Remediation of arsenic from contaminated seawater using manganese spinel ferrite nanoparticles: Ecotoxicological evaluation in Mytilus galloprovincialis

Please cite this article as: Coppola, F., Tavares, D.S., Henriques, B., Monteiro, R., Trindade, T., Soares, A.M.V.M., Figueira, E., Polese, G., Pereira, E., Freitas, R., Remediation of arsenic from contaminated seawater using manganese spinel ferrite nanoparticles: Ecotoxicological evaluation in Mytilus galloprovincialis, Environmental Research (2019), doi: https://doi.org/10.1016/j.envres.2019.04.008.


Arsenic quantification 201
The quantification of As in water samples collected from each condition (Table 3)

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Total As concentrations in M. galloprovincialis whole soft tissues (Table 4)

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where higher values correspond to higher mussels' response.  Table 5.

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For As concentrations and each biomarker, the null hypotheses (H0) tested were: i) no 311 significant differences exist among CTL and all the contaminated conditions (CTL, A, B and C).

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p-values are presented in Table 5, with significant differences highlighted in bold; ii) no 313 significant differences exist among decontaminated conditions (CTL, a, b, and c). p-values are 314 presented in Table 5, with significant differences highlighted in bold; iii) no significant 3.2 Arsenic quantification in seawater and mussels' tissues 336 Concentrations of As in water samples revealed that real and nominal concentrations 337 were similar, both for A and a conditions. In water samples from conditions without As (B and b) 338 the concentrations of this metalloid were lower than the quantification limit (1.5 µg L -1 ).

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Concentration of As in water after decontamination was 55 ± 13 µg L -1 . Because sorption of As 340 by the NPs is extremely rapid, As was not possible to quantify in water from condition C ( Table   341 3).

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The results obtained from As quantification in mussels showed a significant difference 343 between organisms exposed to CTL and those exposed to A and C conditions (Table 4).

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No significant differences were found between M. galloprovincialis submitted to CTL and 345 the organisms exposed to conditions a, b and c (Table 4).

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and C vs c) (Table 4). Organisms exposed to condition A accumulated more 76% of As than 349 those exposed to condition a, while the contents of As in the mussels exposed to condition C 350 were 62% higher than those in condition c.

Biochemical markers 353
Metabolic capacity and energy-related biomarkers 354 The ETS activity was significantly higher at control (CTL) in comparison to the values 355 obtained in mussels exposed to As contaminated seawater (conditions A, B, C; resembling 356 initial concentrations, measured before decontamination), with the lowest values at condition A 357 ( Figure 2A, Table 5). ETS activity was significantly higher at control (CTL) in comparison to the 358 values obtained in mussels exposed to decontaminated seawater (conditions a, b, c) ( Figure   359 2A, Table 5).

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The ETS activity was significantly higher in organisms exposed decontaminated seawater 361 (conditions a, b, c) in comparison to organisms exposed to As contaminated seawater 362 (conditions A, B, C) ( Figure 2A).

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The GLY content was significantly lower in mussels exposed to control (CTL) in 365 comparison to the values observed in mussels exposed to As contaminated seawater 366 (conditions A, B, C) ( Figure 2B, Table 5).

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Significantly lower GLY content was obtained in organisms exposed to decontaminated 368 seawater (conditions a, b, c) in comparison to organisms exposed to As contaminated seawater 369 (conditions A, B, C) ( Figure 2B).

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The PROT content was significantly lower in mussels exposed to control (CTL) in 372 comparison to values observed in mussels exposed to As contaminated seawater (conditions A, Table 5).

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The PROT content was significantly lower in organisms exposed to decontaminated higher values were obtained in mussels exposed to condition A in comparison to organisms 383 exposed to conditions B and C ( Figure 3A, Table 5).

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The SOD activity was significantly lower in organisms exposed to decontaminated 385 seawater (conditions a, b, c) in comparison to organisms exposed to contaminated seawater 386 (conditions A, B, C) ( Figure 3A).

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The activity of GPx was significantly lower at CTL in comparison to values obtained in 389 mussels exposed to contaminated seawater (A, B, C) ( Figure 3B, Table 4). Significant 390 differences were observed between organisms exposed to A and C conditions ( Figure 3B, 391 Table 5).

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Regarding to organisms exposed to decontaminated seawater, significantly higher GPx  Table 5). No significant differences were observed between organisms exposed to conditions a 395 and b ( Figure 3B, Table 5).

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The GPx activity values were significantly lower in organisms exposed decontaminated 397 seawater (conditions a, b, c) in comparison to organisms exposed to contaminated seawater 398 (conditions A, B, C) ( Figure 3B).

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The GSTs activity was significantly lower at CTL in comparison to values obtained in 401 mussels exposed to contaminated seawater (A, B, C) ( Figure 3C, Table 5). No significant 402 differences were observed between organisms exposed to A and C conditions ( Figure 3C, 403 Table 5).

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Organisms under control (CTL) conditions showed significantly lower GSTs activity than 405 those exposed to decontaminated seawater (condition c) ( Figure 3C and Table 4). No The GSTs activity values were significantly lower in organisms exposed to 409 decontaminated seawater (conditions a, b, c) comparatively to organisms exposed to 410 contaminated seawater (conditions A, B, C) ( Figure 3C).

Cellular damage biomarkers 413
The LPO levels were significantly lower at control (CTL) in comparison to values obtained 414 in mussels exposed to contaminated seawater (conditions A, B, C) ( Figure 4A, Table 4).

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Significantly lower LPO values were found in organisms exposed to condition B in comparison 416 to organisms exposed to conditions A and C ( Figure 4A, Table 5).

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Significantly lower LPO levels were observed in organisms exposed to CTL compared to 418 organisms exposed to condition a, b and c ( Figure 4A and Table 5). No significant differences 419 were observed among organisms exposed to a, b and c conditions ( Figure 4A, Table 5).

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The LPO levels were significantly lower in organisms exposed to decontaminated 421 seawater (conditions a, b, c) in comparison to organisms exposed to contaminated seawater 422 (conditions A, B, C) ( Figure 4A).

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The PC levels were significantly lower in mussels exposed to control (CTL) in comparison 425 to values observed in mussels exposed to contaminated seawater (conditions A, B, C) ( Figure   426 4B, Table 5).

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The PC levels in mussels exposed to control (CTL) were significantly lower than those 428 observed in mussels exposed to conditions a and b ( Figure 4B, Table 4). No significant 429 differences were observed among organisms exposed to a, b and c conditions ( Figure 4B, 430 Table 5).

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The PC levels were significantly lower in organisms exposed to decontaminated 432 (conditions a, b and c) seawater comparatively to organisms exposed to contaminated seawater 433 (conditions A, B, C) ( Figure 4B).

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The GSH/GSSG values were significantly higher in mussels exposed to control (CTL) in M A N U S C R I P T

A C C E P T E D ACCEPTED MANUSCRIPT
Significantly higher GSH/GSSG values were observed in mussels exposed to control 439 (CTL) in relation to the values observed in mussels exposed to decontaminated seawater 440 (conditions a, b, c) ( Figure 4C, Table 5). No significant differences were observed between 441 organisms exposed to a and c conditions ( Figure 4C, Table 5).

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The GSH/GSSG ratio was significantly higher in organisms exposed to decontaminated 443 seawater (conditions a, b, c) than in organisms exposed to contaminated seawater (conditions 444 A, B, C) ( Figure 4C).

Neurotoxicity biomarker 447
The AChE activity was significantly higher in mussels exposed to control (CTL) in 448 comparison to the values observed in mussels exposed to contaminated seawater (conditions 449 A, B, C) ( Figure 5, Table 5).

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Significantly higher AChE values were observed in mussels exposed to control (CTL) in 451 comparison to those observed in mussels exposed to decontaminated seawater (conditions a, 452 b, c) ( Figure 5, Table 5).

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Significantly higher AChE values were observed in organisms exposed to 454 decontaminated seawater (conditions a, b, c) than in organisms exposed to contaminated 458 IBR values showed the highest score (16.7) for the mussels exposed to condition B, Regarding As bioaccumulation in the whole soft tissues, as it was expected, the present 487 study demonstrated that the higher contents of As were found in the mussels exposed to

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clearly evidenced that mussels exposed to As at a concentration equal to the maximum Furthermore, at a smaller scale, the results also demonstrated that mussels exposed to As in a the present study demonstrated that seawater contaminated with As at initial (condition A) and 502 final (condition a) concentration levels (1000 and 70 µg/L, respectively) induced biochemical 503 alterations in mussels that resulted in a general oxidative and neurotoxic status, with higher 504 impacts when organisms were exposed to the highest As concentration (condition A). Mussels exposed to As initial concentration (1000 µg/L) clearly reduced their metabolism, preventing the 506 use of energy reserves. However, when exposed to seawater at As concentration equal to that 507 of decontaminated seawater (70 µg/L), albeit minor, the organisms had a metabolic capacity 508 close to those of control indicating that higher impacts on mussels metabolism result from the 509 exposure to the highest As concentration. The decrease of mussels' metabolism may be related . The present results also demonstrated that mussels exposed to As 1000 µg/L 515 contaminated seawater strongly increased their antioxidant defences, which may result from the 516 overproduction of reactive oxygen species due to the stress induced by As, which were efficient 517 in limiting the occurrence of LPO. Nevertheless, at this condition, mussels clearly revealed 518 oxidative damages with lower GSH/GSSG ratio compared to control organisms and damages in inhibited AChE activity, with higher injuries when organisms were exposed to the highest As

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with higher impacts when organisms were exposed to the highest NPs concentration (condition 544 B). In particular, the present findings demonstrated that mussels decreased their metabolic 545 capacity and reduced energy expenditure when exposed to NPs concentration of 50 mg L 548 Gosling, 2003). Nevertheless, when mussels were exposed to NPs at final concentration ETS Our results also demonstrated that mussels exposed to NPs increased their It is known that the presence of NPs (TiO2, Au and CuO NPs) increases the production of ROS, . As a result of increased antioxidant defences in 559 mussels exposed to NPs at concentration of 50 mg L -1 damages of the cellular membrane were 560 prevented. Nevertheless, at this condition, mussels clearly revealed oxidative damages with 561 lower GSH/GSSG ratio compared to control organisms and damages in proteins revealed by 562 higher PC values compared to control organisms. When organisms were exposed to residual 563 levels of NPs (condition b) still oxidative damages were observed, with mussels revealing a 564 limited capacity to eliminate the excess of ROS that originated peroxidation of membrane lipids. , respectively) reduced mussels' metabolism, increased oxidative stress and 579 neurotoxicity compared to control organisms. In particular, organisms exposed to condition C The present study demonstrated that organisms exposed to the decontaminated water 605 (condition c, As 70 µg/L and non-quantifiable concentration of NPs) changed their biochemical 606 performance in comparison to control organisms, namely reducing their metabolism, increasing 607 their oxidative stress and neurotoxic status. In comparison to organisms exposed to conditions 608 a and b, where each contaminant was acting individually, the impacts induced were similar, with 609 no significant differences for most of the biomarkers analysed among conditions (a, b, c).

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Nevertheless, the impacts induced in organisms exposed to decontaminated seawater M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT decontaminated seawater presented higher metabolism than organisms exposed to the water antioxidant capacity, which probably was not activated due to low stress induced at this condition, originating in turn higher LPO levels and lower GSH/GSSG values at this condition.

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Furthermore, greater inhibition of AChE was observed when organisms were exposed to 618 condition C compared to condition c, indicating the highest neurotoxic potential of As+NPs initial 619 conditions. 620 621

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The present study demonstrated that As decontaminated seawater (condition c) still 623 generates oxidative stress in mussels, with increased cellular damage and oxidative stress in 624 comparison with the control conditions (CTL), but contaminated conditions A, B and C clearly higher increase in antioxidant defences, neurotoxicity and reduction in metabolism followed by 627 increase of energy reserves. Overall, these results are innovative since, up to our knowledge, 628 no published information is available on the ecotoxic effects induced in mussels when exposed

CTL
Seawater with As 0 µg L -1 + NPs 0 mg L -1 Water before As decontamination A Seawater with As 1000 µg L -1 B Seawater with NPs 50 mg L -1 C Seawater with As 1000 µg L -1 and NPs 50 mg L -1 Water after As decontamiantion a Seawater with As 70 µg L -1 b Seawater after 24h in contact with NPs (50 mg L -1 ), which were afterwards separated from seawater c Seawater previously contaminated with As (1000 µg L -1 ), then remediation using NPs (50 mg L -1 ) during 24 h (which were afterwards separated from seawater). M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Table 3. Arsenic concentration (µg L -1 ) measured in water samples collected immediately after the weekly water renewal. Results correspond to the mean value and standard deviation of the four weeks.
*Because sorption of As by the NPs is extremely rapid, its quantification in this condition was not performed.
[As] water µg L -1 CTL <1.5 As A 947 ± 17 a 82 ± 15 NP B <1.5 b <1.5 As + NP C * c 55 ± 13 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT