The influence of temperature and salinity on the impacts of Lead in Mytilus

25 Mussels, such as the marine bivalve Mytilus galloprovincialis are sentinels for marine pollution but they are also excellent bioindicators under laboratory conditions. 27 For that, in this study we tested the modulation of biochemical responses under 28 realistic concentrations of the toxic metal Lead (Pb) in water for 28 days under different 29 conditions of salinity and temperature, including control condition (temperature 17±1.0 30 ºC and salinity 30±1.0) as well as those within the range expected to occur due to 31 climate change predictions (±5 in salinity and +4ºC in temperature). A comprehensive 32 set of biomarkers was applied to search on modulation of biochemical responses in 33 energy energy reserves, 34 in lipids, proteins as well as neurotoxicity signs. The application of an integrative 35 Principal Coordinates Ordination (PCO) tool was successful and demonstrated that Pb 36 caused an increased in the detoxification activity mainly evidenced by glutathione S-37 transferases and that the salinities 25 and 35 were, even in un-exposed mussels, 38 responsible for cell damage seen as increased levels of lipid peroxidation (at salinity 39 25) and oxidised proteins (at salinity 35). 40 superimposed as supplementary variables, namely biochemical data (r > 0.75): PC, CAT, PROT, GPx, Pb, GSTs, LPO.

Abstract 25 Mussels, such as the marine bivalve Mytilus galloprovincialis are sentinels for 26 marine pollution but they are also excellent bioindicators under laboratory conditions.

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For that, in this study we tested the modulation of biochemical responses under transferases and that the salinities 25 and 35 were, even in un-exposed mussels, 38 responsible for cell damage seen as increased levels of lipid peroxidation (at salinity 39 25) and oxidised proteins (at salinity 35).

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Environmental pollution by potential toxic elements, such as metals, has been a topic 46 of concern over the last decades, with several studies highlighting not only the accumulation of these elements in different aquatic compartments but also their impacts on freshwater and

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After the exposure time, the whole soft tissue of 9 mussels per condition (3 per

Lead concentrations in water and organisms 273
Concentrations of Pb measured in water collected immediately after spiking showed 274 neither significant differences among non-contaminated temperature and salinity conditions nor 275 among contaminated ones (Table 1). Trace amounts of Pb were also detected in water of 276 unexposed conditions (1.1-8.2 μg/L), while in those exposed to Pb concentrations ranging 277 between 63.1 and 74.2 µg/L, slightly higher than the targeted nominal concentration (Table 1).

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The concentrations of Pb in mussel's soft tissues showed significantly higher (about 3-279 4 fold) values in organisms exposed to Pb in comparison to non-contaminated ones, with no 280 significant differences among mussels exposed to different conditions ( In non-contaminated mussels, significantly lower ETS values were observed in those 287 maintained to salinity 35 in comparison to the remaining conditions. In Pb exposed mussels,

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significantly lower ETS values were observed at salinities 30 and 35 at control temperature (17 289 ºC). At salinity 30, ETS increased at 21ºC but decreased at 17ºC in Pb exposed mussels in 290 respect to non-contaminated ones ( Figure 1A).

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GLY content was only significantly lower at salinity 35 in respect to 30 in non-292 contaminated mussels. By contrast, in the presence of Pb, GLY was significantly enhanced at 293 the salinity 35. At the control salinity (30), lower GLY content was observed in contaminated 294 mussels maintained at both tested temperatures ( Figure 1B).

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PROT content in mussels at 17 ºC was significantly higher at salinity 25 both for non-296 contaminated and Pb contaminated conditions. When considering the temperature influence at 297 salinity 30, PROT reserves were higher in non-contaminated specimens maintained both at 17 3.2.2 Antioxidant and biotransformation defences 301 SOD activity in non-contaminated mussels differed at the three tested salinities at 17 302 ºC, with the lowest activity seen at 25. In Pb exposed mussels held at the same temperature,

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SOD was significantly increased only at salinity 30. At this salinity, the effect of temperature was 304 inverse, while SOD increased in Pb exposed mussels held at 17 ºC, it decreased in 305 contaminated mussels at 21 ºC (Figure 2A).

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CAT activity was little affected by salinity and it only increased in unexposed mussels 307 at the higher salinity of 35 at 17 ºC. At 21 ºC and salinity 30, CAT activity was significantly 308 higher in non-contaminated mussels than in those exposed to Pb at the same temperature and 309 those held at 17 ºC at the same salinity ( Figure 2B).

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GPx activity was highly salinity dependent, with significantly higher values at salinity 311 25 in non-contaminated mussels; while in all the Pb exposed groups this activity was 312 significantly lower at this salinity condition. In regard to the influence of temperature at salinity 313 30, Pb exposed mussels displayed significantly lower GPx activity than non-contaminated 314 mussels at the two temperatures; with significantly higher GPx values at 17 ºC ( Figure 2C).

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GSTs activity was significantly lower in non-contaminated and contaminated mussels 316 at salinity 25 and temperature 17 ºC. Mussels maintained at salinity 30 and different 317 temperatures (17 and 21 ºC) showed the same response, with significantly higher GSTs values 318 in contaminated mussels (Figure 3).

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LPO values were significantly higher in non-contaminated mussels maintained at 322 salinity 25 but they were significantly lower in the Pb exposed group held at the same condition.

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At 21 ºC and salinity 30 LPO values significantly increased in Pb exposed mussels, while at 17 324 ºC and salinity 30 an opposite response was observed ( Figure 4A).

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Oxidised proteins measured as PC significantly increased at salinities of 25 and 35 326 even in the non-exposed mussels. Oxidised proteins content was significantly higher in mussels 327 at 21 ºC and salinity 30 in comparison to organisms maintained at the same salinity but 17 ºC mussels, while an opposite response was observed in non-contaminated mussels with

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The parameters related to energy metabolism such as ETS, which corresponds to the 369 overall mitochondrial activity in relation to energy production, was not a mechanism that 370 significantly contributed to the differences observed as it did not show a correlation >75% with 371 all tested conditions, reason why it did not appear as an explanatory vector in the PCO. Neither 372 did the GLY content account for explaining differences among tested conditions. Despite the 373 limited influence of ETS in the overall responses, the highest salinity alone decreased mussel's 374 metabolic capacity regardless of Pb exposure. However, under Pb contamination, mussels 375 significantly increased their metabolism at the salinity 25 and the highest temperature (21 ºC).

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Thus, two strategies were seen adopted by mussels: one, by decreasing their metabolism at

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both strategies can be alternatively adopted in bivalves. The neurotoxicity marker AChE did not 387 seem to be a mechanism that significantly contributed to the identification of differences among 388 tested conditions (correlation <75%). Salinities 5 units over and under the control value (salinity 389 30) either decreased (non-contaminated mussels) or increased (Pb exposed mussels) this

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As a consequence of mussel's efficent activation of their defence mechanisms, in 431 general, no LPO or PC ocurrence was observed in Pb contaminated mussels. Only one 432 exception being LPO elevation in Pb exposed mussels reared at higher temperature and 433 highlights this as the worst case situation. Efficent defence response were also observed in M. oxidised proteins (measured as PC at salinity 35) even in uncontaminated mussels.

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Temperature alone had more influence in modulating the responses in non-contaminated 448 mussels (separated in the PCO) than those exposed to Pb since the presence of the   contaminated and non-contaminated mussels at salinities 30, 25 and 35 and at temperature 17 ºC, Pb-contaminated and non-contaminated mussels at salinity 30 and at temperature 21 ºC. Values are presented as mean + standard deviation. Significant differences (p ≤ 0.05) among salinity and temperature conditions are represented with different letters: lowercase letters for non-contaminated mussels and uppercase letters for contaminated mussels. Significant differences (p ≤ 0.05) between non-contaminated and contaminated mussels for each salinity and temperature condition are represented with an asterisks. White bars represent non-contaminated mussels. Grey bars represent contaminated mussels.  Figure 1). Values are presented as mean + standard deviation. Significant differences (p ≤ 0.05) among salinity and temperature conditions are represented with different letters: lowercase letters for non-contaminated mussels and uppercase letters for contaminated mussels. Significant differences (p ≤ 0.05) between non-contaminated and contaminated mussels for each salinity and temperature condition are represented with an asterisks. White bars represent non-contaminated mussels. Grey bars represent contaminated mussels.  Figure 1). Values are presented as mean + standard deviation. Significant differences (p ≤ 0.05) among salinity and temperature conditions are represented with different letters: lowercase letters for non-contaminated ACCEPTED MANUSCRIPT mussels and uppercase letters for contaminated mussels. Significant differences (p ≤ 0.05) between non-contaminated and contaminated mussels for each salinity and temperature condition are represented with an asterisks. White bars represent noncontaminated mussels. Grey bars represent contaminated mussels.  Figure 1). Values are presented as mean + standard deviation. Significant differences (p ≤ 0.05) among salinity and temperature conditions are represented with different letters: lowercase letters for non-contaminated mussels and uppercase letters for contaminated mussels. Significant differences (p ≤ 0.05) between non-contaminated and contaminated mussels for each salinity and temperature condition are represented with an asterisks. White bars represent noncontaminated mussels. Grey bars represent contaminated mussels.  Figure 1). Values are presented as mean + standard deviation. Significant differences (p ≤ 0.05) among salinity and temperature conditions are represented with different letters: lowercase letters for non-contaminated mussels and uppercase letters for contaminated mussels. Significant differences (p ≤ 0.05) between non-contaminated and contaminated mussels for each salinity and temperature condition are represented with an asterisks. White bars represent noncontaminated mussels. Grey bars represent contaminated mussels.  Figure 1). Black letters represented contaminated mussels while grey letters represent non-contaminated mussels. Pearson correlation vectors are mussels.
 Overall, exposure to Pb increased detoxification activity measured as GSTs.
 Antioxidant defences failed to prevent LPO at the lowest salinity in controls.
 Damaged proteins occurred at the highest salinity in unexposed mussels. Table 1. Mean Lead concentrations (µg/L) in water samples weekly and immediately sampled after spiking during the experimental period (28 days), at each condition. Non-contaminated (mussels exposed to 0 µg/L of Lead) and contaminated (mussels exposed to 50 µg/L of Lead)

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conditions. For non-contaminated and contaminated mussels, significant differences (p ≤ 0.05) among different salinity and temperature conditions are represented with different lower case letters.  Table 2. Mean Lead concentrations (µg/g) in mussel's soft tissues collected at the end of the experimental period (28 days), at each water condition. Non-contaminated (mussels exposed to 0 µg/L of Lead) and contaminated (mussels exposed to 50 µg/L of Lead) conditions. For noncontaminated and contaminated mussels, significant differences (p ≤ 0.05) among different salinity and temperature conditions are represented with different lower case letters. BCF-Bioconcentration factor.