3.1 Effect of nZVI and AC dosage on PAHs removal
As reported in Supplementary Figure S3, results demonstrated that the nZVI addition induced high PAHs degradation both in sediment and seawater. When considered W + PAHs + nZVI condition, already after 21 hours (T3) of treatment, the degradation of pollutants was total (Supplementary Figure S3). Since the loss of total PAHs by evaporation at T3 was only 20%, this result can be considered completely due to the treatment with nZVI. Considering the aqueous solution of SED1 and SED2 (W + SED1 + nZVI and W + SED2 + nZVI, respectively), no PAHs have been detected at all studied time (from T0 to T6) with only T3 and T4 exception of W + SED1 + nZVI (see also Supplementary Figure S3), where little concentrations of 9 and 8 µg/L have been shown, respectively.
In the case of sediment of Sarno (SED1), the removal of hydrocarbons was total already after 3 hours of treatment (T1); whereas taking in the consideration the sediment of Bagnoli (SED2), at the end of experiment (after 21 days, T6) a residue of about 26 µg/Kg is still measured in the sediment (Supplementary Figure S3). In assessing the removal of individual compounds from SED2 (Supplementary Figure S4), the results showed that this amendment was not able to completely remove the fluoranthene, pyrene, benzo(b)fluoranthene, benzo(a)anthracene, Indeno(1,2,3-cd)pyrene and benzo(a)pyrene.
The addition of 3% AC reduced the aqueous concentration of PAHs, with pollutants concentrations below detection limits already after 21 h (T3) of amendment (Supplementary Figure S5). Also in this case, since the loss of total PAHs by evaporation at T3 was only 20%, this result can be considered completely due to the treatment with AC. Moreover, considering the aqueous solution of SED1 and SED2 (W + SED1 + AC and W + SED2 + AC, respectively), no PAHs were detected at all studied time (from T0 to T6; see also Supplementary Figure S5). The results obtained from sediment treatments with AC were almost similar to those obtained for nZVI. In fact, total removal of PAHs was found for SED1; whereas a residue of about 25.7 µg/Kg was still measured in the sediment of Bagnoli (SED2) from T1 to the end of experiment (after 21 days, T6) (Supplementary Figure S5). In assessing the removal of individual compounds from SED2 (Supplementary Figure S6), the results showed that this amendment was not able to completely remove the fluoranthene, benzo(a)anthracene, dibenzo(a,h)anthracene, Indeno(1,2,3-cd)pyrene and benzo(a)pyrene. The removal efficiencies of two remediation methods demonstrated that they were much more efficient for PAHs removal from aqueous solutions than sediments (Table 1). The degradation efficiencies were 98.3% and 99.6% for total PAHs removal from aqueous solutions (W + PAHs + nZVI or AC; see Table 1), and 98.9% from Sarno sediment (SED1) plus amendments (SED1 + nZVI or AC). The percentage of total PAHs removal from Bagnoli sediment was 60.6% and 49.3% for AC and nZVI methods, respectively (Table 1).
Table 1
Removal efficiencies achieved with different remediation methods. n.a. = not available.
| AC | | nZVI |
| W + PAHs + AC (µg/L) | | W + PAHs + nZVI (µg/L) |
| T0 | T6 | Removal efficiency (%) | | T0 | T6 | Removal efficiency (%) |
Acenaphthylene | 0 | 0 | n.a | | 0 | 0 | n.a |
Acenaphthene | 0.1 | 0.1 | 0 | | 157.7 | 0.25 | 99.8 |
Fluorene | 10.6 | 0.1 | 99.1 | | 176 | 0.25 | 99.9 |
Anthracene | 6.35 | 0.1 | 98.4 | | 13 | 0.25 | 98.1 |
Phenanthrene | 0 | 0 | n.a | | 0 | 0 | n.a |
Fluoranthene | 6.89 | 0.1 | 98.5 | | 88 | 0.25 | 99.7 |
Pyrene | 7.63 | 0.1 | 98.7 | | 32 | 0.25 | 99.2 |
Benzo(a)Antracene | 0 | 0 | n.a | | 0 | 0 | n.a |
Chrysene | 5.24 | 0.1 | 98.1 | | 2.3 | 0.25 | 89.1 |
Benzo(b)Fluorantene | 0 | 0 | n.a | | 0 | 0 | n.a |
Benzo[k]fluoranthene | 3.68 | 0.1 | 97.3 | | 1 | 0.25 | 75 |
Benzo[a]pyrene | 0 | 0 | n.a | | 0 | 0 | n.a |
Indeno[1,2,3-cd]pyrene | 0 | 0 | n.a | | 0 | 0 | n.a |
Dibenz[a,h]anthracene | 0 | 0 | n.a | | 0 | 0 | n.a |
Benzo[ghi]perylene | 0 | 0 | n.a | | 0 | 0 | n.a |
Total PAHs | 2.70 | 0.05 | 98.3 | | 31.3 | 0.1 | 99.6 |
| SED1 + AC (µg/Kg) | | SED1 + nZVI (µg/Kg) |
Acenaphthylene | 4.18 | 0.5 | 88.0 | | 4.18 | 0.5 | 88.0 |
Acenaphthene | 0 | 0 | n.a | | 0 | 0 | n.a |
Fluorene | 0 | 0 | n.a | | 0 | 0 | n.a |
Anthracene | 11.75 | 0.5 | 95.7 | | 11.75 | 0.5 | 95.7 |
Phenanthrene | 0 | 0 | n.a | | 0 | 0 | n.a |
Fluoranthene | 74.5 | 0.5 | 99.3 | | 74.5 | 0.5 | 99.3 |
Pyrene | 63.15 | 0.5 | 99.2 | | 63.15 | 0.5 | 99.2 |
Benzo(a)Antracene | 56.97 | 0.5 | 99.1 | | 56.97 | 0.5 | 99.1 |
Chrysene | 59.96 | 0.5 | 99.2 | | 59.96 | 0.5 | 99.2 |
Benzo(b)Fluorantene | 85.26 | 0.5 | 99.4 | | 85.26 | 0.5 | 99.4 |
Benzo[k]fluoranthene | 35.46 | 0.5 | 98.6 | | 35.46 | 0.5 | 98.6 |
Benzo[a]pyrene | 67.13 | 0.5 | 99.3 | | 67.13 | 0.5 | 99.3 |
Indeno[1,2,3-cd]pyrene | 51.59 | 0.5 | 99.0 | | 51.59 | 0.5 | 99.0 |
Dibenz[a,h]anthracene | 12.55 | 0.5 | 96.0 | | 12.55 | 0.5 | 96.0 |
Benzo[ghi]perylene | 44.82 | 0.5 | 98.9 | | 44.82 | 0.5 | 98.9 |
Total PAHs | 37.8 | 0.4 | 98.9 | | 37.8 | 0.4 | 98.9 |
| SED2 + AC (µg/Kg) | | SED2 + nZVI (µg/Kg) |
Acenaphthylene | 6.5 | 2 | 69.2 | | 15.6 | 8.39 | 46.2 |
Acenaphthene | 2 | 2 | 0.0 | | 2 | 0.47 | 76.5 |
Fluorene | 2 | 2 | 0.0 | | 2 | 0.96 | 52.0 |
Anthracene | 85.2 | 11.2 | 86.9 | | 19.7 | 15.04 | 23.7 |
Phenanthrene | 35.4 | 10.4 | 70.6 | | 11.4 | 4.87 | 57.3 |
Fluoranthene | 125 | 54.6 | 56.3 | | 130.8 | 77.61 | 40.7 |
Pyrene | 146 | 35.6 | 75.6 | | 101.2 | 60.75 | 40.0 |
Benzo(a)Antracene | 85.6 | 54.6 | 36.2 | | 57.8 | 30.62 | 47.0 |
Chrysene | 64.7 | 10.3 | 84.1 | | 47.7 | 26.87 | 43.7 |
Benzo(b)Fluorantene | 32.4 | 5.6 | 82.7 | | 112.6 | 45.45 | 59.6 |
Benzo[k]fluoranthene | 78.9 | 26.8 | 66.0 | | 42.4 | 21.92 | 48.3 |
Benzo[a]pyrene | 65.4 | 35.6 | 45.6 | | 81.1 | 35.73 | 55.9 |
Indeno[1,2,3-cd]pyrene | 74.5 | 45.3 | 39.2 | | 70.4 | 30.65 | 56.5 |
Dibenz[a,h]anthracene | 104.3 | 85.6 | 17.9 | | 13.4 | 4.53 | 66.2 |
Benzo[ghi]perylene | 68.9 | 3.6 | 94.8 | | 60.7 | 25.93 | 57.3 |
Total PAHs | 65.1 | 25.7 | 60.6 | | 51.3 | 26.0 | 49.3 |
3.2 Toxicity effect of nZVI and AC on nauplii
As reported in Fig. 2, after 48 h of exposure to different percentage of aqueous solutions of all experimental conditions of nZVI, an increase of toxicity was observed at higher tested percentage, represented by 50% and 100%.
Considering W + nZVI condition at T0 (Fig. 2A), a little percentage of dead nauplii (about 6.6%) has been shown already at the lowest percentage (6.25%). These data were statistically significant respect to the control (p < 0.05) and others used concentrations (p < 0.0001; see also Supplementary Table S2). At 100%, significant increase of toxicity (about 30%) respect lower (0% and 12.5%; p < 0.0001 and p < 0.001, respectively) and higher (25% and 50%; p < 0.001) tested concentrations has been shown. Taking into consideration T1, at 25%, 50% and 100% a significant decrease of survival (about 13%, 20% and 23%, respectively) respect lower concentrations (0%, 6.25% and 12.5%; p < 0.0001 (Supplementary Table S2) was detected. Moreover, the data reported at 25% were statistically significant respect to 50% (p < 0.05) and 100% (p < 0.001). Considering T2, T3 and T4 only at 50% and 100% a significant decrease of survival (about 10% and 20%, respectively) respect all lower concentrations (p < 0.0001) and among them (p < 0.001; Supplementary Table S2).
As reported Fig. 2B, W + PAHs condition at T0 and T1 caused a little survival decrease (about 10%) already at 6.25%, which was statistically significant respect to control (p < 0.0001) and others concentrations ((p < 0.0001; Supplementary Table S2). At 25%, a significant decrease of about 50% respect lower concentrations (p < 0.0001 (Supplementary Table S2) and higher concentrations (50% and 100%; p < 0.0001) was detected. From T2 to T6, the results were similar (Fig. 2B). In fact, at 25%, a significant decrease of about 30% respect lower concentrations (p < 0.0001 (Supplementary Table S2) and higher concentrations (50% and 100%; p < 0.0001) has been shown.
Considering W + PAHs + nZVI condition at T0 and T1 (Fig. 2C), a low percentage of dead nauplii (about 25%) was detected at 12.5%. At 100%, significant increase of toxicity (about 80%) respect lower (0%, 6.25% and 12.5%; p < 0.0001) and higher (25% and 50%; p < 0.0001) tested concentrations was measured (Supplementary Table S2). Considering T2, T3, T4 and T5 only at 50% and 100% a significant decrease of survival (about 10%) respect all lower concentrations (p < 0.0001) and among them (p < 0.0001; Supplementary Table S2). At T6, no toxicity was found.
Take into the consideration W + SED1 + nZVI condition at T0, T1 and T3 (Fig. 2D), a low percentage of dead nauplii (about 10%) was showed at 50%. At 100%, significant increase of toxicity (about 20–30%) respect lower (0%, 6.25% and 12.5%; p < 0.0001) and higher (25% and 50%; p < 0.0001) tested concentrations was showed (Supplementary Table S2). At T4, T5 and T6, no toxicity was detected.
Similar scenario can be described for W + SED2 + nZVI condition (Fig. 2E). In fact, from time 0 to time 4 only at 50% and 100% a significant decrease of survival (about 10% and 20%, respectively) respect all lower concentrations (p < 0.0001). At T5 and T6, no toxicity was showed.
As reported in Fig. 3, after 48 h of exposure to different percentage of aqueous solutions of all experimental conditions of AC, the scenario was a little different.
Considering W + AC condition at T0 and T1 (Fig. 3A), at 100%, significant percentage of dead nauplii (about 10–20%) was detected in comparison with the others concentrations (0%, 6.25%, 12.5%, 25% and 50%; p < 0.0001, see also Supplementary Table S3). Taking into consideration T2, T3, T4, T5 and T6 no toxicity was detected. At the same manner, take into consideration W + PAHs + AC and W + SED1 + PAHs (Fig. 3C-D), only at 100%, significant percentage of dead nauplii (about 10–20%) was showed respect the others concentrations (0%, 6.25%, 12.5%, 25% and 50%; p < 0.0001, see also Supplementary Table S3).
Only when considered W + SED2 + AC (Fig. 3E), from T0 to T3, at 100%, significant percentage of dead nauplii (from about 10 to 30%) was displayed respect others concentrations (p < 0.0001, see also Supplementary Table S3). At T4, T5 and T6, no decrease of survival nauplii was observed.
3.3 Toxicity effect of nZVI and AC on adults
When evaluated the toxicity on adults, the scenario remains almost similar to that presented for nauplii. As reported in Fig. 4, after 48 h of exposure to different percentage of aqueous solutions of all experimental conditions of nZVI, an increase of toxicity was observed at higher tested percentage, represented by 50% and 100%.
Considering W + nZVI condition at T0 (Fig. 4A), a little percentage of dead adults (about 6.6%) has been shown already at the lowest percentage (6.25%). These data were statistically significant respect to the control (p < 0.05) and others used concentrations (p < 0.0001; see also Supplementary Table S4). At 100%, about 30% of dead adults have been observed respect lower (0% and 12.5%; p < 0.0001 and p < 0.001, respectively) and higher (25% and 50%; p < 0.001) tested concentrations has been shown. On the basis of toxicity observed for T1, at 25%, 50% and 100% a significant decrease of survival (about 13%, 20% and 23%, respectively) respect lower concentrations (0%, 6.25% and 12.5%; p < 0.0001 (Supplementary Table S4) was detected. Moreover, the data reported at 25% were statistically significant respect to 50% (p < 0.05) and 100% (p < 0.001). Considering T2, T3 and T4 only at 50% and 100% a significant decrease of survival (about 10% and 20%, respectively) respect all lower concentrations (p < 0.0001) and among them (p < 0.001; Supplementary Table S4).
W + PAHs condition at T0 and T1 caused a little survival decrease (about 10%) already at 6.25% that was statistically significant respect to control (p < 0.0001) and others concentrations ((p < 0.0001; Fig. 4B; Supplementary Table S4). At 25%, a significant decrease of about 50% respect lower concentrations (p < 0.0001; Supplementary Table S4) and higher concentrations (50% and 100%; p < 0.0001) was detected. From T2 to T6, the results were similar. In fact, at 25%, a significant decrease of about 30% respect lower concentrations (p < 0.0001 (Supplementary Table S4) and higher concentrations (50% and 100%; p < 0.0001) has been shown.
Considering W + PAHs + nZVI condition at T0 and T1 (Fig. 4C), a low percentage of about 25% of dead nauplii was showed at 12.5%. At 100%, significant increase of toxicity (about 80%) respect lower (0%, 6.25% and 12.5%; p < 0.0001) and higher (25% and 50%; p < 0.0001) tested concentrations was detected (Supplementary Table S4). Considering T2, T3, T4 and T5 only at 50% and 100% a significant decrease of survival (about 10%) respect all lower concentrations (p < 0.0001) and among them (p < 0.0001; Supplementary Table S4). At T6, no toxicity was found.
Take into the consideration W + SED1 + nZVI condition at T0, T1 and T3 (Fig. 4D), a low percentage of dead nauplii (about 10%) was found at 50%. At 100%, significant increase of toxicity (about 20–30%) respect lower (0%, 6.25% and 12.5%; p < 0.0001) and higher (25% and 50%; p < 0.0001) tested concentrations was showed (Supplementary Table S4). At T4, T5 and T6, no toxicity was displayed.
W + SED2 + nZVI condition (Fig. 4E), from time 0 to time 4, only at 50% and 100% caused a significant decrease of survival (about 10% and 20%, respectively) respect all lower concentrations (p < 0.0001). At T5 and T6, no toxicity was found.
As reported in Fig. 5, after 48 h of exposure to different percentage of aqueous solutions of all experimental conditions of AC, the scenario was a little different.
Considering W + AC condition (Fig. 5A), at all experimental time, no toxicity was showed. At T0 and T1, take into consideration W + PAHs + AC (Fig. 5C), only at 100%, significant percentage of dead adults (about 10%) was measured respect others concentrations (0%, 6.25%, 12.5%, 25% and 50%; p < 0.0001, see also Supplementary Table S5). When considered the exposure to solutions collected at T2, T3, T4, T5 and T6, no dead organism was observed (Fig. 5C). At same manner, take into consideration W + SED1 + AC (Fig. 5C), only at time 0 and 1, significant percentage of dead adults (about 10%) was showed, testing 100% of solutions respect others concentrations (0%, 6.25%, 12.5%, 25% and 50%; p < 0.0001 and p < 0.05 at To and T1, respectively; see also Supplementary Table S5).
Only when considered W + SED2 + AC (Fig. 5E), at 100% of T0 solutions, significant percentage of dead adults (from about 10%) was observed respect others concentrations (p < 0.0001, see also Supplementary Table S5). At T1, T2, T3, T4, T5 and T6, no decrease of survival adults was observed.
3.4 Effects of nZVI and AC on Gene Expression by Real-Time qPCR
The expression levels of nine genes (Chen et al., 2009), involved in different physiological processes, were followed by Real Time qPCR after nZVI remediation experiment (Fig. 6; see also Supplementary Table S6 for the values). Considering the stress response (Fig. 6A), hsp 60 and COXIII were targeted in all experimental conditions. Specifically, hsp 60 was down-regulated by W + nZVI, W + PAHs, W + SED1 + nZVI and W + SED2 + nZVI, but was up-regulated by W + PAHs + nZVI; COXIII was down-regulated by W + nZVI, W + PAHs, W + PAHs + nZVI and W + SED2 + nZVI, but was up-regulated by W + SED1 + nZVI. Moreover, hsp 26 and hsp70 were down-regulated and up-regulated, respectively, in all conditions with exception of W + nZVI. Finally, COXI was up-regulated only by W + SED1 + nZVI (see Table S6).
Take into the consideration the genes involved in developmental processes (Fig. 6B), four genes were targeted by all conditions with exception of W + nZVI. Common molecular targets for all conditions were HAD-like and CDC48, of which HAD-like was down-regulated by W + PAHs + nZVI, W + SED1 + nZVI and W + SED2 + nZVI and up-regulated only by W + PAHs; whereas CDC48 was down-regulated by all conditions. The gene tcp was up-regulated by W + SED1 + nZVI, and down-regulated by W + PAHs; and UCP2 was up-regulated by W + PAHs + nZVI and W + SED1 + nZVI (see Table S6). Also, after AC remediation experiment, these nine genes expression levels were evaluated (Fig. 6; see also Supplementary Table S7 for the values).
Evaluating the stress response, three heat shock proteins were targets for almost all test conditions. In particular, hsp 60 was down-regulated by W + PAHs, and up-regulated by W + AC, W + SED1 + AC and W + SED2 + AC; whereas hsp 70 was down-regulated by W + SED1 + AC, and up-regulated by W + PAHs, W + PAHs + AC and W + SED2 + AC. The gene hsp26 was molecular target only for W + PAHs showing a down-regulation. Moreover, COXI and COXIII were up-regulated by W + PAHs + AC, W + SED1 + AC and W + SED2 + AC; and only COXIII was down-regulated by W + PAHs (Fig. 6C). Considering the impact on developmental processes (Fig. 6D), also in this case, four genes were targeted by all conditions with exception of W + AC. Common molecular target for all conditions was tcp, which showed an up-regulation after W + PAHs + AC, W + SED1 + AC and W + SED2 + AC treatment, and down-regulation after W + PAHs exposure. HAD-like was up-regulated by W + PAHs, W + SED1 + AC and W + SED2 + AC; whereas CDC48 was down-regulated by W + PAHs and up-regulated by W + SED1 + AC and W + SED2 + AC. Finally, UCP2 was up-regulated by W + PAHs + AC, W + SED1 + nZVI and W + SED2 + AC (see Table S7).
As shown in Fig. 7, among the five genes analysed in adults of A. franciscana, all genes were targeted by all experimental conditions. Specifically, hsp26, hsp60, COXI and COXIII were up-regulated by all conditions of nZVI treatments (see Fig. 7A and Table S8), whereas hsp70 was up-regulated only by W + nZVI, W + PAHs and W + PAHs + nZVI and down-regulated by W + SED1 + nZVI and W + SED2 + nZVI (Supplementary Table S8). As shown Fig. 7B, all tested genes in adults were up-regulated by all experimental conditions (W + AC, W + PAHs, W + PAHs + AC, W + SED1 + AC and W + SED2 + AC treatments; Table S9).