respectively) with respect to lower tested concentrations (4.41, 9.91, 20.4, and 45.6 mg/L; *p* < 0.0001; Supplementary Table S2) has been shown. After 48 h, at 0.81 mg/L, a significant increase of toxicity (about 40%) has been displayed with respect to 0 (*p* < 0.0001) and 0.29 mg/L (*p* < 0.001; see also Supplementary Table S2). However, the toxicity increases of about 80% (2.14 and 4.41 mg/L). These data were statistically significant respect to compared to the three lowest (0, 0.29, and 0.81 mg/L; *p* < 0.0001) and the highest (20.4, 45.6, 91.6, 179, and 325 mg/L; *p* < 0.001; Supplementary Table S2).

When we considered BkF toxicity (Figure 1D) after 24 h, no effect has been recorded. Only after 48 h, at 2.4, 5.3, 10.4, 19.5, 41.7, and 84.6 mg/L, a significant increase in toxicity (about 10%) was shown with respect to the control (*p* < 0.01; Supplementary Table S2).

## *3.3. Naphthalene, Phenanthrene, Fluoranthene, and Benzo(k)fluoranthene Toxicity on Adults*

After 24 h of exposure, NAP and PHE and BkF did not affect the survival of *A. franciscana* at all tested concentrations (Figure 2).

**Figure 2.** After 24 h and 48 h, the percentage of dead adults in control (0 mg/L) and treated samples with (**A**) NAP at the concentrations of 0.025, 0.05, 0.1, 0.2, 0.4, 0.5, 1, 2.5, 5, and 10 mg/L; (**B**) PHE at the concentrations of 0.36, 1, 2, 3, 4, 5, 10, 57.5, 115, and 230 mg/L; (**C**) FLT at the concentrations of 0.41, 1, 2.5, 5, 12.5, 25, 50, 97.5, 195, and 390 mg/L; and (**D**) BkF at the concentrations of 0.025, 0.5, 1, 1.5, 3, 6, 12, 23.5, 47, and 94 mg/L was regarded. Data are reported as mean ± standard deviation two-way ANOVA by Tukey's test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001).

However, only FLT showed toxic effects already after 24 h of exposure (Figure 2C). An increase of the percentage of dead (about 60%) with respect to control was already recorded from 9.91, 20.4, 45.6, and 91.6 mg/L. The data reported at these concentrations were statistically significant compared to the four lowest (0, 0.29, 0.81, and 2.14 mg/L; *p* < 0.0001; Supplementary Table S3) and the two highest (179 and 325 mg/L; *p* < 0.0001; Supplementary Table S3) concentrations. At 179 and 325 mg/L, significant increase of toxicity (about 95% and 100%, respectively) with respect to all other concentrations (*p* < 0.0001; Supplementary Table S3) was shown. After 48 h, at 0.29 and 0.81 mg/L, a significant increase of toxicity (about 45%) was displayed with respect to 0 (*p* < 0.0001). However, toxicity increases of about 66% (2.14 and 4.41 mg/L) were shown. These data were statistically significant compared to the three lowest (0, 0.29 and 0.81 mg/L; *p* < 0.0001) and the highest (20.4, 45.6, 91.6, 179, and 325 mg/L; *p* < 0.05; Supplementary Table S3), where a percentage of about 100% was registered.

After 48 h, NAP caused a statistically significant mortality starting from 1.45 mg/L (30%), with a maximum effect of 60% at 10.1 mg/L (Figure 2A).

As shown in Figure 2B, PHE induced an increase of the percentage of dead (about 26.6%) with respect to control already from 0.71 mg/L. The data reported at this concentration were statistically significant compared to the two lowest (0 and 0.21 mg/L; *p* < 0.001) and other concentrations (*p* < 0.0001; Supplementary Table S3). At 1.15, 2.26, 3.45, 4.23, and 7.48 mg/L, significant increase of toxicity (about 30%, 30%, 30%, 33%, and 45%, respectively) with respect to lower (0, 0.21, and 0.71 mg/L; *p* < 0.0001) and higher (48.7, 98.6, and 223.4 mg/L; *p* < 0.0001; see also Supplementary Table S3) tested concentrations was shown. At 48.7 and 98.6 mg/L, the toxicity (about 66%) was statistically significant compared to

other used concentrations (*p* < 0.0001). At 223.4 mg/L, 100% of mortality was registered (*p* < 0.0001; Supplementary Table S3).

When we considered BkF toxicity (Figure 2D) after 24 h, no effect was recorded. Only after 48 h, at 2.4, 5.3, 10.4, and 19.5 mg/L, a significant increase in toxicity (about 25%) was shown with respect to the three lowest concentrations (0, 0.016, 0.41 mg/L; *p* < 0.01) and the highest concentration (84.6 mg/L; *p* < 0.01; Supplementary Table S3), where a percentage of about 50% was registered.

#### *3.4. Lethal Concentrations after 24 and 48 h of Exposure*

Considering nauplii exposure, the NAP solution has a LC50 value of 1.73 mg/L (1.52–46.28 mg/L) and 0.60 mg/L (0.21–90.38 mg/L) after 24 h and 48 h, respectively; PHE solution has a LC50 value of 4.44 (3.66–56.76 mg/L) and 3.07 mg/L (1.32–81.01 mg/L) after 24 h and 48 h, respectively; FLT solution has a LC50 value of 1.30 (0.45–107.5 mg/L) and 0.09 mg/L (0.01–99.1 mg/L) after 24 h and 48 h, respectively (Supplementary Table S4). When considering adults, the NAP solution has a LC50 value of 0.11 mg/L (0.02–12.06 mg/L) and 44.31 mg/L (5.81–268.12 mg/L) after 24 h and 48 h, respectively; PHE solution has a LC50 value of 1.68 mg/L (1.35–234.09 mg/L) after 48 h; FLT solution has a LC50 value of 32.03 mg/L (0.10–120.08 mg/L) and 0.77 mg/L (0.10–103.67 mg/L) after 24 h and 48 h, respectively; and BkF solution has a LC50 value of 28.67 mg/L (0.5–36.67 mg/L) and 6.12 mg/L (0.05–48.72 mg/L) after 24 h and 48 h, respectively (see also Supplementary Table S4).
