Transcriptomic and Physiological Responses of Chlorella pyrenoidosa during Exposure to 17α-Ethinylestradiol

17α-ethinylestradiol (17α-EE2) is frequently detected in water bodies due to its use being widespread in the treatment of prostate and breast cancer and in the control of alopecia, posing a threat to humans and aquatic organisms. However, studies on its toxicity to Chlorella pyrenoidosa have been limited to date. This study investigated the effects of 17α-EE2 on the growth, photosynthetic activity, and antioxidant system of C. pyrenoidosa and revealed related molecular changes using transcriptomic analysis. The cell density of algae was inhibited in the presence of 17α-EE2, and cell morphology was also altered. Photosynthetics were damaged, while reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) content increased. Further transcriptomic analysis revealed that the pathways of photosynthesis and DNA replication were affected at three concentrations of 17α-EE2, but several specific pathways exhibited various behaviors at different concentrations. Significant changes in differentially expressed genes and their enrichment pathways showed that the low-concentration group was predominantly impaired in photosynthesis, while the higher-concentration groups were biased towards oxidative and DNA damage. This study provides a better understanding of the cellular and molecular variations of microalgae under 17α-EE2 exposure, contributing to the environmental risk assessment of such hazardous pollutants on aquatic organisms.


Introduction
Endocrine-disrupting compounds (EDCs) have been drawn much attention in the past few decades due to their endocrine-disrupting effects on humans and wildlife even at low levels [1,2]. Estrogens are a major group of EDCs that are widespread in nature and potentially harmful, causing the feminization of aquatic organisms and affecting the development of their fertilized eggs [3][4][5]. The most common synthetic estrogen is 17αethinylestradiol (17α-EE 2 ) and it has been used as a treatment for prostate and breast cancer, chronic hemospermia, and the induction of labor since 1938 [6]. Consumption of 17α-EE 2 is huge worldwide and it has been detected from time to time in aquatic environments in some countries in Southeast Asia, as well as in China, Brazil, Argentina, Kuwait, and Portugal [6]. Compared with natural estrogens such as estradiol (E 2 ), estrone (E 1 ), and 17β-estradiol (β-E 2 ), 17α-EE 2 was shown to possess more persistence and a high estrogenic activity [7]. The half-life of 17α-EE 2 was reported to be as long as 81 days [8]. Moreover, the harm caused by 17α-EE 2 is transmitted through the food chain and ultimately damages human health [9,10]. As a result, it was included in the EU Resolution 2015/495/EU watchlist as a priority EDC contaminant for regulation [11,12] and there is a strong need to study the biological risks and ecotoxicological effects of 17α-EE 2 .
Microalgae belong to the trophic level at the bottom end of the aquatic ecosystem and are more sensitive to pollutants, acting as important indicator species for evaluating toxicity

Characterization by SEM and FTIR Spectroscopy
As shown in the SEM results, the control group exhibited an intact cell morpholog with no damage to the cell wall in the 96 h period ( Figure 1B). In contrast, within 96 h 17α-EE2 stress, the algal cells appeared to be agglomerated, and the integrity of the ce membrane was disrupted, resulting in the outflow of the cell contents ( Figure 1C).
FTIR spectroscopy analysis was used to determine the dynamic relative changes the levels of proteins, carbohydrates, lipids, and other biomolecules in C. pyrenoidosa cel exposed to 17α-EE2 ( Figure 1D). At 96 h, the transmittance of the peaks at 3309 cm −1 , 296 cm −1 , and 1633 cm −1 was greater in the treatment group than in the CK group, with th peak at 3309 cm −1 representing the stretching vibration of the N-H bond in proteins an

Characterization by SEM and FTIR Spectroscopy
As shown in the SEM results, the control group exhibited an intact cell morphology with no damage to the cell wall in the 96 h period ( Figure 1B). In contrast, within 96 h of 17α-EE 2 stress, the algal cells appeared to be agglomerated, and the integrity of the cell membrane was disrupted, resulting in the outflow of the cell contents ( Figure 1C).
FTIR spectroscopy analysis was used to determine the dynamic relative changes in the levels of proteins, carbohydrates, lipids, and other biomolecules in C. pyrenoidosa cells exposed to 17α-EE 2 ( Figure 1D). At 96 h, the transmittance of the peaks at 3309 cm −1 , 2960 cm −1 , and 1633 cm −1 was greater in the treatment group than in the CK group, with the peak at 3309 cm −1 representing the stretching vibration of the N-H bond in proteins and the O-H bond in carbohydrates. The peak at 2960 cm −1 represents the -CH 3 antisymmetric stretching vibration in proteins, carbohydrates, and lipids, and that at 1633 cm −1 represents the amide I of the β-sheets, indicating a decrease in the levels of biomolecules, such as proteins and lipids, and the MD group decreased more compared with other treatment groups. The peak for the C=O stretching vibration in the protein carboxylic acid group appeared at 1456 cm −1 , and the intensity of the peak was higher in all treatment groups than in the CK group, but it was highest in the MD group than in the other groups. The peak at 1243 cm −1 was mainly derived from the nonvibrational stretching vibrations of the P=O group of phospholipids, with a decrease in peak intensity compared to the CK group indicating a decrease in intracellular phospholipid content, with the greatest decrease in the MD group. The absorption peak associated with the sugar skeleton at 1157 cm −1 had the highest passage rate in the CK group, while the HD group had a higher transmission than the other treatment groups, indicating that sugar synthesis in the algal cells was affected.

Effects on Photosynthesis
The chlorophyll a and b and carotenoid levels of C. pyrenoidosa were affected by exposure to 17α-EE 2 for 96 h, as shown in Figure 2A-C. There was no obvious pattern of change in the chlorophyll content relative to the CK group within 24 h, but the chlorophyll a and b levels both began to decrease between 48 and 72 h, with both being lower than the level in the CK group, and the inhibition increased with increasing 17α-EE 2 concentrations. The higher the treatment concentration was, the lower the chlorophyll content. There were no significant differences in carotenoid content up to 48 h. From 72 h onwards, there was a significant decrease in all treatment groups. The carotenoid content decreased by 10.53%, 13.47%, and 17.98%, respectively, at 72 h in comparison with the CK group, while the decreases were 20.83%, 26.49%, and 29.59% at 96 h of treatment, respectively.
The effects of different concentrations of 17α-EE 2 on the photosynthetic system of C. pyrenoidosa during exposure were investigated in this paper, and the results are shown in Figure 3. The rapid chlorophyll fluorescence-induction kinetics indicated the change in fluorescence from the O to P points, reflecting changes in the primary photochemical reaction of photosystem II (PSII) and the structure and state of the photosynthetic machinery. The chlorophyll fluorescence parameters mainly reflect the absorption, conversion, utilization, and distribution of light energy in plants and can reflect the photochemical reaction activity in algal cells and their self-protection ability in real time (Table S1). The chlorophyll fluorescence curves (OJIP curves) of the leaves and their parameters changed significantly after 96 h of treatment with different concentrations of 17α-EE 2 (Table S2), indicating that 17α-EE 2 severely disrupted the photosynthetic organ structure and function of the leaves. K-steps occurred at both 72 h and 96 h, with even more inflection points occurring in this period. The Fv/Fm of C. pyrenoidosa was inhibited irrespective of 17α-EE 2 concentration and exposure time. The photosynthetic activity of the algae decreased with increasing concentrations of 17α-EE 2 , and the higher the concentration was, the more rapidly the activity decreased. Different concentrations of 17α-EE 2 -stress significantly reduced the corresponding photosynthetic parameters (ABS/RC, RC/CS 0 , ET 0 /ABS, ψ O , RC/CS 0 , DI 0 /RC, ET 0 /RC, and TR 0 /RC) and inhibited the photosynthetic activity of C. pyrenoidosa over a 96 h period.
2.4. Variation in the MDA Levels, SOD Activity, and ROS Generation in Response to 17α-EE 2 MDA, as one of the end products of cellular lipid peroxidation, is often used to characterize whether lipid peroxidation occurs in cell membranes under environmental stress. Figure 4A shows the changes in the MDA content of C. pyrenoidosa cells in response to different 17α-EE 2 exposure concentrations and durations. The results showed that the MDA content of the CK group remained stable and relatively low over time. The MDA content of algal cells in the treated groups increased with the addition of 17α-EE 2 from 48 h onwards. At 72 h, the MDA content of algal cells in the LD, MD, and HD treatment groups increased by 37.57%, 43.30%, and 57.55%, respectively, compared to that of the CK group under the effect of 17α-EE 2 . After 72 h of exposure, the percentage increase in MDA content decreased over time in the LD group, while it continued to increase in the MD and HD groups. The MDA content increased by 47.26% and 63.20% after 96 h of exposure in the MD and HD groups, respectively, compared to the CK group; however, it only increased by 30.43% in the LD group. The effects of different concentrations of 17α-EE2 on the photosynthetic system of C. pyrenoidosa during exposure were investigated in this paper, and the results are shown in

Variation in the MDA Levels, SOD Activity, and ROS Generation in Response to 17α-EE2
MDA, as one of the end products of cellular lipid peroxidation, is often used to characterize whether lipid peroxidation occurs in cell membranes under environmental stress. Figure 4A shows the changes in the MDA content of C. pyrenoidosa cells in response to different 17α-EE2 exposure concentrations and durations. The results showed that the MDA content of the CK group remained stable and relatively low over time. The MDA content of algal cells in the treated groups increased with the addition of 17α-EE2 from 48 h onwards. At 72 h, the MDA content of algal cells in the LD, MD, and HD treatment groups increased by 37.57%, 43.30%, and 57.55%, respectively, compared to that of the CK group under the effect of 17α-EE2. After 72 h of exposure, the percentage increase in MDA The intracellular antioxidant enzyme system was able to regulate intracellular reactive oxygen radicals and reduce peroxidative damage to cells. In this study, changes in SOD activity in C. pyrenoidosa were examined in the presence of 17α-EE 2 ( Figure 4B). The SOD activity in algal cells in the CK group remained basically stable, and the SOD content in algal cells at different time points was higher at different concentrations of 17α-EE 2 than that in the CK group. At 24 h, the SOD content increased significantly in the MD and HD groups. The SOD activity continued to increase in all three groups from 48 to 96 h, with the highest value occurring at 72 h when the activity in the HD group increased by 0.56 U/µg protein compared to that in the CK group. content decreased over time in the LD group, while it continued to increase in the MD and HD groups. The MDA content increased by 47.26% and 63.20% after 96 h of exposure in the MD and HD groups, respectively, compared to the CK group; however, it only increased by 30.43% in the LD group. The intracellular antioxidant enzyme system was able to regulate intracellular reactive oxygen radicals and reduce peroxidative damage to cells. In this study, changes in SOD activity in C. pyrenoidosa were examined in the presence of 17α-EE2 ( Figure 4B). The SOD activity in algal cells in the CK group remained basically stable, and the SOD content in algal cells at different time points was higher at different concentrations of 17α-EE2 than that in the CK group. At 24 h, the SOD content increased significantly in the MD and HD groups. The SOD activity continued to increase in all three groups from 48 to 96 h, with the highest value occurring at 72 h when the activity in the HD group increased by 0.56 U/μg protein compared to that in the CK group. DCFH is oxidized at high fluorescence intensities and is itself nonfluorescent. Intracellular ROS and other peroxides cause DCF production, so ROS production can be measured by determining the fluorescent DCF level. The increase in ROS and MDA levels was not consistent. There was a greater degree of ROS production at all concentrations, with the stress increasing over time, but the maximum ROS stress level remained at approximately 1.36 times that of the control ( Figure 4C).

Differentially Expressed Genes
A total of 24,241 transcripts were detected in C. pyrenoidosa. The correlation among the three replicate-gene-expression patterns of each group was significant ( Figure 5A). Principal component analysis (PCA) showed that the gene-expression clusters in the control, LD, MD, and HD groups were different ( Figure 5B), which was consistent with the results of the heatmap assay ( Figure 5C). We identified 346 (126 up-and 220 down-regulated), 8 of 17 2121 (793 up-and 1328 down-regulated), and 3518 (1293 up-and 2225 down-regulated) differentially expressed genes (DEGs) in each group after low-, medium-, and high-level exposure to 17α-EE 2 , respectively ( Figure 6A). The Venn diagram shows the number of identical and unique DEGs between different concentration groups ( Figure 6B), and circle plots show the gene abundance ( Figure 6C). The number of DEGs increased significantly with increasing stress concentration, with the HD group having the highest number of DEGs. To validate the RNA-Seq results, the expression levels of three DEGs from different pathways were measured by qRT-PCR (Table S3). The results showed that the direction and size of the changes in the mRNA expression of these genes were consistent with the results of the transcriptome analysis ( Figure S1). the stress increasing over time, but the maximum ROS stress level remained at approximately 1.36 times that of the control ( Figure 4C).

Differentially Expressed Genes
A total of 24,241 transcripts were detected in C. pyrenoidosa. The correlation among the three replicate-gene-expression patterns of each group was significant ( Figure 5A). Principal component analysis (PCA) showed that the gene-expression clusters in the control, LD, MD, and HD groups were different ( Figure 5B), which was consistent with the results of the heatmap assay ( Figure 5C). We identified 346 (126 up-and 220 down-regulated), 2121 (793 up-and 1328 down-regulated), and 3518 (1293 up-and 2225 down-regulated) differentially expressed genes (DEGs) in each group after low-, medium-, and highlevel exposure to 17α-EE2, respectively ( Figure 6A). The Venn diagram shows the number of identical and unique DEGs between different concentration groups ( Figure 6B), and circle plots show the gene abundance ( Figure 6C). The number of DEGs increased significantly with increasing stress concentration, with the HD group having the highest number of DEGs. To validate the RNA-Seq results, the expression levels of three DEGs from different pathways were measured by qRT-PCR (Table S3). The results showed that the direction and size of the changes in the mRNA expression of these genes were consistent with the results of the transcriptome analysis ( Figure S1).

Analysis of Transcriptional Changes
After extensive analysis of the transcriptome, changes in the expression of specific pathways and transcription factors were investigated. Upon combining the top 20 enriched pathways from the GO and KEGG analysis results ( Figure 6D,E), DNA synthesis and repair and photosynthesis were found to be the most significantly enriched pathways (with 109 and 88 down-regulated and seven and 11 up-regulated, respectively). The KEGG annotation of the photosynthesis-related metabolic pathways, and changes in gene expression in response to DNA damage are shown in Figure 7A,B. Genes related to photosynthesis (petJ, psaD, psbB, and psbI) and the carotenoid-regulated gene crtz were down-regulated (Table S4). While in the DNA replication pathway, genes such as mcm4 significantly down-regulated and rpa1D genes responsible for mismatch repair were significantly up-regulated. Several specific pathways exhibited various behaviors at different levels ( Figure S2). Ribosome biogenesis in eukaryotes, photosynthesis, and oxidative phosphorylation were the three most significantly enriched pathways (22 genes significantly down-regulated and six genes significantly up-regulated, p < 0.05) in the KEGG analysis of the LD group compared to the CK group (Figures S3-S5). The genes in the photosystem II pathways, psbA, psbB, psbC, and psbE were significantly down-regulated while psb28 was up-regulated. The down-regulated genes atpA, atpG, and cox1 were related to oxidative phosphorylation. When the MD group was compared with the CK group, pyrimidine metabolism, ribosome biogenesis in eukaryotes, porphyrin and chlorophyll metabolism, nitrogen metabolism, and DNA replication were the pathways that were found to have the most enriched and significant genes, with a total of 102 significant DEGs (p < 0.05) (Figures S6-S10). Changes in the expression of KEGG-annotated genes in nitrogen metabolism pathways showed that the down-regulated genes gdh2 and kin14n were related to the nitrogen cycle and ribosomal protein. In response to oxidative damage, the genes sodA and por were up-regulated. When the HD group was compared to the CK group, the most important pathways were DNA replication and mismatch repair and homologous recombination; 87 of the 89 DEGs were down-regulated, algal cell growth was severely inhibited, and the DNA damage was severe ( Figures S11-S13). The genes on the mismatch repair pathway, such as msh1, were up-regulated, while the others, such as exo1 and mus1, were significantly down-regulated.

Analysis of Transcriptional Changes
After extensive analysis of the transcriptome, changes in the expression of specific pathways and transcription factors were investigated. Upon combining the top 20 enriched pathways from the GO and KEGG analysis results ( Figure 6D,E), DNA synthesis and repair and photosynthesis were found to be the most significantly enriched pathways (with 109 and 88 down-regulated and seven and 11 up-regulated, respectively). The KEGG annotation of the photosynthesis-related metabolic pathways, and changes in gene expression in response to DNA damage are shown in Figure 7A,B. Genes related to pho-

Discussion
Exposure to 17α-EE 2 inhibits the growth of C. pyrenoidosa and disrupts the cell structure. The reduction in chlorophyll content in algal cells leads to less efficient photosynthesis in microalgae, which can lead to a lack of optical energy and thus the suppression of the cellular energy transfer and metabolism, which can ultimately inhibit algal growth [32]. Changes in the carotenoid content in algal cells exposed to 17α-EE 2 are among the mechanisms by which algae resist environmental stress; carotenoids are photoprotective during photosynthesis, and their biosynthesis is a direct indicator of photosynthetic capacity [24]. Carotenoids can act as antioxidants to mitigate oxidative damage [33]. These protective mechanisms allow C. pyrenoidosa to remain active after 17α-EE 2 stress; however, the rise in carotenoid levels only briefly counteracts the hormonal effects, after which they remain inhibited, leading to a reduction in photosynthetic activity. Carotenoids exert antioxidant activity by quenching the excessive intracellular accumulation of ROS, protecting against photodamage caused by quenching singlet oxygen and trilinear chlorophyll, and dissipating excess absorbed energy by reacting with excited chlorophyll molecules to reduce the extent of cellular lipid peroxidation [34]. The variation in the chlorophyll content was greater than that in the carotenoid content because of the higher sensitivity to oxidative damage and greater susceptibility to breakdown by ROS under environmental-stress conditions [35].
The chlorophyll fluorescence curves (OJIP curves) of C. pyrenoidosa and their parameters changed significantly after 1 h of treatment with different 17α-EE 2 levels, indicating that 17α-EE 2 severely disrupted the photosynthetic organ structure and function of the algal cells. Duress leads to the appearance of the K-step (300 µs), which is caused by the inhibition of the hydrolysis system and the inhibition of part of the receptor side prior to QA, during which it is the oxygen emission complex (OEC) that is injured, so the K-point can be used as a specific marker of OEC injury. Reduced photosynthetic quantum production due to the attenuation of the OEC state impairs photosynthesis in algal cells [36]. PSII is the first pigment protein complex in the electron transport chain in the chloroplast vesicle-like membrane, consisting of more than 20 protein subunits. It catalyzes the lightdriven cleavage of water and the oxidation of quinones [37]. In this study, the PSII activity (Fv/Fm) of the algal cultures decreased significantly with increasing concentrations of 17α-EE 2 , and decreases in parameters such as RC/CSo and ψ O suggested that inhibition led to the closure of PSII reaction centers [38]. At the same time, the fluorescence yield and photosynthetic electron transport were reduced, mainly due to oxidative damage caused by increased ROS levels in the body.
The production and accumulation of substances within microalgae cells are susceptible to environmental influences. Under stress conditions, cells are able to respond to adverse growth factors by altering the content of cellular macromolecules [39]. Exposure to 17α-EE 2induced oxidative stress was confirmed by increased levels of ROS and MDA in cells and altered antioxidant enzyme activity. The elevation in the MDA content occurred due to the imbalance of the free radical metabolism and the accumulation of ROS in algal cells, which may have caused or exacerbated membrane lipid peroxidation, resulting in a significant increase in MDA content [32]. Excessive production of ROS in algal cells may occur due to the inhibition of photosynthesis, enhancing excess excitation energy in chloroplasts [27]. The excessive accumulation of ROS is an important source of photosynthetic pigment damage, and if the damage is severe, the photobleaching of plants can occur (Miller et al., 2010). SODs are the first antioxidant defense system of the cell when plants are exposed to oxygen stress. The three most common types of SODs in plants are Mn-SOD, Cu/Zn-SOD, and Fe-SOD, the main function of which is to scavenge O 2− [40]. The increase in SOD activity may have occurred due to the progressive production of superoxide, which is thought to be a central component of signal transduction, the level of which increases with the activation of the enzyme pool or with increased SOD gene expression [41]. Some oxidative damage was caused to C. pyrenoidosa, and the inhibition and damage increased with increasing hormone concentrations and treatment times. Treatment with 17α-EE 2 altered gene expression in C. pyrenoidosa, which can respond to 17α-EE 2 toxicity by up-regulating or down-regulating some genes to maintain normal growth. The 17α-EE 2 was found to have a significant inhibitory effect on genes regulating algal growth, photosynthetic pigments, photosynthetic efficiency, antioxidant systems, and DNA replication after 96 h of exposure. At the transcriptional level, algal cells mitigate damage and maintain growth by activating repair mechanisms, a process that includes the repair and replacement of reaction center proteins and the up-regulation of ROS scavenger gene expression, corroborating biochemical phenomena. In the previous study, Microcystis aeruginosa (M. aeruginosa) under a mixed antibiotic treatment up-regulated three genes targeting ROS scavenging (MAE_36510, MAE_58300, and trxA). During redox, reactive oxygen species scavenging genes were enriched to maintain cyanobacterial cell redox homeostasis. In addition, the up-regulation of photosynthesis-related genes may help to increase the photosynthetic activity of antibiotic-treated algal cells, thereby promoting the growth of M. aeruginosa through increased photosynthetic energy production [42].
In algal cells, gene regulation changes with increasing stress levels, with higher stress leading to more severe damage. Low concentrations of 17α-EE 2 mainly disrupt the photosystem, impede electron transfer, and affect energy conversion. During the photoelectron transfer process, both petB and petF were also significantly down-regulated (Table S4), while in previous research, the expression of the genes petF and petH, which are related to the regulation of photosynthetic electrons, and the two PSII subunit genes psbO and psbP, was increased in the irradiated mutants of Nannochloropsis oceanica, leading to enhanced electron transfer during the photoreaction and possibly accelerating the water cleavage process and the rate of oxygen production in the mutants [43].
Genes related to photosynthesis (petJ, psbB, and psbI) were down-regulated in the medium-and high-level treatment groups, and the carotenoid-regulated gene crtz was down-regulated. Nitrogen metabolism pathways in the medium-concentration groups, nita and gdh2, were down-regulated, and the kin14n genes in the ribosome biosynthesis were significantly down-regulated. Algal cell growth will be affected by the reduction of carbon and nitrogen compounds. Similar phenomena have been found in previous studies. Chromochloris zofingiensis cultured in heterotrophic conditions in the dark demonstrated that the "lipid biosynthesis proteins" and "ribosome biosynthesis" pathways are significantly down-regulated and that the biosynthesis of major C-containing compounds (lipids, starch, and proteins) is inhibited when algal cells are exposed to energy limitation [44].
The most severe effect was DNA damage, with genes such as mcm4 being significantly down-regulated and with rpa1D genes responsible for mismatch repair significantly upregulated. DNA damage is one of the most severe forms of oxidative stress, and its repair is linked to cell survival and prognosis [45]. DNA mismatch repair prevents the occurrence of permanent mutations during cell division by correcting the DNA mismatches that occur during DNA replication. Similar DNA damage was observed when transcriptome analysis was performed on erythromycin-stressed Raphidocelis subcapitata [46]. Dunaliella salina shows the up-regulation of most DNA replication-related genes in the presence of molybdenum disulfide nanoparticles (key genes such as DNA polymerase alpha subunit A, mcm6), in order to promote cell division and overcome environmental stresses [47]. It is concluded that 17α-EE 2 primarily impaired the photosynthesis of C. pyrenoidosa, then induced antioxidant system response and DNA damage as its concentration increases.

Algal Cultures
The freshwater green microalga C. pyrenoidosa (No. FACHB-5) was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). The algal cells were transferred aseptically to freshly sterilized BG11 medium (pH = 7.1). Then, they were incubated in a light incubator at a constant temperature of 25 ± 1 • C with a light intensity of 40 µE m −2 s −1 and a 12 h:12 h alternating light/dark light cycle until the logarithmic phase of growth. The algal cultures were shaken at least three times a day to keep the algal cells in suspension and to promote the transfer of carbon dioxide [22].

17α-EE 2 Exposure
The 17α-EE 2 was dissolved in dimethyl sulfoxide and sonicated for 1 h. The stock solution was obtained and then sterilized by membrane filtration (average pore size, 0.2 µm). Then, it was diluted with sterilized culture medium to achieve a series of concentrations, including 0 mg/L (control), 2 mg/L (low dose), 4 mg/L (moderate dose), and 8 mg/L (high dose). The initial absorbance at 680 nm of the C. pyrenoidosa culture was approximately 0.13, and the cell density was approximately 1.80 × 10 6 cells/mL. Three independent assays were accomplished for each treatment group. The culture was incubated for 96 h with the above culture conditions and shaken three times a day to ensure that the cells were well mixed.

Determination of Cell Density and Photosynthetic Pigment Content
The growth of C. pyrenoidosa was measured every 24 h using a UV/VIS spectrophotometer (UV2204PC, Shanghai Jipu Instrument Co. LTD, Shanghai, China) to determine the absorbance at 680 nm. The cell density was estimated by hemocytometry under a microscope. A standard curve and regression equation were established between absorbance values and algal density: cell density (10 6 cells/mL) = 3.6831 × OD680 − 0.652 (R 2 = 99%).
Photosynthetic pigment measurements were carried out using methods that have been described elsewhere [34]. Algal cultures were centrifuged at 8000 rpm for 10 min. Then, the supernatant was discarded, and the algal bodies were collected. Five milliliters of 95% ethanol were added, and the algal cells were extracted for 24 h in a refrigerator at 4 • C and shaken 2-3 times to allow the cytochromes to fully dissolve. The supernatant was centrifuged (8000 rpm, 10 min), and the absorbance values D665, D649, and D470 were determined spectrophotometrically to calculate the chlorophyll a and b and carotenoid levels.

Measurement of Chlorophyll Fluorescence
The chlorophyll fluorescence parameters of C. pyrenoidosa were measured every 24 h with a portable plant efficiency analyzer (PEA, Hanstech, UK). The OJIP test analysis was performed according to available methods and described as follows: samples were placed in the instrument after 15 min of dark adaptation and were measured after 5 s of recording time. The measurements were carried out at room temperature with an excitation light intensity of 50% of the maximum light intensity (1500 µE/m 2 s) [2,48]. The fluorescence parameters were calculated by a previous study [49].

Analysis of Contents of MDA, SOD, and ROS
The MDA and SOD levels were measured within 96 h. A total of 5 mL of fresh algal solution was centrifuged (8000 rpm, 10 min), the algal bodies were collected and washed three times with phosphate buffer (0.1 mol/L, pH 7.8), and 5 mL of phosphate buffer (0.1 mol/L, pH 7.8) was added. The mixture was ultrasonicated in an ice bath and centrifuged at low temperature, and the supernatant was used as the crude enzyme extract for testing. To measure the MDA content, 2 mL 10% trichloroacetic acid and 2 mL 0.6% thiobarbituric acid were added to the crude enzyme solution. The mixed liquor was then incubated in boiling water bath for 20 min, and the supernatants were measured at absorbances of 440, 532, and 600 nm after centrifugation. The nitro blue tetrazolium photoreduction method was used to determine SOD, and absorbance values at 560 nm were measured and calculated [50].
ROS generation was analyzed by fluorescence spectrophotometry (dichloro-dihydrofluorescein diacetate (DCFH-DA) method) [51]. The culture samples were centrifuged at 8000 rpm and 4 • C for 10 min. The supernatants were discarded and the cell pellets were resuspended with pure water. DCFH-DA was then added, and the solutions were shaken before being incubated in the dark for 30 min. The fluorescence content of the algal samples was measured using a fluorescence spectrophotometer (F-4500, Hitachi, Tokyo, Japan) with an excitation wavelength of 485 nm and emission wavelength of 535 nm.

Morphological Examination
The morphological characteristics of the algal cell surface after exposure to 17α-EE 2 were identified using field emission SEM (Zeiss SIGMA, Cambridge, UK) and observed according to the method described in a previously reported method [52]. Algae exposed for 24 h were selected, and the algal solution was pretreated, freeze-dried, and coated with gold for SEM observation.

Determination of Surface Characteristics
Fourier transform infrared (FTIR) spectroscopy analysis was used to identify changes in biomolecules, including proteins, carbohydrates, and lipids, within algal cells under 17α-EE 2 exposure to assess the extent of cellular damage. The samples and associated spectra were analyzed according to the available method [53].

Quantitative Real-Time PCR
To validate the transcriptome sequencing results, real-time quantitative polymerase chain reaction (qRT-PCR) was used to determine the expression levels of three key genes, namely, psaD, sodA, and por. The qRT-PCR methods are detailed in the Supplementary Information.

Statistical Analysis
The experimental data were analyzed using one-way ANOVA (p < 0.05) and least significant differences between control and treatment groups were determined by Duncan's multiple-range test (p < 0.05) in IBM SPSS 23.0. Standard errors were calculated using Excel software. Data were the results of multiple parallel experiments.

Conclusions
Exposure to 17α-EE 2 significantly inhibited the growth of C. pyrenoidosa. It reduced the cell density, damaged the cell structure, decreased the content of photosynthetic pigment, and diminished the photosynthetic system activity. It down-regulated the relevant photosynthetic genes and ultimately impaired photosynthesis. At the same time, 17a-EE 2 induced oxidative stress in algal cells, causing oxidative damage. This was followed by an increase in ROS and MDA levels and antioxidant enzyme activity in C. pyrenoidosa, stimulating the antioxidant system and up-regulating some of the relevant regulatory genes as a means of providing protection against oxidative damage. Transcriptomic analysis showed that the common pathways affected in algal cells exposed to different treatment concentrations of 17a-EE 2 were photosynthesis and DNA synthesis and repair. However, certain specific pathways exhibited different behaviors at different concentrations. The pathways of ribosome biogenesis in eukaryotes, photosynthesis, and oxidative phosphorylation were affected at low doses of 17a-EE 2 , while that of ribosome biogenesis in eukaryotes, porphyrin and chlorophyll metabolism, nitrogen metabolism, and DNA damage repair were affected at moderate doses and higher doses of 17a-EE 2 . This study contributes to the understanding of the molecular mechanisms underlying the response of C. pyrenoidosa to 17α-EE 2 and provides a dataset for a relevant ecological risk assessment.