Environ Sci Pollut Res DOI 10.1007/s11356-014-2539-y
RESEARCH ARTICLE
Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test Arnold V. Hallare & Paulo Lorenzo S. Ruiz & J. C. Earl D. Cariño
Received: 28 July 2013 / Accepted: 8 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, nonformation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.
Keywords Zebrafish . Danio rerio . Embryotoxicity . Jatropha curcas . Phorbol esters . Seed cake . ZFET test Responsible editor: Henner Hollert A. V. Hallare (*) : P. L. S. Ruiz : J. C. E. D. Cariño Department of Biology, CAS, University of the Philippines, Manila, Padre Faura St., Manila 1000, Philippines e-mail: [email protected]
Introduction The nonrenewable nature of fossil fuels as an energy resource and the growing concern on the issue of climate change as induced by greenhouse gases (GHGs) emissions have presented the twenty-first century with the challenge on environmental sustainability (Ahmad et al. 2011; Karaj and Muller 2011; Sahoo and Das 2009). The constant pressure on the environment is feared to outdo ongoing mitigation programs, which could lead to unsustainable environment. Confronted with the specters of fossil fuel depletion and environmental degradation, the recent years have seen substantial development in the areas of renewable energy resource and environmental protection (Kumar et al. 2012a). Currently being viewed as an alternative for conventional fossil fuel is the biodiesel (Kouame 2011). Biodiesel is a renewable and biodegradable substitute and is expected to benefit the environment by alleviating impacts caused by pollution and the global climate change (Kumar et al. 2012b; Hill et al. 2006). GHG emissions from its combustion are reportedly lower than conventional types of transport fuels (da Cruz et al. 2012). The optimistic potential in biodiesel presented an impetus to what is presently a rising interest in the global scale expansion of biodiesel production, which is perceived by a number of legal policies to coincide with the concept of sustainable environment, that is, meeting the needs of the present generation concomitant to preserving the environment for the generations that follow (Bluhm et al. 2012; Silitonga et al. 2011). These merits are expected to mark an appreciation in biodiesel production, which is through solvent-catalyzed transesterification of vegetable oils, animal fats, and recycled oil (Mondala et al. 2009). Among the many advantages of biodiesel as a replacement for conventional fuel is the wide array of available feedstock (Atadashi et al. 2010). The plant Jatropha curcas has been gaining considerable attention as a sustainable biodiesel crop
Environ Sci Pollut Res
(Makkar and Becker 2009). Biodiesel production from nonedible J. curcas seeds is a promising approach because it raises no competition with food resources, particularly edible oils that presently serve as the most common feedstock (Shuit et al. 2010). Furthermore, the Jatropha species is able to meet two primary requirements in selecting for a feedstock: lowcost and large-scale production (Atabani et al. 2012). The full extent of environmental benefits by J. curcas is overwhelming and would remain as an active area of research in the coming years. At present, however, biosafety considerations are being raised on the utilization of Jatropha seed cake (Pandey et al. 2012). The seed cake is a by-product of biodiesel production, which, in part, involves the mechanical pressing of Jatropha seeds to acquire the oil (Francis et al. 2005). The merits of biodiesel production, in general, have been operating on the assertion of biodegradability and exhaust emissions reduction (Leme et al. 2012). Variables such as post-production risk consideration, among others, may have been sidestepped. Makkar and Becker (1997) have mentioned that a hectare of Jatropha plantation would produce an estimated mass of 1 t of seed cake. In the perceivable future, India alone is expected to contribute 20 million tons of Jatropha seed cake annually (Gubitz et al. 1999). In 2017, approximately 95 % of total J. curcas production for biodiesel is expected to come from developing countries in Asia (Makkar et al. 2008; Siang 2009). This would constitute a significant volume of residual organic biomass that could present serious problems on safe waste disposal (Gubitz et al. 1999). While several strategies for seed cake recycling and processing are standing, such as a valuable source of organic manure (Francis et al. 2005), feedstock of biogas production (Subramanian et al. 2005), briquette for industrial and domestic combustion (Singh et al. 2008), straight soil fertilizer (Phasukarratchai et al. 2012), and edible crop fertilizer (Gubitz et al. 1999), the quagmire on the fate of the seed cake, as presented by its sheer volume, is further confounded by its carcinogenic components. The seed cake retains phorbol esters, which are identified as the main toxic constituent of J. curcas (Kumar et al. 2012a; Punsuvon et al. 2012; El Rafei et al. 2011; Karaj and Muller 2011; Rakshit et al. 2008; Menezes et al. 2006). Phorbol esters mainly activate the enzyme protein kinase C (Ratnadass and Wink 2012). They are implicated in cell proliferation and tumor promotion (Goel et al. 2007; Gubitz et al. 1999; Adolf et al. 1984). For a very recent review on the chemistry of phorbol ester toxicity in Jatropha seed, the readers are directed to Wakandigara et al. 2013. Makkar et al. (2008, 2009) have reported that 70–75 % of phorbol ester comes with the oil during mechanical extraction from seeds, while the remainder is retained in the seed cake. Bluhm et al. (2012) have posited that assessment efforts should not only unilaterally cover the positive potentials of biodiesel, such as sustainability and emissions reduction, but also take a hard look on the accompanying risks including
ecotoxicological ramifications, which arise from production and may pose as environmental problems themselves. On another light, Devappa et al. (2012) have recognized the lapse on the end of biodiesel producers in developing countries where expertise and facilities for the biological screening of J. curcas seed ecotoxicity is concerned, particularly in the context of advanced fields of chromatography and spectroscopy. A fast and useful approach that they recommended involves the application of biological test systems or bioassays that can screen for toxic phytochemicals in mixture forms, to which the Jatropha seed cake belongs. Heger et al. (2012) even suggested the use of a whole battery of biotests for investigations on biofuel safety. In response to the above suggestions, we have utilized the potential of disposed seed cake extracts to induce teratogenic or embryotoxic effects on aquatic organisms using zebrafish as a vertebrate model. Since much is already known concerning the carcinogenic nature of phorbol esters, it becomes more practical to use other toxicity endpoints to contribute to a broader view of the whole range of toxicity mechanisms for this toxicant. The assessment of embryotoxic potential offers very high ecotoxicological relevance since effects on embryo growth and ontogeny could be extrapolated to assess effects at population levels (Hallare et al. 2006; Hollert et al. 2003). The evaluation of embryotoxic effects of environmental contaminants that impose exogenous stresses in development of organisms currently relies in systems using zebrafish (Danio rerio) embryos (Bui et al. 2012; Kaiser et al. 2012; Li et al. 2012; Hsu et al. 2010; Oliveira et al. 2009; Chen et al. 2008). As a model organism, zebrafish embryos are useful in testing for chemicals with putative inhibitory or interfering effects on developmental processes (Berry et al. 2007). This suitability as a vertebrate model system in studying developmental toxicity is based on the many molecular, cellular, biochemical, morphological, and physiological characteristics that are conserved in and shared by zebrafish with mammals (Ali et al. 2011; Carlsson et al. 2011; Reimers et al. 2006), with particular emphasis on similarities in embryonic development (Weigt et al. 2011). Broad homologies in the genome and neural system also mutually exist between the two groups (Ali et al. 2012). Following detailed characterization of zebrafish development, there is also room for easier comparison and verification between results obtained through models utilizing the zebrafish embryo and studies on mammalian developmental toxicity (Reimers et al. 2004). Extensive information from past and ongoing researches has led to the elucidation of normal zebrafish biological parameters, which is important in the comparison against aberrations that are induced experimentally (Segner 2009). Zebrafish have many properties that emerge as integral and advantageous aspects of testing for developmental toxicity. They are characterized with high fecundity and rapid
Environ Sci Pollut Res
development (Cowden et al. 2012). Zebrafish embryos are easily accessed and manipulated, so that experimental variables administered at the molecular and cellular levels are capable of being observed in the intact, multicellular embryo stage (Krone et al. 2005). These embryos are mostly transparent that, coupled with an external type of development, prove suitable for continuous and unobstructed observation (Li et al. 2009; Pyati et al. 2007; Reimers et al. 2006). Their relatively small size makes rearing in micro-wells and micro-titer plates used in screening possible (Cowden et al. 2012). Moreover, they can be produced in large clutch sizes out of economic husbandry requirements. It cannot be stressed enough that on top of these attributes is another crucial property, i.e., high sensitivity of the early developmental stages of zebrafish, consistent with several species of fish, that enables them to readily absorb chemicals (Morin et al. 2011; Selderslaghs et al. 2009; Osman et al. 2007; Hallare et al. 2006; Marguerie et al. 2006). The study aimed to determine the inherent embryotoxicity of J. curcas seed cake on zebrafish. Specifically, the study aimed to determine the (1) LC50 of the J. curcas seed cake and the (2) morphological aberrations in zebrafish embryos induced by the J. curcas seed cake.
Materials and methods Ethical considerations The study (protocol no. 2012–015) was submitted for review to the University of the Philippines Manila–National Institutes of Health (NIH) Institutional Animal Care and Use Committee and had been granted approval for implementation on 9 October 2012.
Procurement and maintenance of zebrafish broodstock A breeding stock composed of 20 male and 10 female zebrafish, aged about 4 to 6 months, was procured from a tropical fish farm maintained and located at Purok 5, Barangay Pansol, Pila, Laguna. The spawners were placed in oxygensaturated Calypso™ bags. It was ensured that they were free of macroscopically discernable symptoms of infection, and not treated with any pharmaceutical (acute or prophylactic) agents 2 months prior to spawning. In the laboratory, the spawners underwent initial acclimation with careful addition of dechlorinated water equal to about half the original volume of water in the transportation bags. The procedure was carried out twice between a 5-min interval. Spawners were maintained in an aquarium with a minimum loading capacity of 1-L water per fish. Initial preparations included cleaning the aquarium interior with detergent and rinsing for several times with tap water. Approximately 40-L tap water dechlorinated with 5-mL NutraFin Aqua Plus™ was used to fill the aquarium. A fixed 12:12 h light photoperiod was observed in the duration of the experiment. Oxygen saturation of about 80 % was maintained; water temperature was monitored at 26±1 °C; and pH was recorded at 6.6–8.5. Excess filtering rates that may have caused heavy perturbation of the water was avoided. The semi-static condition guaranteed that traces of ammonia, nitrite, and nitrate levels were kept below the critical limit for toxicity. Filters were cleaned once every 2 weeks and tank water was changed on a weekly basis. In the process of water replacement, one-fifth of the original tank water volume was replaced with dechlorinated tap water. Fish were fed with commercially available diets (TetraMin™ flakes) thrice daily. Overfeeding was strictly monitored to avoid pollution of the tank water.
Collection of test substance
Preparation of test solutions
By the time of collection, the Department of Science and Technology (DOST) was undertaking pilot tests on the efficiency of J. curcas as a biodiesel source. The procedures included procurement of seeds and extraction of J. curcas oil. The seed cake is a residual by-product of mechanical oil extraction. Seed cake samples were stacked at the DOST– Industrial Technology Development Institute (ITDI)’s Jatropha Oil/Biodiesel Processing Facility and Analytical Laboratory (DOST-PAFC joint project) located at the DOST Compound, General Santos Avenue, Bicutan, Taguig City. This study utilized J. curcas seed cake samples that were immediately collected after oil extraction in ITDI. The samples were contained within black plastic bags and stored securely at 4 °C in a polystyrene jar throughout the duration of the biotest.
Reconstituted water was prepared based on the protocol set by ISO 7346/3. Each liter of distilled water contained 294.0 mg CaCl2.H2O, 123.3 mg MgSO4.7H2O, 63.0 mg NaHCO3, and 5.5 mg KCl. Parameters like pH (maintained at 6.5–8.5 using 1-M HCl or NaOH as necessary), conductivity, and temperature (kept at 26±1 °C) were monitored after the preparation. Conductivity and pH were measured using a Eutech™Cyberscan Series 600 portable meter. On the other hand, J. curcas seed cake samples were ground and disaggregated using mortar and pestle. For every exposure test, a stock solution was prepared by dissolving 50-g seed cake in 1-L reconstituted water (initially aerated for at least 12 h). Test solutions of varying concentration were prepared from the stock solution by dilution. Reconstituted water served as dilution water. An exposure test
Environ Sci Pollut Res
was defined as a setup inclusive of test solutions and control treatments. Collection of zebrafish embryos On the day prior to exposure, a tray covered with plastic mesh (1.25 mm) was placed at the bottom of the aquarium before the onset of darkness. Artificial plants made of green plastic were fixed to the grid and served as spawning stimuli. The tray was removed 30 min after the onset of light in the morning when mating, spawning, and fertilization have already taken place. Exposure tests A series of range-finding tests was performed. Five concentrations were derived for the proper test, which was repeated independently thrice. The highest concentration was preferred to cause 100 % lethality while the lowest concentration was estimated to give zero toxicity. A constant factor between the concentration groups not exceeding 2.2 was considered. Pure reconstituted water served as both negative control and internal control. For the positive control, a solution of 3,4Dichloroaniline (4.4 mg dissolved in 1-L Millipore MilliQ™ water) was used. Collected eggs were rinsed with reconstituted water and transferred to petri plates not later than 1-h post fertilization (hpf). The plates served as preliminary test chambers for the test solutions of varying concentration and controls. At about 2 hpf, viable eggs from each petri plate were replaced to a 24-well plate using a micropipette with a widened tip. Each 24-well plate served as proper test chamber. Twenty eggs were transferred individually to each well, which contained 2 ml of the test solution with same concentration as the source petri plate. The remaining four wells served as internal control containing reconstituted water. Separate plates for the positive control and external negative control were provided for each exposure test. All 24-well plates were pre-saturated with assigned treatments for 24 h. Prior to egg transfer, the wells were emptied and added with 2-ml freshly prepared treatment. Viable eggs corresponded to zebrafish embryos undergoing cleavage with no apparent irregularity, such as asymmetry or vesicle formation. Viability was screened under a stereomicroscope. Evaluation of embryotoxicity The toxicity of J. curcas on zebrafish embryos was evaluated based on observable morphological parameters. Three arbitrary time points of observation following exposure were identified as follows: 24, 48, and 72 hpf. An embryo was considered dead if it demonstrated any of four lethal endpoints, namely coagulation, non-formation of somites, and
non-detachment of tail (observable beginning 24 hpf), and absence of heartbeat (observable from 48 hpf). Observations were conducted in an Olympus™MIC-D digital microscope (×80 magnification) connected to a laptop computer. Scores were treated in binominal convention. Surviving embryos received a score of 0, while an embryo that presented with a lethal endpoint earned a score of 1. A score for overall mortality was a summation of the counts of death recorded from the four lethal endpoints. Percent mortality for a test solution was calculated as the number of dead embryos over the number of embryos at the start of exposure (n=20). Observable sublethal effects, which could refer to lack of pigmentation, yolk sac edema, pericardial edema, and spinal deformation, were recorded at 72 hpf. Statistical analysis LC50 estimates with 95 % confidence for the three time points of observation were computed using Probit Analysis. The statistical significance of mortality arising as an effect of concentration was tested using the Kruskal-Wallis ANOVA among groups. The Mann–Whitney U test was used to test for statistical significance between groups. These were performed with the SPSS v21 statistical software.
Results Reconstituted water parameters Throughout the test, the pH of reconstituted water ranged from 8.0 to 8.1, while temperature remained fairly constant at 26±1 °C. These were within acceptable limits specified by the zebrafish embryo toxicity (ZFET) standard operation procedure (OECD 2006). The pH for the test solutions range from 6.6 to 7.6, which is in accordance with the optimal pH tolerance range for zebrafish set at 6.0 to 8.0. Oxygen was ensured to be at optimal levels by fully aerating the test solutions prior to exposure. Conductivity for the test solutions was consistent between 510 and 540 uS/cm. Test solutions The series of range-finding tests, performed in order to pinpoint five mass concentrations of J. curcas seed cake used in the test proper, revealed a range between 2.15 and 1.00 g/L with a constant factor of 1.21. This was based on actual mortalities on exposed zebrafish embryos recorded at 24 hpf, following observations throughout the tests that percent mortalities at the two subsequent time points (48 and 72 hpf) presented no apparent increase. All percent mortalities were taken as observed deaths relative to the total number of embryos at the start of exposure.
Environ Sci Pollut Res
Overall toxicity and LC50
Lethal endpoints
As reasonably established by preliminary testing, zebrafish embryos exposed to 2.15 g/L of J. curcas seed cake (highest concentration) exhibited full toxicity, while embryos tested in 1.00 g/L (lowest concentration) showed zero toxicity. Decreasing partial toxicity was observed in the three consecutive concentrations bounded by the highest and lowest groups, respectively. The mean values of percent mortality experienced by zebrafish embryos following exposure to J. curcas seed cake for the three time points of observation are summarized in Table 1. The effect of concentration on mortality among the five groups was found to be highly significant (p= 0.008). All concentration groups, except the lowest, were statistically significant (p< 0.05) from the negative control. Toxicity was seen to manifest at the earliest time point of observation (24 hpf). For the later time points (48 and 72 hpf), all surviving embryos remained alive, but most of them manifested abnormality. Mortality in the five concentrations therefore remained constant across all time points for the three independent tests performed. This concomitantly corresponded to a single value of mean LC50 for the three observation times. The mean LC50 estimate was determined at 1.61 g/L between an interval of 1.51 and 1.71 g/L with 95 % confidence (Fig. 1). Per ZFET Validation Report specifications (OECD 2011), the mean partial toxicities of 65, 28.3, and 8.3 %, which were respectively elicited by the three consecutive concentration groups (1.78, 1.47, 1.21 g/L), held as informative, i.e., provided good information on the LC50.
As indicated earlier, embryotoxicity is a consideration that answers to a positive outcome in any of four morphological endpoints namely, coagulation, non-formation of somites, and non-detachment of tail, observable beginning 24 hpf, as well as the absence of heartbeat observable beginning 48 hpf. Data on these parameters, which cumulatively consisted of the overall embryotoxicity, are summarized in Table 1. Embryo coagulation was notable as the most frequent parameter recorded from higher concentrations in the wider range-finding tests (data not shown). In the test proper, it accounted for 8 of 60 deaths observed in the three independent tests for the highest concentration (mean mortality of 13.3 out of 100 %; N=3, 20 samples per N). The frequency of coagulation gradually decreased in the lower concentrations. Effect of concentration was insignificant statistically (p=0.144). Non-formation of somites represented 63.3 % of full toxicity in zebrafish embryos (mortality equals 100 %) in the highest concentration, 18.3 in 65 % of observed deaths in the second highest concentration, and 1.67 in 28.3 % of mortalities in the third highest concentration. This endpoint was not elicited by the two lowest concentrations, but its concentration-dependent occurrence tested significantly (p= 0.013). The three highest concentrations were statistically significant (p
RESEARCH ARTICLE
Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test Arnold V. Hallare & Paulo Lorenzo S. Ruiz & J. C. Earl D. Cariño
Received: 28 July 2013 / Accepted: 8 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, nonformation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.
Keywords Zebrafish . Danio rerio . Embryotoxicity . Jatropha curcas . Phorbol esters . Seed cake . ZFET test Responsible editor: Henner Hollert A. V. Hallare (*) : P. L. S. Ruiz : J. C. E. D. Cariño Department of Biology, CAS, University of the Philippines, Manila, Padre Faura St., Manila 1000, Philippines e-mail: [email protected]
Introduction The nonrenewable nature of fossil fuels as an energy resource and the growing concern on the issue of climate change as induced by greenhouse gases (GHGs) emissions have presented the twenty-first century with the challenge on environmental sustainability (Ahmad et al. 2011; Karaj and Muller 2011; Sahoo and Das 2009). The constant pressure on the environment is feared to outdo ongoing mitigation programs, which could lead to unsustainable environment. Confronted with the specters of fossil fuel depletion and environmental degradation, the recent years have seen substantial development in the areas of renewable energy resource and environmental protection (Kumar et al. 2012a). Currently being viewed as an alternative for conventional fossil fuel is the biodiesel (Kouame 2011). Biodiesel is a renewable and biodegradable substitute and is expected to benefit the environment by alleviating impacts caused by pollution and the global climate change (Kumar et al. 2012b; Hill et al. 2006). GHG emissions from its combustion are reportedly lower than conventional types of transport fuels (da Cruz et al. 2012). The optimistic potential in biodiesel presented an impetus to what is presently a rising interest in the global scale expansion of biodiesel production, which is perceived by a number of legal policies to coincide with the concept of sustainable environment, that is, meeting the needs of the present generation concomitant to preserving the environment for the generations that follow (Bluhm et al. 2012; Silitonga et al. 2011). These merits are expected to mark an appreciation in biodiesel production, which is through solvent-catalyzed transesterification of vegetable oils, animal fats, and recycled oil (Mondala et al. 2009). Among the many advantages of biodiesel as a replacement for conventional fuel is the wide array of available feedstock (Atadashi et al. 2010). The plant Jatropha curcas has been gaining considerable attention as a sustainable biodiesel crop
Environ Sci Pollut Res
(Makkar and Becker 2009). Biodiesel production from nonedible J. curcas seeds is a promising approach because it raises no competition with food resources, particularly edible oils that presently serve as the most common feedstock (Shuit et al. 2010). Furthermore, the Jatropha species is able to meet two primary requirements in selecting for a feedstock: lowcost and large-scale production (Atabani et al. 2012). The full extent of environmental benefits by J. curcas is overwhelming and would remain as an active area of research in the coming years. At present, however, biosafety considerations are being raised on the utilization of Jatropha seed cake (Pandey et al. 2012). The seed cake is a by-product of biodiesel production, which, in part, involves the mechanical pressing of Jatropha seeds to acquire the oil (Francis et al. 2005). The merits of biodiesel production, in general, have been operating on the assertion of biodegradability and exhaust emissions reduction (Leme et al. 2012). Variables such as post-production risk consideration, among others, may have been sidestepped. Makkar and Becker (1997) have mentioned that a hectare of Jatropha plantation would produce an estimated mass of 1 t of seed cake. In the perceivable future, India alone is expected to contribute 20 million tons of Jatropha seed cake annually (Gubitz et al. 1999). In 2017, approximately 95 % of total J. curcas production for biodiesel is expected to come from developing countries in Asia (Makkar et al. 2008; Siang 2009). This would constitute a significant volume of residual organic biomass that could present serious problems on safe waste disposal (Gubitz et al. 1999). While several strategies for seed cake recycling and processing are standing, such as a valuable source of organic manure (Francis et al. 2005), feedstock of biogas production (Subramanian et al. 2005), briquette for industrial and domestic combustion (Singh et al. 2008), straight soil fertilizer (Phasukarratchai et al. 2012), and edible crop fertilizer (Gubitz et al. 1999), the quagmire on the fate of the seed cake, as presented by its sheer volume, is further confounded by its carcinogenic components. The seed cake retains phorbol esters, which are identified as the main toxic constituent of J. curcas (Kumar et al. 2012a; Punsuvon et al. 2012; El Rafei et al. 2011; Karaj and Muller 2011; Rakshit et al. 2008; Menezes et al. 2006). Phorbol esters mainly activate the enzyme protein kinase C (Ratnadass and Wink 2012). They are implicated in cell proliferation and tumor promotion (Goel et al. 2007; Gubitz et al. 1999; Adolf et al. 1984). For a very recent review on the chemistry of phorbol ester toxicity in Jatropha seed, the readers are directed to Wakandigara et al. 2013. Makkar et al. (2008, 2009) have reported that 70–75 % of phorbol ester comes with the oil during mechanical extraction from seeds, while the remainder is retained in the seed cake. Bluhm et al. (2012) have posited that assessment efforts should not only unilaterally cover the positive potentials of biodiesel, such as sustainability and emissions reduction, but also take a hard look on the accompanying risks including
ecotoxicological ramifications, which arise from production and may pose as environmental problems themselves. On another light, Devappa et al. (2012) have recognized the lapse on the end of biodiesel producers in developing countries where expertise and facilities for the biological screening of J. curcas seed ecotoxicity is concerned, particularly in the context of advanced fields of chromatography and spectroscopy. A fast and useful approach that they recommended involves the application of biological test systems or bioassays that can screen for toxic phytochemicals in mixture forms, to which the Jatropha seed cake belongs. Heger et al. (2012) even suggested the use of a whole battery of biotests for investigations on biofuel safety. In response to the above suggestions, we have utilized the potential of disposed seed cake extracts to induce teratogenic or embryotoxic effects on aquatic organisms using zebrafish as a vertebrate model. Since much is already known concerning the carcinogenic nature of phorbol esters, it becomes more practical to use other toxicity endpoints to contribute to a broader view of the whole range of toxicity mechanisms for this toxicant. The assessment of embryotoxic potential offers very high ecotoxicological relevance since effects on embryo growth and ontogeny could be extrapolated to assess effects at population levels (Hallare et al. 2006; Hollert et al. 2003). The evaluation of embryotoxic effects of environmental contaminants that impose exogenous stresses in development of organisms currently relies in systems using zebrafish (Danio rerio) embryos (Bui et al. 2012; Kaiser et al. 2012; Li et al. 2012; Hsu et al. 2010; Oliveira et al. 2009; Chen et al. 2008). As a model organism, zebrafish embryos are useful in testing for chemicals with putative inhibitory or interfering effects on developmental processes (Berry et al. 2007). This suitability as a vertebrate model system in studying developmental toxicity is based on the many molecular, cellular, biochemical, morphological, and physiological characteristics that are conserved in and shared by zebrafish with mammals (Ali et al. 2011; Carlsson et al. 2011; Reimers et al. 2006), with particular emphasis on similarities in embryonic development (Weigt et al. 2011). Broad homologies in the genome and neural system also mutually exist between the two groups (Ali et al. 2012). Following detailed characterization of zebrafish development, there is also room for easier comparison and verification between results obtained through models utilizing the zebrafish embryo and studies on mammalian developmental toxicity (Reimers et al. 2004). Extensive information from past and ongoing researches has led to the elucidation of normal zebrafish biological parameters, which is important in the comparison against aberrations that are induced experimentally (Segner 2009). Zebrafish have many properties that emerge as integral and advantageous aspects of testing for developmental toxicity. They are characterized with high fecundity and rapid
Environ Sci Pollut Res
development (Cowden et al. 2012). Zebrafish embryos are easily accessed and manipulated, so that experimental variables administered at the molecular and cellular levels are capable of being observed in the intact, multicellular embryo stage (Krone et al. 2005). These embryos are mostly transparent that, coupled with an external type of development, prove suitable for continuous and unobstructed observation (Li et al. 2009; Pyati et al. 2007; Reimers et al. 2006). Their relatively small size makes rearing in micro-wells and micro-titer plates used in screening possible (Cowden et al. 2012). Moreover, they can be produced in large clutch sizes out of economic husbandry requirements. It cannot be stressed enough that on top of these attributes is another crucial property, i.e., high sensitivity of the early developmental stages of zebrafish, consistent with several species of fish, that enables them to readily absorb chemicals (Morin et al. 2011; Selderslaghs et al. 2009; Osman et al. 2007; Hallare et al. 2006; Marguerie et al. 2006). The study aimed to determine the inherent embryotoxicity of J. curcas seed cake on zebrafish. Specifically, the study aimed to determine the (1) LC50 of the J. curcas seed cake and the (2) morphological aberrations in zebrafish embryos induced by the J. curcas seed cake.
Materials and methods Ethical considerations The study (protocol no. 2012–015) was submitted for review to the University of the Philippines Manila–National Institutes of Health (NIH) Institutional Animal Care and Use Committee and had been granted approval for implementation on 9 October 2012.
Procurement and maintenance of zebrafish broodstock A breeding stock composed of 20 male and 10 female zebrafish, aged about 4 to 6 months, was procured from a tropical fish farm maintained and located at Purok 5, Barangay Pansol, Pila, Laguna. The spawners were placed in oxygensaturated Calypso™ bags. It was ensured that they were free of macroscopically discernable symptoms of infection, and not treated with any pharmaceutical (acute or prophylactic) agents 2 months prior to spawning. In the laboratory, the spawners underwent initial acclimation with careful addition of dechlorinated water equal to about half the original volume of water in the transportation bags. The procedure was carried out twice between a 5-min interval. Spawners were maintained in an aquarium with a minimum loading capacity of 1-L water per fish. Initial preparations included cleaning the aquarium interior with detergent and rinsing for several times with tap water. Approximately 40-L tap water dechlorinated with 5-mL NutraFin Aqua Plus™ was used to fill the aquarium. A fixed 12:12 h light photoperiod was observed in the duration of the experiment. Oxygen saturation of about 80 % was maintained; water temperature was monitored at 26±1 °C; and pH was recorded at 6.6–8.5. Excess filtering rates that may have caused heavy perturbation of the water was avoided. The semi-static condition guaranteed that traces of ammonia, nitrite, and nitrate levels were kept below the critical limit for toxicity. Filters were cleaned once every 2 weeks and tank water was changed on a weekly basis. In the process of water replacement, one-fifth of the original tank water volume was replaced with dechlorinated tap water. Fish were fed with commercially available diets (TetraMin™ flakes) thrice daily. Overfeeding was strictly monitored to avoid pollution of the tank water.
Collection of test substance
Preparation of test solutions
By the time of collection, the Department of Science and Technology (DOST) was undertaking pilot tests on the efficiency of J. curcas as a biodiesel source. The procedures included procurement of seeds and extraction of J. curcas oil. The seed cake is a residual by-product of mechanical oil extraction. Seed cake samples were stacked at the DOST– Industrial Technology Development Institute (ITDI)’s Jatropha Oil/Biodiesel Processing Facility and Analytical Laboratory (DOST-PAFC joint project) located at the DOST Compound, General Santos Avenue, Bicutan, Taguig City. This study utilized J. curcas seed cake samples that were immediately collected after oil extraction in ITDI. The samples were contained within black plastic bags and stored securely at 4 °C in a polystyrene jar throughout the duration of the biotest.
Reconstituted water was prepared based on the protocol set by ISO 7346/3. Each liter of distilled water contained 294.0 mg CaCl2.H2O, 123.3 mg MgSO4.7H2O, 63.0 mg NaHCO3, and 5.5 mg KCl. Parameters like pH (maintained at 6.5–8.5 using 1-M HCl or NaOH as necessary), conductivity, and temperature (kept at 26±1 °C) were monitored after the preparation. Conductivity and pH were measured using a Eutech™Cyberscan Series 600 portable meter. On the other hand, J. curcas seed cake samples were ground and disaggregated using mortar and pestle. For every exposure test, a stock solution was prepared by dissolving 50-g seed cake in 1-L reconstituted water (initially aerated for at least 12 h). Test solutions of varying concentration were prepared from the stock solution by dilution. Reconstituted water served as dilution water. An exposure test
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was defined as a setup inclusive of test solutions and control treatments. Collection of zebrafish embryos On the day prior to exposure, a tray covered with plastic mesh (1.25 mm) was placed at the bottom of the aquarium before the onset of darkness. Artificial plants made of green plastic were fixed to the grid and served as spawning stimuli. The tray was removed 30 min after the onset of light in the morning when mating, spawning, and fertilization have already taken place. Exposure tests A series of range-finding tests was performed. Five concentrations were derived for the proper test, which was repeated independently thrice. The highest concentration was preferred to cause 100 % lethality while the lowest concentration was estimated to give zero toxicity. A constant factor between the concentration groups not exceeding 2.2 was considered. Pure reconstituted water served as both negative control and internal control. For the positive control, a solution of 3,4Dichloroaniline (4.4 mg dissolved in 1-L Millipore MilliQ™ water) was used. Collected eggs were rinsed with reconstituted water and transferred to petri plates not later than 1-h post fertilization (hpf). The plates served as preliminary test chambers for the test solutions of varying concentration and controls. At about 2 hpf, viable eggs from each petri plate were replaced to a 24-well plate using a micropipette with a widened tip. Each 24-well plate served as proper test chamber. Twenty eggs were transferred individually to each well, which contained 2 ml of the test solution with same concentration as the source petri plate. The remaining four wells served as internal control containing reconstituted water. Separate plates for the positive control and external negative control were provided for each exposure test. All 24-well plates were pre-saturated with assigned treatments for 24 h. Prior to egg transfer, the wells were emptied and added with 2-ml freshly prepared treatment. Viable eggs corresponded to zebrafish embryos undergoing cleavage with no apparent irregularity, such as asymmetry or vesicle formation. Viability was screened under a stereomicroscope. Evaluation of embryotoxicity The toxicity of J. curcas on zebrafish embryos was evaluated based on observable morphological parameters. Three arbitrary time points of observation following exposure were identified as follows: 24, 48, and 72 hpf. An embryo was considered dead if it demonstrated any of four lethal endpoints, namely coagulation, non-formation of somites, and
non-detachment of tail (observable beginning 24 hpf), and absence of heartbeat (observable from 48 hpf). Observations were conducted in an Olympus™MIC-D digital microscope (×80 magnification) connected to a laptop computer. Scores were treated in binominal convention. Surviving embryos received a score of 0, while an embryo that presented with a lethal endpoint earned a score of 1. A score for overall mortality was a summation of the counts of death recorded from the four lethal endpoints. Percent mortality for a test solution was calculated as the number of dead embryos over the number of embryos at the start of exposure (n=20). Observable sublethal effects, which could refer to lack of pigmentation, yolk sac edema, pericardial edema, and spinal deformation, were recorded at 72 hpf. Statistical analysis LC50 estimates with 95 % confidence for the three time points of observation were computed using Probit Analysis. The statistical significance of mortality arising as an effect of concentration was tested using the Kruskal-Wallis ANOVA among groups. The Mann–Whitney U test was used to test for statistical significance between groups. These were performed with the SPSS v21 statistical software.
Results Reconstituted water parameters Throughout the test, the pH of reconstituted water ranged from 8.0 to 8.1, while temperature remained fairly constant at 26±1 °C. These were within acceptable limits specified by the zebrafish embryo toxicity (ZFET) standard operation procedure (OECD 2006). The pH for the test solutions range from 6.6 to 7.6, which is in accordance with the optimal pH tolerance range for zebrafish set at 6.0 to 8.0. Oxygen was ensured to be at optimal levels by fully aerating the test solutions prior to exposure. Conductivity for the test solutions was consistent between 510 and 540 uS/cm. Test solutions The series of range-finding tests, performed in order to pinpoint five mass concentrations of J. curcas seed cake used in the test proper, revealed a range between 2.15 and 1.00 g/L with a constant factor of 1.21. This was based on actual mortalities on exposed zebrafish embryos recorded at 24 hpf, following observations throughout the tests that percent mortalities at the two subsequent time points (48 and 72 hpf) presented no apparent increase. All percent mortalities were taken as observed deaths relative to the total number of embryos at the start of exposure.
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Overall toxicity and LC50
Lethal endpoints
As reasonably established by preliminary testing, zebrafish embryos exposed to 2.15 g/L of J. curcas seed cake (highest concentration) exhibited full toxicity, while embryos tested in 1.00 g/L (lowest concentration) showed zero toxicity. Decreasing partial toxicity was observed in the three consecutive concentrations bounded by the highest and lowest groups, respectively. The mean values of percent mortality experienced by zebrafish embryos following exposure to J. curcas seed cake for the three time points of observation are summarized in Table 1. The effect of concentration on mortality among the five groups was found to be highly significant (p= 0.008). All concentration groups, except the lowest, were statistically significant (p< 0.05) from the negative control. Toxicity was seen to manifest at the earliest time point of observation (24 hpf). For the later time points (48 and 72 hpf), all surviving embryos remained alive, but most of them manifested abnormality. Mortality in the five concentrations therefore remained constant across all time points for the three independent tests performed. This concomitantly corresponded to a single value of mean LC50 for the three observation times. The mean LC50 estimate was determined at 1.61 g/L between an interval of 1.51 and 1.71 g/L with 95 % confidence (Fig. 1). Per ZFET Validation Report specifications (OECD 2011), the mean partial toxicities of 65, 28.3, and 8.3 %, which were respectively elicited by the three consecutive concentration groups (1.78, 1.47, 1.21 g/L), held as informative, i.e., provided good information on the LC50.
As indicated earlier, embryotoxicity is a consideration that answers to a positive outcome in any of four morphological endpoints namely, coagulation, non-formation of somites, and non-detachment of tail, observable beginning 24 hpf, as well as the absence of heartbeat observable beginning 48 hpf. Data on these parameters, which cumulatively consisted of the overall embryotoxicity, are summarized in Table 1. Embryo coagulation was notable as the most frequent parameter recorded from higher concentrations in the wider range-finding tests (data not shown). In the test proper, it accounted for 8 of 60 deaths observed in the three independent tests for the highest concentration (mean mortality of 13.3 out of 100 %; N=3, 20 samples per N). The frequency of coagulation gradually decreased in the lower concentrations. Effect of concentration was insignificant statistically (p=0.144). Non-formation of somites represented 63.3 % of full toxicity in zebrafish embryos (mortality equals 100 %) in the highest concentration, 18.3 in 65 % of observed deaths in the second highest concentration, and 1.67 in 28.3 % of mortalities in the third highest concentration. This endpoint was not elicited by the two lowest concentrations, but its concentration-dependent occurrence tested significantly (p= 0.013). The three highest concentrations were statistically significant (p
