Warming coupled with elevated pCO2 modulates microplastic inhibition in a commercial red alga Pyropia haitanensis

1、Introduction：

Plastics have become ubiquitous in modern society due to their ease of manufacture, low cost, and versatility (Geyer et al., 2017). Among the most widely used polymers are polyethylene (PE), polypropylene (PP), and polyvinyl chloride (PVC), which are found in a vast array of con- sumer products and industrial applications (Zhang et al., 2022). In 2022, global plastic production surpassed 400.3 million tons, with single-use plastics comprising approximately two-thirds of this total output (Pilapitiya and Ratnayake, 2024). The rapid accumulation and disposal of these materials have led to unprecedented levels of plastic waste in the environment. Notably, an estimated 1.15–2.41 million tons of plastic waste are transported annually to the oceans via rivers, highlighting the

extensive scope of plastic pollution in aquatic systems (Lebreton et al.,2017). Plastics exposed to the marine environment can be decomposed through physical fragmentation, photodegradation, or thermal oxida-tion into micro-particles, which were smaller than 5 mm in diameter (Hidalgo-Ruz et al., 2012). Microplastics exhibited morphological accumulation in macroalgae, with a previous study identifying five modalities: wrapping, attachment, embedment, entanglement and entrapment (Li et al., 2022). As a rising recognized class of environ-mental contaminants, microplastics have garnered significant research interest due to their potential to induce complex ecological impacts.Previous study indicates that Polyamide (PA) fibers have generally exhibited negligible effects on growth rate and photosynthetic activity even as the contents of malondialdehyde of Caulerpa lentillifera and

Gracilaria tenuistipitata (Li et al., 2023). In contrast, the polystyrene (PS) particles at high concentrations of 100 mg L_ 1 significantly inhibits growth and photosynthetic oxygen evolution while increasing oxidative damage, as shown by elevated malondialdehyde (MDA) content and decreased EPS in both seaweeds (Li et al., 2023). Similarly, a high concentration (100 mg L_ 1) of PE showed significantly decreased photosynthetic activity as well as growth in Ulva prolifera by shading the light and inhibiting the exchange of nutrients, CO2 and O2 (Feng et al., 2020). Despite identical exposure conditions, sensitivity to microplastics can vary substantially among algal genera. Greater concentrations of MPs led to significant reductions in both growth and net photosynthesis by increasing the reactive oxygen species (ROS) in Chondrus sp., whereas Grateloupia turuturu was not markedly affected under the same condi- tions due to the different surface characteristics (Jung et al., 2023). Emerging evidence also suggests that the effects of microplastics can be modulated by other environmental factors. Under elevated temperature conditions, microplastic exposure can enhance the expression of genes associated with carbon and nitrogen metabolism in Phaeodactylum tri- cornutum, thereby promoting nitrogen utilization efficiency (Sun et al., 2023). Therefore, understanding the ecological consequences of microplastics requires considering their interactions with co-occurring environmental stressors.
Among anthropogenic climate change, ocean acidification and warming have emerged as critical global challenges driven by increasing atmospheric CO2 concentrations. Since the Industrial Revolution, CO₂ emissions have sharply increased from human activities, primarily the burning of fossil fuels and land-use changes (Feely et al., 2009). The ocean, serving as Earth's largest carbon sink, absorbs approximately one- third of anthropogenic carbon dioxide emissions, playing a vital role in regulating global climate (Sabine et al., 2004). This uptake of CO₂ leads to a decrease in seawater pH, a process known as ocean acidification (OA) (Caldeira and Wickett, 2003). According to projections by the Intergovernmental Panel on Climate Change (IPCC), atmospheric CO₂ concentrations could reach 1000 ppm by 2100, resulting in roughly a 0.4-unit decline in average pH of global ocean (Gruber et al., 2012). This further drop in pH and calcium carbonate availability impacts critical processes such as photosynthesis, nitrogen fixation, and calcification (Doney et al., 2009). Compounding the challenge, rising atmospheric CO₂ is also the principal driver of global warming. Over the past half century, ocean surface temperatures have risen by about 0.1 O C per decade, which would further increase by 1.8–4 O C by the end of this century (Gattuso et al., 2015; Wu et al., 2018). Ocean acidification and warming together challenge the physiological adaptability of founda- tional marine organisms, particularly primary producers such as phytoplankton and macroalgae, and thereby influence primary pro- duction and carbon cycling (Hutchins and Fu, 2017).
Evidence suggests that ocean warming increases the relative growth rate, content of chlorophyll a and carotenoid in Neopyropia yezoensis, while inhibiting the accumulation of soluble carbohydrates (Wu et al., 2024). Likewise, Ocean acidification can stimulate the growth of U. compressa, but it may simultaneously weaken its antioxidant defenses and reduce nutritional value (Vinuganesh et al., 2022). Beyond single- factor responses, ocean warming and acidification can also interact, producing coupled effects on algae. At 20 O C, U. fasciata showed significantly increased growth and photosynthesis, yet acidification at this temperature intensified photoinhibition and declined growth. Moreover, Sargassum horneri exhibited higher growth and DOC release at low temperature (15 O C) than at high temperature (20 O C), and acidification at the lower temperature further promoted its growth during the early stage (Wang et al., 2024). In Cystoseira tamariscifolia, acidification alone enhanced photosynthetic rates, antioxidant capacity and phenolic content, but warming counteracted these benefits and only the combined treatment of warming and acidification increased biomass in nutrient-replete thalli (Celis-Pl et al., 2017). Even sluggish alter- ations of in the marine environment have the potential to propagate through food webs, disrupting ecosystem functioning and stability.

Hence, studying multi-stressor scenarios is necessary to realistically evaluate the impacts of human activities on aquatic life.
Pyropia haitanensis, an economically valuable macroalgae, is exten- sively cultivated along the Chinese coasts, including major aquaculture areas in Fujian, Zhejiang, and Jiangsu provinces, and plays a funda- mental role in local economies and food security (Jiang et al., 2018). Its high commercial value and increasing demand have brought research interest to both its physiological resilience and its potential vulnerabil- ities under environmental change. Despite its prevalence in coastal aquaculture, most current studies on the effects of microplastics have focused on microalgae, with few addressing macroalgae (Su et al., 2023; Wang et al., 2023). Moreover, more attention has been given to the single effect of microplastics, leaving the combined effects with co- occurring environmental stressors largely unexplored.
Therefore, this study employs P. haitanensis as a representative macroalga to examine physiological responses to combined exposure to microplastics, ocean acidification, and warming under controlled labo- ratory conditions. By assessing growth, photosynthetic efficiency and key biochemical components (pigments, carbohydrates, and proteins), this research aims to uncover the underlying response mechanisms of P. haitanensis to these stressors. The findings will advance mechanistic understanding of macroalgal ecophysiology in changing oceans and inform conservation strategies for coastal ecosystem health amid esca- lating environmental pressures.

2、Materials and methods：

2.1. Experimental materials
The samples of Pyropia haitanensis was collected on November 5, 2024 from cultivation area of Gaogong island in Jiangsu Province (119.53OE; 34.91ON), China, and transported to the laboratory in a 4 OC cooling box within 2 h. Healthy thalli were selected and cleaned several times using autoclaved seawater to remove sediments. The thalli were pre-cultured in 500 mL balloon flasks containing sterile seawater (salinity 30) enriched with von Stosch's enrichment (VSE) Medium. The seawater medium were changed every day and aerated continuously by fresh outdoor air. The flasks were cultured in an illumination incubator (GXZ-500B, Ningbo, China) at 20 O C, under 80 μmol photons m_ 2 s_ 1 light intensity with a 12 L:12 D photoperiod for 3 days. The thalli were then cut into 1 cm x 1 cm squares for subsequent experiments.
Polyvinyl chloride (PVC) microplastics with a particle size of 1 μm were purchased from Dongguan Huachuang Materials Co. Ltd. (Guangzhou, China). To prepare the stock solutions of PVC microplastics (25 g L_ 1), an appropriate amount of microplastics was dissolved in pure water with sonication for 30 min and stored at room temperature (Xu et al., 2024).
2.2. Experimental design
After the pre-culture, algal segments (0.05 g fresh weight, FW) were selected randomly and placed in balloon flasks with 500 mL sterile artificial seawater supplemented with VSE medium. Based on pro- jections of Representative Concentration Pathway (RCP) 8.5 by the end of century, two temperature levels (LT: 20 O C; HT: 24 O C) and two CO2 concentrations (LC: 418 μatm; HC: 1000 μatm) were set in this study. Meanwhile, five microplastic concentration gradients (0.025, 2.5, 25, 50 and 100 mg L_ 1) were prepared to evaluate the influence of micro- plastic. Notably, the initial microplastic concentration (0.025 mg L_ 1) was the baseline value in coastal seawater (Feng et al., 2020; Green et al., 2017). The LC treatment was achieved by pumping outdoor ambient air, while the elevated CO2concentration was generated using a customized CO2 enricher (CE100D, Wuhan, China) by mixing pure CO2 with outdoor air. The target CO2 concentrations were continuously aerated with 0.45-μm disposable filter to minimize the influence of airborne microplastics during the experiment. The medium was

replaced every 3 days, and was supplemented with microplastics to the target concentrations, pre-equilibrated with target CO2 levels at respective temperatures. The carbonate chemistry parameters in the cultures were shown in Table S1. All treatments were performed in three replicates for 7 days.

2.3. Growth measurement
At the beginning and end of the experiment, the fresh weight was measured after using absorbent paper to remove excess water on the surface of P. haitanensis. The relative growth rate (RGR) of P. haitanensis was calculated using the following formula:
RGR (%day_ 1 ) = [Ln(Wt /W0) ]/t × 100%
where W0 and Wt represent the fresh weight of thalli at the initial time and t days, respectively.

2.4. Chlorophyll fluorescence parameters
During the middle of the photoperiod within 2 h, the maximum quantum yield (Fv/Fm) of the algae were measured using a chlorophyll fluorometer (Aquapen AP100, Czech). The saturation pulse was set at 3000 μmol photons m_ 2 s_ 1, and lasted for 800 ms. The thalli were dark- adapted for 15 min before measuring the Fv/Fm, with the actinic light intensity set equal to or close to the cultivation light intensity. The Fv/Fm was calculated according to the equation:
Fv /Fm = (Fm _ F0)/Fm
where the F0and Fm were minimum chlorophyll fluorescence and the instant maximum fluorescence after dark-adaption.

2.5. Pigment content measurement
Approximately 0.02 g of fresh thalli was weighted for Chlorophyll a (Chl a) and carotenoids (Car) measurement. The thalli were cut into small fragments and placed in 2 mL of anhydrous methanol. After extraction overnight in darkness at 4 ◦ C, the supernatant after centri- fugation at 5000 ×g for 10 min was measured at 470, 653 and 666 nm for absorbance by a UV spectrophotometer (UV-1800, Shimadzu, Japan). The photosynthetic pigment content was calculated using the following formulae (Wellburn, 1994):
Chl a (mg g_ 1 FW) = (15.65 × A666 _ 7.53 × A653) × V/W/1000
Car (mg g_ 1 FW) = (1000 × A470 + 1403.57 × A666 _ 3473.87 × A653) /211 × V/W/1000
where the V was the volume of anhydrous methanol, and W repre- sented the fresh weight of thalli.

2.6. Phycoerythrin and phycocyanin content measurement
Approximately 0.01 g FW of thalli were ground in a pre-cooled mortar (kept on ice during grinding). Then, 2 mL phosphate buffer (0.1 M, pH 6.8) was added, and the mixture was centrifuged at 5000 ×g for 15 min (Eppendorf 5417R, Germany). The supernatant was scanned at 455, 564, 593, 618 and 645 nm to determine the phycobiliprotein contents. The phycoerythrin (PE) and phycocyanin (PC) contents were calculated as follows (Beer and Eshel, 1985):
PE (mg g_ 1 FW) = [(A564 _ A592) _ (A455 _ A592) × 0.2 ] × 0.12 × V/W PC (mg g_ 1 FW) = [(A618 _ A645) _ (A592 _ A645) × 0.51 ] × 0.51 × V/W

2.7. Soluble carbohydrate and protein content measurement
The soluble carbohydrate content of thalli was determined using the anthrone sulfuric acid colorimetric method (Deriaz, 1961). Briefly, 0.05 g FW of samples were cut into small pieces and homogenized with 5 mL phosphate buffer and boiled for 1 h. After centrifugation at 5000 ×g for 10 min, 1 mL supernatant was mixed with 4 mL anthrone sulfuric acid solution and boiled for another 10 min. The absorbance of mixture was measured at 620 nm after rapidly cooling to room temperature. The soluble carbohydrate content was calculated using a standard curve.
The Bradford method was used to determine the soluble protein content (Bradford, 1976). Approximately 0.01 g FW of P. haitanensis were rapidly frozen in liquid nitrogen and then ground in a pre-cooled mortar with 2 mL phosphate buffer (kept on ice during grinding). After centrifugation at 5000 ×g for 15 min, the supernatant was reacted with Coomassie Brilliant Blue G-250 for chromogenic reaction, and then was recorded at 595 nm. The soluble protein content was calculated using a standard curve.
2.8. Data analysis
Experimental data are presented as mean ± SD triplicate analyzed using Origin 2024 software (OriginLab Corporation, Northampton, MA, USA). Three-way ANOVA was performed to determine the main and interactive effects of temperature level, CO2 concentration, microplastic concentration, and their interactions among treatments. One-way ANOVA and Tukey's test were employed to analyze the statistical dif- ferences among different treatments with a significance level of 0.05 (p < 0.05). Prior to analysis, all data were tested for homogeneity of variance (Levene test) and normality (Shapiro-Wilk test).