New insights on the impacts of e-waste towards marine bivalves: The case of the rare earth element Dysprosium.

With the technological advances and economic development, the multiplicity and wide variety of applications of electrical and electronic equipment have increased, as well as the amount of end-of-life products (waste of electrical and electronic equipment, WEEE). Accompanying their increasing application, there is an increasing risk to aquatic ecosystems and inhabiting organisms. Among the most common elements present in WEEE are rare earth elements (REE) such as Dysprosium (Dy). The present study evaluated the metabolic and oxidative stress responses of mussels Mytilus galloprovincialis exposed to an increasing range of Dy concentrations, after a 28 days experimental period. The results obtained highlighted that Dy was responsible for mussel's metabolic increase associated with glycogen expenditure, activation of antioxidant and biotransformation defences and cellular damage, with a clear loss of redox balance. Such effects may greatly impact mussel's physiological functions, including reproduction capacity and growth, with implications for population conservation. Overall the present study pointed out the need for more research on the toxic impacts resulting from these emerging pollutants, especially towards marine and estuarine invertebrate species.


97
Although it is reported the increasing presence of REEs in marine coastal areas, their 98 toxicological understanding, an in particular for Dy, in such aquatic systems is still almost 99 unknown but of increasing concern. Nevertheless, the emergence of Dy in the aquatic systems 100 has raised attention into the scientific community related to its effects in the living organisms. REEs, including Dy. The results obtained showed that P. lividus embryos had a decreased 104 mitotic activity and an increased aberration rate. Sperm exposed to these elements showed 105 decrease in fertilization success along with increase in offspring damage. These authors

116
revealed that H. azteca is 1.4 times more sensitive than D. pulex. In this study, it was also 117 verified the toxicity modifying influence of Ca, Na, Mg, pH and dissolved organic matter (DOM) 118 in the presence of Dy with a more sensitive organism, H. azteca. It was concluded that additions 119 of Ca and Na, low pH and DOM provided protection of the organisms against Dy toxicity, while 120 on the contrary the addition of Mg increase the toxicity of Dy.

121
From the literature available it is possible to recognize that no knowledge exists on the 122 toxic effects of Dy towards marine or estuarine bivalves, namely on species with high ecological 123 and economic relevance. Nevertheless, marine coastal systems are frequently final destination 124 of these pollutants putting at risk inhabiting animals and public health in the case of bivalves 125 associated with human consumption. Therefore, the present study aimed to investigate the 126 biochemical alterations induced in the mussel species Mytilus galloprovincialis, when exposed 127 to an increasing exposure gradient of Dy, resembling low to highly contaminated areas.

128
Although no studies are known on the impacts of Dy in bivalves, and in particular in mussels, 129 recent studies demonstrated the negative impacts of other REEs (e.g. Neodymium, Lanthanum,

152
Animals were collected in September 2018, at the Ria de Aveiro lagoon (Portugal).

153
Mussels with similar size (5.7±0.7 cm length; 3.0±0.4 cm width) were selected to avoid 154 differences in biological responses.

155
Bivalves were transported from the field to the laboratory where they were placed in 156 aquaria for depuration and acclimation to laboratory conditions for two weeks. During this 157 period, mussels were maintained under constant aeration in different aquaria with artificial 158 seawater (Tropic Marin® SEA SALT) at temperature, pH and salinity values resembling the 159 sampling site conditions (18.0 ± 1.0 ºC; 8.0 ± 0.1, 30 ± 1, respectively). Seawater was renewed 160 every day during the first seven days and then every three days until the end of the acclimation 161 period.

162
After this period, mussels were distributed in different aquaria (with four aquaria per 163 condition with 3 L of seawater each) and exposed to the following conditions for twenty-eight

168
To evaluate the stability of Dy in the water medium a parallel experiment was conducted,

169
in the absence of mussels. For this, glass containers with 500 mL of artificial seawater were 170 spiked with 2.5 and 40 µg L -1 of Dy (10 containers per concentration) and, during seven days 171 (corresponding to the period between water renewals along the twenty-eight days experimental 172 assay), aliquots of 5 mL were daily collected to assess concentrations of Dy in the water.

8
During the experimental period (twenty-eight days), water was changed every week and 174 the medium conditions re-established, including Dy concentrations and seawater parameters

177
Every week, immediately after water renewal, water samples were collected from each 178 aquarium for Dy quantification, to assess real exposure concentrations. During this period, 179 mussels were fed with Algamac protein plus (150,000 cells/animal) three times per week.

180
Mortality was also daily checked, with 100% of survival recorded during all the experimental 181 period.

182
At the end of the exposure period, mussels were frozen individually with liquid nitrogen

Biochemical markers 205
The whole tissue of mussels was used for biomarkers determination.

257
Absorbance was measured at 450 nm and the extinction coefficient of 22,308 M −1 cm −1 was 258 used to calculated PC levels, which were expressed in nmol per g of FW.

260
Metabolic capacity and energy reserves 261 The ETS activity was measured based on King and Packard (1975)

265
For GLY quantification the sulphuric acid method was used, as described by Dubois et al.

267
Absorbance was measured at 492 nm after incubation during 30 min at room temperature.

268
Results were expressed in mg per g FW.

269
The PROT content was determined according to the spectrophotometric Biuret method  Table   290 format.

291
The matrix gathering the Dy concentrations in mussels soft tissues and biochemical 292 results per condition were used to calculate the Euclidean distance similarity matrix, which was .

Dysprosium concentrations in seawater and mussel tissues 300
Results concerning the stability of Dy in seawater medium showed that, in the absence of 301 mussels, concentrations were maintained along seven days' exposure period, with results

302
showing that the mean±STDEV values after exposure to 2.5 and 40 µg/L of Dy were,

306
In what regards to Dy concentrations in seawater from the experimental exposure assay, 307 values obtained in water samples collected immediately after spiking revealed that measured 308 and nominal concentrations were similar, for all the conditions and weeks, validating the Dy 309 spiking process (Table 1).

310
The results obtained from Dy quantification in mussel's tissues showed significant 311 difference among animals exposed to tested conditions, with increasing Dy levels along the 312 increasing exposure concentration (Table 1).

316
The activity of SOD was significantly lower at control and at 2.5 µg/L of Dy in comparison 317 to mussels exposed to higher concentrations ( Figure 1A, Table 2). The activity of CAT was 318 significantly higher in mussels exposed to 20 and 40 µg/L of Dy in comparison to the remaining 319 conditions ( Figure 1B, Table 2). The activity of GPx was significantly higher in mussels exposed 320 to 40 µg/L of Dy in comparison to non-contaminated mussels and the ones exposed to 5.0 and 321 10 µg/L of Dy ( Figure 1C, Table 2).

324
The activity of GSTs increased with the increase of Dy exposure concentration, with 325 significantly higher values in mussels exposed to 20 and 40 µg/L of Dy in comparison to animals 326 under control and exposed to the lowest Dy concentration (Figure 2, Table 2). The GSH/GSSG values were significantly lower in contaminated mussels compared to 330 control ones, with the lowest values in animals exposed to concentrations 2.5 and 5.0 µg/L of 331 Dy (Figure 3, Table 2).

333
Cellular damage 334 Levels of LPO were significantly higher in contaminated mussels compared to control 335 ones, with the highest values in mussels exposed to 20 µg/L of Dy. No significant differences 336 were observed among mussels exposed to 2.5, 5.0 and 10 µg/L of Dy ( Figure 4A, Table 2). The

337
PC levels increased in mussels exposed to Dy, with significant differences between control and 338 Dy exposed mussels. Although higher PC levels were obtained in animals exposed to 10 µg/L 339 of Dy, no significant differences were observed among contaminated mussels ( Figure 4B, Table   340 2).

342
Metabolic capacity and energy reserves

343
The ETS activity showed no significant differences among conditions, although higher 344 values were observed at the highest Dy exposure concentrations ( Figure 5A, Table 2). The GLY 345 content decreased in Dy exposed mussels, with significant differences between the control and 346 mussels exposed to 5, 10, 20 and 40 µg/L of Dy ( Figure 5B, Table 2). As for the ETS activity, no 347 significant differences were observed among tested conditions in terms of PROT content,

348
although higher values were noticed at higher Dy concentrations ( Figure 5C, Table 2).

Integrated Biomarker Response 351
Integrated Biomarker Response (IBR) values showed the highest score (3.4) for 352 mussels exposed to the highest Dy exposure concentration. The lowest IBR values were 353 observed for mussels exposed to the concentrations 2.5 and 5.0 µg/L of Dy (Table 3).

Principal Coordinates Analysis 356
The Principal Coordinates Analysis (PCO) representation revealed that PCO1 explained 357 64.2% of the total variation among the data, separating mussels exposed to control and to the 358 15 two lowest Dy exposure concentrations (2.5 and 5 µg/L of Dy) in the positive side from the 359 mussels exposed to higher concentrations (10, 20 and 40 µg/L of Dy) in the negative side.  present results highlight that mussels exposed to Dy were able to develop a defence strategy to

409
Thus, previous studies and the presents findings highlight the efficiency of this group of 410 enzymes to detoxify mussels from REE.

411
Besides antioxidant and biotransformation enzymes, low molecular weight scavengers 412 are also able to neutralize ROS by direct reaction with them. The most abundant cytosolic 413 scavenger is GSH. In particular, GSH can be oxidized to GSSG (oxidized glutathione) by GPx.

414
For this, H 2 O 2 acts also as substrate for GPx, using GSH as electron donor to catalyse the

419
results obtained in the present study also demonstrated that mussels exposed to Dy strongly

438
Similarly, several other studies used LPO as a marker of the oxidative stress generated by the 439 exposure of bivalves to different pollutants, including in mussels, exposed to classical pollutants

450
Although less used as oxidative stress biomarker in bivalves, and especially in clams and

467
evidencing that under Dy exposure conditions mussels may use this energy reserve. In aquatic 468 species, including bivalves, ETS activity has been used also to assess the oxygen demand to

478
Overall the present study clearly demonstrated a dose-dependent response, with 479 mussels showing higher biological impacts at to higher exposure concentrations. Nevertheless, 480 at intermediate exposure concentrations, namely at 5 µg/L of Dy, it seems that mussels were 481 able to avoid injuries by efficiently activating their defence mechanisms (including increase of 482 antioxidant enzymes activities), while at the lowest concentration these strategies were not 483 activated due to low stress levels. At higher concentrations, although enzymes activities 484 increased mussels these defence mechanisms were not enough to avoid injuries, leading to

488
IBR as a useful tool for quantitative assessment of stress levels in mussels exposed to different distinction between: i) mussels exposed to control and the two lowest Dy concentrations (PCO 494 axis 1, positive side), associated to lower cellular damage and the maintenance of redox 495 balance; ii) and mussels exposed to the three higher Dy concentrations (PCO axis 1, negative 496 side), close associated with higher metabolic capacity, higher antioxidant and biotransformation 497 defences, and higher protein content.

501
The present study clearly demonstrated the impacts of Dy in M. galloprovincialis, with 502 increasing antioxidant defences, cellular damages and oxidative stress in Dy contaminated 503 mussels. The results obtained further demonstrated that mussels exposed to Dy increased their