IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 The Impact of Ascending Levels of Crude Oil Pollution on Growth of Olive ( Olea europaea Linn) Seedlings B. A. A . Ali , H . H. Ali and G . A. Shaker Iraq natural History Museum and Research Centre , University of Baghdad Abstract A study on the impact of ascending levels of crude oil on the growth of transplanted seedlings ( March2005 ) of Olive (Olea europaea Linn) was carried out at the experimental area of Iraq Natural History Museum and Research centre / Baghdad University (Bab-Al-Madham –Baghdad) grown under field condition and continued till April 2008.The experiment was laid out in complete randomized design ( CRD ) with five levels of pollution (0.0 , 0.5 , 1.0 , 2.0 and 3.0 liter / seedling ) poured at the soil surface , each seedling represented one replicate and was replicated four times . Data collected from the experiment were visual symptoms , percents of seedlings death, plant height and total dry weight of harvested plants. The results indicated that crude oil pollution caused an adverse effect on the Olive (Olea europaea Linn). The crude oil led to leaf chlorosis , dryness and death of seedlings. The adverse effects were proportional with the levels of pollution. Plant heights and total dry weights were significantly reduced (p< 0.05) as a consequence of pollution. Results of the study indicated that the effect of pollution clearly appeared after three weeks at the high levels then the symptoms extended to lower levels during the first six months. The study showed that treated plants with low levels of crude oil pollution were less growth but survived and stayed alive, while the higher levels led to seedlings death. It was obvious that the adversely affected plants could not be able to recover even after 27 months after pollution, the time of the experiment. Introduction Olive tree (Olea europaea Linn) , is a small tree , mostly not exceeding 6 t0 9 m in height , and bearing small lanceolate leaves and axillaries flowers oblong. While the production of oil is the main aim of planting this species ; a specific orchard management should be applied to maintain acceptable levels of productivity, oil quality , and cost of production (1). It was introduced to Iraq since a long time ago and extensively cultivated in different parts of Iraq( 2 ) especially in the upper parts of the country. The tree is considered as tolerant for drought, and for nitrogen deficiency (3 ).Recently the Iraqi Ministry of Agriculture considers the tree as one of the important economic plants and adopted many projects to expand planting areas and encouraged farmers to grow the tree in their private plantations. Soil protection and remediation of oil polluted soil sites became an important part of the environmental protection policy in many oil producing countries. Research in preventing and recuperating oil pollution became an integral part of petroleum industry activities because the crude oil has considered as the most lasting and persistent environmental pollutants affecting the soil ecosystems. Crude oil pollution is the most frequent one in Iraq than in any other producing countries due to the wars , fighting and unnatural security conditions , where pipe lines , oil transporting trailers and tanks were mostly exposed to attack by explosions causing an intensive crude oil - soil pollution . IBN AL- HAITHAM J. FO R PURE & APPL. SC I VOL.22 (3) 2009 Plants respond differently to pollution depending on many factors such as type and level of pollutant , age and size of the plant and Its inherent characteristic, in addition to environmental conditions under which the plant is grown. Oil pollution has adversely effects on different p lant species ( 4) ,( 5) , (6) and (7) referred to crude oil as phytotoxic for many plants and considered the poor growth of oil treated plants as attributed to suffocation of plants caused by exclusion of air by the oil or exhaustion of oxygen by the increased microbial activity and toxicity from some oil compounds . In spite of the adverse effects of oil pollution on the plants, no much works were done in Iraq and due to the shortage of the available literatures related to this subject current study had been done and aimed to investigate the performance of Olive tree (Olea europaea ) growth under different levels of crude oil pollution . Materials and Methods Fifty seedlings of Olive (Olea europaea ) nearly 30-40 cm tall and free of insects or dieses infections obtained from a local nursery and were transplanted in a clay soil with distances of one by one meter between seedlings at the experimental area of the Iraq Natural History Research Centre & M useum / Baghdad University on May of 2005 . Twenty healthy seedlings and nearly equally height of them were selected on December 2005 . Five levels of Basrah light crude oil obtained from Iraqi Oil Ministry used ( 0.0 (L0) , 0.5 ( L1), 1.0 (L2) , 2.0 (L3) and 3.0 (L4) liter of oil / seedlings) were distributed randomly on the selected seedlings and each level was repeated four times .Soil Crude oil pollution was simulated by pouring the crude oil over the surface soil and inorder to ensure that the amounts in all treatment were distributed over the same area, a ring of a plastic tube with diameter of 30 cm was made , and was put on the soil to surround the seedling, then the crude oil was distributed equally and evenly inside the ring. Resulted trees were harvested on April 2008. The impact of the seedling as affected by crude oil was studied depending on the visual symptoms and observations appeared on the plants, percents of death seedlings( percent of death seedling to the 4 replications of each treatment) were collected during the experiment time and on the measurements of the heights and total dry weight of harvested trees. Complete randomized design (CRD) was used as an experimental design and the results were were statistically analyzed by using Statistica program (99 edition)(8). Results and Discussion The treated surface soils were turned immediately to oily with brown color after adding the crude oil. The resulted color was proportional with oil level used. The surface soil of L1 , L2 and L3 was able to absorb the crude oil amounts directly after the addition , while soil of L4 took more time to absorb and became saturated with it. The intensity of the brown color of the surface soils started to the reduce gradually with in time and this could be due to the degradation of the oil or the penetration within the soil. The treated surface soils were able to recover to the original color during the first six months of the experiment . The consumed time to recover the original soil color was also proportional with the levels of crude oil used . All the seedlings of Olea europaea exposed to crude oil responded negatively to pollution in comparison with control treatment ( L0) Fig (1). Chlorosis (yellowing of plant leaves) was the early distinguished symptoms observed on all of the crude oil treated seedlings . Although the chlorosis was a common feature for all of the treate seedlings, their yellow color intensity was different and ranging between light to intensive depending on levels of pollution being used . The chlorosis was firstly observed in some of the old leaves and then developed with in time to speared all over the plant and eventually led to the dryness and death of seedlings in some cases especially in the higher levels of pollution ( L3 and IBN AL- HAITHAM J. FO R PURE & APPL. SC I VOL.22 (3) 2009 L4). In sp ite of no obvious cases of infection were observed during the time of the experiment, it was possible to ensure that the dryness and death followed the chlorosis were due to pollution and not for other reasons and therefore, put the dead seedlings under a close visual and microscopic examination . First case of seedling dryness and death was recorded on L4 treatment after three weeks, and then the phenomena appeared on other treatments within the first six months after pollution. Crude oil pollution caused death to some replications of the treated seedlings and the percent of death was directly proportional to the level of pollution and ranged between 75 to 100 % Fig( 2 ). The increase of the death percentage of Olea europaea with the increase of the levels of crude oil pollution was clearly indicated to an adverse effect of oil on this species. It is well known that the visible symptoms have often provided the first indication of other problems which might be resulted from the impaired metabolism within the plant. Crude oil pollution led to different symptoms on seedling during the first six months after exposing to crude oil. Old leaves of treated seedling became pale in comparison to control and this could be due to the breakdown in chlorophyll production. Such symptom had similar aspects of nitrogen deficiency, where specific symptoms of nitrogen deficiency first appear on the oldest leaves. Plant suffering from nitrogen deficiency is pale in color in comparison to healthy one due to the breakdown in chlorophyll production. Such observations were noticed by (9) where they referred that the specific symptoms of nitrogen deficiency first appeared ( as with deficiencies of phosphorus and potassium) on the oldest leaves, and these become paler then turned to chlorosis ( M arked yellowing) . It is well known that crude oil pollution leads to increase soil organic carbon as well as the populations of total heterotrophic microorganisms and also, a reduction in nitrate-nitrogen and available phosphorus (10).The shortage of nutrients availability ,especially the nitrogen as a result of the increase of the total soil heterotrophic could be expected according to the current study since no nitrogen fertilizer was added. For this reason, the nitrogen deficiency could be as one cause of the chlorosis and can not be rolled out. The adverse effects of crude on Olea europaea trees continued for more than 27 months after pollution . No more death happened for trees after the first six months but the survived trees from crude oil pollution possessed weaker growth in comparison with control as reflected by trees height and total dry weight .The mean height for control trees was 203.7 cm , but due to pollution it was reduced to 79.5 , 36.5 , 14.7 , 0.0 cm for L1, L2, L3 and L4 respectively table (1). Total dry weigh also was reduced from 675.5 to 195.87, 30.62 , 12.25 , 0.0 gm for the same three levels Table (2). Current results clearly refer to the adverse effects of crude oil on plants growth and were in an agreement with to the results of (4),(5),(7), (11), (12), and (13) . Since uptake of water and nutrients is carried out by the root system, untreated seedlings of Olea europaea with sound, healthy roots grew normally while the treated seedlings could suffer from anatomical aberration that may caused by presence of oil films in a specific parts of plant leading to a negative effect on the growth. In anatomical study (14) noticed the presence of oil films in the epidermal and cortical regions of root, stem and leaves of the plant . The increase of death percent of Olea europaea seedlings with increasing of pollution as occurred in the current study, could be due to one or a combination of following factors; disruption of root-soil-water relationship , direct impact on plant metabolic processes , toxicity of living cells, reduced oxygen exchange between the atmosphere and soil , nutrients immobilization and an alteration in the physical, chemical and biological properties of soil. Such factors could have observed visual symptoms , reduction in biomass or deaths of seedlings. From the foregoing, it is clear that crude oil may affect plant by causing direct death due to coating the roots and soil particles by it or through the affect of toxic components, where the oil or its components interfere with cellular processes, resulting the plant death . Conclusion Some conclusions could be mentioned according to this experiment ; (a): Crude oil application to soil IBN AL- HAITHAM J. FO R PURE & APPL. SC I VOL.22 (3) 2009 has adverse effects on the growth of Olea europaea seedlings ,(b): The effect being proportional to the concentration of crude oil is applied, (c): Oil may affect the plants by causing direct death in high levels of pollution , (d): In some levels of pollution olive plants may undergo a weakness in growth and reduction of biomass producing a stunted plants, and (e) the effect of pollution by crude oil on Olea europaea may last more than 27 months (the period of the experiment). Acknowledgements The authors are grateful to Iraqi ministry of oil and the management of Dorra refinery for providing the Basroh crude oil that was used in the current study. References 1-Michelakis,N.(2002).ISHS Acta Horticulturae. 586 :239-24 http://www.actahort.org/books/586/586_45.htm 2 - Chakravarty , H.I. 1976. Plant wealth of Iraq. Vol (1), Botany Directorate, Ministry of Agriculture and Agrarian Reform, Baghdad, Iraq. 3 - Fernández-Escobar ,R., Sÿnchez-Zamora ,M.A., Uceda, M. and Beltr ,G. (2002).ISHS Acta Horticulturae 586: 158-163. http://www.actahort.org/members/showpdf?booknrarnr=586_88 4 - Agbogidi, O.M , Onosode,A.T and Ofuoku,A.U. (2006). European. J. of Scientific Research, 13 (2). :165-171. 5 - Akaninwor, J.O. , Ayeleso, A.O. and Monago , C.C ( 2007). Scientific Research and Essay. 2(4): 127-131. 6 - Ogbonna,D.N , Iwegbue,C.M.A , T.G. Sokari and Akoko, I.O.( 2007). Earth and Environmental Science. 27( 2) :303-309. 7 -Udo,E.J. and Feyemi,A.A.A. (1975). 4:537-540. 8-Statistica for windows "99 Edition" , (1999) . Kernel release 5.5 A , StatSoft,Inc . 9 - Snowball, K. and Robson , A.D (1983) .Symptoms of nutrient deficiency . Department of soil science and plant nutrition, Institute of Agriculture, University of Western Australia. 10- Okolo, J.C. , Amadi, E.N and Odu, C.T.I (2005). Applied ecology and environmental research 3 (1) : 47-53. 11 - Issoufi, I , Rhykerd, R.L and Smiciklas, K.D (2006). J Agron Crop Sci, 192 ( 4 ):310-317. 12-Baek, Kyung-Hwa , Kim, Hee-Sik , Hee-Mock, Oh , Yoon, Byung-Dae,Yoon , Kim,Jaisoo and Lee, In-Sook. (2004). J.of Environ sci and Health. A39 (9 ): 2465-2472. 13 - Gill,L.S. , Nyawuame,H.G.K. and Ehihametelor, A.G. (1992). Newsletter 5:46-50. 14 - Anoliefo, G.O. (1991). Forcados blend oil effect in respiration,metabolism, elemental composition and growth of Citrullus vulgaris (Schard).Ph.D. Thesis Benn.pp. 6-30. In Gill,L.S. , Nyawuame,H.G.K. and Ehihametelor, A.G. (1992). IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 Table (1): Plant height (cm) at the end of the experiment as influenced by five levels of crude oil 0.0 refer to dead plant. Means with different superscripts are significantly different (LSD) at p< 0 Table( 2): Plant dry weight ( gm) at the end of the experiment as influenced by Five levels of crude oil in soil Mean Dry Weight (gm) Replications Treatments (Levels o f soil pollution) 4th 3d 2nd 1St 675.5 a 195.87 b 30.62 b 12.25 b 0.00 b 661 0.0 0.0 49.0 0.00 686 0.0 61.25 0.00 0.00 980 416.5 61.25 0.00 0.00 735.0 367.0 0.00 0.00 0.00 L0 L1 L2 L3 L4 0.0 refer to dead plant . Means with different superscripts are significantly different (LSD) at p< 0.05 Plant Height (cm) Replications Treatments Mean 4th 3d 2nd 1St Levels of soil pollution 203.7a 79.5b 36.5 b 14.7 b 0.0 b 186.0 0.0 0.0 59.0 0.0 192.0 0.0 52.0 0.0 0.0 228.0 166.0 94.0 0.0 0.0 0 .209 152.0 0.0 0.0 0.0 L0 L1 L2 L3 L4 IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (3) 2009 Fig (1): Effect of five levels of crude oil on the growth of Olive seedling af ter six month from pollution Fig (2): Effect of five levels of crude oil pollution on the percent of death of olive plan L0 L1 L2 L3 L4 100 75 50 50 Levels of crude oil Percent of Death (%) 2009) 3( 22المجلد للعلوم الصرفة والتطبیقیة مجلة ابن الھیثم Olea europaea نمو شتالت في النفط الخام ب التلوث من متزایدةتأثیر مستویات الزیتون شاكر عبد الوهاب حسن حسین علي و كونر،باسم عباس عبد علي د ،مركز بحوث و متحف التاریخ الطبیعي جامعة بغدا الخالصة في المساحة التجربیة في 2005في اذار نمو شتالت الزیتون المزروعة في مختلفة من النفط الخام مستویاتتأثیر درس الى 2005من آذار المدة تحت الظروف الحقلیة) بغداد –باب المعظم (جامعة بغداد /مركز بحوث و متحف التاریخ الطبیعي ط الخام وهي فصممت التجربة الحقلیة على التصمیم العشوائي الكامل باستخدام خمسة مستویات من الن. 2008نیسان كل شتلة اذ ،أربعة مكررات عمالباست و اضیفت على سطح التربة )الشتلة / لتر نفط 3.0و 2.0، 1.0، 0.5 ، 0.0( ة الو ،عراض المرئیة التي ظهرت على النباتاتاأل ت تسجیل من التجربة شملالتي جمعت البیانات . مثلت مكرر نسبة المئوی كلي للنباتات ،و أطوال النباتات ،لموت النباتات أشارت النتائج إلى أن التلوث النفطي أدى إلى . بعد الحصاد و الوزن الجاف ال االصفرار على أعراضطي إلى ظهور فالتلوث الن أدى .ر سلبیة على نباتات الزیتون تناسبت مع مستویات التلوثاثآظهور نتیجة 0.05على مستوى احتمال ائیااحص الجافة هاأوزان النباتات و أطوال انخفضت . احیانا و الجفاف ثم موت النبات األوراق تلوث ظهر واضحا بعد ثالثة اسابیع الدراسة الى اننتائج اشارت . لتلوث النفطيل م فياثر ال المستوى االعلى من التلوث ث ت البحث ت نتائجكما أظهر .المستویات االدنى وذلك خالل االشهر الستة االولى امتد ظهور االعراض الىبعدها ان النباتا نجو ولكن و اً نمو كانت اقل القلیلة من النفط الخام مستویات الالمعاملة ب وث ها استطاعت ان ت تبقى حیة في حین ادى التل كان واضحا ان النباتات التي تأثرت سلبیا بالتلوث استمر معها التأثیر السلبي ولم دوق. الى موت الشتالتبالمستویات العالیة .استمرار التجربةمدة ، شهرا من بدء التلوث النفطي 27تتعاف حتى بعد مرور