1. INTRODUCTION

Environmental pollution is currently the most significant challenge facing humanity, leading to increased rates of mortality and morbidity. Pollution caused by industrial and human activities leads to the decline in the quality of the natural environment (Landrigan, 2017). Environmental degradation is an inevitable consequence of contemporary economic development that relies on fossil fuel use and industrial output (Haas, 2001). In recent years, the natural environment has undergone changes, becoming more vulnerable, delicate, and exceedingly intricate. Humanity carries a profound responsibility to protect the natural environment (Butnariu, 2018).

Likewise, the genesis of environmental contamination frequently lies in the introduction of harmful materials, such as sewage, industrial discharges, agricultural runoff, and electronic waste into aquatic environments; toxic materials, particulate matter, and gaseous pollutants into the air; along with actions that contribute to soil contamination, including unlawful dumping, mining, deforestation, and landfills (Ukaogo et a., l2020).

Soil pollution apart from earthquakes, erosion, and other natural disasters that tend to damage the soil, centuries of anthropogenic activities have given rise to widespread issues related soil pollution around the globe (Koul,2018). The main human caused sources are the chemicals used in or produced as a by-product of industrial, domestic and municipal wastes including crude oil products (Rodríguez et al., 2018).

Fossil fuels from electric-generating plants, petrochemical plants and petroleum refineries also endorse soil pollution (Ukaogo et al, 2020). Entry of pollutants either directly or indirectly has been proved to contaminate wide areas of groundwater bodies and soil resources influencing crop production likewise human and animal health through food contamination and the environment in general (Saha et al, 2017).

Crude Oil contains many different kinds of hydrocarbons, the most prevalent ones are paraffin, naphthene as well as arenes, additionally, a proportion of asphalt compounds. These hydrocarbons have different properties and uses such as lubricants, fuel and chemical feedstock. In order to obtain these products crude oil, undergo process which involve refining, petroleum exploration, and distribution through road transport that consequently led to soil contamination (Ekperi et al., 2021). The above-mentioned situation demands prompt attention and resolution. Although cleaning up terrestrial and aquatic ecosystems with conventional techniques considered efficient, however, a high cost with a specialized staff and equipment are needed. Bioremediation and phytoremediation proven to be an eco-friendly and a cost-efficient solution (Hussein et al, 2022). Furthermore, phytoremediation is one of the rehabilitation techniques relied on for the sites contaminated with total petroleum hydrocarbons (TPH), by utilizing green plants with the aim of restoring the biological, chemical and physical features of the environment demolished by anthropogenic activities. Nowadays, a wide range of plants identified for Phyto-remediating the area polluted with petroleum hydrocarbons (Masu et al, 2016) due to their ability to absorb, accumulate and detoxify pollutants from the soil (Ayuba et al, 2020). Many studies have been conducted on the cleaning of crude oil polluted soils with phytoremediation using different plant species (Wang et al., 2008, Moubasher et al., 2015; Xiao et al., 2015, Tang & Angela 2019). Nevertheless, limited number of studies have investigated the use of trees for remediation (Ayuba et al., 2020; Oyedeji et al., 2022; Abdallah et al., 2023). In spite the fact that Iraq is one of the largest crude oil producers in the world that transport, produce and refine oil, few studies on trees have been reported (Al-Obaidy et al., 2016., Al-Obaidy et al., 2018, and Al-Obaidy et al., 2019). This study examined to investigate the effect of crude oil contamination on soil properties and plant growth parameters, also to test the effectiveness of Catalpa bignonioides tree in phytoremediation of crude oil-contaminated soil, measured by the reduction of total petroleum hydrocarbons (TPH) and polycyclic aromatic hydrocarbons (PAH) in soil samples, as part of the broader environmental pollution concerns already highlighted in previous studies in the Duhok region (Khaled et al., 2023).

2. MATERIALS AND METHODS

Tree Description :

Catalpa bignonioides is known as Indian Bean Tree, Cigar Tree or Southern Catalpa. It’s a deciduous tree of medium-sized, growing 15–18 meters tall, with a trunk reaching 1 meter diameter. The tree characterized with its brown to gray bark, evolving into sturdy plates or ridges as it matures (Keeler, 2005). Additionally, it has a short thick trunk which form a broad and irregular head. While, the roots are fibrous, fleshy and poisonous. The foliage is of vibrant green, large sized with a heart shaped form (Sun et al, 2024).

Catalpa trees endure broad range of soil conditions, encompassing acidic to alkaline ranges with wet clay soils, sandy terrain, loamy compositions and more. Good drainage is constantly preferred; however, Catalpa can subsist both flooding and further drought periods (Keeler, 2005).

Study Area:

This study was carried out in the lath house of the College of Agricultural Engineering Sciences within Duhok city area which is situated 430-540m above sea level in the north of Kurdistan region (N 36˚ 52ʹ 03ʺ E 42˚ 59ʹ 34ʺ) (Mohammed, 2013). The ongoing study was conducted during February and persisted further till December of the year 2023.

Crude Oil Used in the Study:

The crude oil used in the experiment was obtained from local oil refineries in Kwashe industrial area in Duhok city, the Compositional Analysis Stabilized Oil by Gas Chromatography Technique Based On ASTM D5307 showed the following hydrocarbon in the crude oil used in the study; Propane (N-C3), iso-Butane (N-C4), n- Butane (N-C4), iso-Pentane (N-C5), n-Pentane (N-C5), Hexanes (N-C6), Heptanes (N-C7), Octanes (N-C8), Nonanes (N-C9), Decanes (N-C10), Undecanes (N-C11), Dodecanes (N-C12), Tridecanes (N-C13), Tetradecanes (N-C14), Pentadecanes (N-C15), Hexadecanes (N-C16), Heptadecanes (N-C17), Octadecanes (N-C18), Nonadecanes (N-C19), Eicosanes (N-C20), Henicosanes (N-C21), Docosanes (N-C22), Tricosanes (N-C23), Tetracosanes (N-C24), Pentacosanes (N-C25), Hexacosanes (N-C27), Heptacosanes (N-C27), Octacosanes (N-C28), Nonacosanes (N-C29), Triacontanes (N-C30), Hentriacontanes (N-C31), Dotriacontanes (N-C32), Tritriacontanes (N-C34), Tetratriacontanes(N-C34), Pentatriacontanes(N-C35), and Hexatriacontanes (N-C36)

Soil Preparation :

Soil was collected from the fields of the College of Agricultural Engineering Sciences. Physical and chemical properties of soil and water used in the study are represented in the tables 1 and 2 respectively.

Crude oil was incorporated into the air dried soil at concentrations of 0%, 1%, and 2% (weight: weight), ensuring uniform mixing. Four kilograms of the contaminated soil were placed into pots, which were then set up at the experimental site. The soil was irrigated for two weeks to stabilize before planting. Uniform one- and two-year-old Catalpa seedlings were sourced from the Directorate of Nurseries in Duhok. Careful handling ensured minimal root damage as seedlings were removed from pots. Their roots were rinsed with water to clear soil residues and immediately planted into the experimental pots. Watering was conducted as needed to support seedling establishment.

Seedlings Selection:

Catalpa tree species was chosen for the current study. The seedlings of both ages one and two years old were selected and transferred from Malta plants nursery to the study site. The initial and final height measurements were taken for both before planting. The seedlings were planted in April 2023, in a polyethylene bags (each of 4 kg of soil). Throughout the entire experiment duration, the seedlings were consistently watered with well water.

Design of the Experiment :

The Randomized Completely Block Design (RCBD) was followed during the current study. Catalpa seedlings species of the two different ages (one- and two-years old), with three crude oil concentrations (0%, 1% and 2%), three replicates and four plants within each experimental unit.

Accordingly, the experimental unit’s number = 1 plant X 2 ages X 3 concentration X 3 replicates = 18 experimental units X 4 plants in each experimental unit = 72 seedlings

Measurement of Seedling Height Increase :

At the beginning and end of the study, the height of each seedling was measured from the soil surface to the apex using tape. The increase in seedlings’ height was computed by subtracting the initial measurement from the final one (Figure 3).

Measurement of Chlorophyll A, Chlorophyll B and Total Chlorophyll:

Before plants rooting up Chlorophyll a, b, and total chlorophyll (mg/g fresh weight) were measured by extracting one gram of leaves in 98% ethanol, measuring absorbance at 645 nm and 665 nm, calculating concentrations using formulas from Wintermans and DeMots (1965).

Chlorophyll a:

mg chl. a/ml solution = (13.7) (A 665 nm) – (5.76) (A 645 nm)

Chlorophyll b:

mg chl. b/ml solution = (25.8) (A 645 nm) – (7.6) (A 665 nm)

Total chlorophyll:

Total chlorophyll = (chlorophyll a) + (chlorophyll b)

Root Biomass (Seedling Uprooting)

After 8 months of experimental study exactly in 2022, November all seedlings were rooted up. Each plant was delicately uprooted and the shoot system was separated from the root system. Soil attached to the roots was meticulously cleared, and both shoot and root systems were washed individually. Subsequently, they were left to air dry 24 to 48 hours. After the drying period, the plants were 72 hours oven dried at 70°C. Finally, each experimental unit was individually placed in paper bags.

Analysis of Soil Samples:

Soil samples underwent a natural air-drying process at room temperature. While for those retaining moisture an overnight oven treatment at 60 °C was applied. Subsequently all soil samples were crushed and sieved using a mesh with 2-mm aperture size and further ground into a fine powder which was then preserved in sanitized jars (for digestion). EC, pH, chlorophyll content, shoot and root biomass and removed oil were analyzed at Research Center of Duhok Agricultural Engineering Science College. While, OM and NPK samples were analyzed at College of Agriculture and Forestry, University of Mosul.

Soil Electrical Conductivity (EC, µs/Cm)

The electrical conductivity of the studied soil was measured in soil extracts of (1:2.5) utilizing EC meter model (Microprocessor Conductivity/TDS meter HANNA-HI-9635) as performed by (Rowell, 2014).

Organic Matter Content :

The soil (O.M) content was determined during this research, as documented by Walkley and Black technique using K2Cr2O7 (1N) according to (Allison, 1965).

O𝐫𝐠𝐚𝐧𝐢𝐜 𝐦𝐚𝐭𝐭𝐞𝐫 (%) = 𝐎𝐂 (%) × 𝟏. 𝟕𝟐

Determination of Ph:

ESMART Pen Type Water Quality Meter was used to measure the pH of all the soil samples. The soil pH of extracts (1:2) was determined, utilizing pH meter model (HI 9023) as described by (Jackson, 1958).

Digestion of Shoots and Soil Samples :

Shoots and Soil samples were digested following the guidelines outlined by Tandon in 1999.

Determination of Total Nitrogen in Shoots and Soil:

The total Nitrogen was determined by classic Microkjelhdahls, following the procedure of (Ryan & Astafan, 2003).

N%=V.HCl×N.HCl×14.00710001005500.5

Determination of Potassium in Shoots and Soil :

By following Flame-phtometer JENWAY PFP 7, and the equation below:

ppm=sample result1.12350100010000.5

P%=ppm/10000

Determination of Total Phosphate in Shoots and Soil:

The Phosphate in shoots and soil samples were detected by colorimetric method, 6705 UV/Vis. Spectrophotometer. Based on the procedure of (Ryan & Astafan, 2003) with using the following formula:

ppm=ABs0.077×50×50510.5

P%=ppm/10000

Determination of Total Nitrogen % in Plant:

Soil Nitrogen determined by Kjeldal apparatus according to the steps previously applied by (AOAC., 2004)

Determination of Phosphorus % in plant:

Phosphate was determined by using colorimetric methods based on the method of (AOAC., 2004)

Determination of Potassium in Plant:

Potassium in leaves was Measured using the flame photometer method at 589 nm wavelength. (AOAC., 2004).

Fraction Analysis of Total Petroleum Hydrocarbon in Soil by: GC-Gas Chromatography

The determination of Total Petroleum Hydrocarbons fraction was conducted using an Agilent 7890A Gas Chromatograph coupled with a Flame Ionization Detector (GC-FID). The system utilized an HP-5 capillary column (30 m × 0.32 mm × 0.25 µm), operating with a detector temperature of 350 °C, an inlet temperature of 320 °C, and a carrier gas flow rate set at 1 mL/min. Samples were initially stored at 4 °C, then dried using anhydrous sodium sulfate (Na₂SO₄) to remove moisture. Extraction of Total Petroleum Hydrocarbons was achieved using a combination of hexane and acetone as solvents. Prior to GC-FID analysis, samples underwent fractionation and cleanup into aromatic and aliphatic components using silica gel adsorbents. This approach is consistent with contemporary methods for analyzing TPHs in environmental matrices (TPHCWG, 1998).

Percentage of Petroleum Degradation :

Percent of petroleum decomposition in the soil was evaluated using an updated weight loss protocol (Li et al., 2021). A 10 g soil sample contaminated with crude oil was extracted with 20 ml dichloromethane (DCM) in a container of known weight. The mixture was gently agitated to enhance oil extraction and then allowed to sit at room temperature for 24 hours to ensure full solvent evaporation. The container's weight with the residual oil was measured, and the percentage of oil degradation was calculated using the formula:

Analysis of Data:

The data were analysed by using Microsoft Statistical Analysis System (SAS) 2002. The variations among the treatment means and ANOVA (Analysis of variance) were tested with Duncan Multiple Range Test at 0.05 probability level (Duncan, 1955).

3. RESULTS AND DISCUSSION

Water Parameters:

Irrigation water parameters in table 1; The measured pH of the water is 7.4; the value indicates a slightly alkaline condition which, according to WHO guidelines falls within the optimal range for agricultural use, most freshwater organisms, and human consumption (WHO, 2017). The electrical conductivity (EC) of 892.7 µS/cm indicates moderate salinity suitable for various daily activities. This result aligns with WHO standards (<1000 µS/cm) and poses minimal risk to crops and soil health (Ayers & Westcot, 1985 and WHO, 2017). The total dissolved solids (TDS) value of 450.7 mg/L falls within the acceptable range for necessary daily uses, as recommended by WHO with a maximum limit of 1000 mg/L for palatability (WHO, 2017). The total alkalinity of 386.0 mg/l implies a high buffering capacity, supporting irrigation and aquatic life while stabilizing pH by controlling acidity-related stress (Chapman, 1996). Lastly, the total hardness of 480.4 mg/l reflects a high concentration of magnesium and calcium ions, classifying it as hard water. This hardness positively contributes to plant nutrition by providing essential minerals (Ayers & Westcot, 1985).

Soil Ph, Electrical Conductivity and Organic Matter :

Soil pH is vital for plant health as it controls nutrient availability, microbial activity, and prevents element toxicity, with most nutrients being accessible at a pH range of 6.0–7.5. (Wang et al., 2013). From the interaction involving two years old seedlings, soil pH exhibited minimal variation the with the highest value of 7.958 at 0% crude oil and the lowest of 7.722 at 1% oil concentration illustrated in table 3. These minor differences reflect the limited influence of crude oil on soil alkalinity (Brown & Lee, 2020). This study supports evidence from previous observations of Barua et al. (2011), who demonstrated that contaminated soils are modestly more acidic as a result of the formation of toxic acids in the oil spill.

Electrical Conductivity (EC) is a measure of a soil's capacity to conduct electrical current, it is directly connected to the soluble salts’ concentration in soil. From the interaction regarding EC, higher values demonstrate elevated salinity, which can be due to crude oil contamination (Chukwu, et al., 2022). EC values varied across treatments, with the highest EC values (840.667 µS/cm and 836.333 µS/cm) observed under 2% and 1% crude oil treatments, respectively. The lowest EC value (752.000 µS/cm) was found in two-year-old seedlings under 0% crude oil as appeared in table 3. These discrepancies indicate that crude oil contamination exacerbates salinity or ion concentration in the soil environment, particularly affecting younger seedlings (Fu et al., 2022). The results of other research has reported reductions in EC under crude oil contamination, suggesting that hydrocarbon coatings might limit ion exchange in certain soils (Saikia et al., 2023). These variations indicate that while increasing crude oil levels generally raise EC, the extent of this effect depends on soil type and specific contamination characteristics.

Organic matter (OM) content peaked in two-year-old seedlings (2.123) and was lowest in one-year-old seedlings (1.613). Crude oil contamination appeared to slightly increase OM through the accumulation of decomposed hydrocarbons and organic debris in the topsoil. The more extensive root systems in older seedlings may correlate with higher organic contributions and microbial activity, contributing to elevated OM levels (Fageria and Moreira 2011).

Degradation Percentage of Petroleum Hydrocarbons in Soil:

Petroleum hydrocarbons residues in soil determinate after 8 months, and percentages degradation was calculated as compared with initial crude oil concentration 1 and 2% (Table 3). According to the data, we observe that the phytoremediation process by Catalpa plants degraded most of the crude oil, with a percentage degradation range of 71.131-84.040 %. This range is comparable to what researcher (Abdallah et al., 2023) found when he planted seedlings of Acacia sieberiana Tausch in soils polluted with 0.5, 1, 1.5, and 2% crude oil; he recorded 49-79% oil degradation after 3 months. Both the current results and those of other researchers highlight the potential of using trees for soil remediation in oil-contaminated areas. Additionally, other plants are used for crude oil polluted soils phytoremediation such as cotton that removed 50.66% of the TPHs from soil polluted with 5% crude oil after 5 months (Al-Obaidy, 2018). Furthermore, Cyperus laxus Lam has been utilized in phytoremediation of soil contained with 6 % hydrocarbons and 55 % hydrocarbons removed after 6 months (Escalante-Espinosa et al., 2005)

There were no significant differences between one- and two-year-old seedlings in the process of crude oil phytoremediation from the soil. The higher removal rates are possibly linked to increased microbial activity in the rhizosphere, as root exudates enhance hydrocarbon breakdown. This highlights the role of plant-microbe interactions in oil remediation, influenced by localized soil texture, aeration, and nutrient availability around the seedlings (Lee and Park, 2019 and Brown et al., 2020).

Table 3: Effects of seedling age, crude oil contamination and their interaction on pH, EC, OM and oil percentage degradation.

Treatments		pH	EC (µS/cm)	OM %	Oil degradation%
Seedling age	1-yr	7.844a	817.222a	1.871111 a	77.956
Seedling age	2-yr	7.805a	797.444a	1.907778 a	75.350
Crude oil conc.	0 %	7.923a	763.333b	1.748333 c	Why there is not value here
	1%	7.794ab	826.167a	1.865 b	82.175
	2%	7.758b	832.500a	2.055 a	71.131
Age X Oil conc.	1-yr X 0 %	7.888ab	774.667bc	1.753333 c	Why there is not value here
	1-yr X 1 %	7.866ab	836.333a	1.873333 ab	84.02
	1-yr X 2 %	7.778ab	840.667a	1.986667 ab	71.895
	2-yr X 0%	7.958a	752.000c	1.743333 c	Why there is not value here
	2-yr X 1%	7.722b	816.000ab	1.856667 ab	80.333
	2-yr X 2 %	7.737b	824.333a	2.123333 a	70.367

For each factor means with same letters are not significantly different at 5% level based on Duncan’s multiple range Test.

Table 4: Effect of crude oil contamination on N, P, and K (%) in soil and Catalpa shoots

Treatments		N in shoots	N in soil	P in shoots	P in soil	K in shoots	K in soil
Seedling age	1 yr	1.592a	0.0062a	0.228a	0.00072b	0.609a	12.082a
Seedling age	2 yr	1.496b	0.0057b	0.221a	0.00079a	0.477b	12.159a
Crude oil conc.	0 %	1.453b	0.0056b	0.223a	0.00072b	0.540a	11.956a
	1%	1.597a	0.0057b	0.221a	0.00074b	0.544a	11.586a
	2%	1.582a	0.0066a	0.229a	0.00079a	0.546a	12.818a
Age X Oil conc.	1yr X 0 %	1.380e	0.0059c	0.204a	0.00063d	0.550c	11.884ab
	1yr X 1 %	1.700a	0.0058c	0.242a	0.00068cd	0.612b	10.695b
	1yr X 2 %	1.697a	0.0068a	0.236a	0.00084a	0.665a	13.666a
	2yr X 0%	1.527b	0.0054d	0.241a	0.00081ab	0.530c	12.029ab
	2yr X 1%	1.493c	0.0055d	0.200a	0.00081ab	0.476d	12.478ab
	2yr X 2 %	1.467d	0.006b	0.221a	0.00074bc	0.426e	11.970ab

For each factor means with same letters are not significantly different at 5% level based on Duncan’s multiple range Test.

CONCLUSION

Depending on the results of this study, it was concluded that Catalpa bignonioides seedlings can be grown in soil polluted with crude oil and can tolerate oil concentration up to 2%. Additionally, there was 84.02 % loss of TPH at the end of the eighth month. The results revealed that soil acidity decreased with crude oil contaminated, on the other hand, electrical conductivity and organic matter increased. Crude oil treatment of the soil resulted in a significant increase in total nitrogen concentration, while no significant changes were observed for phosphorus (P) and potassium (K). Moreover, two-year-old seedlings showed better growth performance and higher nutrient uptake than one-year-old seedlings. The species also contributed to the complete removal of light hydrocarbon fractions (C3–C14), indicating its efficiency in degrading low-molecular-weight hydrocarbons. Based on the obtained results, it can be concluded that Catalpa begnonoides is suitable for phytoremediation as a phytoextraction technique of hydrocarbon-contaminated soil.

Acknowledgments:

The authors extend their sincere gratitude to the College of Agricultural Engineering Sciences at the University of Duhok for providing the necessary facilities and support for this research. Special thanks to the staff of the research laboratories and the Directorate of Nurseries in Duhok for their assistance in seedling provision and analysis.

Author Contributions:

All authors contributed equally to the conception, design, experimentation, data analysis, and writing of this manuscript. All authors have read and approved the final version of the manuscript.

Declaration of Competing Interest:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Funding:

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Water parameters	Values	Units
pH	7.40
Electrical conductivity	892.7	(µs.cm^-1)
Total dissolved Solids	450.7	mg. l^-1
Total alkalinity	386.0	mg. l^-1
Total hardness	480.4	mg. l^-1

Soil parameters	Values	Units
Clay	41.3	%
Silt	20.5	%
Sand	38.2	%
Texture	Sandy clay loam	---
pH	7.88	---
EC	790	µs.cm^-1
Total CaCO₃	16.43	%
Total organic matter	1.13	%
Total nitrogen	0.0053	mg/kg
Total phosphorus	0.00081	mg/kg
Total potassium	12.34	mg/kg