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Comparative sensitivity of Crassostrea angulata and Crassostrea gigas embryo-larval development to As under varying salinity and temperature

30 Oysters are a diverse group of marine bivalves that inhabit coastal systems of 31 the world’s oceans, providing a variety of ecosystem services and represent a major 32 socioeconomic resource. However, oyster reefs have become inevitably impacted from 33 habitat destruction, overfishing, pollution and disease outbreaks that have pushed 34 these structures to the break of extinction. In addition, the increased frequency of 35 climate change related events promise to further challenge oyster species survival 36 worldwide. 37 Oysters’ early embryonic development is likely the most vulnerable stage to 38 climate change related stressors (e.g. salinity and temperature shifts) as well as to 39 pollutants (e.g. arsenic), and therefore can represent the most important bottleneck that 40 define populations’ survival in a changing environment. In light of this, the present 41 study aimed to assess two important oyster species, Crassostrea angulata and 42 Crassostrea gigas embryo-larval development, under combinations of salinity (20, 26 43 and 33), temperature (20, 24 and 28 ºC) and arsenic (As) exposure (0, 30, 60, 120, 44 240, 480, 960 and 1920 µg. As L -1 ), to infer on different oyster species capacity to cope 45 with these environmental stressors under the eminent threat of climate change and 46 increase of pollution worldwide. 47 Results showed differences in each species range of salinity and temperature 48 for successful embryonic development. For C angulata , embryo-larval development 49 was successful at a narrower range of both salinity and temperature, compared to C. 50 gigas. 51 gigas had a greater influence on embryos’ sensitivity to As. This pattern was mostly noticed 56 for C. gigas , with lower salinity inducing higher sensitivity to As. Results were 57 discussed considering the existing literature and suggest that C. angulata populations 58 are likely to become more vulnerable under near future predictions for temperature 59 rise, salinity shifts and pollution.


222
The zygote suspensions from each species that remained unused from 223 embryotoxicity assays were analysed for trace elements (As,Cd,Cr,Cu,Hg,224 Ni, Pb, Sn and Zn), following an adaptation of Weng and Wang (2017)  were represented in Contour plots using SigmaPlot version 11.0.

248
To assess As embryotoxicity, data from As exposures were corrected for 249 the effects observed in the respective negative control (salinity and 250 temperature) using the Abbott's formula (ASTM, 2004     As toxicity measured in terms of EC50 considering standard toxicity 379 assay conditions with oyster embryos (24 ºC, 33 salinity, 24 h) for C. angulata 380 was 39.2 µg L -1 As (18.7 µg L -1 As at 48 h) (Table I). To our knowledge, this is 381 the first study on the effects of contaminants to C. angulata embryonic 382 development. The toxicity of As (EC50) measured in the present study for C.
between C. angulata (Spanish origin) and C. gigas (French origin), regarding 417 each progenies' maturation traits as well as mortality (Soletchnik et al., 2002). 418 Also, Huvet et al., (2002) suggested that C. gigas could present overall better 419 gamete quality than C. angulata, while studying hybrid crosses between both 420 species. To our knowledge this is the first study on C. angulata embryotoxicity, 421 and our data add to the body of evidence that show differences in 422 ecophysiological traits between these closely related species (Soletchnik et al., 423 2002;His et al., 1972;Goulletquer et al., 1999;Heral, 1996;Moreira et al., 424 2016).

425
Other factors such as broodstock origin or parental exposure history 426 could also have influenced each species sensitivity to As but were unlikely 427 given our results and the existing literature as follows.

428
Gamain and co-workers (2017) studied C. gigas from different origins 429 (hatchery, cultivated and wild) and found no major differences in embryotoxicity 430 of metolachlor to embryos of different parental origins. However, and in contrast 431 with our study, progeny from hatchery oysters were more sensitive to Cu than 432 wild and cultivated ones. Considering this, it would be unlikely that oysters 433 (hatchery C. gigas, and cultivated C. angulata) from our study, would have 434 presented the observed differentiated response to As, solely based on 435 broodstock origin (hatchery and cultivated). temperature levels (Fig. 4).

522
These results are in line with previous studies using C. gigas exposed active transport processes (Connel, 1989;Grosell et al., 2007). Another factor 530 contributing to higher sensitivity to As at lower salinities, could be related to 531 differences in As speciation with salinity variation, however we discarded such 532 possibility, because under a similar range of salinity (and temperature) 533 conditions, As speciation analysis showed no important differences in prevailing

536
The effect of varying temperature on C. gigas embryos' sensitivity to As, 537 inferred by pairwise comparisons of EC50 values among temperature levels, 538 were variable within each salinity level and incubation period (Fig. 4) was significantly lower at 20 ºC, comparing to the remaining temperature levels, 542 after 24 h incubation. At salinity 33 after 24 h embryo development, the highest 543 EC50 value was observed at 24 ºC, with significant differences towards that 544 obtained at 20 ºC. (Fig. 4). 545 These findings suggest that higher sensitivity (lower EC50s) observed at 546 lower temperatures (20 ºC, 24 hours) for all tested salinity levels likely resulted 547 from an additive effect of delayed development induced by both As and low 548 temperature, considering that the developing rate of oyster larvae decrease at 549 lower temperatures (His et al., 1989;Dove and O´Conner, 2007), and that As Results further revealed that after 48 h no significant differences were 552 observed among temperature levels at salinity 26 and 33 (Fig 4). These findings 553 demonstrate that the arresting effect of As on C. gigas larvae development to values obtained after 24 h. It is important to note that at salinity 20 at 20 ºC, 568 EC50 was only possible to calculate after 48 h exposure, since after 24 h 569 embryo development was not completed in these conditions ( Fig. 2B and D).

607
Comparisons on As toxicity measured as EC50 values between 608 species, showed that C. angulata was at least 10 times more sensitive to As 609 than C. gigas and these differences were likely species related. Our results 610 suggest that the survival of C. angulata strongly depends on a narrower range 611 of abiotic factors compared to its closely related congener, and therefore the 612 future of this population may be endangered considering the future projections 613 on climate change and pollution worldwide.

614
Other issues rise from the present study, namely the observations that     n.c -EC50 value not calculated due to low or zero percentage of developed larvae (Dshape) n.d -95% confidence interval not determined due to few points for curve fit parameter estimation