The Effect of Global Warming and Acidication on PAHs Bioaccumulation in Pearl Oyster Pinctada Radiate

Warming and acidication are expected impact of climate change that will affect marine areas in the future. These areas are, furthermore, vulnerable to strong anthropogenic stresses such as chemical pollutants. Nevertheless, the consequences of both stressors for marine ecosystems and organisms are still unidentied. The present study aims to examine, for the rst time, the effect of temperature and CO2 pressure increase on bioaccumulation of phenanthrene as a PAHs model in four tissues, gills, digestive gland, muscle and mantle of a commercially important pearl oyster Pinctada radiata. Oysters were exposed to various combination of the ambient temperature and pH currently measured in Persian Gulf (T = 24 ºC and pH = 8.1) and the expected ocean warming and acidication (T = 28 ºC and pH = 7.6), as well as proper PhE concentration (0.8 ng.l − 1 ) during 28 days. In all exposures, higher PhE contents were observed under hypercapnia and warming condition in the digestive gland and gills, followed by the mantle and muscle. Generally, the results visibly reveal that longer exposure period led to promote PhE bioaccumulation in all tissues under ocean warming and acidication environment which was time-dependent pattern of PhE accumulation in P.radiata. Present-day PhE environmental concentrations, which combined with ocean warming and acidication, may lead to rigorous interruption of physiological functions can be extra threatened the ecological tness of pearl oysters.


Introduction
The notable rise of the anthropogenic effects on the earth such as continuous population growth, extreme use of natural resources, and excessive production of pollutants, remarkably since the mid-20th century, has led to climate change ). Since industrial revolution era, relevant atmospheric carbon dioxide concentrations have been increasing (≃400 CO2 µatm in our time), and are predicted that by the year 2100, it will be reached nearly 1000 CO2 µatm ( Adult pearl oyster, Pinctada radiate, (mean ± SD, 4.8 ± 0.6 cm shell length) were acquired in November 2018 from Hendorabi Island of Persian Gulf, Iran. They were transferred to the Shrimp Research Institute established in Busher province via land for 12h, in a container containing oxygen-saturated air and seawater. Upon arrival, samples were categorized and acclimatized in several 300 l tanks for two weeks with aerated ltered seawater at following local environmental conditions: salinity (35 PSU), temperature (24°C), pH (8.1), O 2 concentration (4.5 ± 2.1 mg.l − 1 ), total alkalinity (2521.34 ± 1.52 µmol.kg − 1 ) and natural photoperiod.
Following 14 days of laboratory acclimation, Oysters were exposed during 28 days to four crossed treatment combinations of two pH levels (7.6 and 8.1, ΔpH = 0.5 units), two temperature levels ( The hypercapnic condition was regulated by blending seawater (SW) with pH 8.1 with small amounts of CO2-saturated seawater, attained through bubbling pure CO2 into the ltered seawater for at least 24 h, to reach the desired pH (7.6) Schulz et al. 2013;Nardi et al. 2017) To ensure steadiness for the duration of the experiment, the pH of each level in the experimental aquaria was measured three times daily before and after water exchange using a pH meter (HQ 40d, Hach) calibrated with standard buffers. Before adding into the aquaria, seawater was reached to targeted temperature and seawater temperatures in raised up temperature was kept by aquarium heather in each experimental aquaria.
Phenanthrene (PhE) (98% purity, Sigma-Aldrich) was dissolved in acetone and then added to each experimental exposure aquaria (PhE, A-PhE, W-PhE, A-W-PhE ), to reach a nal nominal the PhE concentration of 0.8 ng.L − 1 and nal acetone concentration was lower than of 0.01% of the aquaria volume. Temperatures were measured three times daily with a mercury thermometer. Water was exchanged every day, and oysters were fed 12 h before renewing the water with a commercial algae concentration of cheatocerous. No mortalities were observed in any of the control and treated groups after 28 days.

Phenanthrene Analysis In Seawater And Oysters
After 24 h, 48h and 28 days of exposure to increased temperature, decreased pH and PHE, ve animals from each experimental tank were randomly selected for PhE measurement in chosen soft tissues of the animals. Five oysters from each treatment aquarium were sacri ed and gills, digestive gland (D.g), mantle and adductor muscle were instantly frozen in liquid nitrogen and then individually kept at -80 ºC for phenanthrene content.
In order to validate the phenanthrene concentration variations in the exposure aquaria during the experiment, 1 L of seawater was collected from each treatment immediately after adding the initial phenanthrene and before exchanging the seawater. One liter seawater sample was taken in separotory funnel and extracted by typical liquid-liquid extraction with dichloromethane at volumes of 50, 30, and 20 ml. The extract was condensed to nearly 5 mL by a rotary evaporator. Under mild ow of puri ed nitrogen gas, the condensed extract volume was compressed and then, the extract was redisssolved into 1 ml of acetonitrile.
Phenanthrene extraction from tissue oyster samples were carried out using ultrasonication procedure Then, each prepared sample was powdered and extracted by ultrasonic bath using 50 ml mixture hexaneacetone (1:1 v/v). To eliminate sulfuric compounds in the sample, approximately 5 g activated Cu powder was put into the extract and left for 24h. After that, the extracts were let to sediment and solvent layer was ltered via a Whatman 41 lter paper containing anhydrous sodium sulphate. Rotary evaporator was employed for condensing the solvent extract to get approximately 2 mL. Subsequently, the analyte was concentrated to 0.5 ml under gentle N2 ow and then, it was dissolved in 1mL acetonitrile. To measure the analyte of interest, the extract was injected into HPLC with uorescence detector (Hodgeson et al. 1990). PhE was identi ed via the retention time comparison with standard solution and analytical procedures were examined processing bland and reference samples (Mussel Tissue Standard Reference Material GBW0871, EERC-CAS).

Statistical Analyses
For the PhE concentrations, Shapiro-Wilks test was carried out for Normality and homogeneity of variances assumptions were checked by Bartlett's test. When necessary, data were normalized using the logarithmic transformation. Generalized linear models (GLM) analysis was conducted to determine signi cant differences between group treatments. Levene's and Shapiro-Wilk tests were performed to verify homogeneity of variance and residuals normality respectively. investigative variables or factors were generally used including Temperature (T) in 2 levels: 24 ºC, 28 ºC, pH (pH) in 2 levels: 8.1 unit, 7.5 unit, time sampling (time) in 3 levels: 24 h, 48 h and 28 days and organ tissue sampled (Tissue) in 4 levels: Gills, Digestive gland, Muscle and Mantle, according to phenanthrene bioaccumulation as a dependent variable. The Akaike Information Criterion (AIC), a prevalent indicator, was performed to obtain best model selection proper for our data that equilibrates model complication with model quality of tness (Quinn and Keough 2002). Parameters that did not affect data difference were eliminated and thus, models were simpli ed. Gaussian family was used to t the data. To con rm nal models, model residuals were controlled for homogeneity of variances, independence and leverage. Finally, to provide F and p values for factors with more than two levels, ANOVA test was performed. All statistical analyses were carried out for a signi cance level of 0.05, on R studio software (version 1.3.1093). To visualize the differences among various treatments and tissues, GraphPad Prism software (version 8.0.1) was applied.

Results
After 28 days of experimental exposure, no mortality was recorded in any exposure aquarium. The PhE concentration at different treatments were calculated in water of each aquaria. The concentrations of PhE in water retained at contaminant condition (PhE), hypercapnia condition and PhE, warming condition and PhE + hypercapnia + warming conditions were, respectively, 60. Bioaccumulation of PhE showed signi cant differences between different tissues (GLM, t= -9.311, p < 0.001) which revealed tissue-speci c trend (ANOVA F test, F = 37.54, p < 0.001). PhE concentration was higher in the digestive gland (D.g& Mantle/ D.g & Muscle p < 0.001 GLM analysis Table 2) and muscle tissue showed lower PhE concentration (D.g & Muscle/ Gill& mantle p < 0.001 GLM analysis Table 2, Fig. 1.B). Phenanthrene concentrations in gill tissue revealed signi cant differences between pH levels (t=-3.5, p < 0.01 GLM analysis in Table 3, ANOVA F test, F = 12.22, p < 0.01) and temperature levels (t = 11.52, p < 0.001, GLM analysis in Table 3, ANOVA F test, F = 132.72, p < 0.001).
PhE accumulation increased in oysters gill in the time of exposure for 24, 48 h and 28 days to 0.8 ng.L − 1 of phenanthrene (Time1&Time2/Time1&Time3/Time2&Time3, GLM analysis, Table 3). As seen in Table  3, signi cant additive effect on phenanthrene bioaccumulation between increasing temperature and decreasing pH interaction was not detected. Regarding the results, there was no signi cant interactive effect on bioaccumulation when both stressors and the time of exposure combined. In other words, isolated stressors caused to increase and affect PhE concentrations in gill tissue.  Table 4, ANOVA F test, F = 8.56, p < 0.01, for pH, F = 106.3, p < 0.001 for temperature). It follows from our results ( Table 4) that the phenanthrene bioaccumulation in P. radiata digestive gland considerably increased over the exposure time (Table 4). At the end of the experiment, the bioaccumulated PhE was higher by 2-fold than at the rst of the experiment. A signi cant extra effect between rising temperature and the exposure time was observed, leading to higher PhE accumulation in the tissue (Temp28°C & time2, t = 3.14, p < 0.01, GLM analysis in Table 4, ANOVA F test, F = 6.006, p < 0.01). The impact of elevated CO2, warming and the exposure of time on phenanthrene accumulation in the oyster mantle is shown in Table 5. PhE concentrations were differently affected by various stressors in the tissue (GLM analysis in Table 5). The contaminant reported higher levels in the exposure time 3 (28 days, t = 22.32, p < 0.001 and t = 28, p < 0.001, GLM analysis in Table 5, ANOVA F test, F = 135, p < 0.001) and lower in the interaction of pH 8.1 time 1(24 h, positively) and pH 8.1 time2 (48 h, negatively) (t = 0.13 p < 0.001, t= -0.13 p < 0.001 respectively. GLM analysis in Table 5), but these were not signi cant. Looking at Table 5, PhE concentration was positively affected by pH 7.6 with the exposure time 3(28 days) and temperature 28ºC with the exposure time 3(28 days) (t = 3.76, t = 4.22 p < 0.001 GLM analysis in Table 5). Nevertheless, pH 8.1 and the exposure time 3(28 days) interactively was negatively affected showing an antagonistic relation within the mantle (t=-3.76, p < 0.01, GLM analysis in Table 5). The phenanthrene concentrations in muscle tissue were also signi cantly affected by temperature, pH and the exposure time between treatment exposures (Table 6). According the ndings, P.radiata muscle analysis revealed higher effect on PhE concentration in the exposure time 3 (28days, GLM analysis in Table 3) in comparison to other times and stressors in all treatments. Lower effect on PhE concentration was seen in normal condition (pH = 8.1, temp = 24C) in time 2 (48h) but it was not signi cant (ANOVA F test, F = 0.039, p = 0.84, t=-0.11, p = 0.9, GLM analysis in Table 6). presented new evidence that future predictions of elevated pCO2 and increased temperature in Persian Gulf waters can modify pollutant accumulation of phenanthrene in the pearl oyster P.radiata.
Our ndings revealed that the P. radiata were able to concentrate low molecular weight PhE from seawater. The reduction in phenanthrene concentrations in water after 24 h is probable to display the bioavailability and uptake consequences of experiments revealed higher signi cant concentration of phenanthrene in the gills digestive gland of p. radiata exposed to this contaminant than mantle and muscle. Our ndings showed no difference of phenanthrene uptake in between digestive gland and gills of oysters exposed to the chemical at lower pH and/or higher temperature, as well as between mantle and muscle. These ndings revealed that the impact of enhancing temperature and pCO2 on phenanthrene bioconcentration cannot be popularized, depending on biota and the PAHs compounds, hence it is di cult to forecast only from the chemical model. higher than two other tissues (mantle, muscle) during the exposure time; there was no statistically signi cant differences between digestive gland and gills with mantle and muscle. Bioaccumulation of organic pollutants in aquatic organisms is a balance between principally passive processes of uptake and depuration and elimination of contaminants through biotransformation pathways ( Livingstone 1991). Nonetheless, PAHs metabolism are signi cantly less than uptake rates in mollusks, resulting in strong bioaccumulation (Livingstone 1998;Liu et al. 2014).
Since oysters are lter-feeder and sessile creatures frequently exposed to PAH compounds in their habitat In the present study, GLM analysis in different organs revealed the time-dependent pattern of PhE uptake.
time-dependent pattern of B[a]P uptake was con rmed in scallop's ovary (Tian et al. 2014). It is plausible to assume that this pattern could be proceeded to saturate the organs completely during the experiment or feasibly the PhE levels were saturated in all tissues of P.radiata before the exposure time has been nished. Similar saturation patterns were also found for other marine biota exposed to various contaminants and temperatures. Oyster Crassostrea brasiliana exposed to phenanthrene at 18, 24 and 32°C (Lima et al. 2018), mussels M. galloprovincialis exposed to cadmium at 20 and 25°C (Nardi et al. 2017), and sh P. avescens exposed to Ni at three temperature levels of 9, 20 and 28°C (Grasset et al. 2016) presented similar elevated levels of contaminants in high temperatures during the time of experiments.

Conclusion
In present study, chronic exposure of pearl oysters to conditions mimicking the climate changes which may occur in 50-100 years later and accomplished by phenanthrene as a contaminant model, is able to affect the bioaccumulation levels of PhE in oyster soft tissues con rming pollutant accumulation would be in uenced by increasing temperature and PCO2. In our experimental conditions oyster tissues showed signi cant PhE accumulation, partially at least, in uenced by the increased and modulated metabolic activity under elevated seawater temperature and PCO2 in isolation and combination in the marine organism. In particular, bioaccumulation changes appeared to be dependent on duration of exposure to PhE and the condition of the experiments. Thus, uptake of components such as PhE can be different in a species and tissue-speci c aspect, emphasizing the need of further investigation to explain the effect of several stressors, principally in species with high commercial or ecological importance.

Declarations
Acknowledgments: the authors acknowledge ENG Hossein Rameshi for providing pearl oyster for the trail and Shrimp Research Institute in Busher province, Iran for providing the place for conducting the experiment.
Ethics approval and consent to participate: All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Consent to Participate: not applicable Consent for publication: not applicable Availability of data and material: Raw data were obtained by authors derived data supporting the nding of this study is available from the corresponding author on request.
Competing interest: We (all authors) declare that we have no signi cant competing nancial, professional, or personal interest that might have in uenced the performance of the work described in this manuscript.