*Article* **Seasonal Variation in Fecal Glucocorticoid Levels and Their Relationship to Reproductive Success in Captive Populations of an Endangered Parrot**

**Brian Ramos-Güivas 1,2,\*, Jodie M. Jawor 2 and Timothy F. Wright 2**

**Abstract:** Many species are threatened with extinction, and captive breeding programs are becoming more common to avoid this outcome. These programs serve to prevent extinction and produce individuals for eventual reintroduction to natural populations in historical habitat. Captive animals experience different energetic demands than those in the wild, however, and as a result may have different levels of glucocorticoid hormones. Glucocorticoids help with responses to energetically expensive and potentially stressful situations. Elevated glucocorticoid levels can also potentially alter reproduction and other key behaviors, thus complicating successful captive breeding. The Puerto Rican parrot (*Amazona vittata*) is a critically endangered parrot that currently exists in only two wild and two captive populations. Its recovery program provides a good platform to better understand how glucocorticoid levels may relate to reproductive success under captive conditions. We validated a corticosterone assay in this species and used non-invasive techniques of measuring fecal glucocorticoid metabolites of males and females from two captive populations (Rio Abajo and El Yunque) of Puerto Rican parrots over two consecutive breeding seasons, 2017 and 2018, and the prebreeding season of 2018, which occurred just after Hurricane Maria struck Puerto Rico. Our results show that levels of fecal glucocorticoid metabolites of males measured during the breeding season of 2018 negatively correlated to the number of total eggs and fertile eggs laid by pairs. In contrast, there was a positive relationship of female fecal glucocorticoid metabolite levels during the pre-breeding season of 2018 with total eggs laid. In males from the Rio Abajo population, we found seasonal differences in fecal glucocorticoid metabolite levels, with higher levels during the pre-breeding season of 2018 compared to both 2017 and 2018 breeding seasons. There was no difference in the mean value of male fecal glucocorticoid metabolites between the 2017 breeding season and 2018 breeding season which started four months after Hurricane Maria struck Puerto Rico. We did find sex differences during the pre-breeding season of 2018 in birds from the Rio Abajo population. Adjustments in the care routine of both populations that could reduce circulating baseline glucocorticoids and avoid frequent, sudden elevations of glucocorticoids should be considered. These results provide a baseline for future comparison with reintroduced populations of this endangered species and other species with captive breeding programs.

**Keywords:** captive populations; endangered species; glucocorticoids; parrot; reproductive success; seasonality

### **1. Introduction**

Changes in the local environment of an organism can promote responses or changes in both physiological processes and behavior, specifically an increase in glucocorticoids [1]. Environmental challenges can be either predictable or unpredictable, but in either case, can have a major impact on individual fitness and evolutionary adaptation [1]. One of the primary mechanisms to promote adaptive responses to environmental challenges

**Citation:** Ramos-Güivas, B.; Jawor, J.M.; Wright, T.F. Seasonal Variation in Fecal Glucocorticoid Levels and Their Relationship to Reproductive Success in Captive Populations of an Endangered Parrot. *Diversity* **2021**, *13*, 617. https://doi.org/10.3390/ d13120617

Academic Editors: José L. Tella, Guillermo Blanco and Martina Carrete

Received: 13 October 2021 Accepted: 21 November 2021 Published: 25 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in vertebrates is the elevation of glucocorticoid hormone levels by the hypothalamicpituitary-adrenal (HPA) axis [2,3]. Glucocorticoids primary role in an organism is to aid the metabolism of protein and lipids into carbohydrates for energy consumption and multiple other functions [4,5]. During a disturbance, an animal will release glucocorticoid hormones, resulting in a process of prioritization of energy for a survival response [2,6,7] and subsequent recovery. The frequency and duration of perturbations can cause an animal to have either persistently high glucocorticoid levels, or repeated short-term elevations of glucocorticoids, sometimes termed 'allostatic overload', with potentially negative consequences [1,5]. For example, in birds, elevated levels of corticosterone (the primary avian glucocorticoid) may inhibit the production of luteinizing hormone and prolactin [8–10] and reduce affiliative behaviors, potentially affecting reproductive success [8,11,12].

Captivity is a prime example of an environment that can potentially alter normal glucocorticoid levels and reproductive success [8]. Captive breeding programs are becoming more common as human-mediated habitat changes and other anthropogenic disturbances threaten more species with population reduction and even extinction. A high production of individuals from captive breeding populations of endangered species can allow managers to develop better strategies to enhance reintroduction efforts and sustain wild populations [13,14]. But under captive conditions, animals may have altered levels of glucocorticoids compared to individuals in the wild, modifying behavior otherwise characteristic of the species [15]. In captivity, individuals may experience increased social interactions, atypical photoperiods and limited space overall [16], which can lead to an increase in glucocorticoid levels and altered behavior [17]. Captive conditions for individuals born and raised in the wild may cause a drastic increase in glucocorticoids levels, especially when these individuals spend long periods of time in captivity, with substantial effects on reproduction [17]. In black-legged kittiwakes (*Rissa tridactyla*) artificial increase of the glucocorticoid corticosterone, reduced the production of prolactin, significantly reducing reproductive success [8]. Furthermore, increased glucocorticoids could negatively impact reproductive success by altering parental behaviors, such as feeding patterns of offspring, incubation consistency in birds and the timing of nesting [8,15]. Although the endocrine system is well conserved across vertebrates, the reaction to captivity may vary depending on the species [16]. There may also be physiological differences in responses among populations and individuals [18–23]. Some species may never be able to reproduce effectively in captive conditions even after generations, while in others, artificial selection can lead to changes in reproductive physiology [16]. Therefore, studying the effects of glucocorticoid levels on parental care behavior and reproductive success in captive breeding individuals and understanding the difference among populations, sex and seasonal variation can help program managers develop improved techniques to increase reproduction.

Another factor to consider in species conservation is ongoing anthropogenic-induced climate change which has led to unpredictability in weather, potentially increasing the frequency, duration, and severity of climate events. In particular, hurricanes can be a major threat to vulnerable and threatened populations of wildlife [24]. For wild populations, direct wind effects during the hurricane can kill individuals, and those that survive must deal with limited food resources [25,26], potentially experiencing altered glucocorticoid levels. Furthermore, species that coordinate their breeding with local fruiting patterns can experience reduced reproductive success after such events [26]. Although captive populations are buffered from many weather events, hurricanes may still affect them. During and after an extreme weather event, housing facilities may suffer damage and loss of power, and caretaking staff may have limited access to facilities. Food scarcity, overcrowding and isolation from normal light cycles could alter the physiology of captive individuals in these situations. Despite these potentially important effects, there are few studies examining changes in glucocorticoid levels in captive populations after hurricanes or other natural disasters [27].

The parrots and cockatoos (Order Psittaciformes) are one group for which increased knowledge of the relation of glucocorticoid levels to captive reproduction is critical. Psittaciformes have a worldwide species distribution with 419 known species, 42.2% of which are classified from near threatened to critically endangered [28]. Major threats include habitat loss and capture for the pet trade, leading to captive breeding being increasingly used as a conservation tool to protect these species [29–33]. One endangered species in which captive breeding programs play a critical role is the Puerto Rican parrot (*Amazona vittata*). This species is endemic to the island of Puerto Rico and underwent a drastic decline during the 20th century due primarily to habitat loss as the island's native forest was converted to agriculture [34]. The periodic threat of hurricanes combined with low population numbers has stalled population growth in the wild [35,36] and potentially perpetuates the genetic bottleneck observed in the species [37].

The Puerto Rican Parrot Recovery Program is an excellent model in which to test the relationship of glucocorticoids to parental care and reproductive success in captivity. The program currently consists of two captive populations (Rio Abajo and El Yunque) in different locations on the island that are closely monitored with good record-keeping practices. Climate and husbandry methods differ between each population. The urgent need for the establishment of wild populations in historical habitat demands high productivity of individuals from the captive populations for release. Birds are reintroduced at both wild populations' sites at least once a year, with the number of released birds varying among releases depending on the yearly production of the captive populations. Captive breeding is an essential part of the plan to save this species, but no study to date has investigated the relationship between glucocorticoid levels and reproductive success in this, or any other, captive breeding program for parrots.

In this study, we validate a commercial corticosterone assay and use it to examine fecal glucocorticoid metabolite levels in both captive populations of Puerto Rican parrot in 2017 and 2018 and relate these measures to reproductive success of captive individuals. Assessment of fecal glucocorticoid metabolites is non-invasive and can provide a broader picture of general circulating levels of glucocorticoids in animals [38]. We predicted that high glucocorticoid metabolite levels during the pre-breeding season and the beginning of the breeding season would have a negative relationship to reproductive success. If so, we expected to see females with lower levels of fecal glucocorticoid metabolites during the pre-breeding season produce more fertile eggs and males with lower levels of fecal glucocorticoid metabolites during the breeding season produce more chicks and fledglings. Additionally, we were provided with an opportunity to test for variation of glucocorticoid metabolites of captive individuals before and after a major environmental perturbance in the form of a hurricane. We also explore the effects of Hurricane Maria, a Category 5 hurricane that caused extensive damage to local forests and impacted the captive breeding facilities and their staff, on fecal glucocorticoid metabolites levels before and during the following breeding season, while also testing for differences among season, populations, and sexes.

### **2. Materials and Methods**

The Puerto Rican parrot has a monogamous mating system with biparental care in which only the females incubate, and the male provides food to the female and the chicks. Chicks hatch asynchronously, and once the youngest can thermoregulate (at approximately 14 days post-hatch), the female joins the male in foraging for food for the chicks. In captivity, first eggs are laid from the end of January to the beginning of February. Hatching time is on average 26 days after egg laying and a range of 55–75 days until chicks fledge from the nest [34]. Clutch size and the number of chicks that fledge from nests vary among mated pairs, but typically 3 eggs are laid per pair each year in a single clutch [34]. The production of fledglings varies with population. Fledgling production from 2015 to 2018 in the Rio Abajo captive population was 1.02 per pair, and in the Rio Abajo wild population was 1.29 per pair. In the El Yunque captive population fledgling production was 0.71 per pair. The El Yunque wild population was extirpated by Hurricane Maria in 2017 but releases have

been ongoing since summer 2019. The birds produced in captivity are used for eventual reintroductions into the wild or as new breeding individuals in the captive populations.

Each captive population houses around 220 individuals in outside cages with yearly fluctuations due to births, deaths, translocations, and reintroductions. Each year there are at least 80 to 120 individuals that form mated pairs at each captive population. Housing conditions and daily care routines differ somewhat between the two captive populations. At Rio Abajo, the captive breeding season starts between January 15–25 when the breeding pairs are each placed in their own breeding cages and ends when the last chicks fledge in July-August. During the breeding season at Rio Abajo, personnel enters the breeding areas in the morning to feed the birds and in the afternoon to collect food dishes. All nests are inspected on Monday mornings by personnel, with secondary nest checks done on some nests on Thursday or Friday mornings. After all the chicks have fledged from the nest, mated pairs are placed in retention cages in which some pairs are maintained together in the same cage and other pairs are separated with males and females placed in individual cages next to each other. All pair members at Rio Abajo remain in these cages until the beginning of the next breeding season.

At El Yunque, the captive breeding season also starts between January 15–25 and ends when the last chick fledges from the nest in July-August. El Yunque personnel feed the birds in the morning and nest checks are done throughout the week at variable times, with personnel potentially entering breeding areas multiple times during a day. At the end of the breeding season, the nest entrance is closed until the initiation of the next breeding season and pairs remain in these breeding cages year-round except for a week-long period, during which their cage is washed and prepared for the next breeding season. The activity of cleaning the breeding cages can occur at any moment between August to December.

### *2.1. Ethical Considerations*

The Puerto Rican parrot is classified as critically endangered by the IUCN Red List [28]. This status imposes particular ethical considerations regarding the type of handling, sampling, and experimental manipulations that might be conducted in a study of this type. For example, frequent bleeding for measurement of circulating hormone levels or experimental manipulation of the HPA axis via adrenocorticotropic hormone or dexamethasone challenges, are considered overly invasive and are not permitted by the two managing authorities for this species (Puerto Rico Department of Natural and Environmental Resources and United States Fish and Wildlife Service). To minimize disruption to reproductive efforts in this species we primarily used non-invasive sampling of fecal glucocorticoids metabolites collected as part of the regular daily care routine already established at the two breeding facilities. This study was conducted under NMSU IACUC protocols 2014-030 and 2021-014, approved by the Puerto Rican Parrot Recovery Program Interagency Operational Team, supported by the management under the Puerto Rico Department of Natural and Environmental Resources and conducted under the United States Fish and Wildlife Service Permit TE125521-4.

### *2.2. Reproductive Information*

We collected reproductive information for the pairs that were included in our study from captive population managers. This information included the production of total eggs, fertile eggs, first egg fertility, chicks, and fledglings.

### *2.3. Pair Selection and Breeding Stages*

For this study, we randomly selected 46 pairs from all the breeding pairs in the two captive populations as follows: 21 pairs for the 2017 breeding season (Rio Abajo = 12 and El Yunque = 9), 12 for 2018 breeding season (Rio Abajo) and 13 for the intervening pre-breeding season of 2018 (Rio Abajo). We resampled 10 pairs from 2017 in the 2018 breeding seasons. At the Rio Abajo captive population, the breeding areas are clusters of breeding pairs, and each area is located in a different section of the facility. To minimize the potential effect of breeding area the pairs selected were placed in Area I. At the El Yunque captive population, all breeding cages are in close proximity compared to the structure used at the Rio Abajo captive population, for this reason, there was no potential effect of area. We define the breeding season for the captive population as the period of the year from January when pairs are placed in their breeding cage (Rio Abajo) or when the nest box is opened (El Yunque) up to the date the last chick in the population fledged. We define pre-breeding as the time before pairs are placed in their respective breeding cages (November to December in both populations); during this period, it is common to see birds showing pair-bonding behaviors (synchronized flights, duets, allopreening, copulations, allofeeding) and territorial displays.

### *2.4. Assay Validation*

We used the DetectX Corticosterone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor MI, USA) to measure fecal glucocorticoids. To validate this assay for the measurement of corticosterone in plasma and fecal glucocorticoids in the Puerto Rican parrot, we conducted a small study with a limited subset of birds and under supervision of a veterinarian. We employed two different manipulations that were each anticipated to be stressful to the birds and collected blood and fecal samples at regular intervals after each to determine whether we could detect a rise in plasma corticosterone and fecal glucocorticoids following these stressful events. The first manipulation was to capture birds from a group flight cage and transfer them to individual cages, with subsequent collection of fecal samples to monitor fecal glucocorticoids. The second manipulation was capture and immediate blood collection followed by 30 min of restraint and then a second blood collection. Individuals were returned to cages and additional fecal samples were collected following blood collection. This manipulation allowed us to compare baseline and stress response circulating corticosterone to fecal glucocorticoid metabolites measured before and after the stressful event. Assessment of circulating corticosterone and levels to which it elevates with handling were key to assessing later secretion of glucocorticoids into fecal material and to confirm that general handling procedures are seen as stressful to this species.

To conduct our validation, we selected twelve birds from the captive population at Rio Abajo, six males and six females. All birds were two years old at the time of the study.  This method of capture, while standard in the facility, is thought to be stressful for the birds as it involves chasing them with the net, during which time they often produce alarm calls. We captured birds at 7:00 and then collected fecal samples every 2–3 h on the day of capture and the following 4 days to assess changes in fecal glucocorticoid levels after this putatively stressful event. For this study, we only used the day of capture and the following day after capture. If this event caused an increase in circulating glucocorticoids, as anticipated, then we also expected to see a transitory rise in fecal glucocorticoids later on the day of capture compared to samples collected in following days.

For the second part of the validation, we collected blood samples and performed a capture restraint on the same 12 birds after two weeks of individual housing in the same cages. Approximately 0.3 cc of whole blood was collected from the jugular vein from each bird between 8:00 am and 9:30 am. Samples were collected in under 3 min from approaching each cage, and again after 30 min of restraint in a small wooden box to measure baseline and stress-response circulating corticosterone. The blood samples were centrifuged, and plasma extracted and frozen immediately. We collected fecal samples for our baseline values before the restraint protocol was conducted. We then continued to collect fecal samples from birds for the remainder of the day after blood samples were collected. For the stress response analysis, we used from each individual the fecal sample with the highest value after restraint collected between 13:30 to 18:00.

### *2.5. Fecal Sampling and Analysis*

For our main study, we collected fecal samples from males in both the breeding and pre-breeding seasons and from females during the pre-breeding season only. Pre-breeding (November to January) samples were collected only at Rio Abajo where the members of each pair are separated, which allowed us to distinguish which individual produced each sample. Breeding season samples were collected from males in both populations due to their presence outside nesting cavities while females incubate. To collect the fecal samples, we placed a clean PVC plank under a perch where individuals roosted at night after sunset between 7:30 to 8:00 PM and collected the plank before the following sunrise between 5:30 to 6:00 AM. The droppings collected at sunrise were thus collected no more than 10 h after defecation and when no rainfall occurred overnight. A study examining the effects of environmental changes on fecal glucocorticoid metabolites found no effect of room temperature for 12h on fecal glucocorticoid metabolite concentration but it did find effects of rainfall on the concentration of fecal glucocorticoid metabolites [39]. Samples were collected in a 2ml microcentrifuge tube and then stored at −20 ◦C until drying and analysis.

Fecal samples were dried using a gravity convection oven (Fisher Scientific, No: 3511FS) preheated to 90 ◦C, the samples remained in the oven for at least 2 h until fully desiccated. A study examining the effect of this procedure on glucocorticoid metabolites levels found that samples that are frozen and then dried in conventional ovens do not change significantly in glucocorticoid metabolite concentration [40]. After drying the samples were pulverized and centrifuged, then stored at −20 ◦C until analysis. Rainfall and small amounts of fecal material in the samples of some individuals, lead to some individuals having low sample collection during the pre-breeding season of 2018. We randomly selected 3 samples per individual during the pre-breeding and a range of 3 to 15 samples per individual during the breeding seasons that had ≥0.2 g of dry feces to measure glucocorticoids metabolites. We extracted glucocorticoid metabolites from fecal samples following the DetectX Steroid Extraction Protocol (Arbor Assay), after drying, 2 mL of ethanol was added to the solid to initiate the extraction of the glucocorticoids. We did minor adjustments to the centrifugation step during which extracted samples were centrifuged at 4000 rpm for 20 min. We used a SpeedVac (Eppendorf) centrifuge to dry down extracted samples, after which samples were stored at −20◦ until they were assayed (within 24 h), sample recovery was assumed to be 100% in subsequent analyses. We adjusted the kit recommended reconstitution procedure slightly and dissolved extracted samples in 50 μL of ethanol followed by 600 μL of Assay Buffer. Fecal glucocorticoid metabolite levels were then analyzed using the DetectX® Corticosterone Enzyme Immunoassay kit (EIA, Arbor Assays, Inc., #K014-H5). This kit has high affinity for corticosterone (100%), with much lower crossreactivity for other glucocorticoids and their metabolites (Desoxycorticosterone—12.30%, Tetrahydrocorticosterone—0.76%, Aldosterone—0.62%, Cortisol—0.38%, Progesterone— 0.24%, Dexamethasone—0.12%, Corticosterone-21-Hemisuccinate—<0.1%, Cortisone— <0.08%, Estradiol—<0.08%); since in fecal samples there is low presence of corticosterone, we report on glucocorticoid metabolites as opposed to corticosterone.

### *2.6. Statistical Analysis*

Since we analyzed samples in multiple plates and years we corrected our glucocorticoid estimates using a correction factor applied to all samples to account for inter-assay variation that could be linked to variation in kits or lab conditions as glucocorticoid analyses spanned across years [as in 41]. We used the estimated levels of glucocorticoid metabolites from the fourth point of the standard curve as a basis for our correction factor. We used the grand mean for all point 4 data points from all standard curves and divided this by the mean point 4 standard curve point for each individual plate to develop a correction factor [41–47]. We then multiplied each hormone value on each plate by the corresponding correction factor. Inter-assay variation was measured as the coefficient of variation (N = 21, Mean = 1241.44, SD = 131.78, CV = 0.106 equaling an inter-assay variation of 10.6%, intra-assay variation range = 3.22–9.09%). The adjusted glucocorticoid values were then

log-transformed for subsequent analyses. For all analyses, we used the mean glucocorticoid values obtained for each individual across all measures within a season.

From the full set of data available, we used a subset to address different questions depending on the data available. We used twelve males for the 2017 and 2018 breeding seasons, and ten for the 2018 pre-breeding from the Rio Abajo captive population. From the Rio Abajo captive population, nine males had data across all seasons, two males had data only for the 2017 breeding season, only one male had data for pre-breeding and breeding season of 2018. We had data from nine males for 2017 breeding season from the El Yunque captive population. For females, we had data from only eleven females from the Rio Abajo captive population from the 2018-pre-breeding season. After a Shapiro Wilk Test we Log transform our glucocorticoid values for all the analyses. For the determination of the relation of reproductive success measures and glucocorticoid metabolite levels at each season, we used only males (see Table 1) from the Rio Abajo captive population and performed a Spearman correlation analysis. To compare glucocorticoid metabolite levels between the pre-breeding and two breeding seasons we used an ANOVA followed by Tukey post hoc tests using only the males (14) from the Rio Abajo captive population because this was the only population sampled over all three seasons. Since the breeding season of 2017 occurred before Hurricane Maria and the breeding season of 2018 occurred after, we used the results from the previous analysis to compare levels before and after Hurricane Maria. We performed a paired *t*-test to investigate differences among males (10) and females (11) from the Rio Abajo captive population during the pre-breeding season. For population differences in glucocorticoid metabolite levels, we compared the data from the breeding season of 2017 of only males (21) from each population using a paired *t*-test. We used a logistic regression to determine the relationship of glucocorticoids metabolite levels during the pre-breeding to first egg fertility. Statistical analysis was performed using the JMP statistical software package, version 14.0.0 (SAS Institute, Inc. 2018, Cary, NC, USA).

**Table 1.** Relationship between male fecal glucocorticoid metabolite levels and reproductive success variables †.

† All tests Spearman correlations.

### **3. Results**

### *3.1. Validation of Corticosterone Assay*

In the first potentially stressful event of our validation study (capture and placement in cages), we saw a sharp but transitory rise in estimated fecal glucocorticoid metabolite levels in the 12 birds following initial capture from group housing and movement to individual housing. Although we were not able to obtain fecal samples from all individuals immediately upon capture, all samples collected in the 6 h following capture had estimated fecal glucocorticoid metabolite levels under 20,000 pg/mg (Figure 1a). After 6 h there was a general rise in CORT levels that persisted for 30 h after capture, at which time levels decreased (Figure 1a). In our second stressful event (capture and restraint) we saw a significant rise in circulating plasma CORT from baseline to stress-response levels following 30 min of a standard capture restraint protocol for the whole group (Figure 1b, paired *t*-test, df = 20.12, t = 5.92, *p* = 0.001). When we compared levels based on sex, we found that at time zero there was no difference in plasma glucocorticoid levels between the sexes (paired *t*-test, df = 7.41, t = 0.02, *p* = 0.987) but after restraint males showed higher plasma glucocorticoid levels than females (paired *t*-test, df = 9.77, t = 2.76, *p* = 0.021). Both sexes demonstrated a restraint response with an increase of plasma glucocorticoids (paired *t*-test males, df = 11.04, t = 5.59, *p* = 0.0002 and for females df = 4.93, t = 2.86, *p* = 0.036). There was a concomitant rise in estimated fecal glucocorticoid levels in fecal samples collected on the same day before and after the capture restraint (Figure 1c, paired *t*-test, df = 13.09, t = 4.76, *p* = 0.0004). Comparing the sexes, both sexes demonstrated a significant increment of fecal glucocorticoids after the restraint protocol (paired *t*-test for males, df = 5.89, t = 3.48, *p* = 0.0136 and for females df = 5.47, t = 6.59, *p* = 0.0009). Before the restraint protocol females had higher fecal glucocorticoid levels than males (paired *t*-test, df = 10.00, t = −3.82, *p* = 0.003) but there was no significant difference between the sexes after the restraint protocol (paired *t*-test, df = 6.83, t = −1.46, *p* = 0.1998). As a final assessment concerning the ability of this assay to detect fecal glucocorticoids in this species, we used extra, mixed fecal samples from individuals that we then measured as split into two aliquots (*n* = 10). These split aliquots were analyzed on the same plate with final calculated levels of fecal glucocorticoid metabolites compared for similarity. Levels from split samples were highly correlated, supporting the reproducibility of this assay and its ability to detect fecal glucocorticoid metabolites for this species (Pearson Correlation, r = 0.963, *n* = 10, *p* < 0.0001). These results validate that the Arbor Assay Detect-X ELISA Assay is effective in detecting both CORT in plasma and feces of this species. No birds were harmed during this procedure and veterinary monitoring detected no adverse impacts on their health.

**Figure 1.** Glucocorticoids levels in fecal and plasma samples. A total of 12 individuals were used for this test with 6 males and 6 females. (**a**) Fecal glucocorticoids fluctuation same day of capture up to 40 h after first capture. Multiple samples per individual, with a total of 37 samples. (**b**) Plasma glucocorticoids levels before and after bleeding, *n* = 12, 6 males and 6 females. Figure shows means ± SE. (**c**) Fecal glucocorticoids levels before and after restraining for blood capture, *n* = 12, 6 males and 6 females. Figure shows means ± SE.

#### *3.2. Glucocorticoid Metabolite Levels and Measures of Reproductive Success*

We assessed how male fecal glucocorticoid metabolite levels in different seasons related to reproductive success. For the 2018 breeding season we found a significant relationship between fecal glucocorticoid metabolite levels in males and measures of reproductive success in the egg stage (total eggs and fertile eggs), but not at the post-egg stage (chicks and fledglings) (Table 1). Fecal glucocorticoid metabolite levels had a negative relationship with both the number of eggs laid and the number of fertile eggs (Figure 2). In contrast, we did not find any relationship between male fecal glucocorticoid metabolite

levels during either the 2017 breeding season or the pre-breeding season of 2018 with measurements of reproductive success at the egg stage (total eggs laid, number of fertile eggs) or chick stage (number of chicks hatched, number of fledglings).

In females, fecal glucocorticoid metabolite levels during the pre-breeding stage of 2018 were positively related to the total number of eggs laid but were not related to the number of fertile eggs, chicks produced, or fledglings (Table 2, Figure 2). There was no significant relationship between fecal glucocorticoids metabolite levels of either males or females during the pre-breeding stage in 2018 to the fertility of the first egg (Female df = 1, r2 = 0.1748, X2 = 2.1361, *p* = 0.1439; Male, df = 1, r2 = 0.0727, X2 = 0.6927, *p* = 0.4052).

**Table 2.** Relationship between fecal glucocorticoid metabolite levels in females during the prebreeding season and reproductive success variables †.

**Figure 2.** Significant relationships of fecal glucocorticoid metabolites to reproductive success measures. (**a**) Females showed a positive relationship of fecal glucocorticoid metabolites during the pre-breeding to the total eggs laid by female in the following breeding season (2018). Males have a negative relationship of fecal glucocorticoid metabolites to (**b**) total eggs and (**c**) total fertile eggs laid in the following breeding season (2018).

### *3.3. Hurricane Maria and Seasonal Variation*

For the Rio Abajo captive population, we did not identify differences between the 2017 and 2018 breeding seasons (d.f = 16.53, t = −1.018, *p* = 0.323), which represented preand post-Hurricane Maria, respectively (Figure 3). We found a difference in male fecal glucocorticoid metabolite levels between seasons (df = 2, F = 7.015, *p* = 0.003; Figure 3). Here, male birds had higher fecal glucocorticoid metabolite levels during the pre-breeding season of 2018 (N = 10, mean ± SE = 3.83 ± 0.15 ng/g) compared to the breeding season of 2017 (N = 12, mean ± SE = 3.28 ± 0.13 ng/mL) and the breeding season of 2018 (N = 12, mean ± SE = 3.108 ± 0.13 ng/mL). After a Tukey HSD post-hoc comparison, pre-breeding levels in 2018 were significantly different from the 2017 breeding season (df = 12.821, t = −3.795, *p* = 0.0023), as well as the 2018 breeding season (df = 19.144, t = 2.354, *p* = 0.029).

**Figure 3.** Seasonal variation in fecal glucocorticoid metabolite of males at Rio Abajo. During prebreeding 2018 fecal glucocorticoid metabolites were higher relative to both the 2017 and 2018 breeding seasons, which did not differ from each other. The 2017 breeding season occurred prior to Hurricane Maria while the pre-breeding and breeding season of 2018 occurred after Hurricane Maria. Graphs show means ± SE.

### *3.4. Glucocorticoid Metabolite Levels Differed between Sexes and Populations*

We found that the fecal glucocorticoid metabolites ranged from 10.988 to 133.750 ng/g for males and 14.057 to 40.606 ng/g for females. We compared pre-breeding season fecal glucocorticoid metabolite levels in males and females and found that males had higher levels (mean ± SE = 3.827 ± 0.17 log ng/g) than females (mean ± SE = 3.284 ± 0.86 log ng/g; df = 1, t = 2.836, *p* = 0.00138; Figure 4). In addition, we found a trend towards males' glucocorticoid levels differing between populations during the 2017 breeding season (Figure 5). Males at the Rio Abajo captive population had lower glucocorticoid metabolite levels (mean ± SE = 3.285 ± 0.15 log ng/g) than males at the El Yunque captive population (mean ± SE = 3.647 ± 0.11 log ng/g), but this trend was not statistically significant (df = 1, t = −1.895, *p* = 0.0737).

**Figure 4.** Sex difference in fecal glucocorticoid metabolites during the pre-breeding 2017–2018. Males had higher fecal glucocorticoid metabolite levels than females during the pre-breeding season. These samples include individuals from Rio Abajo captive populations (Rio Abajo males = 10, Rio Abajo females = 11). Graphs shows means ± SE.

**Figure 5.** Populations comparison in fecal glucocorticoid metabolites between males in the Rio Abajo (12) and El Yunque (9) captive populations during the 2017 breeding season. Graphs show means ± SE.

### **4. Discussion**

Captive breeding can be an important tool for many conservation efforts. Understanding how a threatened species is affected by captive conditions is critical for conservation success [16]. We evaluated fecal glucocorticoid metabolite levels of captive populations of Puerto Rican parrots using a newly validated commercial corticosterone assay and related this to reproductive success over two breeding seasons and the intervening non-breeding season. We expected to see a negative relationship between the reproductive success of captive breeding pairs and fecal glucocorticoid metabolite levels of males and females during pre-breeding, and glucocorticoid metabolite levels of males in the breeding seasons. Glucocorticoids have been shown to negatively impact reproductive behavior and success in other avian species [8]. Our results showed a negative relationship of male fecal glucocorticoid metabolite levels to total eggs and fertile eggs during the breeding season of 2018 (Figure 2), while female fecal glucocorticoid metabolite levels during the pre-breeding season of 2018 showed a positive relationship to total eggs laid. We also found differences in fecal glucocorticoid metabolite levels between the sexes and seasonal variation in males. Below we discuss the implications of these results and put them in the context of similar work on the physiology of captive populations.

### *4.1. Fecal Glucocorticoid Metabolite Levels and Reproductive Success*

Previous research has found that high levels of glucocorticoids can downregulate reproductive efforts [8]. In a previous study of captive breeding Puerto Rican parrots [48] visual inspection of adrenal gland and testes showed that in some males the adrenal gland was hypertrophied and the testes were small. It was suggested that these findings could be due to high levels of stress [48], however, circulating or excreted glucocorticoids were not assessed. Here we showed that a standard capture restraint protocol produced a rise in circulating corticosterone that was mirrored by a rise in fecal glucocorticoid metabolites. Using this validated assay, we found that male fecal glucocorticoid metabolites during the breeding season of 2018 had a negative relationship to total eggs and fertility of eggs providing support to the previous study's findings related to the testes [48]. For females during the pre-breeding season of 2018, we found a positive relationship of fecal glucocorticoid metabolites levels with total eggs laid. However, our results, for either sex, do not show any relation of fecal glucocorticoid metabolites to the number of chicks and fledglings produced.

In males, increases in glucocorticoids have been documented to suppress production of testosterone [49–51] and luteinizing hormone [49]. Suppression of testosterone and luteinizing hormone can negatively affect reproductive behaviors. On the other hand, in a study of wild populations of house wrens (*Troglodytes aedon*), females whose glucocorticoids levels were artificially elevated before breeding produced heavier eggs and had higher nestling feeding rates, significantly increasing chick body mass [52]. Elevated glucocorticoids levels in females may mobilize energy to prepare for the breeding season. Together, these results suggest that, in some species, males and females are differently impacted by changes in glucocorticoid levels.

The relationship between reproductive success and glucocorticoids may also be affected by a species' natural history [1,53]. Short live species may have mechanisms that suppress the effects of high glucocorticoids while long-lived species may experience selection for survival over reproduction [53]. Like many parrots, the Puerto Rican parrot is a long-lived species and may live for up to 37 years in captivity [54]. Under natural conditions, in long-lived species, individual survival may be prioritized before reproduction and males, in particular, may not have evolved the capacity to modulate the effects of frequent energetic challenges experienced in captivity. On the other hand, females may evolve mechanisms to modulate any negative effects of elevated glucocorticoid just prior to egg-laying [55] and this may be held over into captivity. Additionally, egg production is an energetically demanding process, in which the female must place energetically rich resources into the production of eggs and may use glucocorticoids for the mobilization of that energy. This may explain the positive relationship observed between fecal glucocorticoid metabolite levels in the pre-breeding season and egg production in females seen in this study.

### *4.2. Seasonal Variation*

Many animals have seasonal variation in hormone levels in the wild [53,56–59] but it remains unclear whether that variation is retained under captive conditions. Because of the outdoor housing and seasonal breeding cycle of the Puerto Rican parrot in the captive populations assessed here, we expected to see variation among the breeding and pre-breeding seasons in fecal glucocorticoid metabolite levels. We found that male fecal glucocorticoid metabolite levels in the Puerto Rican parrot were higher during the prebreeding than in the breeding season.

Seasonal variation has also been documented in captive red-tailed parrots (*Amazona brasiliensis*), with mean fecal glucocorticoid metabolite concentrations highest just before breeding [60]. Research in wild populations of northern cardinals (*Cardinalis cardinalis*) has shown that females and males have higher plasma glucocorticoid corticosterone levels in the pre-breeding compared to the breeding season [47]. In northern cardinals, it was suggested that high glucocorticoid corticosterone levels during the non-breeding season could be an adaptation for energy needed to survive cold conditions. In the Puerto Rican parrot, each pair displays territorial behaviors year-round defending their nesting cavity with a peak in the pre-breeding season which could explain higher fecal glucocorticoid metabolite levels in males at that time [34], elevated plasma glucocorticoids could provide the energy needed for expensive territorial activities in this species.

Puerto Rican parrots at Rio Abajo are housed outdoors and exposed to normal daylight changes, temperature changes and rainfall, and social activity of wild parrots, all of which may affect glucocorticoids levels between seasons [1]. In European starlings (*Sturnus vulgaris*) individuals of both sexes placed in indoor housing experienced delayed reproductive behaviors while outdoor-housed individuals initiated breeding earlier [61]. Pairs of European starlings housed indoors had significantly lower sex steroids and higher expression of gonadotropin inhibitory hormone (GnIH). It is possible that the year-round outdoor housing experienced by these birds results in individuals having hormonal seasonal variation similar to what would be seen in wild individuals.

Alternatively, birds at Rio Abajo are housed in different cages during the pre-breeding versus the breeding season; these conditions may prevent the expression of the full range of natural behaviors during this period and may in turn alter glucocorticoid levels [16,61]. The cages used in the pre-breeding period are smaller than breeding cages and they are placed in closer proximity to other individuals. The proximity of the cages provokes frequent territorial displays (B. Ramos-Guivas, pers obs), potentially at a higher rate than when birds are in their breeding cages. This could result in males having higher glucocorticoid levels during the pre-breeding than during the breeding season. In addition, closer proximity to human activity and capturing for medical antiparasitic treatments (twice over two weeks) during the pre-breeding season may contribute to the seasonal glucocorticoid variations observed.

### *4.3. Glucocorticoid Levels Do Not Differ between Breeding Seasons, despite Hurricane Maria*

The breeding seasons of 2017 and 2018 occurred pre and post-Hurricane Maria. At the Rio Abajo captive population, we found no significant differences in fecal glucocorticoid metabolite levels between the two seasons. Although initially surprising, it is important to note that captive conditions were relatively stable compared to those experienced by wild populations. Many wild populations of animals suffer increased mortality both during a hurricane (due to climatic conditions), and afterward due to increased exposure to predators and reduced food availability [26,62]. Indeed, the wild population of the Puerto Rican parrot at El Yunque, which was only 31 birds before Hurricane Maria, was totally extirpated by the hurricane. New releases started in 2019 to re-establish this wild population. The wild population at Rio Abajo fared somewhat better but is thought to have declined from 134 to 110 birds as a result of direct effect of the hurricane. Secondary effects as scarcity of food and exposure to predators reduced the population even more in the following months after the hurricane. In contrast, the Rio Abajo captive population was sheltered from the direct impacts of the storm in a hurricane shelter and had no changes in food availability.

#### *4.4. Males Show Higher Fecal Glucocorticoid Metabolite Levels than Females*

Differences between the physiology of males and females are common, therefore we expected to see a difference in fecal glucocorticoid metabolite levels between males and females during the pre-breeding season even when faced with similar captivity stimuli (variation linked to preparation for breeding). We found that males had significantly higher fecal glucocorticoid metabolite levels than females during the pre-breeding. It could be that lower levels of glucocorticoids in females compared to males represent suppression of glucocorticoid levels during the pre-breeding in preparation for egg-laying [60]. Alternatively, higher energetic demands for males than females during the pre-breeding may be due to increases in courtship or territorial behavior shown by males, which may increase glucocorticoid levels [63,64].

### *4.5. Differences between Captive Populations*

For our study, we collected fecal samples from both extant captive populations during the 2017 breeding season. Because of the difference in location and management techniques we expected to see differences in the glucocorticoid levels between the populations. We found that fecal glucocorticoid metabolite levels among males were somewhat higher at the El Yunque population than at the Rio Abajo population, although the difference was not quite statistically significant. Housing conditions, climate and daily routines differ between the two captive populations. At the El Yunque captive population, the breeding cages are more exposed to personnel activity, so recurrent perturbations by people walking by may stimulate continuous energetic challenges [65]. In addition, the nest checks and daily feeding are not performed in a strict schedule at El Yunque captive population adding an element of unpredictability for individuals. Recurrent intrusion to breeding areas can maintain glucocorticoids levels above baseline, eventually impacting reproductive success [65]. At the Rio Abajo captive population, the breeding cages are located behind vegetation that may serve as a screen from personnel, potentially reducing glucocorticoid levels. Another difference between the populations is the daily feeding routine. At El Yunque, feeding time lasts longer than at Rio Abajo. The longer presence of personnel inside the breeding areas may increase baseline glucocorticoids levels. High levels of glucocorticoids during the breeding season could negatively affect reproduction.

### **5. Conclusions**

Fecal glucocorticoid metabolite analysis provides an opportunity to study species in a minimally invasive fashion. Our results provide insight into the relationship of fecal glucocorticoids metabolites to reproductive success under normal daily routines in captivity. Although it has been reported in previous studies that captive individuals have a more attenuated response to energetic challenges than wild individuals [17,66,67], captive individuals can still be affected by high glucocorticoids levels [68–70]. The negative relationship of fecal glucocorticoid metabolite levels in males to total eggs laid and total fertile eggs may indicate a susceptibility to anthropogenic activities. For endangered species programs, strategies should be developed to reduce perturbations and regularize their occurrence during key periods of the year to minimize the potential negative effects on reproduction. The differences between populations in glucocorticoid levels suggest that managers of captive breeding species should carefully evaluate factors that may be driving these differences (e.g., housing placement and the frequency and predictability of anthropogenic activities during critical stages of the breeding season). Future research should examine the role of glucocorticoids in reproduction of captive populations of other endangered species, and where possible, compared to wild populations of these species.

**Author Contributions:** Conceptualization, B.R.-G. and T.F.W.; methodology, B.R.-G., T.F.W. and J.M.J.; validation, B.R.-G., J.M.J.; formal analysis, B.R.-G. and J.M.J.; investigation, B.R.-G., J.M.J.; resources, B.R.-G., T.F.W. and J.M.J.; data curation, B.R.-G. and J.M.J.; writing—original draft preparation, B.R.-G. and T.F.W.; writing—review and editing, T.F.W. and J.M.J.; visualization, B.R.-G.; supervision, T.F.W.; project administration, B.R.-G. and T.F.W.; funding acquisition, B.R.-G. and T.F.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the World Parrot Trust (grant date 2015, 2016 and 2018).

**Institutional Review Board Statement:** All research activities and animal handling were carried out in full compliance and accordance with the NMSU IACUC protocols 2014-030 and 2021-014, the Puerto Rican Parrot Recovery Program Interagency Operational Team, supported by the management under the Puerto Rico Department of Natural and Environmental Resources and conducted under the United States Fish and Wildlife Service Permit TE125521-4.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data availability in this study are available upon request from corresponding author.

**Acknowledgments:** We thank Marisel López, Jafet Vélez, Iris Rodriguez-Carmona, Gabriel Benitez-Soto, Limary Ramírez-Esquilín, Arelys Johnson-Camacho from the USFWS for their cooperation in collecting the samples at the Iguaca facilities at El Yunque National Forest. We thank Miguel A. García-Bermudez, Ricardo Valentín de la Rosa, Tanya Martínez-Ramirez, Jong Piel Banchs-Plaza and Roseanne Medina-Miranda from the PR DNER for their cooperation during the study. We thank Elisa Boyd for the help provided during the preparation of the samples, Dominique Hellmich, Angela Medina and Grace Smith Vidaure for sharing their knowledge all members of the Wright Lab at the New Mexico State University for the help provided during the analysis process. We thank Antonio Rivera-Guzman, Tomás Medina-Cortés, Magaly Massanet, Valeria Perez-Marrufo, Natasha L. Perez-Nieto, Mariana Travieso-Diffoot, Gabriela Fonseca-Cordero, Adriana Pérez-Vega, Alondra Villalba, Echo Ruff for their labor during the validation.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Permits and Approval Statement:** The study was approved by the Puerto Rico Department of Natural and Environmental Resources Puerto Rican Parrot Program coordinator leadership and by the US Fish and Wildlife Puerto Rican Parrot program coordinator.

### **References**


*Edited by Denis Larrivee*

With the conclusion of the *Decade of the Brain* and *Decade of the Mind*, neuroscience has advanced well beyond single neuron functions, and begun to investigate global properties that emerge from central nervous system operation. Core ethical issues for neural intervention, in consequence, now touch on concerns over how the individual as a whole may be affected. Central to these concerns is the fundamental value of the human being, which lends normative weight to questions, interventions, and practices influencing him or her. Yet, despite wide recognition of the crucial relevance of human value, the derivation of metaethical principles that underwrite this value is by no means uniformly agreed to. Why and how the human being is normatively privileged, accordingly, emerge as core questions that frame issues of ethical praxis. This book tackles this dissonance, and exposes the philosophical foundations that are rooting contemporary divisions in ethical approaches to intervention in the nervous system.

ISBN 978-953-51-7265-9

Neuroethics in Principle and Praxis - Conceptual Foundations

Published in London, UK © 2019 IntechOpen © TimArbaev / iStock


*Article* **Migration Behavior and Influencing Factors of Petroleum Hydrocarbon Phenanthrene in Soil around Typical Oilfields of China**

**Hao Chen 1 , Mengfan Lang 1 , Changjun Liao 2,\* and Xuetao Guo 1,3,\***

**Abstract:** Petroleum spills and land contamination are becoming increasingly common around the world. Polycyclic aromatic hydrocarbons (PAHs) and other pollutants found in petroleum are constantly migrating underground, making their migration in soil a hot research topic. Therefore, it is of great significance to evaluate the migratory process of petroleum hydrocarbons in petroleumpolluted soil to clarify its ecological and environmental risks. In this study, Phenanthrene (PHE) was used as a typical pollutant of PAHs. The soil was gathered from three typical oilfields in China, and a soil column apparatus was built to simulate the vertical migration of PHE in the soil. The migration law and penetration effect of PHE in various environmental conditions of soil were investigated by varying the ionic strength (IS), pH, particle size, and type of soil. According to the literature, pH has no discernible effect on the migration of PHE. The migration of PHE was adversely and positively linked with changes in IS and soil particle size, respectively. The influence of soil type was mainly manifested in the difference of organic matter and clay content. In the Yanchang Oilfield (YC) soil with the largest soil particle size and the least clay content, the mobility of PHE was the highest. This study may reveal the migration law of PAHs in soils around typical oilfields, establish a new foundation for PAH migration in the soil, and also provide new ideas for the management and control of petroleum pollution in the soil and groundwater.

**Keywords:** penetration curve; petroleum pollution; PHE; soil column; typical oilfields; vertical migration

### **1. Introduction**

Petroleum is a complex organic compound composed of various straight paraffins, naphthenes, monocyclic aromatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) [1]. Petroleum hydrocarbons in petroleum are composed of organic components with different molecular weights [2]. As a kind of non-renewable energy, petroleum is often the main goal of geological exploration. In today's world market, the demand and price of petroleum are rising constantly [3], and China's oil industry is also developing rapidly [4]. Therefore, the leakage of hydrocarbons is inevitable [5]. Oil spill on land can cause serious soil diffusion pollution [6], and the high content of toxic compounds in crude oil can also cause physical and biochemical poisoning to soil microbes [7]. Petroleum spills will cause the overflow of PAHs and benzene, ethylbenzene and other benzene series, which are collectively referred to as persistent organic pollutants, and they are extremely harmful to the environment [8,9].

PAHs are one of the typical pollutants of petroleum, formed by incomplete combustion or pyrolysis of organic matter [10], and they contain two or more benzene rings. They are characterized by high hydrophobicity, high toxicity, high lipophilicity, low volatility and high chemical stability [11–13]. The sources of PAHs include natural and human

**Citation:** Chen, H.; Lang, M.; Liao, C.; Guo, X. Migration Behavior and Influencing Factors of Petroleum Hydrocarbon Phenanthrene in Soil around Typical Oilfields of China. *Processes* **2022**, *10*, 1624. https:// doi.org/10.3390/pr10081624

Academic Editors: Guining Lu, Zenghui Diao and Kaibo Huang

Received: 13 July 2022 Accepted: 11 August 2022 Published: 17 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

activities, and the concentration of PAHs in urban soil is much higher than that in places far away from urban areas [14]. Petroleum spills are the most important source of PAHs in soil. As an important area for petroleum exploration, there is a large amount of oil pollution in the oil production site. In this region, large amounts of volatile oil carry a large number of organic pollutants such as PAHs. The transport of oil in the soil, in turn, can have environmental and human health impacts [15]. PAHs can cause carcinogenic [16], teratogenic, mutagenic and genotoxic effects on the environment [17]. The pollution of PAHs in soil to surface soil will be greater than that in deep soil [14], and PAHs in soil will be directly or indirectly exposed to human beings, endangering human health [15]. Studies have shown that the pathogenic factors of human lung cancer are closely related to the excessive inhalation of PAHs [18]. In addition, PAHs in the atmosphere will migrate and deposit on water, soil and plants, eventually threatening human health due to their accumulation in the food chain [19]. Therefore, it is necessary to study the environmental behaviors and characteristics of PAHs.

High hydrophobicity makes PAHs easily adsorbed on environmental media such as water and soil, and finally deposited in the matrix through migration and adsorption [13]. Therefore, the research on migration of PAHs has gradually become the focus of attention. At present, the research on the vertical distribution of PAHs in soil sediments shows that its concentration is inversely proportional to the soil depth [20–23]. However, PAHs have been capable of migrating deeper into the soil profile for a long time [24]. Sun et al. found that PAHs migrate from the soil surface to the ground through pore infiltration in the rainy season and surface dissolution in the dry season [25]. When studying the cotransport of PAHs and other substances, Fang et al. found that the nanoparticles (NPs) in soil with high organic matter are the most effective in transporting phenanthrene (PHE) through columns [26]. In addition, the presence of oxidized multi-walled carbon nanotubes (O-MWCNTs) in the influent significantly improved the mobility of PHE in the chromatographic column [26]. Due to the complex chemical nature of PAHs, its transport and diffusion processes in soil will be affected by adsorption-desorption, volatilization, leaching and bioaccumulation [27]. However, research on the migration of single PAHs in soil, especially in the soil of typical oilfields, has not been carried out systematically.

In order to make up for the deficiency of research on PAHs migration in the soil of typical oilfields, we selected the soil around the typical oilfields in northern China (Shengli Oilfield in Shandong, Nanyang Oilfield in Henan and Yanchang Oilfield in Shaanxi) as the medium. PHE, one of the PAHs, was chosen as the target pollutant. As an internationally recognized priority pollutant, PHE not only has two methods of co-metabolic oxidation and direct oxidation, but also has stable chemical properties. Therefore, it is widely used in the study of degradation, adsorption and migration of PAHs [28].

In order to explore the migration behavior and influencing factors of petroleum hydrocarbon component PHE in the soil around typical oilfields, we simulated the migration process of PHE in soil by building a soil column migration device in the laboratory. The experiments were conducted to explore the effects of different factors on the migration of PHE in soils around oilfields by varying the ionic strength (IS), pH, soil particle size and type, respectively. Our experimental process strictly followed the single-factor variable control principle. The mobility was reflected by measuring the concentration of PHE in the solution after migration. This study could further enhance our understanding of the migration behaviors of PAHs in soils around typical oilfields and the influence of soil environment on their migration effects. It may provide new ideas for the study of the migration of PAHs in soil, thus providing a basis for the pollution management of petroleum pollution in soils around typical oilfields.

### **2. Materials and Methods**

### *2.1. Materials*

PHE with a purity greater than 90% was purchased from Bailingwei Technology Co., Ltd. in Beijing, while other chemicals such as sodium chloride (NaCl, >99.5%), hydrogen

peroxide (30%) and calcium chloride (CaCl2, 96%) were purchased from Guanghua Co., Ltd., a company in Guangdong. Methanol (>99%) was purchased from AMPEL Laboratory Technology Co., Ltd., a company in Shanghai. After washing the quartz sand, it should be thoroughly cleaned with 0.1 M hydrochloric acid and 5% hydrogen peroxide. The two chemicals will remove any contaminants from the surface of the sand particles [29,30]. After a rinse with deionized water, the pH gradually becomes neutral and the sand is dried in a 35 ◦C oven. Guanghua Co., Ltd., a company in Guangdong. Methanol (>99%) was purchased from AMPEL Laboratory Technology Co., Ltd., a company in Shanghai. After washing the quartz sand, it should be thoroughly cleaned with 0.1 M hydrochloric acid and 5% hydrogen peroxide. The two chemicals will remove any contaminants from the surface of the sand particles [29,30]. After a rinse with deionized water, the pH gradually becomes neutral and the sand is dried in a 35 °C oven. *2.2. Sampling and Pretreatment*

ideas for the study of the migration of PAHs in soil, thus providing a basis for the

PHE with a purity greater than 90% was purchased from Bailingwei Technology Co., Ltd. in Beijing, while other chemicals such as sodium chloride (NaCl, >99.5%), hydrogen peroxide (30%) and calcium chloride (CaCl2, 96%) were purchased from

pollution management of petroleum pollution in soils around typical oilfields.

*Processes* **2022**, *10*, x FOR PEER REVIEW 3 of 12

#### *2.2. Sampling and Pretreatment* We selected the uncontaminated soil around three typical oilfields in northern China

**2. Materials and Methods**

*2.1. Materials*

We selected the uncontaminated soil around three typical oilfields in northern China (Shengli Oilfield, Nanyang Oilfield and Yanchang Oilfield). The Shengli Oilfield is located in the Yellow River delta area of Dongying City, Shandong Province, with geographical coordinates of 37◦230 N and 118◦310 E. The Nanyang Oilfield is located in Nanyang City, southwest of Henan Province, with geographic coordinates of 32◦410 N and 112◦340 E. The Yanchang Oilfield is located in Yan'an City, northern Shaanxi Province; the landforms are mainly the Loess Plateau and hilly areas, and the geographical coordinates are 36◦400 N and 109◦330 E. We measured the content of PHE in the sampled soil, and found no accumulation of PHE in these uncontaminated soils. The three soils were named SL, NY and YC, respectively. The geographical location of the three sampled oilfields is shown in Figure 1. (Shengli Oilfield, Nanyang Oilfield and Yanchang Oilfield). The Shengli Oilfield is located in the Yellow River delta area of Dongying City, Shandong Province, with geographical coordinates of 37°23′ N and 118°31′ E. The Nanyang Oilfield is located in Nanyang City, southwest of Henan Province, with geographic coordinates of 32°41′ N and 112°34′ E. The Yanchang Oilfield is located in Yan'an City, northern Shaanxi Province; the landforms are mainly the Loess Plateau and hilly areas, and the geographical coordinates are 36°40′ N and 109°33′ E. We measured the content of PHE in the sampled soil, and found no accumulation of PHE in these uncontaminated soils. The three soils were named SL, NY and YC, respectively. The geographical location of the three sampled oilfields is shown in Figure 1.

**Figure 1. Figure 1.** Geographical location of soil Geographical location of soil sampling in three typical oilfields. sampling in three typical oilfields.

Three types of soils were taken from the laboratory, dried and, firstly, impurities such as small stones, debris, plant and animal residues were picked up with forceps [31,32]. The soils were then placed in a mortar for grinding and sieved using a standard test sieve (Shangyu Dahengqiao laboratory instrument factory, Zhejiang, China). During the sampling process, we found that the main particle sizes of all soils were distributed around 1 mm. When exploring the influence of other variables, it is necessary to control the soil to be in the same particle size range. Moreover, if the particle size is too large or too small, it is not conducive to the penetration of the solution. Taking all into consideration, the soil

particle sizes of SL after sieving were 0.45~1 mm, 0.15~0.45 mm, 0~0.15 mm, and 0~1 mm, while the particle sizes of NY and YC were 0~1 mm.

### *2.3. Preparation of PHE and Electrolyte Solution*

Based on the study of Liu et al. [32], we made appropriate improvements to the preparation of the solution. To make a 1 g/L PHE stock solution, place 0.5 g of PHE solid powder in a beaker, dissolve it in methanol and transfer it to a volumetric flask. PHE solutions of 1000 ppb, 800 ppb, 400 ppb, 200 ppb, 100 ppb and 50 ppb were prepared using the concentration gradient method. In a volumetric flask, CaCl2 (0.555 g) was added, and the original solution of 50 mM CaCl2 was obtained by constant volume with ultrapure water. CaCl2 solutions of 1 mM, 0.5 mM and 0.1 mM were made by diluting the original solution. Similarly, 1 mM, 5 mM and 10 mM NaCl solutions were prepared. In addition, mixed solutions containing the above two electrolytes were prepared with a background concentration of 500 ppb PHE. All solutions are sealed and stored for subsequent column transport experiments.

### *2.4. Column Transport Experiments*

According to existing research [29,31–35], the column transmission experiment was conducted. In order to prevent the sand core from being blocked by soil, quartz sand (1 cm) should be loaded into the bottom of the soil column. The dried treated soil was slowly filled into a clean and dry column and the column was shaken to prevent the formation of large pores. The length of the column used in the experiment was 10 cm and the inner diameter was 10 mm. The soil was filled to a height of 8 cm. A 1 cm layer of quartz sand was added to the top and bottom of the soil column to prevent water droplets from eroding the column.

The experiment made some adjustments to the leaching method and sequence used in the previous study [32]. A peristaltic pump was used to inject about 30 mL of ultrapure water into the column from top to bottom, followed by 30 mL of electrolyte solution to stabilize the chemical properties of the system. Then, 40 mL PHE-electrolyte solution (500 ppb PHE) was injected into the column from top to bottom by peristaltic pump at a flow rate of 0.5 mL/min (2 rpm). Finally, the column was rinsed with 30 mL electrolyte solution until the concentration of PHE solution was stable. The sample of solution collected from the outlet at the bottom of the soil column during injection and flushing was stored in a brown chromatograph bottle, and the eluent was collected every 5 mL during the experiment until the end of the experiment. Finally, the column was washed with ultrapure water [32,36]. The pH value of the leaching solution was adjusted with hydrochloric acid and sodium hydroxide, and the IS was adjusted with calcium chloride and sodium chloride solutions.

The soil column experiment's design procedure was as follows: First, six groups of IS migration experiments were conducted using three different concentrations of NaCl and CaCl2, a neutral pH solution, and soil with SL of 0~1 mm. Permeability was calculated from the concentration of PHE in the solution collected for this experiment. The IS with the best migration effect was selected as the constant condition of the subsequent experiments, and then the solution under three pH (4, 7 and 10) conditions and the soil under three particle sizes (<0.15 mm, 0.15~0.45 mm and 0.45~1 mm) was selected for migration experiments. The former controls the soil particle size to be 0~1 mm, and the latter controls the pH to be neutral. Finally, three different soil migration experiments of SL, YC and NY were carried out, and other solutions and soil conditions were kept the same. After each experiment, the soil column was washed with deionized water and ultrapure water and dried for reuse. The experimental design strictly followed the single-factor variable control principle. Through the comparison of the concentration of PHE in the solution collected under the influence of various factors, the longitudinal migration behavior of PHE in the soil around typical oilfields under different factors was comprehensively analyzed.

### *2.5. HPLC Determination Conditions of PHE*

During High Performance Liquid Chromatography analysis, the analytical methods and parameter conditions used were adjusted and improved on the basis of the research of the previous study [32]. The concentration of PHE was determined using an Agilent High Performance Liquid Chromatography (HPLC, Agilent 1200) equipped with a fluorescence detector (FLD) and an ultraviolet detector (UVD). C18 reversed-phase column (Luna, 250 mm × 4.6 mm) was used as the chromatographic column. At a flow rate of 1 mL/min, the mobile phase was a combination of 90% acetonitrile and 10% aqueous solution. The fluorescence detector's excitation wavelength was 250 nm, and its emission wavelength was 364 nm. The UV detector's wavelength was set at 250 nm. The peak area was quantified using an external standard technique, and the retention duration was qualitative [31,36].

### *2.6. Statistical Analysis*

Potassium dichromate was used as an oxidant to undergo a redox reaction with the organic matter in the soil, and the content of the organic matter in the soil was determined by a titration method. Soil total nitrogen was determined by the more conventional Kjeldahl method (national standard method: HJ 717-2014); total phosphorus was determined by the molybdenum-antimony resistance colorimetric method (NY/T 88-1988 Determination of soil total phosphorus). The specific operation steps of the two can be found in the national standard. Soil nitrate nitrogen (NO3 −–N) and ammonium nitrogen (NH4 +–N) were measured using SmartChem automatic discontinuous chemical analyzer. When measuring, rinse the sample cup three times with the sample before adding the sample. Add the corresponding reagent to the corresponding reagent position. Pour the reagent into the reagent bottle slowly to prevent air bubbles from forming. The composition of soil particle size was sieved and counted by a step-by-step sieving method. According to the corresponding relationship between the permeability and time of PHE, the Origin 2018 software was used to make the penetration curve of PHE under each group of experiments.

### **3. Results**

### *3.1. The Physical and Chemical Properties of Three Soils*

We characterized and measured the physical and chemical properties of three soils. Table 1 shows the physical and chemical properties of the surrounding soil in SL [37], NY and YC.

**Table 1.** The physical and chemical properties of the three soils.

\* OM—organic matter; TN—total nitrogen; TP—total phosphorus.

As can be seen from Table 1, different soils show different structural and compositional characteristics. The contents of inorganic nutrients such as N and P in the three soils differed somewhat, with YC soils having higher contents of NO3 −–N and TP than the other two soils. Moreover, the highest contents of inorganic nutrients in NY soils were TN and NH4 +–N compared to YC and SL. Additionally, in terms of organic matter, NY soil also had the highest content of it. In contrast, SL had the lowest organic matter and N element content. In addition, through the investigation of soil particle size, we determined that the overall particle size of the three kinds of soil was YC > NY > SL. Among the three, YC had the biggest particle size, with 63.16% of the soil particle size in 0.02~2 mm. SL and NY had a more uniform particle size distribution (Table 1).

#### *3.2. The Influence of Types of Soil* After the plateau period, the decline rate from fast to slow was also YC, NY and SL. It can

*3.2. The Influence of Types of Soil*

were TN and NH4

before migration.

Under the condition of soil particle size of 0~1 mm, maintaining the same IS and pH, and using the soil types as a variable, the differences in the vertical migration of PHE in three soils of SL, YC and NY were explored. The penetration curve was shown in Figure 2. Permeability (C/C0) is defined as the ratio of the concentration of PHE in the solution flowing from the bottom of the soil column to the concentration of PHE in the added PHE-electrolyte solution, i.e., the concentration ratio of contaminants after migration and before migration. be seen that the penetration effect of YC was the best, with the highest permeability of 1.00; that is, YC can completely penetrate the soil column. Followed by NY, the C/C0 value of the stable period was 0.93. A little worse than NY was SL, whose permeability C/C0 was basically around 0.9. It is speculated that this may be due to the differences of soil composition and physical and chemical properties in different areas. During the process of migration of pollutants such as PAHs, different physical and chemical conditions in soil will inevitably produce different migration results [38,39].

As can be seen from Table 1, different soils show different structural and compositional characteristics. The contents of inorganic nutrients such as N and P in the

+–N compared to YC and SL. Additionally, in terms of organic matter,

than the other two soils. Moreover, the highest contents of inorganic nutrients in NY soils

NY soil also had the highest content of it. In contrast, SL had the lowest organic matter and N element content. In addition, through the investigation of soil particle size, we determined that the overall particle size of the three kinds of soil was YC > NY > SL. Among the three, YC had the biggest particle size, with 63.16% of the soil particle size in

Under the condition of soil particle size of 0~1 mm, maintaining the same IS and pH, and using the soil types as a variable, the differences in the vertical migration of PHE in three soils of SL, YC and NY were explored. The penetration curve was shown in Figure 2. Permeability (C/C0) is defined as the ratio of the concentration of PHE in the solution flowing from the bottom of the soil column to the concentration of PHE in the added PHE-electrolyte solution, i.e., the concentration ratio of contaminants after migration and

As can be seen from Figure 2, the penetration curve of PHE (expressed as C/C0) in different soil types showed a rapid upward trend at first, then entered a stable period when the pore volume was about 6, and finally decreased rapidly after the pore volume was 10. The initial growth rate of C/C0 from fast to slow is YC, NY and SL, respectively.

−–N and TP

three soils differed somewhat, with YC soils having higher contents of NO3

0.02~2 mm. SL and NY had a more uniform particle size distribution (Table 1).

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NY 15.92 0.83 19.87 2.61 0.47 3.22 44.75 30.32 21.71 \* OM—organic matter; TN—total nitrogen; TP—total phosphorus.

**Figure 2. Figure 2.** The influence of types of soil on vertical migration of phenanthrene (PHE). The influence of types of soil on vertical migration of phenanthrene (PHE).

As can be seen from Figure 2, the penetration curve of PHE (expressed as C/C0) in different soil types showed a rapid upward trend at first, then entered a stable period when the pore volume was about 6, and finally decreased rapidly after the pore volume was 10. The initial growth rate of C/C0 from fast to slow is YC, NY and SL, respectively. After the plateau period, the decline rate from fast to slow was also YC, NY and SL. It can be seen that the penetration effect of YC was the best, with the highest permeability of 1.00; that is, YC can completely penetrate the soil column. Followed by NY, the C/C0 value of the stable period was 0.93. A little worse than NY was SL, whose permeability C/C0 was basically around 0.9. It is speculated that this may be due to the differences of soil composition and physical and chemical properties in different areas. During the process of migration of pollutants such as PAHs, different physical and chemical conditions in soil will inevitably produce different migration results [38,39].

### *3.3. The Influence of IS*

As an important factor in soil, IS will affect the physical and chemical properties of soil and the environmental behaviors of coexisting substances [35]. In this experiment, NaCl and CaCl2 electrolyte solutions were used to simulate the effect of is on the vertical migration of PAHs in soil. The type of soil should be unchanged when exploring the effects of other factors on migration of PHE, so we used SL soils in our studies of IS, pH and soil particle size.

The penetration curves of PHE under different conditions of IS are shown in Figure 3. It can be seen that the migration and dissolution rate of PHE in the soil decreased with the increase of IS, which leads to the widening and delay of the penetration curve. This was consistent with the results of previous studies [32,40]. It could be that the increase in IS leads to the formation of more cations and anions in solution, which compresses the soil particles [41]. Therefore, electrostatic electric double layer particles reduce the repulsion of soil particles. Moreover, the increase of metal ions will adsorb and deposit a certain

amount of PHE, which will enhance its migration resistance. In the presence of the two selected electrolyte solutions, it can be clearly seen that the ability of NaCl to promote PHE migration was significantly stronger than that of CaCl2. Different concentrations of NaCl (1 mM, 5 mM, 10 mM) had little effect on the migration of PHE, but the highest migration of PHE was observed under the electrolyte solution with 1 mM NaCl, up to 93%. However, under the action of CaCl2, its promotion effect was weaker. It can be found that when the concentration of CaCl2 was between 0 and 1 mM, the lower the electrolyte concentration, the higher the degree of PHE migration. soil particles [41]. Therefore, electrostatic electric double layer particles reduce the repulsion of soil particles. Moreover, the increase of metal ions will adsorb and deposit a certain amount of PHE, which will enhance its migration resistance. In the presence of the two selected electrolyte solutions, it can be clearly seen that the ability of NaCl to promote PHE migration was significantly stronger than that of CaCl2. Different concentrations of NaCl (1 mM, 5 mM, 10 mM) had little effect on the migration of PHE, but the highest migration of PHE was observed under the electrolyte solution with 1 mM NaCl, up to 93%. However, under the action of CaCl2, its promotion effect was weaker. It can be found that when the concentration of CaCl2 was between 0 and 1 mM, the lower the electrolyte concentration, the higher the degree of PHE migration.

As an important factor in soil, IS will affect the physical and chemical properties of soil and the environmental behaviors of coexisting substances [35]. In this experiment, NaCl and CaCl2 electrolyte solutions were used to simulate the effect of is on the vertical migration of PAHs in soil. The type of soil should be unchanged when exploring the effects of other factors on migration of PHE, so we used SL soils in our studies of IS, pH

The penetration curves of PHE under different conditions of IS are shown in Figure 3. It can be seen that the migration and dissolution rate of PHE in the soil decreased with the increase of IS, which leads to the widening and delay of the penetration curve. This was consistent with the results of previous studies [32,40]. It could be that the increase in IS leads to the formation of more cations and anions in solution, which compresses the

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*3.3. The Influence of IS*

and soil particle size.

**Figure 3.** The influence of the ionic strength (IS) on the vertical migration of PHE in soil. **Figure 3.** The influence of the ionic strength (IS) on the vertical migration of PHE in soil.

#### *3.4. The Influence of pH 3.4. The Influence of pH*

Figure 4 shows the influence of pH value on the vertical migration of PHE in soil. Under the experimental settings of pH 4, 7 and 10, the three penetration curves kept the same form. Under these three conditions, the fluidity reached the maximum when the pore volume was about 9.8. It can be seen from the penetration curve that change of pH value had no obvious effect on the vertical migration of PHE. It is inferred that the transport of PHE in soil has no significant correlation with the change of pH. Figure 4 shows the influence of pH value on the vertical migration of PHE in soil. Under the experimental settings of pH 4, 7 and 10, the three penetration curves kept the same form. Under these three conditions, the fluidity reached the maximum when the pore volume was about 9.8. It can be seen from the penetration curve that change of pH value had no obvious effect on the vertical migration of PHE. It is inferred that the transport of PHE in soil has no significant correlation with the change of pH. *Processes* **2022**, *10*, x FOR PEER REVIEW 8 of 12

**Figure 4.** The influence of the pH value on the vertical migration of PHE in soil. **Figure 4.** The influence of the pH value on the vertical migration of PHE in soil.

The penetration curves of PHE in soils with different particle sizes are shown in Figure 5. Within the pore volume range of 1~15, there was a peak in the penetration

or partially (0~0.15 mm and 0.15~0.45 mm) pass through the leaching process of soil column. Additionally, the penetration curve with a particle size of 0.45~1 mm appeared

**Figure 5.** The influence of the particle size on the vertical migration of PHE in soil.

*3.5. The Influence of Particle Size of Soil*

to have a stable peak value of 1.00.

#### *3.5. The Influence of Particle Size of Soil* **Figure 4.** The influence of the pH value on the vertical migration of PHE in soil.

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The penetration curves of PHE in soils with different particle sizes are shown in Figure 5. Within the pore volume range of 1~15, there was a peak in the penetration curves of the three particle sizes, which indicated that PHE can completely (0.45~1 mm) or partially (0~0.15 mm and 0.15~0.45 mm) pass through the leaching process of soil column. Additionally, the penetration curve with a particle size of 0.45~1 mm appeared to have a stable peak value of 1.00. *3.5. The Influence of Particle Size of Soil* The penetration curves of PHE in soils with different particle sizes are shown in Figure 5. Within the pore volume range of 1~15, there was a peak in the penetration curves of the three particle sizes, which indicated that PHE can completely (0.45~1 mm) or partially (0~0.15 mm and 0.15~0.45 mm) pass through the leaching process of soil column. Additionally, the penetration curve with a particle size of 0.45~1 mm appeared to have a stable peak value of 1.00.

**Figure 5. Figure 5.** The influence of The influence of the particle size on the vertical migration of PHE in soil. the particle size on the vertical migration of PHE in soil.

### **4. Discussion**

### *4.1. The Influence of Types of Soil*

The soil particle size showed that the particle size of NY and SL was dominated by silt (0.002~0.02 mm), accounting for 44.75% and 54.49%, respectively, while the particle size of YC was mainly 0.02~2 mm, accounting for 63.16% (Table 1). It can be inferred that the particle size composition of the soil significantly affects the migration of PHE, and the large particle size dominates. The larger the proportion of large particle size in the soil, the faster the migration rate of PHE (Figure 2). Therefore, the vertical migration of PHE in soil is positively correlated with soil particle size [34,35,42,43]. Study have shown that the mobility of PAHs in soil is positively correlated with gravel content (0.02~2 mm) and negatively correlated with clay content (<2 µm) [26]. The results of this experiment were consistent with them. In the literature, clay minerals are thought to be an important factor affecting the uptake of organic pollutants in soil [24,44], because the increase of fine-grained clay content will have a larger specific surface area and more adsorption sites, which will adsorb more PHE and reduce its mobility [38,39].

We can find that the soil of YC contained higher NO3 −–N and TP than the other two soils (Figure 2). Rich inorganic nutrients are more conducive to carrying high hydrophobic pollutants such as PHE, and thus, they migrate to the underground [38,39]. In addition, previous studies have shown that the vertical migration of PAHs is negatively correlated with soil organic matter content [26,34,35,42,43]. However, through the analysis of Table 1, it was found that the contents of organic matter in the SL, NY and YC soils are 7.86, 15.92 and 11.14 g/kg, respectively, so the migration rates of PHE should decrease. It was not consistent with the results of YC, NY and SL in Figure 2. Furthermore, it may be inferred that in previous studies, large-scale soil columns were used for long-term migration experiments [26], and the organic matter in the soil had enough time and space to adsorb PAHs, which affected the migration of PAHs. However, in this experiment, a small soil column was used for short-term migration and leaching experiment, so the content of

organic matter in the soil did not play a leading role in the vertical migration of PHE. At the same time, compared with the penetration curve of YC, the penetration curve of NY was lower, which indicates that the positive correlation between soil organic matter content and soil adsorption capacity may be only one aspect of the effect of migration [24,32]. Because the overall soil particle size of NY was smaller than that of YC, its permeability was also lower than that of YC. We speculate that the combined effect of soil particle size and organic matter is the dominant factor affecting the migration of PHE in soil.

### *4.2. The Influence of IS*

From the results in Figure 3 (Section 3.3), the high mobility of PHE at a low electrolyte concentration may be closely related to environmental factors in soil. This may be due to the increase of IS, which leads to the existence of a large number of cations and anions in the solution. Their existence compresses the soil particles, and the electrostatic electric double layer particles reduce the repulsion of soil particles [35,40]. In addition, the increase of metal ions also adsorbed and deposited some PHE, thus enhancing the resistance of PHE migration in soil [42,43]. This was consistent with previous research [32], showing that the decrease of IS leads to an increase in the thickness of the electric double layer and the change of surface charge of the quartz sand and pollutants, thus promoting the release of pollutants. Therefore, it can be speculated that the vertical migration of PHE in soil is negatively correlated with IS, and soil with lower metal ion concentration is more favorable for the migration of PAHs. However, the migration in the solution of monovalent metal ion has no obvious effect, which was consistent with the findings of other studies [29,31–35]. In addition, Liu and Wang found that the influence of divalent Ca2+ on the migration ability of pollutants is significantly higher than that of monovalent K+ [32,36]. Combining these two conclusions, it can be inferred that divalent metal ions have an influence on the migration of PHE in soil. The inhibition of this ability is more obvious than that of univalent metal ions [32].

### *4.3. The Influence of pH*

It is inferred that the transport of PHE in soil had no significant correlation with the change of pH (see in Figure 4). Liu measured the penetration curves of soil colloids under different pH values (5, 7 and 9) and 30 mM NaCl solution, and found that the penetration curves basically overlapped between pH values 7 and 9 [41]. According to present studies, PHE is a highly non-ionizable hydrophobic organic chemical that can maintain a high degree of hydrophobicity across all pH settings. As a result, changes in the pH value had no clear influence on the migration of PHE in saturated sand columns [28,32], so the speciation of PHE does not change with the decrease of pH value, and there was no obvious difference between the penetration curves of PHE. The chemical stability of PHE will not be affected by the difference of pH values. From this experiment, it can be concluded that the pH value has no significant influence on the vertical migration of PHE in soil. According to experimental results, the neutral conditions of PHE were used in the subsequent experiments to explore the influence of other soil conditions.

### *4.4. The Influence of the Particle Size of Soil*

As the soil particle size increased, the pores between the soil particles increased, which was more conducive to the penetration and leaching of the solution, thus speeding up the downward migration of PHE [14,28]. Relevant research shows that as the particle size increases, the inorganic NPs in the soil will move downward at an accelerated rate, so the PHE carried on its surface will move downward completely [32]. Sun et al. confirmed that clay with finer particles has a larger specific surface area and more adsorption sites [38]. Moreover, the ability of fine and dense soil to block and adsorb pollutants is obviously larger than that of coarse-grained soil. As shown in Figure 5, in the fine-grained soil with 0~0.15 mm, the maximum permeability (C/C0) of PHE can reach above 0.6. It is speculated that this is closely related to the related physical and chemical properties of the

soil surrounding the oilfield [45]. Therefore, it can be inferred that the vertical migration of PHE in soil is positively related to the soil particle size. This further corroborates the results of previous studies that the migration rate of PAHs is negatively related to the content of clay in soil [34,35,42,43].

### **5. Conclusions**

To investigate the vertical migration of petroleum pollutants in soil, we chose PHE (one PAHs in petroleum) as the pollutant for soil column migration experiments to simulate this process. The findings revealed that the vertical migration of PHE in soil was inversely linked with IS, and that divalent metal ions have a stronger inhibitory effect than monovalent metal ions.  Furthermore, the migration of PHE in soil was positively connected with soil particle size, because larger pores are more conductive to solution leaching and PHE infiltration. In descending order, the mobility of PHE in different soil types was YC, NY and SL. This is because the soil in YC was mostly made up of large particles, whereas the soil in NY and SL was mainly composed of smaller clay particles. The results reveal that the clay component had a negative impact on penetration rate. This experiment provides a new basis for researching the migration of petroleum pollutants in soil surrounding a real oilfield, as well as a point of reference for estimating the extent of petroleum pollution in deeper soil.

**Author Contributions:** Conceptualization, X.G.; Data curation, H.C.; Formal analysis, H.C. and C.L.; Funding acquisition, X.G.; Investigation, H.C. and M.L.; Methodology, H.C. and M.L.; Project administration, X.G.; Supervision, X.G.; Writing—original draft, H.C.; Writing—review and editing, M.L., C.L. and X.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Program of China (No. 2019YFC1804101).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data discussed in this work are presented in the form of Tables and Figures in the article, and no data are withheld. All the data can be accessed from the journal article.

**Conflicts of Interest:** The authors declare no conflict of interest.

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