J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 Journal of the Nigerian Society of Physical Sciences Physicochemical Characteristics and Toxicity Studies of Crude Oil, Dispersant and Crude Oil-Dispersant Test Media to Marine Organisms E. P. Onokarea, L. O. Odokumab, F. D. Sikokic, B. M. Nziwub, P. O. Iniaghe d,∗, J. C. Ossaie a World Bank Africa Centre of Excellence, Centre for Oil Field Chemicals Research (ACE-CEFOR), Institute of Petroleum Studies, University of Port Harcourt, Choba, Rivers State b Department of Microbiology, University of Port Harcourt, Nigeria c Department of Animal and Environmental Biology, University of Port Harcourt, Nigeria d Department of Chemistry, Federal University Otuoke, Nigeria. e Department of Chemistry, Delta State University Abraka, Nigeria. Abstract In this study, the physicochemical characteristics of crude oil, dispersant (Ecobestr) and crude oil-dispersant testsystems,and their toxicities on representative marine organismswas assessed. The test media included mechanically dispersed crude oil-in-water(MDO) and its water ac- commodated fraction (WAF), chemically dispersed crude oil-in-water (CDO) and its water accommodated fraction (CEWAF), the Dispersant (D), and a reference toxicant, sodium dodecyl sulphate (SDS). These test media were used to carry out toxicity studies on Tilapia guineensis, Palaeomontesafricanus, and bacteria – heterotrophic bacteria and hydrocarbon utilizing bacteria. Physicochemical characteristics of the test me- dia were done using standard methods. The static with renewal bioassay option was employed for toxicity tests involving Tilapia guineensis and Palaeomontesafricanus, while the static without renewal option was used for microbial bioassays.Marine organisms were exposed to the following concentrations: 100%, 50%, 25%, 12.5%, 6.25% and 0% of MDO, CDO, WAF, CEWAF and D, respectively. The 96 h LC50and toxicity factors were determined. Results for physicochemical characteristics of the test media showed that the pH and dissolved oxygen levels were sufficient for sustaining aquatic habitation. Pb metal was present in high amounts in D, but relatively low in CDO and CEWAF. Toxicity data showed that Ecobestr was non-toxic to the test organisms relative to SDS.The 96h LC50 of MDO, CDO, D, WAF, CEWAF and SDS were 89.8, 225.6, 1891.8, 683.9, 528.1 and 1.37 for T. guineensis, 275.5, 137.7, 3800.9, 76.1, 168.3 and 9.99 for P. africanus; 1658.5, 944.1, 17221.9, 228641.0, 1036319.3 and 3.84 for heterotrophic bacteria, and 250.6, 9544.1, 77.2, 141.4, 12780.8 and 3.6 for hydrocarbon utilizing bacteria. SDS exhibited the greatest toxicity, but the dispersant reduced its toxicity by several folds.However, with increased levels of some heavy metalsand polycyclic aromatic hydrocarbons in the test media water, there may be likelihood for bioaccumulation to occur in the tissues of marine organisms. DOI:10.46481/jnsps.2022.427 Keywords: Ecobestr dispersant, Tilapia guineensis, Palaeomontesafricanus, Crude oil, toxicity study Article History : Received: 04 October 2021 Received in revised form: 23 November 2021 Accepted for publication: 24 November 2021 Published: 28 February 2022 c©2022 Journal of the Nigerian Society of Physical Sciences. All rights reserved. Communicated by: E. Etim ∗Corresponding author tel. no: +2347035191298 Email address: po.iniaghe@gmail.com ( P. O. Iniaghe ) 1. Introduction Marine pollution is defined as “the introduction by man, directly or indirectly, of substances or energy into the marine 105 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 106 environment (including estuaries) resulting in such deleterious effects as harm to living resources, hazardsto human health, hindrance to marine activities including fishing, impairment of quality for use of seawater and reduction of amenities” [1]. Oil spills are inevitable during oil exploration activities, and they are a significant threat to the marine environment. Since oil is of a lower density than water, spilled oil floats on surface water. This can cause harm to aquatic organisms, primarily by limit- ing the amount of dissolved oxygen required for respiration. Oil spill response techniques have been developed for remediating the ecological consequences of spilled oil in the marine envi- ronment [2]. Mechanical methods (skimming) and chemical methods (dispersants and sinking agents) are common methods employed for such purposes. With several shortcomings associ- ated with several cleanup methods, the use of dispersants have been seen as a useful alternative [3]. Dispersants are made up of different chemical composi- tions, and are available in the market. They help to break up crude oil into oil-in-water emulsions, which allows for easy degradation by hydrocarbon utilizing bacteria indigenous to the environment [4]. They also help in diluting crude oil rapidly in the water column, thereby preventing a drift of the oil slick into ecologically relevant shorelines. The use of dispersants is still controversial because, although they help in dispersing the spilled oil quickly, they do not necessarily remove the oil [5]. Dispersants could also induce high levels of toxic metals and polycyclic aromatic hydrocarbons in the water column, thereby creating harmful conditions for marine organisms. Many of the earlier used dispersant formulations (prior to 1970) were inherently toxic to aquatic organisms and possessed bioaccumulation potentials (these were historically referred to as first and second generation dispersants, respectively). For instance, the aquatic toxicity of Corexit dispersants to a range of marine organisms in the United States has been reported [6].The toxicity of these early dispersants led to the develop- ment of third-generation dispersants with the belief that they were nontoxic and biodegradable, as against their historical counterparts [7, 8]. The acute toxicity values of some of these third generation dispersants have been reported to range be- tween 190 – 500 mg L−1, as against the earlier formulations, which had acute toxicity values between 20 – 50 mg L−1 [9]. However, the use of these third generation dispersants still needs to be monitored so that they do not cause harm to the environment. The toxicological effects resulting from disper- sant use is not a straightforward task, as it requires at least five components: the dispersant, the oil being dispersed, the nature of the exposure, the age and species of the test organism(s). For instance, Finasol OSR52r, a third generation dispersant was reported to be toxic to sea urchin embryos, and toxicity was enhanced after the dispersant was added [10]. The same disper- sant was also determined to be very toxic to juvenile sea bass, while Finasol OSR51r possessed toxic characteristics to sea urchin embryos [8]. However, Nalco-D4106 was reported to significantly reduce the toxicity of crude oil to T. guineensis and D. trispinosa [10, 11]. Ecobestr dispersant is a third generation dispersant that is about to be introduced into the Nigeria Oil industry for cleanup purposes. It is therefore necessary that its toxicity to organ- isms in the marine environment is evaluated. Presently, there are no studies on the toxicities of this dispersant to tropical ma- rine organisms. Similarly, most studies do not take into account the properties of the habitat water used in their toxicity stud- ies. The aim of this study was to evaluate the physicochemical characteristics of crude oil, dispersant and crude oil-dispersant test media using Ecobestr dispersant, and to study the toxic- ity of its interaction with crude oil on marine organisms using laboratory-scale experimental approach. 2. Research Methodology 2.1. 2.1 Source of Crude Oil, Dispersant and Habitat Water Samples of Bonny light crude oil were obtained from an on- shore operational facility situated in the Niger Delta. Ecobestr oil dispersant was obtained from the National Oil Spill Detec- tion and Response Agency (NOSDRA), while habitat water was brackish water collected at intervals from off the shores of On- nePort. 2.2. Collection of Test Organisms Test organisms recommended by the Department of Petroleum Resources (DPR) of Nigeria were obtained from the African Regional Aquaculture Centre/National Institute of Oceanography and Marine Research (ARAC/NIOMR) in Buguma, Rivers State, Nigeria. They include fish – Tilapia guineensis (tertiary consumer) and crustacean – Palaemon- etesafricanus (secondary consumer). Bacteria (primary con- sumer) was however isolated from brackish water. These test organisms represented the three trophic levels in the food chain within a tropical marine ecosystem. The fish (body weight ranging between 4.80 g and 6.20 g)were caught with nets and transported to the laboratory in transparent polyethylene bags (air bags) containing habitat wa- ter and enough space for air. On reaching the laboratory, organ- isms were introduced into tanks (1 m × 3 m) containing habitat water for acclimatization. Crustaceans were collected during the dry season (January) when the salinity of the river was high for easy acclimatization. They were transported to the labora- tory in the early hours of the morning in transparent polyethy- lene bags (air bags) containing habitat water and enough space for air. Heterotrophic bacteria (HB) and hydrocarbon utiliz- ing bacteria (HUB) were isolated from brackish water using the spread plate technique on Nutrient Agar [37]. Incubation of cultures was 24 to 48 h for HB and 5-7 days for HUB, all at 24 ± 2◦C. 2.3. Acclimatization of Test Organisms 2.3.1. Tilapia guineensis Due to the difference in salinity between the habitat water and sea water, the test organisms were gradually exposed to increasing levels of salinity. They were first acclimatized in 100% habitat water for 72 h. The acclimatization water was then changed every 72 hwith 50:50 of habitat and sea water, followed by 30:70 of habitat and sea water and lastly, 100% sea water for 14 days [13]. 106 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 107 2.3.2. Palaemonetesafricanus The test organisms were introduced directly into the brack- ish water, since its habitat was within the salinity range of the brackish water, and they were acclimatized in the laboratory for 14 days at ambient temperature. 2.4. Preparation of Test Media The test media was made up of five different exposure mix- tures. These were the mechanically dispersed crude oil, chem- ically dispersed crude oil, dispersant, the water accommodated fractions of the mechanically dispersed and chemically dis- persed crude oil, respectively. There was also a control (habi- tat water) set up without crude oil or dispersant, as well as a reference toxicant, sodium dodecyl sulphate (SDS), which is a historically used toxic dispersant. 2.5. Preparation of Test Media 2.5.1. Crude Oil Preparation Crude oil used for the study was bubbled using aerator for 24h at 25oC to mimic external weathering conditions in a trop- ical environment to reduce oil volume by 10 percent [13]. 2.5.2. Mechanically Dispersed Crude Oil Mechanically dispersed crude oil (MDO) was prepared us- ing a loading rate of 1g L−1 made up with habitat water [15]. 10 g of crude oil was diluted with 10 L of habitat water in a 15 L pyrex bottle, and agitated with a magnetic stirrer for 30 minutes at medium energy to mimic field conditions. Five different con- centrations were prepared (i.e. 100%, 50%, 25%, 12.5% and 6.25%, respectively). 2.5.3. Chemically Dispersed Crude Oil Chemically dispersed crude oil (CDO) (containing 10 g of crude oil and 1g of dispersant) was obtained by adding each components respectively into 10 L of habitat water in a 15 L pyrexbottle and agitated for 30 minutes using a magnetic stirrer [14]. Five different concentrations were also prepared. 2.5.4. Dispersant Each gram of dispersant used was mixed with 1 L of water in a glass beaker using a magnetic stirrer for 30 minutes [2]. Five different concentrations were also prepared. 2.5.5. Water Accommodated Fractions The water accommodated fractions were prepared accord- ing to [17]. The water accommodated fraction obtained from mechanically dispersed crude oil (i.e. WAF) was prepared by adding 1 part of crude oil to 9 parts of habitatwater in a 15 L pyrex bottle with gentle stirring using a magnetic stirrer. Mix- ing was done at ambient temperature for 20 h. After mixing, the oil and water phases were allowed to separate for 6 h. The wa- ter phase was then collected and immediately used for analysis. Five different concentrations were also prepared. The same procedure described above was used for preparing the chemically enhanced water accommodated fraction (CE- WAF) from CDO. 2.6. Physicochemical Analysis of Test Media Unstable parameters such as the pH, electrical conductivity (EC), temperature and total dissolved solids (TDS) were mea- sured in-situ. The pH was measured using a hand-held digi- tal pH metre (pHepr Hanna, USA). The EC and TDS were measured using a TDS/conductivity metre. Total solids (TS) and total suspended solids (TSS) were carried out gravimet- rically. Dissolved oxygen (DO), biochemical oxygen demand (BOD5), chemical oxygen demand (COD), nitrate (NO3−), and phosphate were determined using standard methods [18]. 2.7. Heavy Metals Analysis Water samples from the different test media were digested using nitric acid according to [19]. Lead, chromium, vanadium, cadmium, arsenic, mercury, nickel, iron and zinc were quanti- fied using atomic absorption spectrometer (Perkin Elmer 3110). The equipment was calibrated using analytical standards of the respective metals. These standards (1000 mgL−1) were diluted serially to get the working concentrations and subsequently, a calibration graph. 2.8. Total Petroleum Hydrocarbons Total petroleum hydrocarbon (TPH), was determined ac- cordingto ASTM D 7066, D3921. 100 mL of water sample was measured using a graduated cylinder into a separating fun- nel. 1ml of H2SO4 was added, followed by 20 mL of tetra- chloroethylene at 10 mL each. This was sealed, shaken vigor- ously for about 1-2minutes with periodic venting to release the inbuilt pressure, and allowed to stand for 10 mins for separa- tion into organic and inorganic layers. The organic layer (i.e. the lower layer) was collected in a beaker. A filter paper was placed in a filter glass funnel and, approximately, 1 teaspoon of silica gel was added, and the solvent layer was drained through it. A cuvette was filled with the solvent layer and placed on an InfraCal 2 analyzer. “RUN” was selected, and the result was displayed in ppm Result(mg/l) = C × V1 V2 where C = concentration obtained from the instrument, V 1 = Volume of solvent used for extraction, and V 2 = Volume of sample used for extraction. 2.9. Polycyclic Aromatic Hydrocarbons Polycyclic aromatic hydrocarbons (PAHs) was extracted us- ing the US EPA Method 3510 liquid-liquid extraction. 100 mL of water sample was collected into a separatory funnel. A known volume of n-hexane and methylene chloride (3:1) was added, and the sample was spiked with ortho-Terphenyl. This was sealed and shaken vigorously for 1 to 2 minutes with pe- riodic venting to release excess pressure, and the organic layer was allowed to separate from the water phase for a minimum of 5 minutes. The extract was filtered through a glass funnel with glass wool and anhydrous sodium sulphate. The volume of the sample extracted was recorded and the extract was trans- ferred to a Teflon-lined screw-cap vial ready for PAH analysis. 107 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 108 A method blank was similarly carried out. The extracts were quantified using gas chromatograph with a flame ionization de- tector (GC-FID) Model 6890 (Agilent instruments, USA). 2.10. Microbiological Analyses Microbiological characteristics of the sea water and crude oil used for the acute toxicity test was done by adopting meth- ods from [37] and [38]. 2.11. Acute Toxicity Tests Acute toxicity tests were conducted in two steps: first, a pre- liminary range finding test was conducted to evaluate the low- est concentration that would result in 100% mortality (lethal concentration) or 100% inhibition of bioluminescence (effec- tive concentration) and the highest concentration that will cause 0% mortality or 0% inhibition of bioluminescence. Second, the actual toxicity test, which involved exposure of the test organ- isms to varying concentrations of the toxicants, employing the least concentration that would cause mortality or inhibit bio- luminescence, which was obtained from the preliminary range finding test as the highest concentration. Two options were adopted for acute toxicity test. These were the static with renewal option and the static without re- newal option. This test was conducted with reference to [20] and [21] Guidelines in Part III E, Section 4.3.2 of the ‘Envi- ronmental Guidelines and Standards for Petroleum Industry in Nigeria’ 2018. 2.11.1. Acute Toxicity Test for T. guineensis The static renewal option was employed for T. guineensis. The habitat water was renewed every 48 h, while the organisms were fed with commercial feed of 5% body weight every 24 h. The acute toxicity test involved exposure of test organisms to five different concentrations (i.e. 100%, 50%, 25%, 12.5% and 6.25%) of MDO, CDO, D, WAF, and CEWAF. The method is described as follows: 10 healthy accli- matized fish were introduced into an aerated laboratory glass aquarium (18” x 10” x 18”) into each unit of the different- concentrations. Uniformed concentrations were used for all crude oil systems to allow for comparability of results follow- ing CROSERF standards [22]. The setup was done in tripli- cate at room temperature (22 – 27oC). Mortality and impaired movement were the indices for scoring toxicity. Dead organ- isms were removed and counted at 0, 24, 48, 72 and 96 hours. Probit analysis [23] was used to analysze the number of mortali- ties recorded. This was carried out in order to establish the LC50 (median lethal concentration)of the reference toxicant [13]. 2.11.2. Acute Toxicity Test for P.africanus The static renewal option was also employed for P. africanus: 10 healthy acclimatized organisms were introduced into 1000 mL glass jar of each unit of different concentrations of toxicants. Control was SDS for dispersant, and habitat water for crude oil. The set-up was done at room temperature (22-27o C). Mortality was end point for toxicity. It was scored by or- ganisms’ inability to swim and immediately organisms settled at the bottom of the jar. These organisms were counted at 0, 24, 48, 72 and 96 h. Probit analysis [23] was used to analyze the number of mortality recorded. This was carried out in order to establish the LC50 (median lethal concentration). The toxi- city factor was also calculated with respect to the LC50 of the reference toxicant. 2.11.3. Acute Toxicity Test for Bacteria For bacteria, the static nonrenewal method was employed. 100%, 50%, 25%, 12.5% and 6.25% of MDO, CDO, D, WAF and CEWAF were introduced into 1 L glass beakers. The habi- tat water without toxicant was used as control. The experiment was set up in triplicate at 22 – 25oC. Aerobic plate count in nu- trient agar treated with antifungal antibiotics was used to eval- uate toxicity and was done at 0, 24, 48 and 96 hours. Probit analysis was used to analyze the number of mortality recorded. 2.11.4. Toxicity Factor The toxicity factor (TF) for dispersant and synergistic or joint action factor (JAF) for crude oil-plus-dispersant was de- termined using the formula described by Odiete (1999): T F = 96 h LC50 value o f dispersant 96 h LC50 value o f S DS JAF = 96 h LC50 o f dispersant − plus − crude − oil 96 h LC50 value o f cude oil 2.12. Statistical Analysis All analyses were performed in triplicates. Data were ex- pressed as mean±SD. Significant differences among parameters in the different test media were analysed using Analysis of Vari- ance (ANOVA) using SPSS 22.0 version. Data obtained from acute toxicity tests were also subjected to statistical analysis by Probitmethod. 3. Results and Discussions 3.1. Physicochemical Characteristics of the Crude Oil Test Media The physicochemical characteristics of MDO, CDO, CE- WAF, WAF, D and habitat water are presented in Tables 1- 6, respectively. The pH of all test media ranged from near-neutral to slightly alkaline. A gradual reduction in pH was observed with decreasing concentration of toxicant in CEWAF and WAF, while in MDO and CDO, the reduction was observed at toxicant concentration of 50%. A pH range of 7.9 - 8.0 has been previ- ously reported in a similar study [16, 24]. A pH range of 6.09 – 8.45 is reportedly ideal for supporting aquatic life including fish [25]; while, waters with a pH value less than 6.0 may result in stunted, reduced or even absent fish population [26]. The pH of the studied test media will therefore support aquatic habitation. The temperature of the test media fluctuated between 29.0◦C and 31.3◦C. The temperature of surface waters usually range between 0 – 30◦C, but can get to as high as 40◦C, de- pending on the season and the prevailing environmental con- dition. Temperature affects physical, chemical and biological 108 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 109 processes in water bodies and, therefore, influences the concen- tration of many variables. The mean concentration of TDS in the test media ranged as follows: 4365 – 4530 mg L−1in MDO; 4390 – 4435 mg L−1in CDO; 3915 – 4450mg L−1 in CEWAF, 3900 – 3910mg L−1 in WAF and from 4365 – 4410mg L−1 in D. There was no signif- icant difference (p<0.05) in TDS levels across the different test media. In terms of TDS, the water in all test media, including the sea water, can still be classified as fresh water, since their concentrations were less than 5,000mg L−1 [27]. The DO levels ranged from 8.07 – 11.1mg L−1 in MDO; 9.83 – 10.5mg L−1 in CDO; 10.3 – 10.9mg L−1 in CEWAF; 10.1 – 10.2mg L−1 in WAF; and from 10.5 – 13.1mg L−1 in D. A decreasing trend with decreasing concentration of toxi- cant in MDO and D was observed. Comparatively, CEWAF had slightly higher DO levels than WAF; while same trend was observed with MDO and CDO, but at toxicant concentration below 100%. The oxygen level in natural waters usually varies with salinity, temperature, turbulence, the photosynthetic activ- ity of algae and plants, and atmospheric pressure [28]. In fresh- waters, DO at sea level normally ranges from 15 mg L−1 at 0◦C to 8mg L−1 at 25◦C, while concentrations in unpolluted waters are usually close to, but less than, 10mg L−1. The DO levels in all test media indicated that the all media water were effectively aerated. The minimum acceptable DO levels that can maintain fish population in aquatic environment is reported to range be- tween 4 and 5mg L−1, while fish mortality occurs when DO levels are less than 3mg L−1 [29, 30]. The range of DO concen- tration that can support fisheries and aquatic lives is 5 – 9mg L−1 in the EU, 5 – 9.5mg L−1 in Canada, and 4 – 6mg L−1 in Russia [28]. Therefore, the toxicant levels were not sufficient to cause any drastic reduction in oxygen levels and hence, could support fish growth and survival. Biochemical oxygen demand (BOD5) levels in the river wa- ter were relatively high, ranging from 6.23 – 8.25mg L−1 in MDO; 4.02 – 9.17mg L−1 in CDO; 4.03 – 9.41mg L−1 in CE- WAF; 4.01 – 9.45mg L−1 in WAF; and from 4.02 – 6.95mg L−1 in D. High values were observed in the chemically enhanced media (i.e. CDO and CEWAF, respectively). Additionally, an increase in BOD5 levels was obtained with increasing concen- tration of toxicants in the different test media. With respect to BOD5 levels and aquatic pollution status of waters, BOD con- centrations < 1.0mg L−1 have been classified as being unpol- luted; BOD ≥ 2 ≤ 9mg L−1 has been classified as being mod- erately polluted, while BOD > 10 mg L−1 has been classified as being heavily polluted [31, 32, 33]. Similarly, the maxi- mum acceptable limits set by [21] and [34] are 10mg L−1 and 5.0mg L−1, respectively. Introduction of toxicants significantly altered the BOD levels in all test media at toxicant concentra- tions greater than 250 mg L−1. For chemical oxygen demand (COD), the concentrations ranged from 31.8 – 124.1mg L−1 in MDO; 20.0 – 62.4mg L−1in CDO; 21.5 – 64.3 in CEWAF; 20.0 – 64.2mg L−1in WAF; and from 20.0 – 48.3mg L−1in D. Water is considered relatively un- polluted when the COD levels is less than or equal to 20 mg L−1. In this study, the sea water could be said to be relatively unpolluted, with a COD concentration of 20.0 mg L−1. In the different test media and varying toxicant concentrations, as ex- pected, COD levels were all greater those of the sea water, and an increasing concentration was observed with increasing tox- icant concentration in all test media. This shows that there is a positive influence on the amount of oxidisable organic matter in water by the added toxicants. 3.2. Heavy Metals Content in Test Media The concentration of heavy metals at different exposure concentrations in test media are also presented in Tables 1-6 above. Ni had the highest concentration with respect to other metals in all test media. Pb was present in high amounts in D; and its concentration decreased with decreasing concentration of exposure test media. Trace metals such as Ni, V, Cu, Cd and Pb are naturally found in crude oil, and, water contaminated with crude oil may exhibit higher concentrations of these metals. The concentra- tion of Cu was within the 2 mg L−1permissible limit for drink- ing water in all test media and at the different exposure concen- trations. Pb is also a normal constituent of crude oil. Its maxi- mum permissible limit of 0.01 mg L−1 in drinking water was exceeded at all exposure concentrations in D. This indicates that the dispersant itself is a toxin of ecotoxicological impor- tance. In MDO and WAF, exposure concentrations < 50% were within the permissible limit for Pb.In CEWAF, only exposure concentration at 100% exceeded the permissible limit, while in CDO, Pb concentration was within the permissible limit in the different exposure concentrations. For CEWAF and CDO, some form of positive synergy between crude oil and disper- sant, which limited the bioavailability of Pb in the test media, was observed. Cd and Ni, like Pb and Cu, are also natural constituents of crude oil. Their permissible limits (0.003mg L−1 for Cd and 0.1mg L−1 for Ni) were significantly exceeded in all test media and at the different exposure concentrations. This also is in- dicative of some degree of toxicity due to Cd and Ni, to aquatic species. For Cr, its permissible limit of 0.05mg L−1 was slightly ex- ceeded even in sea water. With increasing toxicant concentra- tions, this limit was well exceeded in all test media, which im- plied that there will likely be some level of toxicity to aquatic species. Fe levels were slightly elevated in the different test me- dia compared to control medium. However, Fe concentrations were within the WHO permissible limit of 0.3 mg L−1 in MDO, WAF and D; but were slightly elevated in CDO and CEWAF. Zn, As and V were found in concentrations that were within their respective permissible limits in drinking water, hence, no toxic property is expected from these metals. 3.3. Total Petroleum Hydrocarbons and PAHs in Test Media The total concentration of TPH in the different test media are shown in Figures 1-3. The TPH concentrations did not vary significantly in MDO and CDO, but there were significant vari- ations in TPH levels in WAF and CEWAF test media. A limit value for TPH has been set at 10mg L−1 in many water contam- ination regulations, i.e., the maximum concentration of TPH 109 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 110 Table 1: Physicochemical characteristics of MDO test media Parameters Concentration of exposure medium (%) 100 50 25 12.5 6.25 pH 6.9±0.01a 7.00±0.02a 7.90±0.02a 6.90±0.02a 6.90±0.01a Temp. (◦C) 29.9±1.03a 31.1±0.5a 31.3±0.1a 29.1±0.1a 29.1±0.1a EC (uS/cm) 9060±3.1a 8770±2.5a 8860±2.1a 8810±3.0a 8750±2.1a TDS (mg/L) 4530±2.3a 4385±3.1a 4450±3.0a 4430±2.5a 4415±1.9a TSS (mg/L) 1.20±.0.5a 1.00±0.02a 1.05±0.1a 1.05±0.01a 1.05±0.01a Salinity (PSU) 0.50±0.03a 0.40±0.01a 0.40±0.01a 0.40±0.2a 0.40±0.2a DO (mg/L) 11.1±1.1a 9.80±0.1a 10.6±0.2a 10.9±1.1a 10.8±0.2a BOD (mg/L) 18.2±1.1a 13.0±2.1b 9.40±1.5b 4.70±1.1c 4.30±1.05c COD (mg/L) 124±2.5a 88.4±2.1b 64.3±1.8b 32.2±1.9c 29.7±1.5c Pb (mg/L) 0.17±0.01a 0.06±0.002b ND ND ND Cr (mg/L) 0.17±0.001a 0.13±0.001a 0.13±0.01a 0.13±0.01a 0.10±0.001a Cd (mg/L) 0.13±0.01a 0.11±0.01a 0.09±0.001b 0.08±0.01b 0.06±0.01b Ni (mg/L) 0.54±0.02a 0.75±0.15a 0.97±0.20b 1.19±0.25c 1.20±0.20c Fe (mg/L) 0.25±0.001a 0.39±0.01a 0.35±0.002a 0.30±0.001a 0.39±0.02a Cu (mg/L) 0.06±0.001a 0.16±0.02b 0.32±0.02c 0.45±0.02d 0.51±0.15d Zn (mg/L) 0.04±0.001a 0.05±0.001a 0.04±0.015a 0.04±0.002a 0.03±0001a V (mg/L) 0.09±0.001a 0.09±0.001a 0.09±0.001a 0.09±0.01a 0.09±0.01a values are expressed as mean ± standard deviation. Superscripts with same letters on the same row indicate no significant difference, while superscripts with different letters indicate significant difference at p < 0.05 Table 2: Physicochemical characteristics of CDO test media Parameters Concentration of exposure medium (%) 100 50 25 12.5 6.25 pH 6.82±0.20a 6.63±0.02a 6.86±0.01a 6.83±0.01a 6.81±0.02a Temp. (◦C) 29.1±0.25a 30.5±0.05a 29.0±0.10a 31.0±0.25a 29.0±1.05a EC (uS/cm) 8720±3.00a 8710±1.25a 8830±2.05a 8820±1.95a 8810±2.05a TDS (mg/L) 4410±2.05a 3925±0.50a 4415±1.05a 4415±2.05a 4410±1.85a TSS (mg/L) 1.05±0.50a 1.04±0.01a 1.06±0.01a 1.06±0.02a 1.06±0.01a Salinity (PSU) 0.43±0.10a 0.43±0.01a 0.44±0.01a 0.44±0.01a 0.44±0.02a DO (mg/L) 10.3±0.15a 10.3±0.15a 10.1±1.50a 10.3±1.95a 10.2±0.50a BOD (mg/L) 4.05±1.05a 4.03±1.00a 4.72±0.05a 4.05±0.01a 4.02±0.20a COD (mg/L) 21.9±0.95a 21.5±1.00a 32.1±1.05b 28.6±1.05b 22.9±1.05a Pb (mg/L) ND ND ND ND ND Cr (mg/L) 0.11±0.01a 0.11±0.02a 0.12±0.001a 0.12±0.02a 0.11±0.001a Cd (mg/L) 0.12±0.02a 0.16±0.15a 0.18±0.01a 0.18±0.04a 0.16±0.01a Ni (mg/L) 1.04±0.10a 2.19±0.02b 1.80±0.15b 1.20±0.15a 1.15±0.30a Fe (mg/L) 0.09±0.001a 1.00±0.001b 0.47±0.20c 0.47±0.20c 0.41±0.02c Cu (mg/L) 0.75±0.01a 0.30±0.03b 0.32±0.002b 0.31±0.10b 0.31±0.01b Zn (mg/L) 0.07±0.02a 0.07±0.02a 0.05±0.001a 0.05±0.001a 0.05±0.001a V (mg/L) 0.10±0.03a 0.08±0.001a 0.24±0.01b 0.21±0.001b 0.20±0.01b 110 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 111 Table 3: Physicochemical characteristics of WAF test media Parameters Concentration of exposure medium (%) 100 50 25 12.5 6.25 pH 7.38±0.01a 7.33±0.10a 7.29±0.25a 7.02±0.15a 6.98±0.01a Temp. (◦C) 29.5±1.05a 29.5±0.50a 29.3±1.05a 29.1±1.05a 29.1±0.50a EC (uS/cm) 7820±2.00a 7810±3.05a 7990±2.95a 7970±3.50a 7970±2.50a TDS (mg/L) 3915±1.95a 3910±2.95a 3905±2.50a 3900±2.80a 3900±1.85a TSS (mg/L) 0.55±0.01a 0.55±0.01a 0.49±0.10a 0.42±0.01a 0.40±0.15a Salinity (PSU) 0.39±0.01a 0.39±0.02a 0.43±0.01a 0.43±0.01a 0.43±0.02a DO (mg/L) 10.2±2.05a 10.2±0.10a 10.3±0.05a 10.2±0.05a 10.1±0.10a BOD (mg/L) 9.45±2.05a 9.13±0.20a 7.33±0.10a 4.79±0.10b 4.01±1.50b COD (mg/L) 64.3±1.05a 62.9±0.25a 46.3±0.55b 33.5±0.10c 20.8±0.05d Pb (mg/L) 0.88±0.01a 0.06±0.001b ND ND ND Cr (mg/L) 0.13±0.01a 0.09±0.001a 0.07±0.001a 0.03±0.001b 0.008±0.001c Cd (mg/L) 0.24±0.001a 0.19±0.02a 0.17±0.001a 0.12±0.01b 0.11±0.01b Ni (mg/L) 0.45±0.01a 0.75±0.02a 0.97±0.03b 1.19±0.40c 0.54±0.002a Fe (mg/L) 0.40±0.01a 0.40±0.001a 0.40±0.01a 0.30±0.01a 0.25±0.02a Cu (mg/L) 0.50±0.02a 0.50±0.01a 0.50±0.01a 0.40±0.01a 0.06±0.002a Zn (mg/L) 0.04±0.001a 0.04±0.001a 0.04±0.001a 0.04±0.001a 0.04±0.001a V (mg/L) 0.14±0.001a 0.09±0.001a 0.09±0.001a 0.09±0.001a 0.09±0.001a Table 4: Physicochemical characteristics of CEWAF test media Parameters Concentration of exposure medium (%) 100 50 25 12.5 6.25 pH 7.98±0.15a 6.99±0.02a 6.93±0.15a 6.82±0.15a 6.63±0.02a Temp. (◦C) 31.3±0.10a 29.1±0.05a 29.1±0.10a 29.1±1.05a 30.5±0.05a EC (uS/cm) 8860±2.05a 8810±0.30a 8750±1.55a 8720±2.55a 8710±1.65a TDS (mg/L) 4450±3.15a 4430±1.05a 4415±1.50a 4410±1.50a 3925±1.05a TSS (mg/L) 1.05±0.01a 1.07±0.15a 1.06±0.10a 1.05±0.10a 1.04±0.01a Salinity (PSU) 0.44±0.01a 0.44±0.01a 0.43±0.01a 0.43±0.01a 0.43±0.01a DO (mg/L) 10.6±1.55a 10.9±1.05a 10.8±0.20a 10.3±1.55a 10.3±0.01a BOD (mg/L) 9.41±1.05a 4.70±0.15b 4.25±1.05b 4.06±0.25b 4.03±0.01b COD (mg/L) 64.3±2.05a 32.1±1.55b 29.7±0.20b 21.9±1.05c 21.5±0.50c Pb (mg/L) ND ND ND ND ND Cr (mg/L) 0.10±0.01a 0.10±0.001a 0.12±0.001a 0.12±0.01a 0.10±0.01a Cd (mg/L) 0.16±0.01a 0.15±0.01a 0.18±0.02a 0.15±0.001a 0.15±0.001a Ni (mg/L) 1.40±0.10a 0.97±0.15b 0.80±0.02b 0.80±0.02b 0.57±0.025c Fe (mg/L) 0.43±0.20a 0.20±0.001b 0.13±0.001c 0.09±0.001c 0.08±0.01c Cu (mg/L) 0.27±0.01a 0.16±0.01b 0.13±0.01b 0.13±0.001b 0.12±0.01b Zn (mg/L) 0.04±0.001a 0.04±0.01a 0.04±0.01a 0.04±0.01a 0.04±0.015a V (mg/L) 0.23±0.10a 0.18±0.01a 0.17±0.02a 0.17±0.02a 0.17±0.001a 111 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 112 Table 5: Physicochemical characteristics of Dispersant test media Parameters Concentration of exposure medium (%) 100 50 25 12.5 6.25 pH 7.38±0.01a 7.33±0.10a 7.29±0.25a 7.02±0.15a 6.98±0.01a Temp. (◦C) 29.5±1.05a 29.5±0.50a 29.3±1.05a 29.1±1.05a 29.1±0.50a EC (uS/cm) 7820±2.00a 7810±3.05a 7990±2.95a 7970±3.50a 7970±2.50a TDS (mg/L) 3915±1.95a 3910±2.95a 3905±2.50a 3900±2.80a 3900±1.85a TSS (mg/L) 0.56±0.01a 0.55±0.01a 0.49±0.10a 0.42±0.01a 0.40±0.15a Salinity (PSU) 0.39±0.01a 0.39±0.02a 0.43±0.01a 0.43±0.01a 0.43±0.02a DO (mg/L) 10.2±2.05a 10.3±0.10a 10.3±0.05a 10.2±0.05a 10.1±0.10a BOD (mg/L) 9.45±2.05a 9.13±0.20a 7.33±0.10a 4.78±0.10b 4.01±1.50b COD (mg/L) 64.3±1.05a 62.9±0.25a 46.3±0.55b 33.5±0.10b 20.8±0.05c Pb (mg/L) 0.85±0.25a 0.73±0.25a 0.54±0.01a 0.52±001a 0.08±0.01b Cr (mg/L) 0.13±0.01a 0.08±0.01a 0.07±0.02a 0.07±0.01a 0.08±0.015a Cd (mg/L) 0.20±0.001a 0.19±0.01a 0.16±0.02a 0.14±0.01a 0.13±0.01a Ni (mg/L) 1.04±0.50a 0.99±0.01a 0.88±0.025a 0.80±0.02a ND Fe (mg/L) ND ND ND ND ND Cu (mg/L) 0.45±0.05a 0.40±0.01a 0.36±0.01a 0.40±0.01a 0.30±0.01a Zn (mg/L) 0.05±0.01a 0.04±0.01a 0.04±0.001a 0.04±0.001a 0.03±0.001a V (mg/L) ND ND ND ND ND Table 6: Physicochemical characteristics of habitat water Parameters Concentration of brackish water (%) pH 7.23±0.01 Temp. (◦C) 31.3±0.50 EC (uS/cm) 8780±1.05 TDS (mg/L) 4390±1.00 TSS (mg/L) 1.04±0.01 Salinity (PSU) 0.43±0.01 DO (mg/L) 9.20±0.02 BOD (mg/L) 4.71±0.50 COD (mg/L) 20.0±0.50 Pb (mg/L) ND Cr (mg/L) 0.08±0.001 Cd (mg/L) 0.20±0.01 Ni (mg/L) 0.42±0.02 Fe (mg/L) ND Cu (mg/L) 0.12±0.001 Zn (mg/L) 0.37±0.015 V (mg/L) 0.16±0.001 should be present in waters during the discharge of oilfield wa- ters (Özdemir, 2018). For drinking water, the limit is 0.5 mg L−1 [35] 2008) and 10 mg L−1 [21]. In this study, the TPH level in sea water was 4.43mg L−1, which was within the DPR limit. In the test media, the TPH concentrations ranged from 25.8 – 914.7 mg L−1 in CDO; 19.8 – 239.9mg L−1 in CEWAF; 7.5 – 21.8 mg L−1 in WAF; and from 4.30 – 11.1mg L−1 in D. The levels in D were generally within the limit for TPH in discharged oilfield waters, but well above the drinking water limit. For all crude oil-based media, significant pollution was observed. Results of PAHs concentration in the different test media are presented in Table 7. Only two concentrations of the differ- ent test media (i.e., 100% and 50%) were reported in this study due to the significance of their results relative to the other con- centrations. A decreasing concentration of PAHs was observed in all test media with decreasing concentration of test media. When compared with the PAH concentration of the crude oil (464.3 mg L−1), there was a significant drop in PAHs concen- tration in the crude oil test media. A maximum PAHs concen- tration of 55.1 mg L−1 was observed in CDO test media con- centration of 100%, while the least concentration of 2.04mg L−1 was found in D test media concentration of 50%. All 16 US EPA priority PAHs were detected in WAF test media, with ?16PAHs concentrations of 21.3mg L−1and 12.5mg L−1 at tox- icant concentrations of 100% and 50%, respectively. The least ?16PAHs concentrations was detected in D, with a concentration of 2.04mg L−1.A value of 0.2 µgL−1 (0.0002 mg L−1) has been set as the maximum permissible limit for total PAHs in drink- ing water, while that of benzo{a}pyrene is 0.1 µgL−1 (0.0001 mg L−1) [34]. The results of this study indicated that all test me- dia, including the control, had ?16PAHs that greatly exceeded the permissible limit by several orders of magnitude. This in- 112 Onokare et al. / J. Nig. Soc. Phys. Sci. 4 (2022) 105–116 113 dicated a strong likelihood for significant bioaccumulation of these contaminants in aquatic organisms. For benzo{a}pyrene, which is regarded as the most carcinogenic of all PAHs, it was detected in all test media except CEWAF and the control. Figure 1: Comparing the concentration of TPH in MDO and CDO Figure 2: Comparing the concentration of TPH in WAF and CEWAF Figure 3: Concentration of TPH in D 3.4. Acute toxicity test result for test organisms The acute toxicity of the control toxicant and the different test media on the T. guineensis is presented as the 96h median lethal concentration (LC50) as shown in Tables 8, 9 and 10, re- spectively. The LC50 of the different toxicant test media on T. guineensis followed the order: SDS > MDO > CDO > CE- WAF > WAF > D. Following the description on 96h LC50, the reference toxicant was moderately toxic, with a value of 1.37. The dispersant solution exhibited the least toxicity, and was non-toxic to T. guineensis, having a value greater than 1,000. Several studies have also shown that SDS exhibited the great- est toxicity to marine organisms relative to the dispersant and the dispersant plus crude oil test system [11, 12]. The different exposure media ranged from being non-toxic to being practi- cally non-toxic. The toxicity factors (i.e. the index for assess- ing toxicity) showed that SDS was 1,380 times more toxic to T. guineensis that the dispersant, and 65.7 times more toxic to T. guineensis than MDO. Acute toxicity tests on P. africanus showed that the chem- ically modified test media (i.e., CDO and CEWAF) exhibited slightly greater toxicities to P. africanus than MDO and D; but lesser toxicities compared to the reference SDS toxicant (Table 9). The LC50 of the different toxicant test media followed the order: SDS > CDO > CEWAF> WAF > MDO > D. The disper- sant also exhibited the least toxicity to P. africanus. For the two tested organisms, which are representatives of the secondary and tertiary consumers in the marine food chain, Ecobestr dis- persant was shown to be lesstoxic to both test organisms. How- ever, the LC50 test result suggested that P. africanus was more tolerant to SDS relative to T. guineensis, as seen in the LC50 values for SDS in both organisms. The toxicity of the different dispersants on the two types of microbial populations were determined using the 96hLC50 of the toxicants on the test organisms are presented in Table 9. The toxicity of the toxicant test media followed the order: CE- WAF < D