British Phycological APPLIED PHYCOLOGY Society 2023, VOL. 4, NO. 1, 54–77 Understanding and using algae https://doi.org/10.1080/26388081.2023.2185815 Raymond Sunday Ezenweani and Medina Omo Kadiri Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria ABSTRACT ARTICLE HISTORY Received 16 August 2022 Petroleum pollution can upset the ecological stability and health of a marine ecosystem. The Accepted 3 February 2023 physiological, biochemical and morphological responses of Nannochloropsis oculata and Porphyridium cruentum to three different petroleum fuels, kerosene, diesel and gasoline were KEYWORDS examined. The effect of water soluble fractions (WSFs) of the three petroleum fuels was investi- Environment; marine algae; gated at 0%, 25%, 50% and 100%. The growth response of both species was monitored optically pollution; enzymes; bio- every two days for 14 days using a 721 visible spectrophotometer. Chlorophyll a, morphology and response; petroleum antioxidant enzyme activity of the algae were examined using prescribed methods. In both algae, minimum growth was obtained with 100% WSF of the petroleum fuels. In N. oculata, there was growth stimulation and the maximum growth was obtained at different concentrations (25% and 50%) depending on the test fuels. The maximum growth of P. cruentum was obtained at 10% WSF in all the fuels. ANOVA (p < 0.05) showed significant differences in algal growth with changes in concentration of the test fuels. Unpaired t-tests showed that in all the fuels, there was a significant difference (p < 0.05) between the growth of N. oculata and P. cruentum. N. oculata showed more tolerance to petroleum fuel pollution than P. cruentum. Morphological studies showed that petroleum fuel pollution altered the size of N. oculata and caused severe cell clumping in P. cruentum. Antioxidant concentration assessment showed that whereas N. oculata produced high levels of superoxide dismutase, catalase and peroxidase, P. cruentum produced high levels of superoxide dismutase but was less efficient in catalase and peroxidase production. Clumping and inefficiency in antioxidant production affect the physiological and biochemical response of algae. This study showed that the severity of petroleum fuel pollution is reflected in physiological, morphological, and biochemical responses of the test algae. This research provides baseline information that can be used for the evaluation of the effect of petroleum fuel pollution in marine environment and policy making. Introduction known as the water soluble fraction (WSF). Some of the oil is oxidized, some undergoes bacterial changes and Environmental pollution is one of the most important eventually sinks to the bottom and some is mixed with global problems (Reyes, Schueftan, Ruiz, & González, sediment with serious consequences (Martínez-Go´mez 2018), one source of which is oil spillage. Petroleum is et al., 2010). Petroleum pollution has been noted to pose a dark oily liquid formed from the remains of ancient severe ecological issues and imbalances in aquatic envir- plant and animals (mostly zoo- and phytoplankton) onments (Asif, Chen, An, & Dong, 2022); the severity under conditions of high temperature and pressure depends on the type of oil, extent and time (season and (Hsu & Robinson, 2006). It is a highly toxic mixture of weather) of the spillage, type of shoreline, and the waves different hydrocarbons distilled to make fuels and more and tides in the area of the incidence (Martínez-Gómez volatile fractions of higher economic value (Perez, et al., 2010). The volatile components are well known to Fernandez, & Beiras, 2010; Sattar et al., 2022). When affect aerial life while the dissolution of the less volatile petroleum spillage occurs in a water body it spreads as components results in emulsified water which affects a result of diffusion (Keramea, Spanoudaki, Zodiatis, aquatic organisms (Akpofure, Efere, & Ayawei, 2000). Gikas, & Sylaios, 2021), while the volatile gaseous com- Spillage of petroleum products, especially diesel and ponents evaporate into the air (Keramea, Spanoudaki, gasoline, is a problem in marine ecosystems (López- Zodiatis, Gikas, & Sylaios, 2021). Factors such as time, Rodas et al., 2009). Petroleum pollution is mostly gen- temperature and water agitation caused by waves, wind erated from anthropogenic processes (Atlas & Bragg, and current cause a part of the liquid components to 2009; Haghighat, Akhavan, Mazaheri Assadi, & Pasdar, dissolve and potentially to be absorbed by aquatic 2008), due to its daily usage in marine transportation, organisms (Lu et al., 2021). The dissolved fraction is CONTACT Raymond Sunday Ezenweani firstname.lastname@example.org © 2023 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. APPLIED PHYCOLOGY 55 but oil thefts, illegal refineries and marine transporta- Materials and methods tion also contribute to petroleum pollution in the mar- Procedure for the preparation of water soluble ine environment. When petroleum spillage occurs in fraction of petroleum fuels inland water bodies, this often drains into the marine environment and may affect ecological stability. Gasoline, kerosene and diesel were the test fuels Petroleum pollution is capable of affecting the composi- investigated in this study. The procedure of tion and sustainability of plankton and other aquatic Phaterpekar & Ansari (2000) was followed to obtain plants and animals (Sullivan & Currin, 2000). The the 100% stock solution of the WSF of each of the annual input of petroleum products into the marine fuels. The stock solution was prepared by mixing 1 environment is estimated to be between 1.1 and part of fuel to 9 parts of seawater. The mixture was 7.2 million metric tons (Unite Nations Environmental stirred using a magnetic stirrer hotplate for 24 h. Program, 2006). The actual toxic dose of petroleum The solution was allowed to stand for 24 h in hydrocarbons for plankton is closely related to the a separating funnel after which the aqueous phase amount of dissolved non-volatile components and the was separated and regarded as 100% stock solution bioavailability of the hydrocarbons (Ito et al., 2013). It is of WSF. The seawater used for the preparation of assumed that the WSF is the most environmentally media was collected from the Atlantic Ocean in harmful fraction because it is directly available for Delta State, Nigeria. It was filter-sterilized through uptake by organism (Nayar, Gohb, & Choua, 2004). 0.2 µm omnipore membrane filters (Millipore). F/2 There have been several investigations into the effects medium was added to the filter-sterilized seawater of petroleum fuels on algae (Abdul Hameed & Al in a ratio of 1:1000 ml of seawater (Kadiri & Obaidy, 2014; Fabregas, Herrero, & Veiga, 2021; Jiang Azomani, 2000). et al., 2022; Nayar, Gohb, & Choua, 2004; Perez, Table 1 shows the physico-chemical condition of Fernandez, & Beiras, 2010). Different studies have the seawater. Salinity, pH, conductivity and TDS reported both stimulatory and inhibitory effects of pet- were determined using APERA salinity metre (man- roleum pollution on algae depending on the fuel type ufactured by APERA Instruments LLC, Model num- and composition, type of algae and environmental con- ber: salt20), HANNA pH metre (manufactured by ditions. However, although most studies have been Hanna Instruments Inc., Model number: HI96107), focused on freshwater algae and inland water bodies, ATC conductivity metre (manufactured by Hanna petroleum pollution has its largest impact in marine Instrument Inc., HI98304P) and HANNA TDS ecosystems. metre (manufactured by Hanna Instrument Inc., Algae are the base of the food chain in aquatic ecosys- Model number: HI98301), respectively. tems as the primary producers (Jiang et al., 2022). The unicellular marine algae Nannochloropsis oculata and Procedure for the preparation of culture medium Porphyridium cruentum are useful as bioindicators in the study of the extent and severity of environmental pollu- F/2 medium (Guillard, 1975) was used. The F/2 tion. They are rich source of nutrients for higher organisms medium was obtained from (Algae Research and can play a major role in ecosystem stability (Chislock, Supply, Carlsbad, California, USA). The natural sea- Doster, Zitomer, & Wilson, 2013; Razaghi, Godhe, & water, which was collected from the Atlantic Ocean, Albers, 2014). This study focuses on the determination of Delta State, Nigeria was filter-sterilized through 0.2 the resilience and bioresponse of Nannochloropsis oculata µm omnipore membrane filters (Millipore) and and Porphyridium cruentum to petroleum fuel pollution, used as diluent water in the preparation of the specifically the WSF which is often readily absorbed by culture medium. One ml of f/2 medium was added phytoplankton (Kadiri & Azomani, 2000). In the aquatic to filter-sterilized seawater to make up 1 l of culture environment, oil spills may cause inhibition of plankton medium. growth, shifts in population structure, affecting the abun- dance, diversity and distribution of species (Ko & Day, Table 1. Physico-chemical condition of the seawater used for the 2004; Martínez-Gómez et al., 2010; Peeb et al., 2022), preparation WSF of the fuels and culture medium. which will in turn impact other organisms that depend Parameter Concentration on plankton for food (Akpoghelie, Igbuku, & pH 7.69 −1 Osharechiren, 2021). It is therefore imperative to examine Salinity 23.5 mg l −1 Conductivity 36 050 µS cm the resilience and response of algae to petroleum fuel −1 Total dissolved solid (TDS) 19 085 mg l pollution. 56 R. S. EZENWEANI AND M. O. KADIRI Algal cultures using ocular micrometry. The five largest cells were identified and measured, and five smallest cells were A unialgal culture of Nannochloropsis oculata (Droop) also identified and measured in each mount. The largest D.J.Hibberd was obtained from Algae Research Supply. and smallest lengths and widths were noted to give the This small (2–5 µm in diameter) non-motile species of full size range of the cells. tropical green algae grows in both marine and brackish environments (Assaf-Sukenik, 1989). Nannochloropsis oculata is considered promising for industrial applica- Chlorophyll a determination tions, used as an energy-rich food source for fish larvae and rotifers (Kandilian et al., 2013). This was determined using the method of Talling A unialgal culture of the unicellular red alga (1974). This procedure involved filtration, extraction, Porphyridium cruentum (S.F.Gray) Nageli was also homogenization and spectrophotometry. The sample obtained from Algae Research Supply. Like N. oculata, was filtered using Whatman No. 1 filter paper and this species occurs in both marine and brackish tropical extracted using 90% acetone at room temperature in environments, it is spherical (3–12 µm in diameter) and the dark to avoid exposure to high light intensity. The non-motile (Assaf-Sukenik, 1989). P. cruentum is sample was left for 24 h and reading was taken using 721 a good source of lipids, carbohydrates (up to 57% of visible spectrophotometer at 630 nm, 645 nm, and biomass) and pigments for a broad range of industrial 665 nm, applications and is a good source of energy for aquatic 11:6 Abs 1:31 Abs 0:14 Abs 665 645 630 Chl a ðmg=lÞ¼ � v eq:2 organisms (Razaghi, Godhe, & Albers, 2014). V� I where v = volume of acetone (extractor), V = volume of sample filtered, I = path length of the cuvette (cm). Experimental setup The experimental cultures were grown inside the botany screen house of the Department of Plant Biology, Peroxidase determination (GPx) University of Benin, Benin City, Nigeria in natural con- −2 −1 Peroxidase concentration was determined spectrophoto- ditions (average light intensity: 40 μmol m s , tem- metrically using the method of Kim & Yoo (1996). The perature of the screen house maintained at 28°C). A 2 formation of tetraguaiacol was estimated at 470 nm. The ml of each algal culture was used to inoculate each 200 reaction mixture (3.0 ml) contained 0.9 ml of 0.1 M phos- ml experimental sample and these were kept in 500 ml phate buffer (pH 6.0), 1.0 ml of 15 mM guaiacol, 0.1 ml of cylindrical culture vessels, corked using cotton wool. test sample and 1.0 ml of 3 mM hydrogen peroxide. One The cultures were agitated every morning and evening unit (U) of peroxidase is the amount (concentration) of to improve circulation, aeration and to prevent clump- enzyme that can convert 1 μmol of substrate into product ing (Kadiri & Azomani, 2000). Stock solutions (100% (tetraguaiacol) per time (1 min). The activity of peroxi- WSF) were diluted using 0.2 µm filter-sterilized sea- dase was estimated as shown below. water (culture medium) to 25% and 50% WSF, while the control was 0% WSF (n = 3). The growth of test GPxðU=mlÞ¼ðΔOD=m� Total Assay VolumeÞ= algae was monitored optically using a 721 visible spec- ðE� I� Enzyme extract volumeÞ eq:3 trophotometer (produced by PEC MEDICAL, USA) at where, ΔOD/min = change in absorbance 750 nm (Bianchini, Vieira, & Toledo, 1985; Kadiri & per minute, E = Extinction Coefficient = 2.8 Azomani, 2000) every 2 d for 14 d (see eq. 1), −1 −1 mM . cm , EV = enzyme extract volume (ml), GrowthðAbs Þ¼ Abs Abs eq:1 750nm t 0 and I = diameter of cuvette. where Abs = Abs (time), Abs = Abs (initial) Abs = t o Absorbance. Catalase determination (CAT) Catalase was estimated using the method of Korolyuk, Morphological studies Ivanova, and Majorova (1988) (eq. 4). A 0.1 ml of cell Morphology of all test algae in the treatments was sample was added to 1 ml of 4% ammonium molybdate examined at the end of the experiment (day 14). Cells and 2 ml of 0.03% H₂O₂ solution. One unit of catalase were examined and photographed using Olympus concentration is defined as the amount of enzyme −1 −1 Trinocular Microscope at Magnification × 100. The required to clear 1 μmol of H₂O₂ min ml of sample. sizes (length and width) of the algae were examined The breakdown of hydrogen peroxide in the reaction APPLIED PHYCOLOGY 57 mixture was measured spectrophotometrically at Data analysis 410 nm, PAST (version 4.03) statistical software and Microsoft CATðU=mlÞ¼ðSo=S Þ X 203=1 eq:4 Excel were used to analyse data from three replicates of each treatment. Means and standard errors were derived where So = Abs (std) – Abs (b), where b = blank, std = using Microsoft Excel, and graphs were plotted. A two- ammonium molybdate + Hydrogen peroxide S Abs 3 = way analysis of variance (ANOVA) was used to test for (std) – Abs (t), where t = ammonium molybdate + significant differences in growth response between hydrogen peroxide + test sample after 3 minutes. treatments using PAST. Tukey’s pairwise comparison test was used to compare means. Principal component analysis biplots were used to show the relationship Superoxide dismutase determination (SOD) between growth, chlorophyll a (Chl a), peroxidase This was carried out using the method described by (POD), superoxide dismutase (SOD) and catalase Misra & Fridovich (1972). A 0.2 ml of distilled water (CAT) activity. Unpaired t-tests were used to determine was added to the reference tube, while 0.2 ml of the test significant differences (p < 0.05) between growth of N. sample was added to the sample test tube. To each of oculata and P. cruentum. these, 2.5 ml of the carbonate buffer was added and allowed to reach equilibrium, then 0.3 ml of 0.3 mM adrenaline solution was then added to the reference Results and each of the test solutions and allowed by mixing. Absorbance reading was taken at 420 nm using UV Physiological response (growth) of test algae to the spectrophotometer, WSF of the different petroleum fuels SODðU=mlÞ ¼ %inhibition=50� Y eq:5 The growth response of N. oculata and P. cruentum in different fuels is shown (Figs 1-2). Much faster growth ðOD OD Þ in all the fuels was observed in N. oculata than Ref Test %inhibition ¼ � 100 eq:6 OD P. cruentum (p < 0.05 at day 14). In kerosene (Fig 1a), Ref maximum growth of N. oculata was in 25% WSF, and where y is the volume of sample extract, OD = Ref minimum growth in 100% WSF. In diesel (Fig 1b), absorbance reading of reference tube containing dis- maximum growth of N. oculata was obtained in 50% tilled water, and OD = absorbance reading of test Test WSF, and minimum growth in 100% WSF. In gasoline tube containing algal sample. (Fig 1c), growth of N. oculata was maximum in 25% WSF, and minimum in 100% WSF. An extended lag phase was observed until day 4 in all the fuels. Peak Determination of hydrocarbon content of the WSF growth was observed in N. oculata on day 14 in all the of the different petroleum fuels fuels at 0% (control) to 50% WSF, but it was observed WSFs of the various petroleum fuels were analysed on day 12 in all the fuels at 100%. There was growth using the USEPA 8015 method for the Gas stimulation in N. oculata at 25% and 50% in all the fuels, Chromatography analysis of Diesel Range Organics whereas there was growth reduction at 100% WSF. (DRO) (USEPA, 1996). HP5890 PLUS (manufactured There was a significant difference (ANOVA; p < 0.05) by Agilent Technologies Inc.) was used for this ana- in the growth response of N. oculata at 100% with time lysis. Sample extraction was carried out using n-hex- in all the fuels. ane and was concentrated using blow down. Sample In kerosene (Fig 2a), maximum growth of cleanup was immediately carried out using silica gel. P. cruentum was obtained in 25% WSF, with minimum Detail of procedures used was FID (Flame Ionization growth in 100% WSF. In diesel (Fig 2b), maximum Detector), HP 7353 autosampler, HP-5, 0.32 mm ID × growth of P. cruentum was in 25% WSF, and minimum 0.5 UM, 30 column, 250°C injector temperature, at 100% WSF. In gasoline (Fig 2b), maximum growth of nitrogen carrier gas, hydrogen and compressed air P. cruentum was at 25% WSF, and minimum at 100% (other gas), 350°C detector temperature, 1 µl injector WSF. An extended lag phase was observed up to day 4 volume, FID detector and oven programme (oven and growth reduction and retardation were observed in temperature was set at 50°C then ramped at a rate of all the fuels, the latter more severe with increased con- −1 5°C min to 280°C and held for 6 min), with a total centration of fuels (severe at 100%). ANOVA showed run time of 52 min. Agilent ChemStation quantitative a significant difference (p < 0.05) in the growth response software was used to analyse petroleum hydrocarbons. of P. cruentum at different concentrations with time. 58 R. S. EZENWEANI AND M. O. KADIRI 0% 0.5 0.45 25% 0.4 0.35 50% 0.3 0.25 0.2 100% 0.15 0.1 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 -0.15 Days -0.2 0.5 0.45 0.4 0% 0.35 0.3 25% 0.25 0.2 50% 0.15 0.1 100% 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 Days -0.15 -0.2 0.5 0.45 0.4 0% 0.35 0.3 25% 0.25 0.2 50% 0.15 0.1 100% 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 Days -0.15 -0.2 Figure 1. Physiological response (growth) of test algae to the WSF of the different petroleum fuels with time: a) growth response of Nannochloropsis oculata in kerosene, b) growth response of N. oculata in diesel, c) growth response of N. oculata in gasoline. Growth (Absorbance @750 nm) Growth (Absorbance @750 nm) Growth (Absorbance @750 nm) APPLIED PHYCOLOGY 59 0.5 0.45 0.4 0% 0.35 0.3 25% 0.25 0.2 50% 0.15 0.1 100% 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 Days -0.15 -0.2 0.5 0.45 0.4 0% 0.35 0.3 25% 0.25 0.2 50% 0.15 0.1 100% 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 Days -0.15 -0.2 0.5 0.45 0.4 0% 0.35 0.3 25% 0.25 0.2 50% 0.15 0.1 100% 0.05 -0.05 0 2 4 6 8 10 12 14 -0.1 Days -0.15 -0.2 Figure 2. Physiological response (growth) of test algae to the WSF of the different petroleum fuels with time: a) Growth response of Porphyridium cruentum in kerosene, b) growth response of P. cruentum in diesel, c) growth response of P. cruentum in gasoline. Growth (Absorbance @750 nm) Growth (Absorbance @750 nm) Growth (Absorbance @750 nm) 60 R. S. EZENWEANI AND M. O. KADIRI There was more similarity in the growth trend of WSF of fuels (Table 2). Change in shape of cells from both N. oculata and P. cruentum in kerosene and gaso- spherical to obovoid and oblong also became more line than in diesel. The negative values for P. cruentum severe with increase in concentration of WSF of fuels. (Fig 2a–c) indicate death and reduction of cell number For P. cruentum, there was increase in cell size below day 0 (Initial). (Table 3) accompanied with severe clumping of cells at 100% WSF of the different fuels. Cell clumping was also observed in 25% WSF and 50% WSF of the petroleum fuels except in 25% WSF of kerosene. There was Morphology of test algae in the WSF of the different increase in severity of clumping of cells with increasing petroleum fuels concentration. There was change in shape of cells from Figs 3–8 includes micrographs showing alteration in spherical to ovoid. size and clumping of test algae in the different petro- leum fuels. Both N. oculata and P. cruentum were sphe- rical in shape and uniform in size before the experiment. Antioxidant enzymes concentration of test algae in The microscopic examination after the experiment the WSF of the different petroleum fuels showed that N. oculata reduced in size and was deformed in shape from spherical to obovoid and Fig 9a,b show the peroxidase (POD) concentrations in oblong shape at 25%, 50% and 100% WSF of the differ- N. oculata and P. cruentum in the different petroleum ent petroleum fuels. The reduction in size was not fuels respectively. In kerosene, peroxidase concentra- observed at 50% and 100% WSF of diesel, where tion of N. oculata was highest at 50% WSF (3.46 U −1 −1 minor clumping was observed. Reduction in cell size ml ) and lowest at 100% (1.52 U ml ), in diesel, it −1 became more severe with increase in concentration of was highest at 50% (2.74 U ml ) and was lowest at Figure 3. Morphological alteration of Nannochloropsis oculata in kerosene at the end of the experiment; a) N. oculata before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. APPLIED PHYCOLOGY 61 Figure 4. Morphological alteration of Nannochloropsis oculata in diesel at the end of the experiment; a) N. oculata before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. −1 −1 100% (0.82 U ml ), and in gasoline, it was highest at gasoline the activity was highest at 100% (14.56 U ml ) −1 −1 50% (2.72 U ml ) and was lowest at 100% (1.24 U and was lowest at 0% (12.54 U ml ). There was increase −1 ml ). There was increase in peroxidase concentration in SOD concentration with concentration of petroleum with concentration of petroleum fuels but was fuels up to 100%. decreased at 100%. In kerosene, SOD concentration of P. cruentum was −1 In kerosene, the peroxidase concentration of highest at 50% WSF (8.58 U ml ) and lowest at 0% −1 −1 P. cruentum was highest at 0% WSF (2.73 U ml ) and (4.96 U ml ); in diesel the concentration was highest −1 −1 lowest at 100% (1.53 U ml ), in diesel, it was highest at at 50% (7.55 U ml ) and was lowest at 0% (4.96 U −1 −1 25% (3.27 U ml ) and was lowest at 100% (1.24 U ml ); in gasoline the concentration was highest at −1 −1 −1 −1 ml ), and gasoline, it was highest in 0% (2.73 U ml ) 50% (8.26 U ml ) and was lowest at 0% (4.96 U ml ). −1 and was lowest at 100% (0.48 U ml ). There was There was increase in SOD concentration with concen- decrease in peroxidase concentration with increase con- tration of petroleum fuels but there was a slight reduc- centration of petroleum fuels. tion at 100%. Fig 10a, b show the SOD concentrations in N. oculata Fig 11a, b show CAT concentration in N. oculata and and P. cruentum in the different petroleum fuels respec- P. cruentum in different petroleum fuels respectively. In tively. In kerosene, SOD concentration of N. oculata was kerosene, CAT concentration in N. oculata was highest at −1 −1 −1 highest at 100% WSF (16.53 U ml ) and lowest at 0% 50% WSF (39.09 U ml ) and lowest at 0% (27.63 U ml ), −1 (12.54 U ml ), in diesel the activity was highest at 100% in diesel the concentration was highest at 25% (38.50 U −1 −1 −1 −1 (17.55 U ml ) and was lowest at 0% (12.54 U ml ), in ml ) and was lowest at 0% (27.63 U ml ), in gasoline the 62 R. S. EZENWEANI AND M. O. KADIRI Figure 5. Morphological alteration of Nannochloropsis oculata in kerosene at the end of the experiment; a) N. oculata before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. −1 concentration was highest at 25% (64.73 U ml ) and was catalase in gasoline than in kerosene and diesel. For −1 lowest at 0% (27.63 U ml ). There was higher concentra- P. cruentum, there was higher production of POD in tion of CAT in all the concentration compared to 0% in all diesel than in kerosene and gasoline. There was also the fuels. An increase in fuel concentration resulted in slightly higher production of CAT in diesel than in lowered concentration of CAT in gasoline. gasoline and kerosene. However, less overall production In kerosene, CAT concentration of P. cruentum was of SOD was observed in diesel. −1 highest at 0% WSF (38.73 U ml ) and lowest at 100% −1 (36.11 U ml ), in diesel the concentration was highest −1 at 0% (38.73 U ml ) and was lowest at 100% (37.31 U Effect of fuels on chlorophyll a of test algae in the −1 ml ), in gasoline the concentration was highest at 0% WSF of the different petroleum fuels −1 −1 (38.73 U ml ) and was lowest at 100% (36.52 U ml ). Fig 12a shows the chlorophyll a concentration in There was lower concentration of CAT in all the con- N. oculata in the different fuels. The Chl a concentration centration compared to 0% in all the fuels and an of N. oculata was highest at 50% WSF of kerosene and increase in fuel concentration resulted in lowered con- diesel. In gasoline, the highest concentration was recorded centration of catalase. at 25% WSF and the lowest concentration was recorded at Comparatively, for N. oculata, there was slightly 100%. Comparatively, the overall highest Chl higher production of POD in kerosene than diesel and a concentration was recorded at 50% WSF of kerosene gasoline. There was more efficient production of APPLIED PHYCOLOGY 63 Figure 6. Morphological alteration of Porphyridium cruentum in kerosene at the end of the experiment; a) P. cruentum before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. and the overall lowest was recorded at 100% WSF of fuels. Figs 13a-c are PCA biplots showing the relation- gasoline. ship between growth, Chl a, POD, CAT and SOD of Fig 12b shows the Chl a concentration in P. cruentum N. oculata in kerosene, diesel and gasoline, respectively. in the different fuels. The Chl a concentration in There was positive relationship between growth P. cruentum was highest at 25% WSF in all the fuels, and CAT, POD, Chl a in all the petroleum fuels. while the lowest concentration was recorded at 100% in There was a negative relationship between growth all the fuels. The overall highest chlorophyll and SOD, and between Chl a and SOD in all the a concentration was recorded at 25% WSF of kerosene, petroleum fuels. However, a strong positive relation- while the overall lowest was recorded at 100% WSF of ship was observed between growth, CAT, and POD in the same kerosene. Comparatively, there was no signifi- kerosene; growth, Chl a and POD in diesel; growth cant difference (p < 0.05) in the chlorophyll and CAT in gasoline. a concentration in the different fuels for both algae. The eigen value for component 1, 2 and 3 were 26.574, 3.163 and 0.019, respectively, while percen- tage variability explained were 89.31%, 10.63% and Multivariate analysis 0.06%, respectively, in kerosene. In diesel, the eigen values were 29.688, 3.882 and 0.154, respectively, Principal component analysis (PCA) was done to show while the percentage variability explained were the relationship between growth, Chl a, POD, SOD and 88.03% 11.51% and 0.46%, respectively. In gasoline, CAT activity of test algae in the different petroleum 64 R. S. EZENWEANI AND M. O. KADIRI Figure 7. Morphological alteration of Porphyridium cruentum in diesel at the end of the experiment; a) P. cruentum before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. eigen values were 28.532, 1.927, 0.059, respectively, percentage variability explained were 67.95%, while the percentage variability explained were 28.29% and 3.76%, respectively. In gasoline, the 99.31%, 0.67% and 0.02%, respectively. Figs 14a–c eigen value were 3.219, 0.804 and 0.0385 respectively, are PCA biplots showing the relationship between while the % variability explained was 79.31%, 19.79% growth, Chl a, POD, CAT and SOD of P. cruentum and 0.95%, respectively. in kerosene, diesel and gasoline, respectively. There was positive relationship between growth and POD, Discussion CAT and Chl a in all the petroleum fuels. A negative relationship was observed between growth and SOD This study investigated the resilience and bioresponse of in all the petroleum fuels. However, a strong positive N. oculata and P. cruentum to petroleum fuel pollution. relationship was observed between Chl a, CAT, and The starting total petroleum hydrocarbon (TPH) con- POD in kerosene; growth, Chl a and CAT in diesel; centration shown in Table 4 can be compared to back- and Chl a, POD and CAT in gasoline. ground levels in polluted sites in Southern Nigeria. The eigen value of component 1, 2 and 3 were Daniel & Nna (2016) reported that a part of Cross 3.569, 0.355 and 0.055, respectively, while Percentage River Estuary, Niger Delta, Nigeria has TPH concentra- −1 variability explained were 89.69%, 8.93% and 1.38%, tion ranging between 13,161.81 to 24,854.62 µg l . respectively, in kerosene. In diesel, the eigen value Olufemi, Tunde, & Temitope (2011) reported a higher were 1.562, 0.650, 0.086, respectively, while TPH concentration at Ubeji River, Warri, Nigeria which APPLIED PHYCOLOGY 65 Figure 8. Morphological alteration of Porphyridium cruentum in gasoline at the end of the experiment; a) P. cruentum before experiment, b) 0% WSF, c) 25% WSF, d) 50% WSF and e) 100% WSF of kerosene, respectively. Mag.× 100, arrows highlight algal cells. Table 2. Cell size range and shape of Nannochloropsis oculata at the end of experiment (day 14). Cell size range in (µm) Conc Kerosene Diesel Gasoline Cell clumping Cell shape 0% Length 2.08–4.57 2.08–4.57 2.08–4.57 None Spherical Width 2.08–4.57 2.08–4.57 2.08–4.57 25% Length 2.08–3.74 2.08–3.32 2.08–3.32 None Obovoid, oblong Width 1.25–2.08 1.25–2.08 1.25–2.08 50% Length 1.66–3.32 1.25–3.32 2.08–3.32 Minor clumping in 50% diesel Obovoid, oblong Width 1.25–2.08 2.08–4.98 1.25–2.08 100% Length 1.66–2.08 1.25–4.98 1.66–2.08 Minor clumping in 100% diesel Obovoid, oblong Width 0.83–2.08 2.08–4.98 0.83–2.08 −1 was 73,500.00 µg l . In Indonesia, Sari, oil mining. Some studies have shown that some algae Trihadiningrum, Ni’matuzahroh, and Ni’matuzahroh including green, brown and red algae have the potential (2018) reported a very high TPH concentration (211 to degrade or use some hydrocarbons as a carbon −1 025.73 µg l ) from a surface water sample that was source, indicating their ability to withstand crude oil collected from a small river in Wonocolo public crude pollution (Amran et al., 2022; Naeem & Qazi, 2020). 66 R. S. EZENWEANI AND M. O. KADIRI Table 3. Cell size range of Porphyridium cruentum at the end of experiment (day 14). Cell size range (µm) Conc Kerosene Diesel Gasoline Cell clumping Cell shape 0% Length 3.74–9.96 3.74–9.96 3.74–9.96 None Spherical Width 3.74–9.92 3.74–9.92 3.74–9.92 25% Length 3.32–9.96 3.32–9.96 2.91–9.92 Clumping in 25% WSF of the various fuels Ovoid Width 2.91–7.47 2.91–9.96 2.91–7.47 50% Length 3.74–10.79 3.74–10.79 2.91–10.79 Clumping in 50% WSF of the various fuels Ovoid Width 2.91–9.96 2.91–9.96 2.91–9.96 100% Length 3.74–11.62 3.74–11.62 3.74–11.96 Severe clumping in 100% WSF of all the fuels Ovoid Width 2.91–10.62 2.91–10.62 2.91–11.96 3.5 2.5 0% 2 25% 50% 1.5 100% 0.5 Kerosene Diesel Gasoline 3.5 2.5 0% 25% 1.5 50% 100% 0.5 Kerosene Diesel Gasoline Figure 9. a) Peroxidase concentrations of Nannochloropsis oculata in the different petroleum fuels at the end of the experiment (day 14), b) peroxidase concentration of Porphyridium cruentum in the different petroleum fuels at the end of the experiment (day 14). Growth response of test algae in WSFs of the have considerable effect, stimulatory or inhibitory on different petroleum fuels the growth of the different test algae, depending on concentration, type of fuel and type of algae. Some In this study, after careful analysis of data, result showed other researchers have also reported similar findings. significant responses of the investigated algae as Laura et al. (2018) reported species dependence in expressed by applied biological parameters which may their study of the physiological response of phytoplank- be positive or negative. The different fuels were found to ton species exposed to macondo oil and the dispersant, Peroxidase (U/ml) Peroxidase (U/ml) APPLIED PHYCOLOGY 67 0% 25% 50% 100% Kerosene Diesel Gasoline 0% 25% 50% 100% Kerosene Diesel Gasoline Figure 10. a) Superoxide dismutase concentration in Nannochloropsis oculata in the different petroleum fuels at the end of the experiment (day 14), b) Superoxide dismutase concentration of Porphyridium cruentum in the different petroleum fuels at the end of the experiment (day 14). COREXIT1. The effects of petroleum spillage in marine corroborates the report of Dhull, Soni, Rahi, & Soni environment are gross. There is inadequate real-time (2014) that concentrations of petroleum products in data on the impacts of petroleum spillage in marine a medium increase the growth and biomass produc- ecosystem (Asif, Chen, An, & Dong, 2022; Wang et al., tion of some algae. Stimulation of growth and photo- 2021). synthesis in microalgae exposed to low In N. oculata, growth stimulation was observed in concentrations of hydrocarbons has also been noted concentrations up to 50% WSF. This could be as in Wang, Tang, Li, & Liu (2002). In P. cruentum, a result of its ability to degrade, accumulate and there was reduction and retardation of growth. This use petroleum products as source of carbon. This could be as a result of the high toxicity of petroleum Superoxide Dismutase (U/ml) Superoxide Dismutase (U/ml)) 68 R. S. EZENWEANI AND M. O. KADIRI 0% 25% 50% 100% Kerosene Diesel Gasoline 38.5 37.5 0% 25% 36.5 50% 100% 35.5 34.5 Kerosene Diesel Gasoline Figure 11. a) Catalase concentration of Nannochloropsis oculata in the different petroleum fuels at the end of the experiment (day 14), b) catalase concentration of Porphyridium cruentum in the different petroleum fuels at the end of the experiment (day 14). fuels above the tolerance level of the algae or its low and high concentrations. The resilience and effi- inefficiency in metabolizing petroleum hydrocarbon cient growth of N. oculata in WSF of the different for heterotrophy. This corroborates the result of petroleum fuels suggests that it could serve as Fabregas, Herrero, & Veiga (2021) who observed a potential agent for the bioremediation of petroleum that petroleum oil stimulated the growth of hydrocarbon. The negative values recorded the Tetraselmis suesica at low concentration and inhib- growth response of P. cruentum depict death and ited the growth at high concentrations. Jiang et al. reduction of cell number over time. (2022) reported that the specific growth rates of Extended lag phase observed at high concentration can Skelotenema costatum were significantly inhibited by be attributed to cell adaptation as a result of the change in a hydrocarbon component (phenanthrene) at both medium condition. This is in line with the result of Catalase (U/ml) Catalase (U/ml) APPLIED PHYCOLOGY 69 4.5 3.5 B. EXP 2.5 0% 25% 50% 1.5 100% 0.5 kerosene Diesel Gasoline 1.6 1.4 1.2 B. EXP 0% 0.8 25% 0.6 50% 100% 0.4 0.2 kerosene Diesel Gasoline Figure 12. a) Chlorophyll a concentration of Nannochloropsis oculata in the different petroleum fuels at the end of the experiment (day 14), b) chlorophyll a concentration of Porphyridium cruentum in the different petroleum fuels at the end of the experiment (day 14). Rajabnasab, Khavari-Nejad Ra, Shokravi, Nejadsattari, & oil-producing microalgae from subtropical coastal and Khavari-Nejad (2018) where they reported that extended brackish waters also reported extension of the lag phase. lag phase in some cyanobacteria species as a result of The comparative assessment of the average growth of change in environmental conditions. This is further sub- test algae in WSF of the different fuels which showed that stantiated by the study of Sushama, Reena, Arun, & for N. oculata, lowest growth occurred in diesel corrobo- Madhavi (2008) on the effect of Bombay crude oil on rates the findings of Kadiri & Enoma (2013) where they Thalassiosira sp where WSF fraction was capable of hav- reported that diesel caused higher growth inhibition in ing negative growth rate on 10%, 20% and 40% WSF at Selenastrum capricornutum than kerosene and gasoline. 24 h, indicating effect, and after 24 h, growth rate in 10% For P. cruentum in this study, the same trend was also and 20% increased, while that of 40% remain low for 6 observed. This effect could be as result of diesel being days. Lim et al. (2012) in the isolation and evaluation of a heavier petroleum fuel than kerosene and gasoline. This -1 -1 Chlorophyll a (µg mL ) Chlorophyll a (µg mL ) 70 R. S. EZENWEANI AND M. O. KADIRI Chl a 50% 1.5 1.0 CAT 25% 0.5 POD Growth 0% -8.0 -6.4 -4.8 -3.2 -1.6 1.6 3.2 -0.5 -1.0 -1.5 -2.0 SOD -2.5 100% -3.0 Component 1 2.4 25% 1.6 Chl a POD CAT 0.8 50% Growth 0% -8.0 -6.4 -4.8 -3.2 -1.6 1.6 3.2 -0.8 -1.6 -2.4 SOD -3.2 100% -4.0 Component 1 Chl a 1.5 25% 1.0 POD 0% 0.5 50% Growth CAT -30 -25 -20 -15 -10 -5 5 10 15 -0.5 -1.0 SOD -1.5 -2.0 100% -2.5 -3.0 Component 1 Figure 13. a) Principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Nannochloropsis oculata in kerosene, b) principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Nannochloropsis oculata in diesel, c) Principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Nannochloropsis oculata in gasoline. study showed that WSF of different fuels have different Morphological studies of test algae in WSF of the different petroleum fuels potential environmental damage depending on the type of fuel and the concentrations of WSF. Extensive evi- This study showed morphological alterations of test dence also exists on the effects of petroleum hydrocar- algae and its relationship with concentrations. bons among some other groups of algae (Kadiri & Kerosene, diesel and gasoline were capable of causing Eboigbodin, 2012). This study showed that WSF of pet- change in the shape of N. oculata from spherical to roleum fuels have different potential environmental obovoid and oblong. In P. cruentum, the petroleum damages depending on the type of fuel and the concen- fuels were capable of causing increased cell size and trations of WSF. heavy clumping. Clumping became more severe at Componen t 2 Componen t 2 Componen t 2 APPLIED PHYCOLOGY 71 50% 0.64 SOD CAT 0.48 POD Chl a 0.32 0.16 0% Growth -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 -0.16 25% -0.32 -0.48 -0.64 100% -0.80 Component 1 POD 0.8 50% SOD 0.6 25% 0.4 0.2 Chl a CAT Growth -2.0 -1.6 -1.2 -0.8 -0.4 0.4 0.8 1.2 1.6 -0.2 -0.4 0% -0.6 -0.8 100% -1.0 Component 1 SOD 50% POD 0.6 CAT 0% Chl a 0.3 Growth 25% -2.0 -1.5 -1.0 -0.5 0.5 1.0 1.5 2.0 2.5 -0.3 -0.6 -0.9 -1.2 100% -1.5 -1.8 Component 1 Figure 14. a) Principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Porphyridium cruentum in kerosene, b) principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Porphyridium cruentum in diesel, c) principal component analysis biplot showing the relationship between growth, chlorophyll a (Chl a), peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) activity of Porphyridium cruentum in gasoline. higher concentrations. Increase in size of algae under that the microscopic examination of algal cells in severe toxicity is accompanied with cell clumping. These response to crude oil pollution indicated that the present observations are in good agreement with crude oil led to an increase in algal biomass, although Soltani, Amira, Sifi, & Beldi (2012) where they reported it caused heterocyst separation from the filament of Componen t 2 Componen t 2 Componen t 2 72 R. S. EZENWEANI AND M. O. KADIRI Table 4. Petroleum hydrocarbon component of the WSF of the different petroleum fuels. –1 Concentration (µg l ) Petroleum hydrocarbon Kerosene (B. EXP.) Diesel (B. EXP.) Gasoline (B. EXP.) N-octane <0.01 <0.01 22.99 n-nonane 656.15 50.57 260.51 n-decane 2256.17 268.11 2034.09 n-undecane 1563.64 389.99 1239.29 n-dodecane 5676.2 2083.16 2322.04 n-tridecane <0.01 152.31 <0.01 n-tetradecane 2777.86 2966.49 2846.44 n-pentadecane 206.45 193.92 43.94 n-hexadecane 1654.67 2121.15 1159.47 n-heptadecane 176.15 1068.64 114.95 Pristine 137.05 615.57 55.14 n-octadecane 911.01 1868.29 761.72 Phytane 184.81 1120.91 178.11 n-nonadecane 138.65 250.45 71.21 n-eicosane 442.03 110.63 392.11 n-heneicosane 63.95 57.78 32.33 n-docasane 197.41 382.14 166.88 n-tricosane 22.89 56.2 <0.01 n-tetracosane 95.15 321.61 80.68 n-pentacosane 5.6 32.55 354.83 n-haxecosane 20.92 39.28 12.41 n-heptacosane 20.21 268.61 21.71 n-octacosane 19.09 45.14 19.4 n-nonacosane 23.09 97.05 29.1 n-tricotane 18.35 <0.01 31.32 n-hentriacotane 7.42 24.22 9.44 n-dotriacotane <0.01 <0.01 <0.01 n-tritriacontane 24.63 5.27 <0.01 ∑TPH 17299.57 15590.08 16444.46 Anabaena sp. The clumping observed in P. cruentum The comparative assessment of the morphological corroborates the observation of Soltani, Amira, Sifi, & effects of the WSF of the different fuels which showed Beldi (2012) that the cells of Oscillatoria sp. were aggre- that diesel caused minor cell clumping in N. oculata gated in clusters like a ball covered with oil. Gamila, explains why it could pose higher environmental treat Ibrahim, & El-Ghafar (2003) also observed the same in and caused higher growth inhibition than kerosene and Oscillatoria sp. in their study of the effect of crude oil. gasoline. In P. cruentum, diesel was also observed to Soto, Hutchinson, Hellebust, & Sheath (2011) also cause worst cell clumped than kerosene and gasoline. observed induced morphological abnormalities in the These resulted in great physiological stress of both algae cell of Chlamydomonas angulosa in aqueous crude and in WSF of diesel. fuel oil extracts. Similarly, Gaur & Singh (1990) found that Assam crude oil had a serious effect on heterocyst Chlorophyll concentration of test algae in WSF of differentiation in Anabaena doliolum and that micro- the different petroleum fuels scopic examination of Tetraselmis suecica cells indicated abnormal cellular morphology. Chlorophyll is a photosynthetic pigment which serves as Nechev et al. (2002) postulated that diesel fuel causes a biomass indicator for microalgae and it is the most a disruption of the optimal physical state of the cytoplas- frequently measured biochemical parameter in toxicity mic membranes of algae, thus increasing the permeability studies (Martínez, Kinet, Bajji, & Lutts, 2005; of these membranes, which in turn facilitate the entry of Somruthai, Rungcharn, & Nuttha, 2021). Chl a study diesel fuel into the cells and the accumulation of a high in N. oculata revealed that low (10% WSF) to moderate quantity of hydrocarbons. This could lead to obstruction (50% WSF) petroleum fuel did not have any effect on in biochemical and molecular processes such as cell divi- the chlorophyll concentration. However, at very high sion, thereby suggesting a reason for the increase in cell concentration (100%), the petroleum fuels enhanced size of P. cruentum. The resultant cell clumping then reduction of chlorophyll concentration in the algae. leads to inhibition in growth and productivity of the This corroborates the result reported by Nayar, Gohb, cell. Abdul Hameed & Al Obaidy (2014) suggested that & Choua (2004) that hydrocarbon was capable of a clear reduction of the growth and chlorophyll of increasing productivity and may not affect chlorophyll Microcystis flos-aquae could be due to the cellular struc- production at certain concentrations. Nagwa, Yean- ture changes as a result of the toxic effects of crude oil. Chang, Abd-El-Ruhman, & Rania (2005) reported that APPLIED PHYCOLOGY 73 a positive effect of the oil pollutant was seen on the total changes. The reduction of growth in algae under condi- yield and production of Chl a of algae. High chlorophyll tions of contamination with petroleum fuel can be −2 concentration in alga exposed to petroleum hydrocar- attributed to production of ROS such as O and H₂O₂ bon may be attributed to its utilization of the petroleum (Chia, Cordeiro-Araújo, Lorenzi, & Bittencourt- products as source of organic compounds. Oliveira, 2016), and the inefficient response of algae to In P. cruentum, Chl a concentration was inhibited the stress. SOD is responsible for converting certain with increase in petroleum fuel concentration. This reactive oxygen species (super oxides) to peroxides could be as a result of high toxicity of petroleum fuel while peroxides and catalase converts H₂O₂, to H₂O to the algae compared to N. oculata. The inhibition of and O₂ (Chia & Kwaghe, 2015). As a result of increased chlorophyll with increase in petroleum fuel concentra- ROS, microalgae tend to up-regulate the biosynthesis tion in P. cruentum is in agreement with the findings of and activities of ROS combating enzymes (Chia & Nayar et al. (2003) in their study of the impact of Kwaghe, 2015). The efficient growth and productivity petroleum hydrocarbons (diesel) on periphyton in an of N. oculata can be attributed to the efficient produc- impacted tropical estuary based on in-situ microcosms, tion of antioxidant enzymes. This study showed that where chlorophyll concentration of the periphyton in difference in fuel type had notable effect on the average the background levels of the petroleum hydrocarbon peroxidase production in P. cruentum. For example, was severely affected. Comparative assessment of the gasoline seems to induce the lowest overall production effect of the different petroleum fuels on Chl of peroxidase in P. cruentum which says more about the a concentration showed that none of the petroleum unique responses from the alga. Difference in fuel type fuels caused a significant difference in both test algae. also had notable effect on catalase production in N. oculata which is a unique effect. The observed higher concentration of catalase at all the concentrations com- Antioxidant enzyme concentration of test algae in pared to 0% (control) in all the fuels for N. oculata WSF of the different petroleum fuels which is contrary to the observed decrease of catalase Reactive oxygen species (ROS) are reactive chemical at all the concentrations compared to 0% in all the fuels species of oxygen that are bi-products of biochemical for P. cruentum explains part of reasons for observed reactions. They are chemically reactive and pose threat tolerance and growth efficiency of N. oculata. to aerobic organisms. They are of different forms, namely, hydrogen peroxide (H O ), singlet oxygen 2 2 Multivariate analysis of data −2 (O ) and hydroxyl radicals (OH-) (Pinto et al., 2003). Halliwell & Gutteridge (1999) noted that ROS can alter In N. oculata, there was positive relationship between the structure and mutagens of cells. Cellular defence the growth, chlorophyll concentration, POD and CAT. against these harmful oxygen products involves the This means that growth and productivity of the test production of antioxidant enzymes such as SOD, POD algae in the condition of petroleum pollution depended and CAT by cells. These are the first line of antioxidant largely on their ability to produce peroxidase and cata- enzymes produced by cells during antioxidant activities lase which are responsible for the conversion of perox- (Chia & Kwaghe, 2015). They are involved in the ides to water and oxygen. While high concentration −2 decomposition of H O and O to water and oxygen (100% WSF) of petroleum fuels slightly inhibited per- 2 2 by the interaction of the amino acids, asparagine at oxidase and catalase, SOD concentration was not position 147 and histidine at position 74, which causes affected by high petroleum fuel concentrations. a proton transfer between the oxygen atoms (Tores, Therefore, reduction of POD and CAT at high concen- McNeill, Gibson, Wayne, & Yates, 2008). Antioxidant tration (100% WSF) of the pollutants could be the enzymes serve as signals of distress at the molecular reason for decrease in growth and productivity at that level and useful as toxicity biomarkers (Pinto et al., concentration. This result corroborated the report of 2003; Tores, McNeill, Gibson, Wayne, & Yates, 2008). Chia & Kwaghe (2015) where it was opined that This study showed that WSFs of petroleum fuels decrease in peroxidase and catalase concentrations induced low production of peroxidase and catalase may be related to inhibition of the enzymes by high with increase in concentration in P. cruentum, thereby concentration of contaminant. It is the view of reducing the ability of the alga in combatting stress Cordeiro-Araújo, Chia, Hereman, Sasaki, & effects of ROS with increase in the fuel concentration. Bittencourt-Oliveira (2015) that catalase inhibition This could have also consequently resulted in the inhi- maybe related to the inactivation via the binding of bition of certain physiological (growth) and biochemical thiol group with the bioactive compounds of the tox- processes, and also may have caused morphological icant investigated. The high overall antioxidant enzymes 74 R. S. EZENWEANI AND M. O. KADIRI concentrations in Nannochloropsis oculata can be attrib- The observed tolerance of N. oculata to the petro- uted to be the reason for observed tolerance of the alga leum fuels could be as a result of its high efficiency in in the petroleum fuels. antioxidant production and absence of cell clumping. Positive relationship between growth, chlorophyll The lowest overall growth observed in diesel for concentration, POD and CAT was also observed in Nannochloropsis oculata could be attributed to minor P. cruentum. The lower production of antioxidant clumping observed. The overall higher tolerance of enzymes in P. cruentum, compared to N. oculata, Nannochloropsis oculata to the fuels compared to could have contributed to the reason for lower produc- Porphyridium cruentum could be attributed to tivity due to inefficiency in the conversion of super- oxides and peroxides to oxygen and water. This is in its overall higher efficiency in the production of line with the finding of (Chia, Cordeiro-Araújo, antioxidant enzymes; Lorenzi, & Bittencourt-Oliveira, 2016), where it was morphological factors such as absence of cell observed that, by the 4th day after exposure to contami- clumping; nant, the microalga was no longer able to withstand the more efficiency in chlorophyll production. effect of the contaminant (stress source), as it inhibited CAT and POD activity. It was then concluded that that The observed differences in the enzymatic responses of the altering of the peroxidase and catalase activities in each alga to the different fuels could be as a result of S. quadricauda exposed to contaminant was an indica- concentration differences in the hydrocarbon compo- tion that the microalga suffered severe oxidative stress nents of the petroleum fuels as shown in Table 4. The with increasing aqueous contaminant concentration. increased aggregation of cells with increase in concentra- SOD had a huge impact on growth as shown in the tion of WSF of fuels observed in P. cruentum could be PCA plots and this could explain the uniqueness of the role attributed to the increased toxicity (cytoplasmic disrup- of the enzyme in combatting stress. According to Chia and tion) of the fuel with increase in concentration. Kwaghe (2015), SOD is an enzyme that converts super The most severe clumping and cell deformation −2 oxides O to H₂O₂. Thereafter, peroxidase and catalase which was observed in diesel could have immensely convert peroxides to water and oxygen. Therefore, the role contributed to the overall lowest growth observed in of SOD is unique and an inadequate production of it will diesel for both algae. have impact on the algal development. For N. oculata, as expressed by the eigen values, two Conclusion principal components (component 1 and 2) explained over 99% of the total variance, in all the fuels. For Petroleum fuel pollution could result in growth retardation P. cruentum, as expressed by the eigen values, two or stimulation on marine algae depending on the fuel type, principal components (component 1 and 2) explained extent of the pollution and composition of the algae com- over 98% of the total variance, in all the fuels. munity or type of algae. There was differential response of the test marine algae to the different fuels. N. oculata displayed tolerance to petroleum fuels, while P. cruentum Comparative assessment was more sensitive. High concentrations of WSF of the The poor overall growth of P. cruentum, compared petroleum fuels caused reduction or retardation of growth, to N. oculata in all the fuels, can be attributed to decreased antioxidant enzyme production, as well as severe cell clumping, poor overall production of reduced Chl a concentration in the test algae. The toler- antioxidant enzymes and high sensitivity of ance of test algae depended largely on antioxidant enzyme P. cruentum. The cell clumping according to activity. Morphological alteration and cell clumping were Nechev et al. (2002) is caused by the disruption of also observed as part of the effects of petroleum fuel the optimal physical state of the cytoplasmic mem- pollution. This study provides useful information for eco- branes of algae, thus raising the permeability of these logical capital, economic consideration of the effect of membranes, which in turn facilitate the entry of fuel petroleum fuel pollution in marine environment and pol- into the cells and the accumulation of a high quan- icy making. tity of hydrocarbons. This leads to cell enlargement and the subsequent clumping of cells. Clumping of Acknowledgements cells could have also affected biochemical and phy- siological developments, therefore, could have con- We appreciate Dr (Mrs) J. E. Ukpebor and all staff of Chemistry tributed to the overall lower production of Department, University of Benin technical staff who assisted in antioxidant enzymes observed in P. cruentum. the analysis of hydrocarbons. We appreciate Dr Jeffrey Uyi APPLIED PHYCOLOGY 75 Cordeiro-Araújo, M. K., Chia, M. A., Hereman, T. C., Ogbebor for his technical assistance. We acknowledge and appreciate the Limnology and Algology Laboratory, Sasaki, F. F., & Bittencourt-Oliveira, M. C. (2015). Department of Plant Biology and Biotechnology, University of Selective membrane permeability and peroxidase activity Benin, Nigeria where this research was carried out. response of lettuce and arugula with cyanobacterial-contaminated water. Environmental Earth Sciences, 74, 1547–1553. doi:10.1007/s12665-015-4147-7 Daniel, I. E., & Nna, P. J. (2016). Total petroleum hydrocar- Disclosure statement bon concentration in surface water of cross River Estuary, Niger Delta, Nigeria. Asian Ecolo Journal of Environmental The authors declare that they have no known competing Ecology, 1, 1–7. doi:10.9734/AJEE/2016/31102 financial interests or personal relationships that could have Dhull, N. P., Soni, R., Rahi, D. K., & Soni, S. K. (2014). appeared to influence the work reported in this paper. 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– Taylor & Francis
Published: Dec 31, 2023
Keywords: Environment; marine algae; pollution; enzymes; bio-response; petroleum