HUNGARIAN JOURNAL OF INDUSTRY AND CHEMISTRY Vol. 49(1) pp. 77–82 (2021) hjic.mk.uni-pannon.hu DOI: 10.33927/hjic-2021-10 DAPHNIA MAGNA ACUTE IMMOBILIZATION TEST: AN OPPORTUNITY TO TEST THE ECOTOXICITY OF ALTERNATIVE FUELS KATALIN ESZTER HUBAI*1 1Center for Natural Sciences, University of Pannonia, Egyetem u. 10, 8200 Veszprém, HUNGARY The increasing need for environmental protection has led to the development of alternative biofuels. While the use of alternative fuels has significantly increased recently, only a few studies have addressed the problem of their ecotoxicity. The main aim of this work was to provide a short review of the Daphnia magna acute immobilization test, which has been the most commonly discussed in the literature. Keywords: Daphnia magna, immobilization, ecotoxicology, alternative fuels 1. Introduction Over recent decades, rapid population growth has been accompanied by a growth in the consumption of energy and use of transport fuels, which has caused irreversible environmental degradation and climate change [1]. De- sires for a green environment have increased the demand for alternative fuels which in turn has necessitated re- searchers and industries to develop renewable alternative and cleaner energy sources worldwide [2]. Biofuels are energy-enriched substances manufac- tured from vegetable oils, recycled cooking grease and oil as well as animal fats through a chemical process known as transesterification, which is described below, to pro- duce chemical compounds known as fatty acid methyl es- ters (FAME) [3, 4]. Biodiesel is the name given to these esters when they meet biodiesel standards such as the American ASTM D6751 or the European EN14214 for use as transport fuels [4]. Biodiesel is an eco-friendly form of fuel and may provide a solution to some prob- lems associated with petroleum diesel [5]. Alternative fuels are key to improving the EU’s secu- rity of energy supply, reducing the impact of transporta- tion on the environment and boosting the EU’s compet- itiveness. They are also an important building block for the EU’s transition towards a low-carbon economy. In 2007, the production of biofuels in the EU reached 8,500 ktoe (kilotonnes of oil equivalent), while in 1996, this fig- ure was less than 500 ktoe [6]. In 2010, 15.5% of power generation and 1.3% of energy consumption worldwide was attributed to renewable energy, while today, it is es- timated that 86,000 kt per year of biofuels are produced, with the USA and Brazil being the primary producers [7]. *Correspondence: hubai.katalin@mk.uni-pannon.hu More studies have shown that the use of biodiesel would reduce emissions of hydrocarbons, carbon monoxide and volatile organic compounds [8,9]. However, the results of analyzing the biological effects related to the presence of biodiesel in the environment are ambiguous [10]. Although the use of alternative fuels has significantly increased recently, relatively few studies have addressed the problem of their ecotoxicity. Therefore, the main ob- jective of this study is to provide a short overview of the Daphnia magna acute immobilization test which has been the most frequently discussed in the literature. 2. Methodology 2.1 Test organism In addition to the chemical characterization of a sub- stance, ecotoxicological tests provide an important tool for ecological risk assessments [11], giving a quantitative estimation of the overall toxic effect of the test organ- isms selected [12]. In general, the Daphnia magna acute immobilization test is amongst the most widely used eco- toxicological methods [13]. International standards apply such as OECD 202:2004 [14] or ISO 6341:1996. The test organisms are the freshwater crustaceans D. magna and D. pulex. For the tests, neonates (newborn, freshly hatched juveniles) are used. (The main purpose of any standard protocol is to increase quality assurance which in turn might increase the credibility of the data produced [15]. In order to minimize any possible er- rors caused by improper maintenance of stock cultures, so-called Toxkits have been developed and marketed by MicroBioTests Inc. (Mariakerke-Gent, Belgium) [16]. The main benefits of using a Toxkit are that they are maintenance-free and user-friendly [17] test organisms https://doi.org/10.33927/hjic-2021-10 mailto:hubai.katalin@mk.uni-pannon.hu 78 HUBAI Figure 1: Freshly hatched D. magna neonate whose genetic material is practically uniform and, prior to testing, juveniles of approximately the same age are reproduced (Fig. 1). 2.2 Implementation of experiments There are several options for conducting alternative fuel toxicology studies. In one part of the research, the fuel was stirred in water before the test organisms were in- troduced into the test chamber [18–20]. In this method, the layer of oil on the top of the wells can cause some problems. In other experiments, aqueous extracts were used, for example, a stock solution was made by adding seawater (depending on the test organism) to the sample and stir- ring the mixture for 10 − 24 h [21–23]. Three different biodiesels, that is, two based on the vegetable oils produced by canola and soybean as well as waste frying oil that originated from animals, were used by Hollebone et al. [24]. Oil-in-water dispersions (OWD) and water-accommodated fractions (WAF) were used for the Daphnia magna assay. Different results were observed during the tests; higher LC50 values were mea- sured in WAFs compared to in OWDs. This suggests that the soluble fraction is of lower toxicity compared to the physical danger of the organisms being smothered by the oily fuel (See Table 1). Müller et al. [23] assessed the toxicity of the water- soluble fraction (WSF) of biodiesel on D. magna in com- parison to the WSF of diesel [24]. The tested sample of biodiesel was a fatty acid methyl ester (FAME) mainly produced by soybean oil (95%). This biodiesel did not elucidate a measurable degree of toxicity either following acute or chronic exposure. On the other hand, in a study by Eck-Varanka et al. [21], the ecotoxicity of a rapeseed biodiesel was profiled using a battery of test organisms and D. magna exhibited an extremely high degree of tox- icity, being the most sensitive assay in the battery. Khan et al. [18] carried out an extensive study to com- pare the ecotoxicity of diesel, neat biodiesel (B100) and blends of both (B50, B20 and B5). B100 was produced from recycled cooking oils and fats. The lowest and high- est levels of ecotoxicity were exhibited by B100 and diesel, respectively, while the ecotoxicity of the blends, expressed both in terms of mortality rates and EC50 val- ues, were in the intermediate range. However, the differ- ences between the measured responses were quite small: the LC50 values of Daphnia magna in neat biodiesel and diesel were 4.65 and 1.78 ppm, respectively. Tjar- into et al. (2014) conducted a similar study on biodiesel produced from waste vegetable oil and reported an EC5 value of 3.157 ppm for Daphnia magna [25]. Heger et al. [26] compared the ecotoxicity of two biofuel candidates (1-octanol and 2-butanone) and found that 1-octanol exhibited a significant level of ecotoxic- ity on D. magna while 2-butanone did not. However, as- says conducted on other test organisms revealed that the metabolites of the tested products could pose a higher risk of toxicity. Heger et al. [27] applied the D. magna acute immobilization test to compare the aquatic toxicity of the two biofuel candidates, namely 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF), and found that the latter induced a significantly higher mortality rate than 2-MTHF (See Table 1). Ecotoxicity, more precisely the ecotoxicity impact, is also included in the life cycle assessments (LCA) of alter- native fuels [28]. Since LCAs follow the whole produc- tion line of a product, Bunzel et al. [29] used a D. magna assay to evaluate pesticide runoff from agricultural fields used for the cultivation of energy crops. Khan et al. [18] stressed that one possible major pur- pose of ecotoxicity testing is assessing the potential risk of fuel spills in aquatic ecosystems. As such, it should be emphasized that Daphnia magna, being a freshwater taxon, cannot represent marine ecosystems, instead ma- rine surrogates are used such as the brine shrimp Artemia salina [30]. Gateau et al. [31] investigated water-soluble frac- tions (WSFs) of four different vegetable oil methyl es- ters. Lower EC50 values (> 1000 mg/L) were calculated for vegetable oil methyl esters than for regular diesel (EC50< 100 mg/L) (See Table 1)). The toxicity of biodiesel blends and crude oils have been investigated in other studies and biodiesel has been found to be less toxic to D. magna than both the biodiesel blends and crude oil (See Table 1). 3. Conclusion In conclusion, it should be emphasized that the number of available studies is surprisingly low. Furthermore, these studies are extremely difficult to compare due to the fol- lowing reasons: since the studies have been conducted on alternative fuels of very different origins, more extensive research on their chemical compositions to determine po- tential toxic effects is required. By taking into consid- Hungarian Journal of Industry and Chemistry DAPHNIA MAGNA ACUTE IMMOBILIZATION TEST 79 Ta bl e 1: R es ul ts of th e D ap hn ia m ag na ac ut e im m ob il iz at io n te st (W A F :w at er -a cc om m od at ed fr ac ti on ;O W D :o il -i n- w at er di sp er si on ) F ue lt yp e M et ho d L C 50 R ef er en ce 1- oc ta no l W A F ; M et ho ds of ac ut e to xi ci ty te st - in g us in g fi sh ,m ac ro in ve rt eb ra te s an d am - ph ib ia ns (U S E PA ) 52 0 m g/ L L eB la nc ,1 98 0 [3 2] 1- oc ta no l O W D ;S ec ti on 5, pa ra .1 “N o. 3 of th e R eg ul at io n on A pp li ca - ti on D oc um en ts an d E vi de nc e un de r th e C he m ic al s A ct " (F ed er al E nv ir on m en ta lA ge nc y) 26 m g/ L K ün h et al ., 19 89 [3 3] R ap es ee d oi lM et hy lE st er s (R M E ) W A F ;O E C D 20 2 > 10 00 W A F m g/ m l G at ea u et al ., 20 05 [3 1] E ru ci c R ap es ee d oi lM et hy lE st er s (E R M E ) > 10 00 W A F m g/ m l S un fl ow er oi lM et hy lE st er s (S M E ) > 10 00 W A F m g/ m l H ig h O le ic S un fl ow er oi lM et hy lE st er s (H O S M E ) > 10 00 W A F m g/ m l D ie se lf ue l < 10 0 W A F m g/ m l ba se d on ve ge ta bl e oi lp ro du ce d fr om ca no la O W D ;E nv ir on m en tC an ad a te st m et ho d "B io lo gi ca l Te st M et ho d: A cu te L et ha li ty Te st U si ng D ap hn ia sp p" 28 0 (2 00 -4 10 ) m g/ L H ol le bo ne et al ., 20 08 [2 4] ba se d on ve ge ta bl e oi lp ro du ce d fr om so il cr op s 37 .8 (2 3. 0- 63 .1 ) m g/ L ba se d on w as te fr yi ng oi lp ro du ce d fr om an im al s 58 2 (3 16 -1 08 0) m g/ L U lt ra -L ow su lp hu r di es el 15 .2 (8 .2 -2 9. 3) m g/ L L ow su lp hu r di es el 17 .9 (1 2. 7- 25 .3 ) m g/ L ba se d on ve ge ta bl e oi lp ro du ce d fr om ca no la W A F (2 5 g/ L fu el (1 :4 0, fu el :w at er ); E nv ir on m en tC an ad a te st m et ho d "B i- ol og ic al Te st M et ho d: A cu te L et ha li ty Te st U si ng D ap hn ia sp p" 24 65 0 (2 50 0- 14 00 00 ) m g/ L ba se d on ve ge ta bl e oi lp ro du ce d fr om so il cr op s 75 00 (5 10 0- 11 00 0) m g/ L ba se d on w as te fr yi ng oi lp ro du ce d fr om an im al s 75 00 (5 10 0- 11 00 0) m g/ L U lt ra -l ow su lp hu r di es el 33 00 (1 80 0- 58 00 ) m g/ L L ow su lp hu r di es el > 25 00 0 m g/ L bi od ie se l( fa tt y ac id m et hy le st er ) W A F ;O E C D 20 2 0. 02 26 % (1 00 % w as 1: 1 w a- te r: bi od ie se l) E ck -V ar an ka et al ., 20 18 [2 1] 2- bu ta no ne (m et hy le th yl ke to ne ) O W D ;O E C D 20 2 21 52 .1 ± 44 .6 m g/ L H eg er et al ., 20 18 [2 6] 2- m et hy lt et ra hy dr of ur an (2 -M T H F ) O W D ;O E C D 20 2 1. 11 6± 0. 10 2 m g/ L H eg er et al ., 20 18 [2 7] 2- m et hy lf ur an (2 -M F ) 0. 03 2± 0. 00 4 m g/ L 49(1) pp. 77–82 (2021) 80 HUBAI eration the practical aspects of the tests, different peri- ods of exposure have been employed (chronic exposures of 24, 48 and even 96 h). Sample preparation protocols also differ: oil-in-water dispersions (OWD) and water- accommodated fractions (WAF) have also been used as alternatives [34]. Generally, the Daphnia magna acute immobilization tests show an appropriate degree of sensitivity to a wide variety of compounds or complex mixtures [35–37]. However, as different components of an ecosystem will exhibit taxon-specific sensitivity to a chemical, a care- fully composed battery of biotests should be used to gain a more comprehensive understanding [38]. It is possible that these tests will represent different functional and/or taxonomic groups as the ecotoxicity of pollutants influ- ences the function and structure of aquatic or terrestrial ecosystems [39], moreover, possible endpoints will dif- fer [40]. The minimum battery should involve the lu- minescent bacteria test, algae and zooplanktonic crus- taceans [41]. Acknowledgments This study was funded by the NTP-NFTÖ-19-B-0148 project. 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