Title: Performance of constructed wetlands and associated mechanisms of PAHs removal with mussels

The role of mussels in laboratory-scale constructed wetlands (CWs) where wastewater was contaminated with five typical polycyclic aromatic hydrocarbons (PAHs) (three 3-ring PAHs and two 4-ring PAHs) was investigated in this study. The CWs performance and PAHs removal mechanisms were well studied. Results indicated that these five PAHs improved removal efficiencies of NO 3 -N but in the case of NH 4 -N accumulation occurred. Of the five added PAHs, the 4-ring PAHs were more refractory with higher concentrations in effluent than 3-ring PAHs. By monitoring the five PAHs concentration in water, mussels had excellent removal efficiency of the five PAHs (97%). According to the mass balance calculation, mussels promoted plant uptake of five PAHs, contributing 15.2% of five PAHs removal in CWs. The PAHs could also accumulate in mussels through ingested substrate. Thus, mussels presented a positive correlation with five PAHs purification at a depth of 0-10 cm in substrate, which was 34.7 μ g/kg lower than the control group. Due to the purification and enhanced aerated degradation, mussels performed better in removing 3-ring PAHs in substrate, decreasing 8.3% for 3-ring PAHs compared to the control. A positive correlation between five PAHs addition and nirS , nrfA genes was observed. However, PAHs showed negative impact on nitrifying bacteria ( amoA ). The significant correlations between mussels and five PAHs made it possible to improve CWs for PAHs treatment with aquatic faunas.


Introduction
Polycyclic aromatic hydrocarbons (PAHs) are important organic contaminants and they are widespread with hyper toxicity, and carcinogenic properties [1]. Over the past 30 years, the PAHs mainly originated from anthropogenic processes in urbanized or industrialized regions, such as vehicle exhausts and coal combustion [2]. PAHs accumulate in the aquatic environment and are difficult to be biodegraded. Due to their low solubility and high hydrophobicity, PAHs contaminants in the aquatic ecosystem are adsorbed onto suspended particles and finally accumulate in sediments instead of dissolving in water [3]. They have been shown to constitute a major threat to a wide variety of fishes, invertebrates, and even natural ecosystems [4,5].
Constructed wetland (CW), as an economic technology, has been used to remove common pollutants such as chemical oxygen demand (COD), nitrogen and phosphorus.
However, not much research has focused on specific organic pollutants such as PAHs in CWs [6,7]. In the presented study, settling and sedimentation onto a substrate were considered to be the main removal processes for PAHs in examined systems, but they could not remove the contaminants permanently. PAHs-organic matter bonding to a substrate reduces the mobility of contaminants and encourages PAHs to be more resistant to degradation in the anoxic environment [8]. Therefore, the contamination caused by the PAHs was much more harmful in the substrate phase than in the water phase. To mitigate the risks to ecological health induced by PAHs contamination, several current research projects have attempted to increase the bioavailability of PAHs through surfactants and co-substrates addition. However, this increases the costs and causes secondary pollution [9]. Although limited by the generally low bioavailability of PAHs, transformation by microorganisms is one of the main contributors to decreased PAH-contaminated sites in deep substrate [10]. Compared to physical and chemical remediation technologies, 4 bioremediation is widely recognized as a 'green' or environmentally friendly technique for remediating PAH-contaminated soils. The bioremediation technologies consist of bioaugmentation and biostimulation [11]. It occurs through microbial degradation of PAHs in aerobic or anaerobic conditions [12,13]. It is more effective, exerts a minimal environmental impact and releases no other pollutants.
The benthic fauna is an essential element for PAHs degradation, especially inhabiting the substrate phase in CWs. Firstly, the bio-accumulation of pollutants by benthic fauna through the aquatic food web is an important pathway of PAHs [14].
Secondly, the PAHs, having low molecular weight, could be degraded by soil microorganisms in aerobic conditions [15], but the deep substrate always lacks oxygen.
Plants could also degrade PAHs through plant uptake and enhanced aerobic degradation through radial oxygen loss [16]. Benthic fauna are of great significance for changing the oxygen transfer through bioturbation and enhancing plant growth [17]. Thirdly, microbial degradation of PAHs depends on various environmental conditions, such as nutrients, chemical properties of PAHs, denitrifying conditions, oxygen levels and microbial composition [15]. Under anaerobic conditions, the efficiency of microbial biodegradation highly depends on electron receptors such as nitrate (NO 3 -N), sulfate or ferric iron [18].
As one of the more favorable electron acceptors, NO 3 -N can be reduced by microorganisms associated with the consumption of a variety of organic carbons, including PAHs. According to our previous research, benthic fauna can change the nitrogen cultures in substrate [19,20]. Thus, benthic fauna are considered to significantly influence PAHs removal in an ecological and sustainable way. Mussels are the typical benthic fauna in CWs. The mussels in an aquatic environment mainly inhabit the deep substrate, with excellent filtration capacity for organic pollutants, and change the nutrient level in substrate [21]. Consequently, it is considered that mussels may have an effect on 5 PAHs removal during metabolism.
The majority of previous research focused on the effect of plants and microbes addition on PAHs degradation in the aquatic environment. This is the first study, to the best of our knowledge, that revealed the importance of mussels involved in determining degradation behavior of PAHs in CWs. Here, we analyzed typical 4-ring and 3-ring PAHs contained in the 16 priority PAHs. The typical 3-ring PAHs of acenaphthene (ACE), acenaphthylene (ACY), fluorene (FLU) and 4-ring PAHs of fluoranthene (FLT) and pyrene (PYR) were selected as the target PAHs. All of the five PAHs have high water quality guidelines that were developed by the U.S. Environmental Protection Agency (EPA) [22]. The specific objectives of this study are to: 1) evaluate the influence of mussels on the purification of five PAHs in CWs; 2) evaluate the influence of these five PAHs on standard water quality variables (COD, NO 3 -N, ammonium (NH 4 -N), total nitrogen (TN)); and 3) determine the contributions of CWs components (plants, substrate, and others) and their relationship to the removal of the five PAHs under the influence of mussels. This research study was crucial for developing CWs treatment technologies utilizing benthic fauna whilst also providing useful information on the design of CWs for PAHs removal.

Laboratory-scale CW systems and chemicals
The CW systems under this study were located in the Environmental Research Institute O. javanica were planted in September. The four experimental units were divided in two parts (CW-C and CW-M), which were constructed without and with mussels. The freshwater mussels used in experiments were Lamprotula leai, with a density of 15 individuals per unit [23]. Processes for the domesticated of mussels were detailed discussed in our previous research [21]. The CW-C and CW-M were further divided into two sections. Two microcosms were spiked with a mix of five PAHs in the influent (CW-CP and CW-MP). Four units in total were operated as: CW-C (without benthic fauna); CW-M (with mussels); CW-CP (contained Σ 5 PAHs and without benthic fauna); and CW-MP (contained Σ 5 PAHs and with mussels).
The ACE (>99.5% purity), ACY (90% purity), FLT (98% purity), FLU (>99% purity), and PYR (99% purity) used in this experiment were purchased from Aladdin Reagent (Shanghai, China). The standard substitute (p-Terphenyl-d14 and 2fluorobiphenyl) used for pretreatment recovery rate calculation of PAHs extraction was obtained from the ANPEL Laboratory Technologies (Shanghai) Inc. The internal standards injected into the sample extracts for the purposes of quantitation were also obtained from ANPEL Laboratory Technologies (Shanghai) Inc.

Experimental procedure
A laboratory-scale sequencing batch-operated procedure was implemented in this experiment. The total experiment period lasted from September 2017 to February 2018, 7 with the air temperature ranging from 20 o C to 4 o C. The hydraulic retention time (HRT) of each system was 5 days, corresponding to the hydraulic loading rate (HLR) of 2 cm/d.

Sample processing and analysis
Water samples were taken from influent and overlying water at the beginning and end of each cycle (every 5 days) using a Teflon polyethylene bottle. After collection, the water samples were filtered through a 0.45 μm cellulose acetate membrane by syringe. Specific parameters, i.e. TN, NH 4 -N, NO 3 -N and COD were determined in the laboratory 8 according to the standard methods [25].
For each unit the substrate samples of 25 g were taken every two cycle. The substrate samples at different depths (0-5 cm and 5-10 cm) below the water-sediment interface were collected using a tapered column. Samples were homogenized from five individuals (same height, equal amount). Then they were powdered to pass through a 2 mm sieve and stored at -20 °C prior to analysis. The NO 3 -N content in the substrate was determined by reacting the sand with 2 M KCl (800 g/L) and this entailed 2 h shaking and 20 min centrifuge [26].
The plant heights were tested monthly. The plant samples were randomly collected from each unit every fourth cycle. Plant samples were carefully rinsed with distilled water several times to remove any adhering sand particles, and separated into stem and leaf tissues. Then the leaf was used for chlorophyll detection according to the methods described by Lan et al. [27].
To understand the effect of five PAHs on mussels' activity, the respiration rate was tested by analyzing the decrease in dissolved oxygen (DO) per unit time per mussel individual in a closed system [28]. Once the testing was completed, the mussels were replaced into the units. Two mussels were picked up for analyzing the element and five PAHs contents every fourth cycle. At the same time the mussels with the same weight were added into the systems. After collection the mussels were dissected and the bivalve tissue was removed from the shell. Then the tissues were rinsed with distilled water and dried at 45 o C for 96 h. The C and N contents in animal samples were determined using an elemental analyzer located in the Qilu University of Technology, Jinan, Shandong Province. 9

Wastewater
Extraction of PAHs in wastewater was subsequently done using a solid-phase extraction method. Before extraction the water was filtered through a quantitative filter paper. 1000 mL water samples with surrogates were passed through a C18 extraction membrane using a solid phase extractor (Extrapid). The organic compounds in the membrane were eluted with dichloromethane (20 mL) for 20 min. The organic phase containing PAHs was then collected, and the water phase remaining in the extracts was absorbed with Na 2 SO 4 . The solvent was further concentrated to 1 mL by a vacuum concentrator (Vortex 600). The sample components were identified and measured by injecting the concentrated extract into a gas chromatography/mass spectrometry (GC/MS) system. The PAHs GC/MS analytical procedure was conducted according to EPA 525.2 on a Shimadzu (Japan) QP2020 GC/MS with a Rxi 5Sil MS column, working in SIM mode [29]. The PAHs ○ R concentrations were calculated by the internal standard method (target ion peak areas were used for the calculation).

Substrate
The PAHs in the substrate samples were extracted using an accelerated solvent extractor (Thermo Fisher ASE 350) based at Shandong Academy of Environmental Science with acetone-hexane mixture (1:1). The instruments used for substrate sample testing were similar as described in section 2.4.1. The PAH GC/MS analysis method for substrate was performed according to EPA 8270C [30]. Furthermore, the PAHs contents in substrate were calculated by the internal standard method.

Plants and aquatic animals
The aboveground plant parts, i.e. the stems were measured. The stem parts of plants were naturally dried by air before extraction. For extraction, 5 g of the dried plant sample was ground and placed into a 50 mL Teflon tube with 25 mL dichloromethane. Then they were extracted using the ultrasound extraction technique for 1 h, shaken for 1 h, and centrifuged at 1600 rpm for 10 min. Following extraction the water phase was removed, and the dichloromethane layer was purified by the silica gel column which contained neutral chlorine dioxide, silica gel, and Na 2 SO 4 . Then the residue was reduced to the final volume of 1 mL using the vacuum concentrator. Internal standard was added prior to GC-MS analysis. The extraction and detection method was implemented according to the description by He et al. [31].
For PAHs determination, the mussels' tissue was cut into small pieces. 5 g samples were placed into a 50 mL Teflon tube with 30 mL mixture of n-hexane: dichloromethane (1:1). They were then extracted by ultrasound extraction technique for 1 h, shaken for 1 h, and centrifuged at 1600 rpm for 10 min. The extraction of mussel samples were subjected to Na 2 SO 4 to remove water. The remaining extraction solvent was cleaned up with an alumina purification column containing silica, alumina and Na 2 SO 4 . Then the residue was reduced to 1 mL for detection. The PAHs analysis was done according to the methods documented by Wang et al. [32].

Microbial study
For microbial analysis, DNA and RNA in substrate were extracted using the MOBIO PowerSand™ DNA and RNA Isolation Kits according to the manufacturer's instructions.
Complementary DNA (cDNA) was generated by RNA using the superscript reverse transcriptase (TaKaRa, Japan). The reverse transcription PCR program was implemented according to the method described by Wang et al. [33]. The DNA and cDNA products were stored at -80 °C before qPCR analysis.
A Roche LC-480 real-time PCR system was used to quantify the 16S rRNA and 11 functional genes such as nir (nirS and nirK), amoA, NSR, and nrfA. The abundance of the targeted genes in each sample was determined by Abs Quant/2nd Derivative Max from Roche LC-480 Install. The reaction mixture and qPCR cycling conditions were established as reported in our previous research [19].

Data calculation and analysis
The mass balance of five PAHs was calculated by using the following equation, The total mass of PAHs in influent and the mass of PAHs in effluent were calculated according to the PAHs concentrations and water volume. The total PAHs contents in plant, mussels and substrate were determined according to their weight. and 0.40 μg/kg (ACY). The pretreatment recovery rate for water analyses ranged from 62%-95%, 74%-100% for the substrate, and 42%-96% for the plant and mussel analyses.

Statistical significance
Data detected and their concentrations were analyzed using Mean ± standard deviation (SD). The statistical significance of difference was determined with a one-way analysis of variance (ANOVA) using SPSS 13.0. Correlations at p<0.05 were considered statistically significant. The sum of all the five PAHs detected in samples represented the total PAHs (expressed as Σ 5 PAH). 12

Pollutants removal performance in different CW units
The  (Table S1). Similar results were also reported by Xu et al. [34]. Two possibilities can account for these: firstly, the organic matter stimulated soil microbiological activity due to the additional source of carbon, which was highly beneficial for denitrification and NO 3 -N degradation [10]; and secondly, the dissimilatory N reduction to ammonium (DNRA) process was the dominant pathway of NO 3 -N removal and NH 4 -N accumulation with rich electron donors, such as organic carbon [34].
The enhanced DNRA process and NH 4 -N accumulation with PAHs were verified by the N isotope experiment with K 15 NO 3 . Results for NH 4 -N production confirmed the improved DNRA process with PAHs addition (Table S2). The CWs containing mussels performed the best at removing NH 4 -N. The mussels enhanced NH 4 -N removal through 13 their filtration, which attributed to lower NH 4 -N concentration in CW-MP than that in CW-CP. But the NH 4 -N concentration in CW-MP was still higher than that without PAHs.

Moreover, the Σ 5 PAHs addition reduced the final COD concentrations than without
PAHs contaminants (Fig. 1C), which was responsible for higher NO 3 -N removal efficiency. Organic carbon abundance emerged as an important factor in the heterotrophic denitrification process, meaning that more COD was utilized for denitrification.
The mean concentration values for total PAHs in the influent and effluent of different CW units are shown in Fig. 1D. Apparently, both CW units could efficiently remove Σ 5 PAHs and revealed no difference with the ∑ 5 PAHs effluent concentrations of 3.76 μg/L and 3.49 μg/L for CW-CP and CW-MP, respectively (p>0.05). The CW-MP has a higher removal rate of ∑ 5 PAHs than that of CW-CP (Fig. S1). More than 96% of the ∑ 5 PAHs were removed from the CW-MP, as the mussels demonstrated excellent capacity for organic removal due to their filtration [35]. and 2.97% in two groups of the total PAHs. This is the likely result of PAHs being used as a carbon source and providing energy for denitrification, through co-metabolization with other carbohydrates via the catalysis of dioxygenase [36]. The 4-ring PAHs had low utilization rates and proved to be more difficult to biodegrade than the 3-ring PAHs [10].
For this reason the 3-ring PAHs were preferred for the removal process. 14

Σ 5 PAHs accumulated in substrate along the height of CWs
Substrate plays a very important role in the removal of various pollutants in CWs, especially at the 0-10 cm depth [37]. Settling and adsorption onto substrates are considered to be the most likely removal processes for PAHs, which leads to less bioavailability towards biodegradation. The organics and nutrients settled on the superficial substrate, stimulated microbial activity at the sediment-water interface, reduced DO levels and restrained NH 4 -N removal (Fig. 1B). In the present study, the Σ 5 PAHs contents in the 0-5 cm and 5-10 cm depths of substrate were detected (Fig. 2). The amount of Σ 5 PAHs deposited in substrate ranged from 41.8-62.8 μg/kg substrate. The CW-MP had lower accumulation of Σ 5 PAHs than the CW-CP, especially in the 5-10 cm depth substrate. Diverse micro-environments along the height of the substrate provided various habitats for benthic fauna and microbes, and this affected pollutants removal. The mussels mainly inhabited the 5-10 cm depth. The mussels could generally take up the organic matter from these depths within the substrate by either direct ingestion or by bacteria within their filtration capacity [14]. Consequently, they were able to purify the Anaerobic biodegradation of PAHs pollutants is an important pathway in deep substrate due to the lack of oxygen and photo degradation. Its efficiency is highly dependent on terminal electron receptors such as NO 3 -N. Thus, higher NO 3 -N contents with mussels may enhance PAHs removal by anaerobic degradation.
Amongst the five PAHs, the percentages of the 3-ring PAHs in 0-10 cm depth substrate amounted to 25.0% and 17.7% in the CW-CP and CW-MP, respectively. This was probably due to the low molecular weight of PAHs (2 to 3-ring PAHs) and their preference for being volatilized into the gas phase, whilst the 4-ring PAHs were predominant in the particulate phase and could generally be deposited in increased proportions [38]. The mussels had 8.3% less proportions of 3-ring PAHs accumulated in the substrate. The 3-ring PAHs were most likely degraded via aerobic microbial degradation. The 4-ring PAHs, being "more difficult" components were mostly degraded after the 3-ring PAHs were degraded [39]. It is generally known that the relatively high concentrations of PAHs in substrate mainly existed due to the limited water exchange [40]. Mussel feeding activity and bioturbation may have mixed and aerated the sediments, thus promoting the biodegradation of light PAHs with 3-rings, and contributed to the removal of PAHs in aquatic environments [41].

Plant uptake of Σ 5 PAHs
The role of plants has been extensively studied for PAHs removal in CWs [42,43]. For our study, the plants were analyzed to investigate their absorption of Σ 5 PAHs amounts over the experimental period (Fig. 3A). The overall plant uptake of Σ 5 PAHs had significantly greater accumulation, which was reported by Wang et al. [44]. In the late experimental period, PAHs in plants were slightly removed and this was attributed to plant growth with transpiration and metabolism, as well as utilization of carbon sources (Fig. S2) [36]. The CW plants were able to accumulate PAHs and this meant that harvesting of plants contributed to mitigation of organic pollutants in the CWs. The five PAHs were detected in plants at 16 high concentrations (51.3-248 μg/g dry weight (DW)) and varied with the average concentrations of 144±28.9 μg/g DW and 206±36.2 μg/g DW in CW-CP and CW-MP, respectively. The Σ 5 PAHs accumulation was higher in CW-MP, which confirmed that the mussels promoted the plants' uptake of PAHs. Plants may enhance the degradation of PAHs in the rhizosphere by: firstly, increasing the activity and density of root-associated microorganisms; and secondly, facilitating the PAHs transport to the roots by releasing the root-associated enzymes [45]. The enhanced plant growth by mussels was reflected in the higher chlorophyll content and plant height in CW-MP (Fig. S3), and facilitated the plants' uptake of Σ 5 PAHs. The presence of roots increased the aeration in substrate, thereby promoting PAHs aerobic degradation in the deep layer and transforming PAHs into more bio-accessible compounds [46]. The mussels promoted plant growth and increased the oxygen level due to root function and further improved the 3-ring PAHs removal in substrate.

Σ 5 PAHs in mussel samples
The factors that affected the accumulation of PAHs in aquatic animals include the magnitude of contamination in their habitats, the trophic status, feeding habits, and metabolic capacities [14]. The Σ 5 PAHs residues accumulated in mussels during the experiment period shown in Fig. 3B. The concentrations of Σ 5 PAHs increased from 0.861 to 17.0 μg/g fresh weight tissue. The mussels accumulated hydrophobic pollutants through ingestion. The lipids of mussels are also an important factor for the bioaccumulation of PAHs [32]. Higher percentages of the 4-ring PAHs were found in the mussels. The proportions of two 4-ring PAHs decreased at the end of the experiment, probably due to enhanced biotransformation as well as reduced gut assimilation in mussels. Therefore, the extra uptake via ingested substrate by mussels was also a pathway for PAHs removal in the substrate, resulting in the relatively flat trend of contaminant concentrations in the CWs. However, the PAHs pollution in the sampling area caused a decrease in filtration rates of the mussels (Fig. S4).

Relationships between microbes and nutrient removal
Few studies have focused on the effect of PAHs on the substrate microbial communities in contaminated aquatic ecosystems [47]. The microbial functional genes involved in key N-cycling processes were studied. Bacterial activity was indicated by determining the RNA copy numbers. The DNA and RNA values were obtained and their Log values are shown in Tables S4 and S5, respectively, which represent the order of microbial magnitude in Log values. In the present study, both the microbial abundance and activity were influenced by the mussels and PAHs contaminants. According to the qPCR results, the relative abundance of nitrifying bacteria (amoA and NSR) decreased with Σ 5 PAHs addition, especially the amoA genes. The activity of nitrifying bacteria was clearly restrained with Σ 5 PAHs pollution, the abundance and activity of nirS did increase in CW-MP, and it was higher than that in CW-M. The CWs with mussels still had higher nitrifying and denitrifying bacteria than the control group. The qPCR data showed that the nrfA genes abundance was enriched by the addition of Σ 5 PAHs. Coupled with NH 4 -N accumulation and NO 3 -N removal (section 3.1), we postulate that the abundant PAHs settled on the substrate and restrained the nitrification process but promoted DNRA and the denitrification processes.

Proposed mechanism of Σ 5 PAHs removal in CWs with the addition of mussels
Except for substrate adsorption, aquatic animal bioaccumulation and plant absorption, the PAHs can also be removed by other mechanisms. These other processes cannot be measured directly. Thus, a mass balance calculation was adopted to estimate the removal contribution by CWs components. According to Fig. 4 CW-MP>CW-CP. To summarize, the addition of mussels could promote the removal of Σ 5 PAHs by enhancing plant absorption and uptake of contaminants in the substrate. The amount of Σ 5 PAHs effluent from wastewater took into account no more than 4.3% of the total PAHs removal, with no significant difference (p>0.05) being observed.
Based on the results presented here, there were significant correlations between functional microbial abundance, environmental factors and PAHs concentration in the CWs with mussel addition (Fig. 5). The mussels in CWs enhanced plant growth and further promoted plant uptake of Σ 5 PAHs. Meanwhile, mussels in CWs had a significant positive correlation with Σ 5 PAHs removal in substrate, especially in the 5-10 cm depth that the mussels inhabited. The purification capacity and ingestion of mussels contributed to the enhanced PAHs removal. Furthermore their bioturbation activity and enhanced plant growth were attributed to an oxygenated substrate, therefore the 3-ring PAHs removal was improved by mussels. The changes in microbial abundance and activity with PAHs were responsible for the different performances in removing NO 3 -N and NH 4 -N [49]. Denitrification, coupled with a carbon source provided by PAHs, was speculated to have occurred resulting in NO 3 -N removal. The inhibited NH 4 -N removal was attributed to the enhanced DNRA and restrained nitrification processes. Our findings confirmed that the use of mussels to remove PAHs in CWs is feasible.

Conclusion
The potential of mussels to influence the removal of five typical PAHs in CWs and the effect of PAHs on CWs performance were studied. The results indicated that the Σ 5 PAHs promoted NO 3 -N removal but increased NH 4 -N concentrations in wastewater in CWs.
Mussels had excellent purification capacity with the Σ 5 PAHs removal efficiency improving up to 97% in wastewater. The 4-ring PAHs had higher concentrations than the 3-ring PAHs for each CW in the substrate, probably because of settlement and recalcitrance. Through mass balance calculation, it was found that the mussels decreased the Σ 5 PAHs accumulation at 0-10 cm depth substrate, especially for 3-ring PAHs. This was mainly due to the ingestion, purification, and enhanced oxygenated environment in the deep substrate where the mussels are located. Mussels highly promoted plant uptake of Σ 5 PAHs. The Σ 5 PAHs were assimilated in the mussels. Regarding the microbial results, Σ 5 PAHs in CWs inhibited the abundance and activity of nitrifying bacteria (amoA and NSR), and increased the DNRA process (nrfA). This contributed to the differences in NH 4 -N and NO 3 -N removing in wastewater. Finally, our reported results suggest that mussels can promote Σ 5 PAHs removal in the CWs.