ABSTRACT 
 
Title of Thesis: POLYCYCLIC AROMATIC 
HYDROCARBONS (PAHS) AND PAH 
DEGRADING BACTERIA IN MEDIA OF A 
STORMWATER BIORETENTION CELL 
  
 Chen Yuan, Master of Science, 2020 
  
Thesis Directed By: Assistant Professor, Birthe Kjellerup, Civil and 
Environmental Engineering 
 
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic pollutions that are 
carcinogenic to humans. A bioretention cell at the campus of University of Maryland, 
College Park, evaluated for their potential to remove PAHs from stormwater. The risk 
of PAH pollution was confirmed by the fact that the highest value of total PAH 
concentration (25.5 ?g/g) and the average concentrations of pyrene (1.78 ?g/g) and 
chrysene (1.40 ?g/g) was higher than their respective probable effect concentrations 
(PECs) (22.8 ?g/g, 1.52 ?g/g and 1.29 ?g/g),. Sources of PAHs in the media 
originated from fossil fuel combustion and petroleum. Human carcinogenicity was 
evaluated by determining the Benzo[a]pyrene Total Toxicity Equivalent (BaP-TEQ), 
and benzo[a]pyrene made most contribution to carcinogenicity. Biotransformation of 
PAH is possible in bioretention cell, because PAH-ring cleaving dioxygenases (PAH-
RCD) gene was found in media. 
 
 
 
 
 
 
 
 
 
 
 
POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) AND PAH 
DEGRADING BACTERIA IN MEDIA OF A STORMWATER BIORETENTION 
CELL    
 
 
 
by 
 
 
 
Chen Yuan 
 
 
 
 
 
 
Thesis submitted to the Faculty of the Graduate School of the  
University of Maryland, College Park, in partial fulfillment 
of the requirements for the degree of 
Master of Science 
2020 
 
 
 
 
 
Advisory Committee: 
Dr. Birthe Kjellerup, Chair 
Dr. Allen Davis  
Dr. Alba Torrents  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
? Copyright by 
Chen Yuan 
2020 
 
 
 
Table of Contents 
 
Chapter 1 .................................................................................................................... 1 
Abstract .................................................................................................................. 2 
1. Introduction ........................................................................................................ 3 
2. Materials and Methods ....................................................................................... 8 
3. Results and Discussion..................................................................................... 15 
4. Conclusions and summary ............................................................................... 29 
5. Acknowledgements .......................................................................................... 30 
6. Tables and figures ............................................................................................ 30 
7. Appendix .......................................................................................................... 46 
References ............................................................................................................ 55 
Chapter 2 .................................................................................................................. 65 
Thesis conclusion ................................................................................................. 66 
References ............................................................................................................ 69 
 
 
 
ii 
 
Chapter 1 
 
 
Polycyclic Aromatic Hydrocarbons (PAHs) and PAH Degrading Bacteria in Media 
of a Stormwater Bioretention Cell 
 
 
 
Chen Yuan, Allen P. Davis, Devrim Kaya and Birthe V. Kjellerup 
Department of Civil and Environmental Engineering, University of Maryland College Park 
 
 
 
 
  
1 
 
Abstract 
This research investigated the distribution of 16 EPA priority PAHs and their toxicity 
in the bioretention cell. The Highest total PAH concentration found in the media was 
25.48 ?g/g, and individual PAHs with highest average concentrations were 
banzo[g,h,i]perylene in February and pyrene in June. The PAH concentrations in surface 
samples did not show a clear trend. Sources of PAHs were fossil fuel combustion and 
petroleum. Ecological impact and carcinogenicity to humans of bioretention cell media 
were assessed by probable effect concentrations (PECs) and benzo[a]pyrene total toxicity 
equivalent (BaP-TEQ) respectively. The results show that average concentrations of 
pyrene and chrysene exceed PECs, and the average BaP-TEQ was 1.64 ?g/g, which 
mainly contributed by benzo[a]pyrene. Results of PCR showed that there was one kind of 
PAH-ring cleaving dioxygenases (PAH-RCD) gene (C12O) in the media, which means 
that the aerobic biodegradation of PAHs is possible in the bioretention cell. 
 
 
 
 
 
 
2 
 
1. Introduction 
Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds that 
consist of two or more aromatic rings in the form of a fused ring compound. More than 
100 compounds exist within the group of PAHs from naphthalene (molecular weight of 
128 g/mol, log Kow of 3.32) to coronene (molecular weight of 300 g/mol, log Kow of 6.75) 
(Lu et al., 2008; Pyrene and Analogues, 1983). PAHs are present in many compartments 
of the environment due to their resistance to biodegradation (Crampon et al., 2014) and 
thus bioaccumulate in the food web (Ribeiro et al., 2005). They are recognized as 
carcinogenic compounds and some are classified as the human carcinogens (class 1) as 
categorized by the International Agency for Research on Cancer (IARC, 2012). As a 
result, PAHs are of significant environmental concern and the U.S. EPA has established 
regulations for the concentration of PAHs in the environment (Haritash and Kaushik, 
2009). The U.S. EPA has issued a list of 16 PAHs that frequently occurred in environment 
and are known to be toxic (Table 1). 
Two known pathways for PAH toxicity are 1) Non-metabolic toxicity and 2) 
metabolic toxicity (Sikkema et al., 1994). The toxicity of PAHs is mainly attributed to 
metabolic toxicity (Gauthier et al., 2014). One manifestation of non-metabolic toxicity is 
membrane damage, which is due to the lipophilicity of PAHs. The distribution of PAHs in 
the cell membrane causes the membrane to expand and increase fluidity. (Sikkema et al., 
1994). Non-metabolic toxicity is also reflected in the fact that PAHs inhibits P-ATPase 
3 
 
(Li et al., 2011). These two effects cause loss of cellular function and cause cell death 
under continuous influence. Through bioactivation, PAHs will enable metabolic toxicity 
that leads to carcinogenicity (Gauthier et al., 2014). As an example, Benzo[a]pyrene 
(BaP) can be epoxygenated to 7,8-dihydrodiol-9,10 epoxide (BPDE). However, it can 
also form radical cations through 1-electron oxidations. Once formed, those products may 
bind to nucleophilic sites on DNA and form DNA adducts (Siddens et al., 2012). Based 
on experiments on animals, BaP was determined to be the most potent carcinogenic PAHs 
(Haritash and Kaushik, 2009; Kim et al., 2013).  
PAHs originate from a wide range of sources such as incomplete combustion of 
organic materials (fossil fuels and wood) and during industrial processes (Kim et al., 
2013). PAHs are also produced by natural processes, such as volcanic eruptions, forest 
fires and oil seeps (Yunker et al., 2002). Many processes contribute to the accumulation 
of PAHs in urban landscapes, such as leakage of oil, and abrasion of road surface and 
tires (El-Mufleh et al., 2013). In addition, PAHs discharged to the atmosphere can be 
accumulated on ground surfaces by dry and wet deposition. (Esen et al., 2008). Pollutants 
settled by dry deposition will remain on the surface of ground until they are washed away 
by rainfall/stormwater (Al Ali et al., 2017). PAHs can also contaminate stormwater 
directly by wet deposition in the form of rain and snow (G?bel et al., 2006). PAHs 
consisting of < four rings are mainly removed from the atmosphere by wet deposition 
since these PAHs tend to present in the gaseous state because of the relatively low log 
4 
 
KOA (Parnis et al., 2015). Dry deposition often takes place for PAHs > four rings, which 
are mainly distributed on particles due to adsorption (Cheng et al., 2018). Log Kow 
values of PAHs also increasing with molecular weights (Table 1), which suggests that 
HMW-PAHs are more likely to be adsorbed on particles in stormwater and adsorbed on 
media particles after storm water flow into bioretention cell, while LMW-PAHs are 
relatively easy to transport with stormwater. Partition of PAHs between dissolved and 
particulate phases was reported by Zgheib (2011), results showed that almost all PAHs, 
especially HMW-PAHs, are distributed in the particle phase.  
PAHs can be transported by particulate matter in stormwater, and if left untreated, 
they will flow into aquatic ecosystems such as rivers and cause pollution (Estebe, 1997). 
As a result, stormwater runoff is recognized by the U.S. Environmental Protection 
Agency (USEPA) as one of the important sources of water quality deterioration (USEPA, 
2017).  Water quality deterioration can be caused by stormwater from parking lots and 
streets (Dietz and Clausen, 2006). Urban runoff has caused deterioration of lake water 
quality and increased concentrations of PAHs (highest concentration: 3.66 ?g/g) in 
sediments (Jung et al., 2008). Howitt (2014) found that PAHs (3.02 to 23.13 ?g/g) 
accumulated in sediments of semi-natural wetland in Victoria, Australia because of 
stormwater inputs. A variety of PAHs were identified with concentrations ranging from 
64 ?g/kg to 26288 ?g/kg in the sediments of stormwater retention ponds that treat 
stormwater from golf courses, low and high density residential development, and 
5 
 
commercial development, in the coastal areas of South Carolina, USA (Weinstein et al., 
2010). The concentration depended on the land use, with the highest levels (10,422 ? 
3944 ?g/kg) detected in commercial development.  
Bioretention is one of the most efficient and cost effective stormwater control 
measures (SCMs) (Li and Davis, 2008). This treatment takes advantage of the porous soil 
media, which consists of soil, sand, and organic matter; stormwater flows through the 
medium to reduce the concentration of pollutants (Li and Davis, 2008, Jones and Davis, 
2013). Several studies have reported the effectiveness of bioretention in the removal of 
phosphorus, nitrogen, heavy metals, organic pollutants, such as PAHs, PCBs and oil, and 
pathogens in stormwater (LaBarre et al., 2016, Hong et al., 2006, Wan et al., 2017, Lau et 
al., 2017, Brown and Hunt 2012, DeBusk et al., 2011). In a study of a bioretention system 
in the semiarid Daly City, California, USA, PAH concentrations in the stormwater were 
reduced by 90% (from 2300 to 235 ng/L) with the installation of a bioretention system 
(David et al., 2014). Diblasi (2008) reported that bioretention cells could reduce 31 to 
99% of the PAH event mean concentration and 86% of the PAH mass load; the reduction 
of PAH concentration was associated with the removal of total suspended solids (TSS).  
In addition to physical removal of PAHs via suspended solids removal, 
microorganisms can enhance the removal of PAHs by degrading the organic compounds 
(Hong et al., 2006; LeFevre et al., 2011). PAHs are biodegradable in both aerobic and 
anaerobic environments (Nzila, 2018; Wongwongsee et al., 2013), where various 
6 
 
enzymes play a key role in the involved pathways.  
There are three pathways involved in aerobic PAH degradation (Figure 1). These 
involve cytochrome P-450 monooxygenase, ligninolytic enzymes and PAH ring-
hydroxylating dioxygenase (PAH-RHD). Both bacteria and fungi can degrade PAHs 
through the cytochrome P-450 monooxygenase pathway (Haritash and Kaushik, 2009). 
Ligninolytic enzymes, such as lignin peroxidases, manganese peroxidases, and laccases, 
provide PAHs degrading pathways for fungi, while bacteria can also degrade PAHs via 
the PAH-RHD pathway (Haritash and Kaushik, 2009; Kadri et al., 2017; Syed et al., 
2010). Low-molecular-weight PAHs (LMW-PAH), which are PAHs with two and three 
rings, are generally biodegradable in anaerobic environments. However, anaerobic 
biodegradation of high-molecular-weight PAHs (HMW-PAH) (> 4 rings) were studied in 
an anaerobic batch reactor (Nzila, 2018). Examples of bacteria and fungi that can degrade 
PAHs are shown in Table 2. 
Current studies of PAH fate in bioretention cells has not provided sufficient 
information regarding the presence or distribution of PAHs and PAHs degrading bacteria, 
and few studies have evaluated the environmental impact of bioretention cell media by 
PAH concentrations and toxicity. To assess the environmental impact, information about 
PAH concentrations and spatial distribution is necessary. To evaluate the biodegradation 
potential of bioretention cells for PAHs, potential PAHs degrading bacteria need to be 
identified. The overall objectives of this study were (1) to investigate the spatial 
7 
 
distribution of PAHs in a bioretention cell, (2) to identify the sources of PAHs on the 
campus of University of Maryland based on diagnostic ratios, (3) to estimate toxicity of 
captured PAHs by calculating the benzo[a]pyrene toxic equivalent quantity (BaP-TEQ) 
and (4) evaluate if biodegradation of PAHs can occur at the site. 
To advance the understanding of the PAH presence and biodegradation in 
bioretention, surface samples as well as core samples were collected two times during a 
year at a UMD bioretention cell. These samples were analyzed for PAH concentrations of 
the 16 EPA required PAHs. The microbial composition of the bioretention surface and 
core samples were analyzed using molecular tools to determine if biodegradation of 
PAHs could potentially be occurring in the system. 
 
2. Materials and Methods 
2.1 Sampling sites 
A bioretention cell that was built in spring 2004 and located at the University of 
Maryland Campus in College Park MD was investigated. Asphalt parking lots and roads, 
as well as concrete walkways, form the drainage area with the impervious surface 
accounting for approximately 90% of the total area. The cell is 50.3 m long and 2.4-4.8m 
wide with a total area of 181 m2. A drainage area of approximately 0.28 ha is managed by 
the bioretention cell (DiBlasi et al., 2009). Four media sampling points were utilized in 
the bioretention cell, which were located at 0, 0.91, 1.83, and 2.74 m from the inlet of the 
8 
 
cell (Figure 2).  
A control sampling point was included in the study. This was located across from the 
bioretention inlet but was not contacted by stormwater at any time. Core samples of the 
bioretention media were collected at the depth of 0 to 30 cm, and at sampling points at 
0.91, 1.83, and 2.74 m using a Hoffer Soil Sampler (JBK Manufacturing, Dayton, U.S.). 
The core samples were divided into three segments each 10 cm in length, representing 
media at average depths of 5, 15, and 25 cm, respectively. To study any seasonal 
differences, samples were taken in February 2019, and June 2019. Surface samples were 
collected by a stainless-steel scoop. Samples were stored in glass containers at -20?C 
after collection to avoid biodegradation of PAHs.   
2.2 Extraction of PAHs 
PAHs were extracted by microwave-assisted extraction (MAE) (MARS 6, CEM, 
City, U.S.), based on the EPA SW-846 Test Method 3546 (EPA, 2007). The samples were 
first air dried for 24 hours in a fume hood. The dried samples were ground and weighed 
to approximately 5 g (exact weight was recorded), and then introduced into Teflon 
extraction vessels (100 mL, CEM, Matthews, U.S.). In vessels used for background 
(blanks), 5 g of clean sea sand (Merck, U.S.) was used. The samples and blanks were 
extracted in triplicate. For MAE, 30 mL of n-hexane (HPLC grade)-acetone (HPLC 
grade) (1:1) (v/v) was added to the extraction vessels, followed by 10 ?L (100 mg/L)of 
deuterated PAHs, specifically Naphthalene-d8, Acenaphthene-d10, Phenanthrene-d10, 
9 
 
Chrysene-d12, and Perylene-d12 (AccuStandard, New Haven, U.S.) were used as 
surrogate standards. MAE was performed at 115? for 10 min at 1000 W. After 
extraction, the vessels cooled to room temperature and the solution was transferred with a 
pipette into 60 mL amber vials. Residues were rinsed by 5 mL of hexane, hexane-acetone 
(1 mL) and acetone separately, and the supernatants were transferred to amber vials. 
Then, the extracts were concentrated to 1 mL by a N-EVAP Nitrogen Evaporator 
(Organomation, Berlin, U.S.). 
2.3 Cleanup of extracts 
After extraction, the extracts were cleaned in an alumina column according to EPA 
publication SW-846, method 3630C (EPA, 1996). Before cleanup, Alumina (Fisher 
Scientific, Pittsburgh, U.S.) was baked at 450? for 24 hours to remove organic matter. 
The activated alumina was then placed in a desiccator and cooled to room temperature. 
At this point the alumina was deactivated by adding 3% DI water and it was subsequently 
equilibrated in the desiccator for 16 hours. Sodium sulfate (Fisher Scientific, Pittsburgh, 
U.S.) was a baked at 450? for 4 hours, then placed in a desiccator. To make extraction 
columns, glass wool (Acros Organics, Morris Plains, Germany) was placed at the bottom 
of 10 mL glass disposable pasteur pipets (Pyrex, Corning, U.S.), followed by 5 g of 
prepared alumina. A layer of sodium sulfate was added at the top of the column to 
remove potential water from the extracts. The column was rinsed with 20 mL hexane 
prior to cleaning the PAH extracts that were added at the top of column. The container of 
10 
 
extracts was rinsed with 20 mL hexane-dichloromethane (3:2) (v/v) and transferred to the 
column. The effluent containing PAHs was collected in a graduated centrifuge tube, 
which was then concentrated to less than 1 mL by a N-EVAP Nitrogen Evaporator 
(Organomation, Berlin, U.S.). The cleaned extracts were spiked with 10 ?L internal 
standards mixture (100 mg/L) (Anthracene-d10, Benzo[a]pyrene-d12) (VWR, Radnor, 
U.S.). Hexane (HPLC grade) was added to the effluent to produce a final volume of 
exactly 1.0 mL. The samples were vortexed for 10 s to ensure mixing, then loaded onto 
the GC for GC/MS analysis. 
2.4 GC/MS analysis 
The analysis method was based on EPA method SW-846 8207. Samples were 
analyzed by gas chromatography/mass spectrometry (GC-MS) (7890B, Agilent 
Technologies, U.S.) with an Agilent fused silica capillary column (HP-5ms) and a mass 
spectrometer detector (5973N, Agilent Technologies, U.S.), which operated in the SIM 
and Scan mode. The carrier gas was helium, and the injection was in splitless mode with 
a volume of 1 ?L. The temperature of the oven was raised from 70?C to 180?C at 7?C 
min-1, from 180?C to 225?C at 1?C min-1, from 225?C to 285?C at 5.8?C min-1 with 
isothermal hold at 285?C for 20 min, then ramped to 300?C at 11.5?C min-1 and held at 
300 ?C for 10 min. Target compounds were purchased from AccuStandard (New Haven, 
Connecticut, U.S.).  
2.5 Quality Control 
11 
 
To avoid contamination of the samples, all the containers were made by glass or 
PTFE. Glassware was cleaned using hexane, methanol, and DI water, and baked at 550? 
for 4 hours before any use in experiments. To avoid contamination between extractions, 
Teflon extraction vessels were ultrasonicated with hexane and methanol and then rinsed 
with DI water after sonication. To avoid contamination in the GC-MS column, a hexane 
injection was added at the first and last of the sequence of run. The recoveries for PAHs 
were determined by five surrogate standards, Napthalene-d8, Acenaphthene-d10, 
Phenanthrene-d10, Chrysene-d12, and Perylene-d12, and the recoveries were used to 
correct the measured concentration of PAHs. Naphthalene-d8 was used to correct the 
concentration of NAP. The recovery of acenaphrhene-d10 was used to correct the 
concentration of ACY, ACE and FLU. PHE, ANT and FLA were corrected by 
phenanthrene-d10, PYR, BaA and CHR were corrected by chrysene-d12. perylene-d12 
was used to correct BbF, BkF, BaP, INP, DBA and BPY. The average recoveries of each 
surrogate standard were calculated, which were 105.90%, 119.35%, 84.53%, 55.87%, and 
73.71% for naphthalene-d8, acenaphrhene-d10, phenanthrene-d10, chrysene-d12, and 
perylene-d12 respectively. Anthracene-d10 was used as internal standard for NAP, ACY, 
ACE, FLU, PHE, ANT and FLA, and benzo[a]pyrene-d12 was used as internal standard 
for PYR, BaA, CHR, BbF, BkF, BaP, INP, DBA and BPY (Table 3). The instrument 
detection limits (IDL) for the tested PAHs ranged from 0.0001 to 0.002 ?g/g (Table 4), 
which were calculated by dividing the instrument detection limits by the sample masses. 
12 
 
The values lower than instrument detection limits were calculated as IDL/2 (EPA, 2006).  
2.6 Data analysis 
Five-point calibration curves for 16 PAHs, with concentrations ranging from 0.01 to 
5 ?g/g and R2>0.98, were used for quantification. To evaluate if significant differences 
were present among the sampling sites, depths, and seasons, respectively, student t-tests 
were performed. The confidence level was set at 0.95, and RStudio was used for 
statistical analysis (REF). Benzo[a]Pyrene Total Toxicity Equivalent (BaP-TEQ), was 
calculated by multiplying benzo[a]pyrene toxic equivalence factors (BaP-TEFs) and 
individual PAH concentrations; these values were used to represent the toxicity of total 
PAHs present in the samples. PAHs and their corresponding BaP-TEFs are showed in 
Table 3. The calculation follows formula 1. 
?????? =  ????? ??=1 ???? ? ????????             (1) 
Where i (i = 1???16) described the type of PAH, TEQ stands for the calculated BaP-
TEQ for samples, Ci is the corresponding concentration of PAHi, TEFi is the the value of 
BaP-TEF for PAHi.  
2.7 DNA extraction and quantification 
DNA of positive control cultures (Pseudomonas putida and Pseudomonas 
fluorescens) were extracted by UltraClean Microbial DNA Isolation Kit (QIAGEN, 
Hilden, Germany). DNA of bioretention cell media were extracted from 250 mg samples 
using a DNeasy PowerSoil Kit (QIAGEN, Hilden, Germany) according to manufacturer?s 
13 
 
instruction. Nanodrop 2000 (Thermo Scientific, Waltham, U.S.) was used to quantified 
extracted DNA. 
2.8 PCR amplification 
Universal primers 341F and 907R were used to amplify the 16S rRNA gene. PAH-
ring hydroxylating dioxygenases (PAH-RHDs) and PAH-ring cleaving dioxygenases 
(PAH-RCDs) primers were used to amplify the functional gene of dioxygenase PAH 
degradation, and primer pairs are listed in Table 5. PAH-RHD GN and GP primers were 
work for gram negative and positive bacterial, respectively. There were three primers for 
PAH-RCDs, namely protocatechuate-3,4-dioxygenase (pca H), catechol-1,2-dioxygenase 
(C12O) and catechol-2,3-dioxygenase (C23O). DNA samples were stored at -20 ?. 
For PCR reaction, 10.2 ?L of nuclease-free water (Ambion, Austin, U.S.), 12.5 ?L 
Dream Taq Green PCR Master Mix (Thermo Scientific, Waltham, U.S.), 0.15 ?L of 
forward and reverse primers (Integrated DNA Technologies, Coralville, U.S.), and 2 ?L 
of template DNA were mixed. AT100 Thermal Cycler (Bio-Rad Laboratories, Hercules, 
U.S.) was used for PCR analysis. The following thermal cycling conditions were used for 
PCR amplification: initial denaturation 95 ?C for 3 min; 35 cycles of denaturation 95 ?C 
for 30 s, annealing 30 s at annealing temperatures of primers (Table 5), extension 3 min at 
72 ?C; and a final extension step at 72?C for 10 min. Fragments of 16 S rRNA gene and 
functional gene of aerobic PAH degradation were separated by gel electrophoresis. 1.5% 
agarose gel was made by 1.8 g agarose, 120 mL 1X TAE buffer, and 2 drop of 0.625 
14 
 
mg/mL ethidium bromide (Genesee Scientific, San Diego, U.S.). 5 ?L of PCR products 
were loaded to gel and run at 100 V until the dye line was 80% ranging from the wall. 
After the gel electrophoresis, the digital pictures of gels were obtained and analyzed by 
FlourChem FC3 (ProteinSimple, San Jose, U.S.). 
 
3. Results and Discussion 
3.1 Total PAH concentrations and spatial distribution of PAHs 
Quantifiable concentrations of the 16 EPA listed PAHs were detected at all sampling 
sites; the total PAH concentration ranged from 1.6?0.31 to 25.5?0.14 ?g/g. The lowest 
concentrations of total PAHs in surface samples was detected at 0 m in February 
2019(9.7?1.8 ?g/g), and the highest value was found at 2.74 m in June 2019 (16.1?0.6 
?g/g). The total PAH concentration of the control samples were 1.5?0.5 ?g/g in February 
and 1.2?0.4 ?g/g in June, similar to the lowest concentration in the bioretention cell and 
approximately 10 times lower than the concentrations of surface samples in the 
bioretention cell (Figure 3). Without the added impact of stormwater, PAHs in the control 
samples were mainly accumulated by wet and dry deposition. This result indicates that 
the major accumulation of PAHs in the bioretention cell originated from input delivered 
via stormwater.  
The concentrations of PAHs in the sampling sites were compared by t test, and the 
results showed that total PAH concentrations of 2.74 m sampling site at June was 
15 
 
significantly higher than that at other sampling sites, and at February the total PAH 
concentration of 2.74 m was significantly higher than 0.91 sampling site (p<0.05). Also, 
the total PAH concentration at 1.83 m in February was significantly higher than that at 0 
and 0.91 m (p<0.05). The p-values were showed in appendix. Statistical evaluation of the 
concentration at other sampling sites didn?t show any significant differences. 
Furthermore, seasonal differences in the concentration of PAHs in the surface samples 
were not observed (p>0.05).  
If the sampling point at 0m is not considered, the total PAH concentration was 
positively correlated with distance, and the correlation coefficients in February and June 
were 0.862 and 0.966, respectively (Figure 4). The distribution of PAHs in particles of 
different sizes may be the reason for the increase of PAH concentrations with distance. 
Herngren (2010) studied the distribution of PAHs with particle size in stormwater (< 
0.45?m, 0.45-75?m, 75-150?m and > 150?m), and the highest PAH concentration was 
found in the 0.45-75?m particles. The distribution of PAH in particles of different sizes 
may be the reason for the increase of PAH concentration with distance. Large particles 
tend to be deposited at the inlet of the bioretention cell, while most PAHs will be 
transported by the stormwater with the fine particles and deposited at a lager distance. 
The PAH concentrations in core samples together with the distributions showed in Figure 
6. At the 0.91 m sampling site, the average of the total PAH concentrations increased with 
the depth of the core samples, but there was not a statistical significance (p>0.05). At the 
16 
 
1.83 m sampling site, the highest concentrations were found at 5 cm (21.3?1.6 ?g/g) in 
February and 15 cm (25.5?1.7 ?g/g) in June. Total concentrations had a decreasing trend 
at 2.74 m in both February and June. The same bioretention cell was studied in 2007-
2008 by DiBlasi et al. (2008). Here, the results showed that the trends for the 2.74 m 
location was similar with regard to the total PAH concentrations in core samples at 1.5 m 
of this study, the surface samples had the highest PAH concentrations and the 
concentrations decreasing with the increasing of depth. The core samples at 0.91 m and 
1.83 m have the concentrations higher than 1.5 m core samples in 2008 (2.96 ?g/g in the 
top 10 cm segment, 0.33?0.64 ?g/g in lower segments), but the concentrations of surface 
samples were in the same range (12.5?22.7 ?g/g), this results shows that PAHs were 
accumulated in core samples during past 10 years. 
Concentrations of PAH concentrations in stormwater management facilities reported 
by other studies is provided in Table 6. The PAH concentrations among studies vary 
greatly; the highest value was 89.4 ?g/g, and the lowest value was 0.064 ?g/g. The total 
PAH concentrations within a study were also had wide ranges. In a study of sediments 
from stormwater management ponds at Baltimore, the highest total PAH concentration 
was 88.9 ?g/g and the lowest value was lower than detection limit (0.214 to 4.280 ?g/kg, 
depending on the PAH). One reason for the variations of PAH concentrations may be the 
surrounding environment of the sampling sites. The relatively high level of PAH 
concentrations of sediments from stormwater management ponds in Baltimore may due 
17 
 
to the residential and commercial development of surrounding area in the 20 years before 
the study (Gallagher et al., 2011). Concentration of total PAH was elevated after coal-tar 
based sealcoat was applied in the watershed (10.9-95.7 ?g/g), compared with the 
concentration before coal-tar based sealcoat was applied (0.54-3.08 ?g/g) (Watts et al., 
2010). Weinstein (2010) detected the highest PAH conceration from a commercial area 
(10.4 ? 3.9 ?g/g), and relatively lower concentrations were detected at a golf course (0.42 
? 0.22 ?g/g), high density residential ponds (347 ? 29 ?g/kg), and low density residential 
area (0.347 ? 0.029 ?g/kg). 
3.2 Individual PAH concentrations 
The concentrations of individual PAHs ranged widely in the bioretention cell media 
(Figure 6). The 16 EPA listed PAHs were detected in all samples, and the average 
concentrations of individual PAHs were ranged from 3.68 ?g/g to lower than detection 
limit (0.0001 ?g/g for ACE). Individual PAHs were sorted from highest to lowest by 
average concentration in all depths and distances. Benzo[g,h,i]perylene was the PAH with 
highest average concentration in February (1.8?0.79 ?g/g), while pyrene had the highest 
average concentration in June (1.8?0.80 ?g/g). The lowest concentration was found for 
acenaphthylene (0.007?0.14 ?g/g in February and 0.014?0.014 ?g/g in June). PAHs 
concentration rankings were same in February and June after CHR, and concentrations of 
same PAH had no significant differences between February and June (p>0.05).  
3.3 PAHs sources analysis 
18 
 
The 16 EPA listed PAHs were divided into groups according to the ring number: 2-
ring (NAP), 3-ring (ACY, ACE, FLU, PHE, ANT), 4-ring (FLA, PYR, BaA, CHR), 5-
ring (BbF, BkF, BaP, DBA), and 6-ring (IDP, BPY). PAHs below the 4-ring were 
classified as LMW-PAH, and PAHs of 4-ring and above were classified as HMW-PAH 
(Brown and Peake, 2006). In February and June, HMW-PAH contributed 93% to the total 
PAH concentrations. Furthermore, the results showed that the 4-ring PAHs accounted for 
the largest part of total PAH (38.8%) of total PAH concentration in February and 39.8% 
in June. The second highest group contributing to the total PAH concentration was 5-ring 
PAHs (29.3% in February and 27.9% in June). (Figure 7). Ring-based PAHs distributions 
did not show significant seasonal differences (p>0.05). This suggests that there is no 
seasonal variation in the source of PAHs, which is discussed in more detail with 
diagnostic ratios. 
Most of the PAHs in the bioretention cell media were HMW-PAH (Figure 7). HMW-
PAHs are less volatile, leachable and degradable, so HMW-PAH are more stable in the 
environment of a bioretention cell (Watts et al., 2010). The high proportion of HMW-
PAH may also be due to the sources of PAHs. Lee (1995) studied PAHs in the urban 
ambient air (traffic-sources) in which concentrations of CHR, BaA, BbF. BkF, BaP, INP, 
DBA, and BPY were 7.8 times and 16.5 times higher than the concentrations in urban 
and rural site. Also, a study of highway stormwater runoff in Los Angeles showed that in 
traffic-sources runoff 4 to 5 ring PAHs account for 97% of total PAHs (Lau et al., 2009). 
19 
 
In a study of impervious surface run-off in Shanghai, China, there were 82.3% HMW-
PAH vs. 17.7% LMW-PAH in the stormwater from asphalt road run-off at urban area 
(Hou et al., 2013). Stormwater in the campus of University of Maryland contained 77% 
HMW-PAH 2007-2008 (DiBlasi et al., 2008), which was lower than the proportion of 
HMW-PAH (93%) in the media in this research. This result shows that the accumulation 
rate of HMW-PAH is faster than that of LMW-PAH, and this may be caused by the 
relatively lower bioavailability and higher Kow of HMW-PAH. DiBlasi also found that in 
the bioretention media 90% to 94% of total PAHs were HMW-PAH, which shows that the 
proportion of HMW-PAH did not change in the past 10 years. In addition, 6-ring PAHs 
were more prevalent in the bioretention media than in control samples (11.3% in 
February and 15.8% in June of control samples), since PAHs in the control samples 
mainly originate from dry and wet deposition of the atmosphere, and most of the PAHs in 
the bioretention media were input by stormwater transportation.  
PAHs with two or three rings are primarily generated by petrogenic sources, whereas 
PAHs generated by incomplete combustion are composed by PAHs with four to six 
aromatic rings (Pies et al., 2008). Diagnostic ratios, which is the specific ratio of PAHs 
and isomer, can be used to identify the sources of PAHs. For example, the ratio of FLA / 
(FLA + PYR) can distinguish petrogenic (<0.4) and pyrogenic sources (>0.4) (Galarneau, 
2008; Yunker et al., 2002; Yunker et al., 2012). BaA/(BaA+CHR), FLA/(FLA+PYR) and 
ANT/(ANT+PHE) were three diagnostic ratios calculated (Table 7). For each diagnostic 
20 
 
ratio, a PAH isomer with less thermodynamic stability is used in the numerator. As a 
result, the higher the diagnostic ratios the more combustion-derived PAHs in the samples 
(Yunker et al., 2002; Yunker et al., 2012). Diagnostic ratios were applied in bivariate form 
(Figure 8). 
The results from the source analysis suggest that PAHs in most bioretention media 
sampling points were generated by a mix of petroleum sources together with fossil fuel 
combustion source. No significant difference between February and June was found for 
both FLA/(FLA+PYR) and BaA/(BaA+CHR) (P>0.05). So, there was no seasonal trend 
when evaluating the sources. The bioretention cell collected stormwater from a drainage 
area with impervious surfaces such as parking lots and campus roads, thus major sources 
of PAHs may be leakage of oil and incomplete combustion of fossil fuel. These results 
are consistent with the results of individual PAH concentrations, which showed that BPY 
and PYR had the highest concentrations in the bioretention cell (Figure 5). BPY and IND 
sources are typically caused by gasoline emission and combustion of heavy oil (Iwegbue 
et al., 2019; Kwon and Choi, 2014; Larsen and Baker, 2003), whereas PYR, BaA, CHR, 
BbF, BkF, IND, and DBA are markers for diesel emissions (Kwon and Choi, 2014; Wang 
et al., 2013; Yang et al., 2013).  
Results of the three diagnostic ratios in February  show (BaA/(BaA+CHR) <0.35, 
FLA/(FLA+PYR) <0.4, ANT/(ANT+PHE) < 0.1) and two diagnostic ratios 
(BaA/(BaA+CHR) < 0.35, FLA/(FLA+PYR) < 0.4) in June showed that PAHs from a 
21 
 
petroleum source or mix sources was observed at the 0 m sampling point. The diagnostic 
ratios had no seasonal difference (Figure 8A), but there were differences in the source 
ratios between depths of the collected samples. For both ANT/(ANT+PHE) and 
BaA/(BaA+CHR), the samples at 0 cm were significantly lower than the samples at 15 
cm and 25 cm (p<0.05), and the samples at 5 cm were also significantly lower than the 
samples at 15 cm and 25 cm (p<0.05), p values are showed in appendix table A3.  
The source of PAHs in the surface samples originated from a petroleum source, and 
as the depth increases, the source of PAHs changed to be from a combustion source 
(Figure 8B). Due to the filtration of the media, the suspended particles was concentrated 
at the inlet of the bioretention cell and surface of the media (DiBlasi et al., 2008). As a 
result, petroleum source PAHs may mainly input into the bioretention cell through 
suspended particles in stormwater. This may be the reason for the observation of more 
PAHs originating from a petroleum source at the surface and the inlet of bioretention cell. 
The source of PAHs in the control samples were derived from a combustion source and 
were different from the source of PAHs in the bioretention media. This indicated that the 
PAH content of the petroleum source in the stormwater was higher than that in the 
atmosphere.  
3.4 Toxicity 
To assess the effect of the bioretention media on the ecosystem, the concentrations of 
total PAH and individual PAHs were compared with threshold effect concentration (TEC) 
22 
 
and probable effect concentration (PEC) as a measure of toxicity (MacDonald et al., 
2000).  
TECs and PECs were determined by the survival or growth of sediment-dwelling 
organisms in the sediments from aquatic environment (i.e., midge, daphnid, amphipod, 
bacteria)(MacDonald et al., 2000). If the concentrations of PAHs were higher than PECs, 
it will have an adverse effect on the survival of the bacteria, which in turn may affect the 
effect of bioremediation. Since there are no PECs and TECs established for the soil at the 
moment, and the media in the bioretention cell is similar to the sediments in the aquatic 
environment, it is assumed that PECs and TECs of sediments are equally effective for 
bacteria in the media.  
Comparison of PAH concentrations with TECs and PECs showed that that mean 
concentrations of PYR (1.78 ?g/g) and CHR (1.40 ?g/g) were higher than PECs (Table 
8), which means that toxicity of those two PAHs will be observed in the bioretention cell. 
FLA, PYR, BaA, CHR, and BaP, had the highest concentrations higher than their PECs. 
As a result, the toxicity of those PAHs will be observed at specific sampling site where 
FLA, PYR, BaA, CHR, and BaP had the highest concentrations. The highest value of 
total PAH concentration (25.5 ?g/g) was higher than PEC (22.8 ?g/g) thus the risk of 
toxicity caused by the 16 EPA listed PAHs at this sampling point (1.83 m, 15cm depth in 
June) was present. In contrast, total PAH concentrations in both control samples were 
lower than TEC, so adverse effects were not expected to occur. Those results show the 
23 
 
negative environmental impact of stormwater with input of PAHs into the bioretention 
cell with subsequent PAH accumulation in the media. The average concentrations of 
pyrene and chrysene in all samples were higher than PECs, which shows that these two 
PAHs in the bioretention cell will affect the survival of bacteria. At 1.83m in June, the 
total PAH concentration at 15cm depth is greater than PEC, which means that at this 
sampling point, the survival of bacteria will be affected by the total PAH concentration. 
This result shows that when bioremediation is used to remove PAHs in media, the effect 
of PAHs concentrations should be considered. 
Other studies have reported similar effects. Howitt (2014) reported that stormwater 
collected from surrounding residential and commercial districts and highway caused 
accumulation of PAHs in sediments of a semi-natural wetland in Victoria, Australia. 
Various PAHs with concentrations exceeded PCEs, which indicate that the toxicity can be 
observed, were found at the sampling site with highest PAH concentrations (ACE: 0.62 
?g/g, PHE: 1.8 ?g/g, FLA: 3.9 ?g/g, PYR: 3.8 ?g/g, BaA: 1.8 ?g/g, CHR: 1.8, BaP: 1.8 
?g/g). A study in Toronto Canada showed that PECs exceeded by PAH concentrations of 
sediments (PHE: 1.4 ?g/g, FLA: 4.3 ?g/g, PYR: 3.3 ?g/g, BaA: 1.1 ?g/g, CHR: 2.4 ?g/g, 
BaP: 1.6 ?g/g) in stormwater management facility (Bartlett et al., 2012). The results of 
those studies indicates that stormwater causes the accumulation of PAHs, and make the 
concentrations of PAHs in bioretention media and sediments to exceed PEC.  
BaP-TEQs were used in this study to assess the carcinogenic potency of samples in 
24 
 
the bioretention media Eq. (1). BaP-TEQs of surface and core samples in February 
ranged from 0.22 ?g/g to 2.96 ?g/g, BaP-TEQs in June ranged from 0.28 ?g/g to 3.56 
?g/g, and there were no seasonal differences between February and June (p > 0.05). The 
highest values of BaP-TEQs were found at 1.83 m at 5cm depth in February and 1.83 m, 
15cm depth in June., The lowest values were found at 2.74 m, 25 cm depth (Table 9). 
BaP made the greatest contribution to the total BaP-TEQ, and the average 
contribution rates was 60.1% (Table 6). Contribution rates of carcinogenic PAHs showed 
in the decreasing order: BaP (60.09%) > DBA (16.59%) > BdF (9.49%) > INP (8.90%) > 
BaA (4.34%) > BkF (0.52%) > CHR (0.09%). BaP had the third highest concentration of 
all carcinogenic PAHs, but it contributed the most to carcinogenicity due to its high BaP-
TRQ. This indicated that BaP is the most important carcinogen in the bioretention cell, 
which means that when assessing the environmental impact of the bioretention cell 
media, BaP should receive most attention. In soils from an industrial district in Shanxi, 
China, BaP accounted for 73.7% of the total BaP-TEQ (Jiao et al., 2017) and in surface 
sediments of San Diego Bay, CA, USA, the contribution rate of BaP for BaP-TEQ was 
over 90% (Neira et al., 2017). Since the main source of BaP is fossil fuel combustion by 
motor vehicle (Dickhut et al., 2000), the carcinogenicity of media in the bioretention cell 
is mainly caused by motor vehicle emissions. This shows that the bioretention cell helps 
to reduce the pollution of receiving waters by BaP by adsorbing BaP from vehicle 
exhaust. However, if the concentration of BaP accumulated in the bioretention cell media 
25 
 
is too high, there is also a risk of environmental impact.  
3.5 Gradient PCR  
Pseudomonas putida and Pseudomonas fluorescens were tested as positive control 
for PAH-RHD GN, C12O and pca H primer, and gradient PCR was performed for its 
DNA samples to find the optimal annealing temperature for PCR. The annealing 
temperature range of gradient PCR were 50? to 60?, and automatically divided into 
eight temperatures by the instrument (50?, 50.7?, 51.9?, 53.8, 56.1, 58.0?, 59.2?, 
and 60?). The electrophoretic profiles of the gradient PCR are showing in appendix 
(figure A2 and figure A3). Bands for PAH-RHD GN were not find on the gel for both 
Pseudomonas putida and Pseudomonas fluorescens, but bands for C12O (470 bp) and 
pca H (395 bp) were found for Pseudomonas putida. There were error bands, which were 
around 600 pb and more than 1000 bp, for C12O. The optimal annealing temperature can 
be determined based on the brightness of the bands. Therefore, the optimal annealing 
temperature for C12O was between 56.1? and 58?, because the bands for C12O had 
relatively high brightness and error bands had low brightness. The optimal annealing 
temperature for pca H was 50 ?, because the brightness of the band is the highest. 
3.6 Gel electrophoresis 
PAH-RCDs can promote the degradation of catechol, which is PAH biodegradation 
intermediate products. C12O gene and pca H gene work for ortho fission and meta fission 
pathway, respectively(Thomas et al., 2016). The gel electrophoresis analysis revealed the 
26 
 
biodegradability of PAHs in the bioretention media (Figure 9). There are 1 lane for 
control sample and 12 lanes for media samples. Bands for C12O genes were found for 
some samples, but pca H gene was not found in the bioretention cell. As a result, bacteria 
in media may be able to degrade catechol through ortho fission pathway. Bands for C12O 
were not appeared at S7 (1.83m 5cm), S8 (1.83m 15cm), S11 (2.74m 15cm) and S12 
lanes (2.74m 25cm). This result is consistent with the dioxygenase degradation pathway 
of PAHs. Because this pathway is an aerobic pathway, the lack of oxygen deep in the 
media makes this degradation pathway difficult. The degradation of catechol by PAH-
RCDs is one of the key steps in the biodegradation of PAHs, but only the presence of the 
PAH-RCD genes in media does not determine that PAH can be biodegraded in 
bioretention cells. In addition, it is also necessary to determine whether the PAH-RHD 
genes, which play a role in the first step of PAH biodegradation, exists in media.  
The distribution of PAH-RCD genes indicates that the PAH biodegradation of the 
dioxygenase pathway may not occur in the deep part of bioretention, but these parts still 
have PAHs that need to be degraded. For example, there was no PAH-RCD gene below 
5cm depth at 1.83m, but the total PAH concentrations were even higher than the total 
PAH concentration of the surface sample. This result reveals the limitations of using the 
dioxygenase pathway for bioretention cell media bioremediation, that is, this approach 
may not be possible in the depths of media. Therefore, when performing bioremediation 
of the bioretention cell, it may be necessary to cooperate with bioventing. 
27 
 
3.7 Recoveries 
The calculation of recoveries has influences on the total and individual PAH 
concentrations and one diagnostic ratio. The distribution of PAHs was hardly affected by 
recoveries. And the figures and tables without the calculation of recoveries are in the 
appendix. 
Without the calculation of recoveries, the total PAH concentrations were ranged from 
1.20 ?g/g (2.74 m 25 cm in February) to 19.37 ?g/g (1.83m 15 cm in June), which lower 
than the values corrected by recoveries (Figure A4). There are larger seasonal differences 
between February and June. Total PAH concentrations in the surface samples at 0.91 m 
and 2.74m have significant seasonal differences (p < 0.05). The highest values of total 
PAH concentrations in the surface samples still be found at 2.74 m. The distribution of 
total PAH concentrations in core samples was the same as the values corrected by 
recoveries (Figure A5). 
Because of relatively low recoveries of HMW-PAHs, HMW-PAHs had lower 
proportion in total PAH concentrations. The proportion of HMW-PAHs reduced form 
93% to 92%, and instead of benzo[g,h,i]perylene, pyrene had highest average 
concentration in June (Figure A6). 
The reduction of PAH concentrations also caused less PAH concentrations exceed 
PECs. Compare with the concentrations corrected by recoveries, the highest 
concentration of benzo[a]anthracene and total PAH were reduced below PECs, and no 
28 
 
average concentration higher than PEC (Table A4). There was little change in the 
contribution of carcinogenic PAHs to BaP-TEQs. Benzo[a]pyrene still made most 
contribution to the total BaP-TEQ, and the proportion was 60.73%. 
Recoveries had influence on diagnostic ratio of FLA/(FLA+PYR) because the 
recoveries of fluoranthene and pyrene were represented by different surrogate standards. 
Without the correction of recoveries, FLA/(FLA+PYR) has larger values, which indicates 
coal, wood, and grass combustion source (figure A7). Other ratios were not influenced by 
recoveries since PAHs on the numerator and denominator had the same surrogate 
standard. 
4. Conclusions and summary 
Studies have shown that in the range of this study, the total PAH concentration was in 
the middle distance and middle depth of the bioretention cell. And through compare the 
results with the study at the same site in 2008, it is revealed that PAHs accumulated in the 
core samples during past 10 years. Studies on individual PAH concentrations found that 
PAHs derived from fossil fuel combustion mainly contributed to total PAH 
concentrations, which is consistent with the results of PAH source analysis based on the 
diagnostic ratios. The distribution of PAH toxicity in bioretention cell is similar to the 
distribution of PAH concentrations, the highest total PAH concentration exceeds PEC, 
and the concentrations of PAHs in some individuals exceeds PECs, which indicates that 
PAH toxicity in media is likely to cause environmental impacts. BaP emitted by motor 
29 
 
vehicles mainly contributed to the carcinogenicity in media.  
5. Acknowledgements 
This study was funded by SERDP Project ER18-C3-1303 to PI Dr. Birthe Kjellerup, 
University of Maryland, Department for Civil and Environmental Engineering, College 
Park. 
 
6. Tables and figures 
Table 1. 16 priority PAHs identified by EPA
Group of carcinogenic 
Compound name Abbreviation LogK  LogK  
activity by IARC1 OA OW
Naphthalene NAP 2B 5.19 3.3 
Acenaphthylene ACY - 6.23 3.93 
Acenaphthene ACE 3 6.28 3.92 
Fluorene FLU 3 6.58 4.18 
Phenanthrene PHE 3 7.33 4.46 
Anthracene ANT 3 7.09 4.45 
Fluoranthene FLA 3 8.32 5.16 
Pyrene PYR 3 8.8 4.88 
Benzo[a]anthracene BaA 2A 9.1 5.76 
Chrysene CHR 2B 9.4 5.73 
Benzo[b]fluoranthene BbF 2B 10.68 5.78 
Benzo[k]fluoranthene BkF 2B 10.73 6.11 
Benzo[a]pyrene BaP 1 10.86 6.13 
Indeno[1,2,3-cd]pyrene INP 2B 11.56 6.7 
Dibenzo[a,h]anthracene DBA 2A 13.67 6.5 
Banzo[g,h,i]perylene BPY 3 11.77 6.63 
1Carcinogenic to human beings: (1) carcinogens to human, (2A) probable carcinogens, 
(2B) possible carcinogens, and (3) evaluation of carcinogenic activity is not possible due 
to limited data, IARC (2012). 
 
30 
 
 
Figure 1. Pathway of PAH biodegradation. 
 
 
 
 
 
 
 
 
 
 
 
 
 
31 
 
Table 2. Examples of bacteria and fungi that can degrade PAHs 
 
Enzyme Kingdom Name of organism PAH References 
Aerobic 
Aaphthalene, 
phenanthrene, Syed and 
P450 monooxygenases fungi Phanerochaete chrysosporium 
pyrene, Yadav,2012 
benzo[a]pyrene 
Phenanthrene, 
Carmichael and 
P450 monooxygenases Bacteria Bacillus megaterium fluoranthene, 
Wong, 2001 
pyrene 
Ligninolytic enzymes Fungi Peniophora incarnate Anthracene, 
Ligninolytic enzymes Fungi Trichaptum abietinum phenanthrene, Lee, Jang et al., 
fluoranthene, 2014 
Ligninolytic enzymes Fungi Mycoaciella bispora 
pyrene 
PAH-RHD Bacteria Pseudomonas salomonii Fluorene, 
Bacosa and 
PAH-RHD Bacteria Sphingobium amiense phenanthrene, 
Inoue, 2015 
PAH-RHD Bacteria Dokdonella koreensis pyrene 
Naphthalene, Dutta, 
PAH-RHD Bacteria Pseudomonas putida phenanthrene, Shityakov et 
pyrene al., 2017 
PAH-RHD Bacteria Mycobacterium vanbaalenii Darmawan, 
Pyrene, 
Nakata et al., 
PAH-RHD Bacteria Burkolderia fungorum fluoranthene 
2015 
Anaerobic 
Himmelberg, 
Phenanthrene 
Bacteria Desulfobacteraceae organism Phenanthrene Br?ls et al., 
carboxylase 
2018 
phenanthrene, 
Liang et al., 
Unknown Bacteria Pseudomonas stutzeri benzo[a]pyrene, 
2014 
fluoranthene 
32 
 
 
Figure 2. Vertical view of the bioretention cell at UMD (Yellow dot: sampling points for 
surface samples; red dot: sampling points for surface samples and core samples.) 
 
 
 
 
 
 
 
 
 
 
33 
 
Table 3. Standards and BaP-TEFs for PAHs 
Compound name Surrogate standard Internal standard BaP-TEFs1 
Naphthalene Napthalene-d8 - 
Acenaphthylene - 
Acenaphthene Acenaphthene-d10 - 
Fluorene Anthracene-d10 - 
Phenanthrene - 
Anthracene Phenanthrene-d10 - 
Fluoranthene - 
Pyrene - 
Benzo[a]anthracene Chrysene-d12 0.1 
Chrysene 0.001 
Benzo[b]fluoranthene 0.1 
Benzo[k]fluoranthene benzo[a]pyrene-d12 0.01 
Benzo[a]pyrene 1.0 
Perylene-d12 
Indeno[1,2,3-cd]pyrene 0.1 
Dibenzo[a,h]anthracene 1.0 
Banzo[g,h,i]perylene - 
1Data source: Provisional guidance for quantitative risk assessment of polycyclic 
aromatic hydrocarbons (EPA, 1993) . 
 
 
 
 
 
 
 
 
 
34 
 
Table 4. IDLs and quantifiable limits of PAHs 
Comp # Compound Name IDL (ppm) IDL (?g/g) Quantifiable Limit (?g/g) 
1 Naphthalene 0.0005 0.0001 0.0002 
2 Acenaphthylene 0.005 0.001 0.002 
3 Acenaphthene 0.0005 0.0001 0.0002 
4 Fluorene 0.0005 0.0001 0.0002 
5 Phenanthrene 0.001 0.0002 0.0004 
6 Anthracene 0.0005 0.0001 0.0002 
7 Fluoranthene 0.0005 0.0001 0.0002 
8 Pyrene 0.0005 0.0001 0.0002 
9 Benzo[a]anthracene 0.005 0.001 0.002 
10 Chrysene 0.0005 0.0001 0.0002 
11 Benzo[b]fluoranthene 0.005 0.001 0.002 
12 Benzo[k]fluoranthene 0.005 0.001 0.002 
13 Benzo[a]pyrene 0.005 0.001 0.002 
14 Indeno[1,2,3-cd]pyrene 0.01 0.002 0.004 
15 Dibenzo[a,h]anthracene 0.01 0.002 0.004 
16 Banzo[g,h,i]perylene 0.005 0.001 0.002 
 
Table 5. Primers and target genes for aerobic PAH degrading 
Annealing Annealing 
Target Primer F/R Reference 
length (bp) temp. (?) 
341 f 
16S rDNA 586 55 Li et al., 2009 
907 r 
f 
PAH-RHD GN 306 57 
r 
PAH-RHD C?bron et al., 2008 
f 
PAH-RHD GP 292 54 
r 
f 
C12O 470 58 
r 
f 
 PAH-RCD C23O 407 58  Thomas et al., 2015 
r 
f 
pca H 395 50 
r 
35 
 
18
Jun Feb
16
* 
14
12
10
8
6
4
2
0
0 m 0.91 m 1.83 m 2.74 m Control
 
Figure 3. Total PAH concentrations in the surface samples at the bioretention cell (Error 
bars show the standard deviation of the samples. One star reflects a significance level of 
at least 0.05 between February and June.) 
 
18
16
R? = 0.862
14
12
10 R? = 0.9662
8
6 Feb
Jun
4
Linear (Feb)
2 Linear (Jun)
0
0 0.5 1 1.5 2 2.5 3
Distance (m)  
Figure 4. Linear correlation between concentration and distance 
36 
 
Concentration (?g/g) Concentration (?g/g)
Concentration (?g/g) Concentration (?g/g)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
0 0
0.91 m Feb 0.91 m Jun
1.83 m Feb 1.83 m Jun
5 2.74 m Feb 5 2.74 m Jun
10 10
15 15
20 20
25 25
30 30
A B
     
Figure 5. Total PAH concentrations of core samples. A: Total PAH concentration of core 
samples in February. B: Total PAH concentration of core samples in June (Error bars 
indicates the standard deviation of the samples) 
 
 
 
 
 
 
37 
 
Depth (cm)
Depth (cm)
Table 6. Reported PAH concentrations in stormwater sediments  
Range 
Location Year Sample Compounds Reference 
(?g/g) 
Kielce, Sediments from stormwater (Sa?ata and 
2015 15 PAHs 39.3-89.4 
Poland drainage system D?bek, 2017) 
2008- Stormwater wet detention (Isteni? et al., 
Denmark 16 PAHs 2.22-0.019 
2009 ponds 2011) 
Toronto, 
2007- Stormwater management (Bartlett et al., 
Ontario, 16 PAHs 6.1-20 
2008 facility sediments 2012) 
Canada 
Warrnambool, 
2008- Wetland sediments impacted (Howitt et al., 
Victoria, 16 PAHs 3.02-23.13 
2009 by stormwater 2014) 
Australia 
College Park, 
Sediments from bioretention (DiBlasi et al., 
Maryland, 2007 16 PAHs < 22.7 
cell 2008) 
U.S. 
Baltimore, 
Sediments from stormwater (Gallagher et al., 
Maryland, 2007 14 PAHs < 88.94 
management ponds 2011) 
U.S. 
Bergen, Sediments from stormwater (Jartun et al., 
2005 16 PAHs < 80 
Norway traps 2008) 
Oxted, Stormwater drainage (Kamalakkannan 
2002 16 PAHs 33.5-64.5 
Surrey, UK sediments et al., 2004) 
Coastal South 2005- Stormwater detention pond 0.064- (Weinstein et al., 
16 PAHs 
Carolina, U.S. 2007 sediments 26.29 2010) 
University of Sediments from a 
2007- (Watts et al., 
New stormwater swale and 16 PAHs 0.54-95.7 
2010 2010) 
Hampshire receiving wetland 
 
38 
 
A 
B 
 
Figure 6. Individual PAH Concentrations in the surface and core samples at the 
39 
 
bioretention cell (A: Concentrations of individual PAHs of samples in February. B: 
Concentrations of individual PAHs of samples in June. The upper and lower ends of the 
lines are the maximum and minimum values, the upper and lower ends of the boxes are 
quartiles, the middle segments of the boxes are the medians, the crosses represent the 
means, and the dots represent the outliers) 
 
 
 
 
 
 
 
 
 
40 
 
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0 m 0.91 m 0.91 m 0.91 m 0.91 m 1.83 m 1.83 m 1.83 m 1.83 m 2.74 m 2.74 m 2.74 m 2.74 m Control
A 5cm 15cm 25cm 5cm 15cm 25cm 5cm 15cm 25cm
2-ring 3-ring 4-ring 5-ring 6-ring
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
0 m 0.91 m 0.91 m 0.91 m 0.91 m 1.83 m 1.83 m 1.83 m 1.83 m 2.74 m 2.74 m 2.74 m 2.74 m Control
5cm 15cm 25cm 5cm 15cm 25cm 5cm 15cm 25cm
B
2-ring 3-ring 4-ring 5-ring 6-ring
 
Figure 7. Proportion of 2-ring, 3-ring, 4-ring, 5-ring and 6-ring PAHs in total PAH 
concentration A: February. B: June 
Table 7. Significations of PAH source diagnostic ratios 
Liquid fossil fuel Grass/wood/coal 
Diagnostic ratios Petroleum References 
combustion combustion 
BaA/(BaA+CHR) <0.20 >0.35 >0.35 
(Yunker et al., 2002; 
FLA/(FLA+PYR) <0.40 0.40-0.50 >0.50 
Yunker et al., 2012) 
ANT/(ANT+PHE) <0.10 >0.10 >0.10 
41 
 
Mixed source Combustion 
0.8
0.7 Jun
Feb Coal/wood/grass Control 
0.6 combustionsamples
0.5 Fossil fuel 
0.4 combustion
0.3
0.2 Petroleum
0.1
0
0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55
A BaA/(BaA+CHR)
 
Mixed source Combustion 
0.3
0 cm
0.25 5 cm
Control 
15 cm samples
0.2 Combustion25 cm
0.15
0.1
0.05 Petroleum
0
0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55
B BaA/(BaA+CHR)
 
Figure 8. Diagnostic ratios of FLA/(FLA+PYR) against BaA/(BaA+CHR) (A) and 
diagnostic ratios of ANT/(ANT+PHE) against BaA/(BaA+CHR) (B) 
42 
 
ANT/(ANT+PHE) FLA/(FLA+PYR)
Table 8. Highest, lowest, and mean values of Total PAH and 16 PAHs, and compared 
with TECs and PECs (?g/g) 
Compound Highest 
Lowest value Mean TEC1 PEC1 References 
name value 
NAP 0.2546 0.0065 0.0676 0.1760 0.5610 MacDonald, et al. 2000 
ACY 0.0521 <0.001 0.0106 0.0059 0.1280 CCME, 1998 
ACE 0.0838 <0.001 0.0205 0.0067 0.0889 CCME, 1998 
FLU 0.1065 0.0124 0.0397 0.0774 0.5360 MacDonald, et al. 2000 
PHE 1.1692 0.0746 0.6393 0.2040 1.1700 MacDonald, et al. 2000 
ANT 0.1701 0.0201 0.0842 0.0572 0.8450 MacDonald, et al. 2000 
FLA 2.6018 0.1276 1.3097 0.4230 2.2300 MacDonald, et al. 2000 
PYR 3.6839 0.2056 1.7751 0.1950 1.5200 MacDonald, et al. 2000 
BaA 1.5541 0.1262 0.6999 0.1080 1.0500 MacDonald, et al. 2000 
CHR 2.7115 0.2070 1.3986 0.1660 1.2900 MacDonald, et al. 2000 
BbF 2.8661 0.1547 1.5878 0.2400 13.4000 Similar as below2 
BkF 1.7517 0.1236 0.8686 0.2400 13.4000 Persaud et al. 1993 
BaP 2.2591 0.1178 1.0507 0.1500 1.4500 MacDonald, et al. 2000 
INP 3.0652 0.1764 1.5712 0.2000 3.2000 Persaud et al. 1993 
DBA 0.5358 0.0524 0.2734 0.0330 1.3000 Persaud et al. 1993 
BPY 3.1367 0.1948 1.7714 0.1700 3.2000 Persaud et al. 1993 
Total PAH 25.4848 1.6031 13.1682 1.6100 22.8000 MacDonald, et al. 2000 
1Values of TEC and PEC normalized by 1% total organic carbon (TOC) content, assumes 
that TOC of samples in bioretention cell and control samples were 1%. 
2There are no guideline values for BbF, assumes that BbF have the same TEC and PEC 
value with BkF because of the similarity of their chemical structures. 
 
 
 
 
 
43 
 
Table 9. BaP-TEQs for carcinogenic PAHs in the media of bioretention cell (?g/g) 
Feb 0m 0cm 0.91m 0cm 0.91m 5cm 0.91m 15cm 0.91m 25cm 1.83m 0cm 1.83m 5cm 
BaA 0.04 0.05 0.06 0.08 0.13 0.07 0.12 
CHR 0 0 0 0 0 0 0 
BbF 0.16 0.21 0.13 0.16 0.2 0.17 0.27 
BkF 0.01 0.01 0.01 0.01 0.01 0.01 0.01 
BaP 0.67 0.8 0.92 1.2 1.64 1.13 1.81 
IDP 0.1 0.07 0.15 0.19 0.22 0.18 0.27 
DBA 0.17 0.13 0.26 0.32 0.39 0.31 0.48 
Total 1.15 1.27 1.53 1.96 2.6 1.88 2.96 
  1.83m 15cm 1.83m 25cm 2.74m 0cm 2.74m 5cm 2.74m 15cm 2.74m 25cm Control 
BaA 0.11 0.08 0.07 0.06 0.05 0.01 0.01 
CHR 0 0 0 0 0 0 0 
BbF 0.24 0.18 0.2 0.16 0.11 0.02 0.02 
BkF 0.01 0.01 0.01 0.01 0.01 0 0 
BaP 1.65 1.24 1.19 1.01 0.72 0.12 0.09 
IDP 0.24 0.19 0.2 0.16 0.11 0.02 0.01 
DBA 0.42 0.33 0.34 0.28 0.2 0.05 0.04 
Total 2.67 2.03 2.02 1.69 1.19 0.22 0.17 
Jun 0m 0cm 0.91m 0cm 0.91m 5cm 0.91m 15cm 0.91m 25cm 1.83m 0cm 1.83m 5cm 
BaA 0.06 0.06 0.05 0.08 0.09 0.06 0.06 
CHR 0 0 0 0 0 0 0 
BbF 0.14 0.14 0.13 0.16 0.18 0.16 0.16 
BkF 0.01 0.01 0.01 0.01 0.01 0.01 0.01 
BaP 0.87 0.93 0.86 1.2 1.34 0.93 1.05 
IDP 0.13 0.14 0.13 0.16 0.19 0.14 0.18 
DBA 0.22 0.24 0.24 0.3 0.34 0.26 0.28 
Total 1.15 1.27 1.48 1.91 2.52 1.81 2.86 
  1.83m 15cm 1.83m 25cm 2.74m 0cm 2.74m 5cm 2.74m 15cm 2.74m 25cm Control 
BaA 0.16 0.08 0.08 0.06 0.03 0.01 0.01 
CHR 0 0 0 0 0 0 0 
BbF 0.29 0.13 0.22 0.15 0.05 0.02 0.01 
BkF 0.02 0.01 0.01 0.01 0 0 0 
BaP 2.26 1.05 1.21 1.04 0.32 0.16 0.07 
IDP 0.31 0.15 0.2 0.18 0.05 0.03 0.01 
DBA 0.54 0.25 0.33 0.28 0.08 0.05 0.02 
Total 2.58 1.95 1.95 1.63 1.15 0.2 0.17 
 
44 
 
 
Figure 9. Gel electrophoresis of PAH-RCD genes 
45 
 
7. Appendix 
 
Figure A1. Molecular structure of 16 EPA PAHs 
 
 
 
 
 
 
46 
 
Table A1. Surface samples in February and p-values (H0: ?row = ?column, HA: ?row > 
?column) 
February 0 m 0.91 m 1.83 m 2.74 m C 
0 m 0.5000 0.2648 0.0667 0.0615 0.9590 
0.91 m 0.7352 0.5000 0.0110 0.0016 0.9998 
1.83 m 0.9333 0.9890 0.5000 0.0614 0.9997 
2.74 m 0.9385 0.9984 0.9386 0.5000 1.0000 
C 0.0410 0.0002 0.0003 0.0000 0.5000 
 
Table A2. Surface samples in June and p-values (H0: ?row = ?column, HA: ?row > ?column) 
June 0 m 0.91 m 1.83 m 2.74 m C 
0 m 0.5000 0.3337 0.1083 0.0019 0.9999 
0.91 m 0.6663 0.5000 0.1663 0.0044 0.9990 
1.83 m 0.8917 0.8337 0.5000 0.0016 1.0000 
2.74 m 0.9981 0.9956 0.9984 0.5000 1.0000 
C 0.0001 0.0010 0.0000 0.0000 0.5000 
 
Table A3. Samples with p-value less than 0.05 (H0: ?1 = ?2, HA: ?1 < ?2) 
 0 cm and 15 cm 0 cm and 25 cm 5 cm and 15 cm 5 cm and 25 cm 
ANT/(ANT+PHE) 0.0260 0.0255 0.0325 0.0284 
BaA/(BaA+CHR) 0.0061 0.0002 0.0069 0.0002 
 
47 
 
 
Figure A2. Electrophoretic profiles of PAH-RHD GN 
 
Figure A3. Electrophoretic profiles of C12O and pca H 
48 
 
14
Jun Feb
12
10
8
6
4
2
0
0 m 0.91 m 1.83 m 2.74 m Control  
Figure A4. Total PAH concentrations in the surface samples at the bioretention cell (Error 
bars show the standard deviation of the samples.) 
 
49 
 
Concentration (?g/g)
Concentration (?g/g) Concentration (?g/g)
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18 20 22
0 0
0.91 m Feb 0.91 m Jun
1.83 m Feb 1.83 m Jun
5 2.74 m Feb 5 2.74 m Jun
10 10
15 15
20 20
25 25
30 30
A B
 
Figure A5. Total PAH concentrations of core samples. A: Total PAH concentration of core 
samples in February. B: Total PAH concentration of core samples in June (Error bars 
indicates the standard deviation of the samples) 
50 
 
Depth (cm)
Depth (cm)
A 
 
B 
 
51 
 
Figure A6. Individual PAH Concentrations in the surface and core samples at the 
bioretention cell (A: Concentrations of individual PAHs of samples in February. B: 
Concentrations of individual PAHs of samples in June. The upper and lower ends of the 
lines are the maximum and minimum values, the upper and lower ends of the boxes are 
quartiles, the middle segments of the boxes are the medians, the crosses represent the 
means, and the dots represent the outliers) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
52 
 
Table A4. Highest, lowest, and mean values of Total PAH and 16 PAHs, and compared 
with TECs and PECs (?g/g) 
Compound Highest Lowest 
Mean TEC1 PEC1 References 
name value value 
NAP 0.1235 0.0087 0.0639 0.1760 0.5610 MacDonald, et al. 2000 
ACY 0.0497 <0.001 0.0107 0.0059 0.1280 CCME, 1998 
ACE 0.0563 <0.001 0.0215 0.0067 0.0889 CCME, 1998 
FLU 0.0670 0.0233 0.0442 0.0774 0.5360 MacDonald, et al. 2000 
PHE 0.9960 0.0678 0.5362 0.2040 1.1700 MacDonald, et al. 2000 
ANT 0.1440 0.0183 0.0708 0.0572 0.8450 MacDonald, et al. 2000 
FLA 2.4717 0.1159 1.0998 0.4230 2.2300 MacDonald, et al. 2000 
PYR 2.0726 0.1061 0.9410 0.1950 1.5200 MacDonald, et al. 2000 
BaA 0.8763 0.0651 0.3679 0.1080 1.0500 MacDonald, et al. 2000 
CHR 1.5273 0.1068 0.7364 0.1660 1.2900 MacDonald, et al. 2000 
BbF 2.3402 0.1298 1.1477 0.2400 13.4000 Similar as below2 
BkF 1.4302 0.1038 0.6356 0.2400 13.4000 Persaud et al. 1993 
BaP 1.8448 0.0990 0.7665 0.1500 1.4500 MacDonald, et al. 2000 
INP 2.5043 0.1480 1.1474 0.2000 3.2000 Persaud et al. 1993 
DBA 0.4376 0.0439 0.1995 0.0330 1.3000 Persaud et al. 1993 
BPY 2.5619 0.1633 1.2945 0.1700 3.2000 Persaud et al. 1993 
Total PAH 19.3721 1.1996 9.0838 1.6100 22.8000 MacDonald, et al. 2000 
1Values of TEC and PEC normalized by 1% total organic carbon (TOC) content, assumes 
that TOC of samples in bioretention cell and control samples were 1%. 
2There are no guideline values for BbF, assumes that BbF have the same TEC and PEC 
value with BkF because of the similarity of their chemical structures. 
 
53 
 
Mixed source Combustion 
0.8
0.7 Jun
Feb Coal/wood/gras
0.6 Control s combustionsamples
0.5 Fossil fuel 
0.4 combustion
0.3
0.2 Petroleum
0.1
0
0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55
BaA/(BaA+CHR)
 
Figure A7. Diagnostic ratios of FLA/(FLA+PYR) against BaA/(BaA+CHR) 
 
7.1 Future works 
Some characteristics, such as total organic carbon, pH, and particle distribution of 
bioretention cell media will be measured to study their relationship with PAH 
concentration. More bacteria will be tested to be the positive control for PAH-RHD GP, 
PAH-RHD GN, and C23O primers. The gradient PCR will also be performed to positive 
cultures to find the optimal annealing temperatures for PAH-RHD GP, PAH-RHD GN, 
and C23O primers. After determining positive control and optimal annealing temperature 
for all primers, PCR will be conducted to all media samples and control samples, to do 
qualitative analysis of the presence of PAH degrading bacteria in the media. And qPCR 
will be used to do quantitative analysis of PAH degrading bacteria. 
54 
 
FLA/(FLA+PYR)
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64 
 
Chapter 2 
 
 
Thesis Conclusion 
 
  
65 
 
 Thesis conclusion 
The study evaluated the effectiveness of a bioretention cell for the removal of PAHs 
in stormwater. This was done by comparing control samples collected near the 
bioretention cell with the bioretention cell media samples. The results showed that the 
bioretention cell contained 9.70 times higher levels of total PAH than the control samples. 
It was found that PAHs accumulated in the media might have a negative impact on the 
environment by being ingested by surrounding organisms and bioaccumulating. A 
comparison between samples collected in this study with samples from the same location 
collected in 2008, it was found that PAHs accumulated in the media 5 cm below the 
surface. This finding provides new information that is relevant for the management of 
bioretention cells. PAHs tend to accumulate in the media due to their relatively low 
bioavailability. If allowed to accumulate, PAHs will eventually break through the media, 
overload the media adsorption capacity and enter the aquatic environment thus causing 
contamination of nearby waterways. Therefore, it is important to determine and 
implement proper maintenance frequency and routines of the bioretention cell. The 
maintenance could be replacing contaminated media with clean media or to further 
biotransformation. Evaluation of the sources of PAHs found those detected in the 
bioretention cell mainly originated from petroleum source and fossil fuel combustion 
source. This corresponds to the location of the bioretention cell, where it collects 
stormwater from parking lots and roads. This result reveals the importance of using 
66 
 
stormwater management facilities alongside roads to prevent PAH pollution. The study of 
the PAH carcinogenicity in the media revealed that benzo[a]pyrene (BaP) contributed the 
most, although it did not have the highest concentration of the individual PAHs, which 
illustrates the importance of BaP as a carcinogen. This result can help to improve the 
management of stormwater bioretention cells by for example evaluating the effect of 
stormwater treatment. Here the BaP concentration can be used as an important indicator. 
Therefore, when designing stormwater management facilities along roadways, the BaP 
treatment capacity should be considered. 
To conduct a more in-depth study of the accumulation of PAHs in bioretention cell, 
more research is still needed. First, the relationship between characteristics of media and 
PAHs needs to be studied by determining the distribution of PAHs associated with media 
particles of different sizes as well as, the relationship between the TOC of the media and 
the concentration of PAHs. Also, in this study the presence of PAH transforming bacteria 
was performed but was cut short due to the University Shutdown in March 2020. 
Therefore, this part of the study will continue by applying molecular biology tools to 
evaluate the potential for in situ PAH degradation in the bioretention media. If the 
bacteria are present, conditions for enhancement of their activity will be determined.  
Based on the current conclusions, it is not clear whether biodegradation of PAHs has 
occurred in the media of the bioretention cell in this study, but the accumulation of PAHs 
in the media indicates that the degradation rates of PAHs should be less than their 
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accumulation rates. As a result, it is necessary to strengthen the biodegradation of PAH in 
media. 
The half-lives of PAHs in soil are related to their molecular weights. The half-life of 
phenanthrene is 16 to 126 days, while the half-life of benzo[a]pyrene is 229 to 2100 days 
(Shuttleworth and Cerniglia, 1995). Biostimulation and bioaugmentation have been 
successfully used to enhance the biodegradation of PAH in soil and solid waste. 
Nagalakshmi (2019) removed 99% PAHs from soil by biostimulation in 56 days. To 
improved biodegradation of PAHs, Llad? (2013) biostimulated industrial polluted soil by 
adding lignocellulosic substrate, soybean oil and Manganese ions, and 76% PAHs were 
removed in 60 days. Sun (2012) combined biostimulation and bioaugmentation to treat 
PAHs-contaminated soil. 43.9% and 55.0% of total PAHs and HMW-PAH were degraded 
in 175 days, respectively. Since the environment of bioretention cell media is similar to 
soil, biostimulation and bioaugmentation can also be used to degrade PAHs in the 
bioretention cell media. The bioremediation methods used in the bioretention cell will 
depend on the microbial community in the media. If there are enough PAH degrading 
bacteria in the media, biostimulation can be used. If there are insufficient PAH degrading 
bacteria in the media, bioaugmentation should be used to add PAH degrading bacteria to 
the media. Biostimulation and bioaugmentation can also be used in combination to to 
enhance the biodegradability of the PAHs in the media. This will improve the life span 
and sustainability of bioretention cell and limit the requirements for management. 
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References 
Haleyur, N., Shahsavari, E., Jain, S. S., Koshlaf, E., Ravindran, V. B., Morrison, P. D., 
Osborn, A. M., and Ball, A. S. 2019. Influence of bioaugmentation and 
biostimulation on PAH degradation in aged contaminated soils: Response and 
dynamics of the bacterial community. Journal of environmental management, 
238: 49-58. 
Llad?, S., Covino, S., Solanas, A., Vi?as, M., Petruccioli, M., and D?annibale, A. 2013. 
Comparative assessment of bioremediation approaches to highly recalcitrant PAH 
degradation in a real industrial polluted soil. Journal of hazardous materials, 
248: 407-414. 
Shuttleworth, K. L., and Cerniglia, E. 1995. Environmental aspects of PAH 
biodegradation. Applied biochemistry and biotechnology, 54(1-3): 291-302. 
Sun, G.-D., Xu, Y., Jin, J.-H., Zhong, Z.-P., Liu, Y., Luo, M., and Liu, Z.-P. 2012. Pilot 
scale ex-situ bioremediation of heavily PAHs-contaminated soil by indigenous 
microorganisms and bioaugmentation by a PAHs-degrading and bioemulsifier-
producing strain. Journal of hazardous materials, 233: 72-78. 
 
 
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