Technical paper Toxicity of Commercial Surfactants to Phosphate-Accumulating Bacterium Tomislav Ivankovic,1* Jasna Hrenovic1 and Ivana Gudelj2 1 University of Zagreb, Faculty of Science, Division of Biology, Rooseveltov trg 6, 10000 Zagreb, Croatia 2 Lakmus d. o. o., Zagrebacka 231, 10370 Dugo Selo, Croatia E-mail address: tivanko@zg. biol. pmf. hr; Tel: +3851 48 77 700; Fax: +3851 48 26 260 Received: 25-03-2009 Abstract The toxicity of twelve commercial surfactants to phosphate-accumulating bacterium Acinetobacter junii was tested. The tested products were selected to cover a variety of surfactant types and classes (anionic, cationic, amphoteric and nonio-nic). One surfactant proved to be highly toxic (ECjj < 1 mg L-1), six surfactants were toxic (ECjj values between 1 and 10 mg L-1), two were harmful (ECjj values between 10 mg L-1 and 100 mg L-1) and three surfactants with ECjj higher than 100 mg L-1 were considered as non-toxic to A. junii. The most toxic commercial surfactant was Kutriacid (linear alkylbenzene sulphonate) with ECj^ value of 0.15 mg L-1. When all of the tested surfactants were compared there could be no generalization in toxicity regarding the class or the length of the alkyl chain of surfactant. One observed feature is that all of the surfactants with branched alkyl chain had least toxic or no toxic effect to A. junii. Keywords: Acinetobacter junii, bacteria, phosphate, surfactants, toxicity, wastewater. 1. Introduction Surfactants generally consist of a hydrophilic head group (charged or non-charged) and a hydrophobic hydrocarbon tail. Depending on the charge of surfactant head group, they are generally classified as anionic, cationic, nonionic and amphoteric. The commercial surfactants are industrially manufactured solutions containing a single type of surfactant or mixtures of multiple structurally similar surfactants, water and impurities. These commercial products are used in industrial manufacture of detergents, personal care products, textiles, pharmaceuticals etc.1 The behaviour, fate and biological effects of surfactants in the environment need to be understood to assess their environmental risk. In a study by Warne et al.^ surfactants were identified as the main contributors of detergent toxicity to aquatic organisms. After use in household or industry, residual surfactants and their degradation products are discharged to sewage system or directly to surface waters, and then dispersed into different environmental compartments (water, sediments, soil). Surfactants can also accumulate in the sludge sewage treatment flow3 and exert an inhibitory effect on the sewage sludge microorganisms. Such effect could have serious consequences, as the effectiveness with which the water treatment plant removes pollutants and breaks down sewage might be compromised. In this study we wanted to investigate the possible toxicity of commercial surfactants to a specific type of microorganism inhabiting the activated sludge; a phosphate-accumulating bacterium Acinetobacter junii. The bacteria from the genus Acinetobacter were isolated from the phosphorus removing activated sludge plant4 and although Acinetobacter spp. were present in extremely low number, their capacity to remove phosphate was the highest among all the phosphate-accumulating isolates.5 These bacteria have become the model organism for enhanced biological phosphorus removal (EBPR) and elucidating the negative effects of surfactants on these bacteria could help to understand or prevent the possible failures of EBPR systems. 2. Materials and Methods 2. 1. Commercial Surfactants The commercial surfactants were obtained from Croatian detergent producing companies. The 12 surfac- tants (Table 1) tested in the study were selected based on their widespread usage in industrial production and to cover a variety of surfactant types and classes (anionic, cationic, amphoteric and nonionic). Commercial products were either used in the experiment immediately after their arrival in the laboratory, or stored at 4 °C. The valid material safety data sheet for each surfactant was provided by the manufacturer. 2. 2. Bacterium The phosphate-accumulating bacterium A. junii (strain DSM 1532) was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweg, Germany and maintained as described previously.6 2. 3. Simulative Wastewater A chemically defined water solution that simulated the real wastewater was used as a growth medium for A. junii. The composition was as follows (in mg L-1 of distilled water): Na-propionate 300; peptone 100; MgSO4 10; CaCl2 6; KCl 30; yeast extract 20; KH2PO4 88. The pH was adjusted to 7.00 ± 0.04 with 1M HCl or 1M NaOH before autoclaving at 121 °C for 20 min. 2. 4. Toxicity Test Design The A. junii was pregrown on nutrient agar plates for 16 h at 30 ± 0.1 °C. The biomass was then resuspended in 9 mL of sterile 0.3% NaCl solution. A 5 mL of such suspension was inoculated into the flasks containing 500 mL of simulative wastewater, after which the starting number of bacterial cells was determined. Appropriate amounts of surfactant commercial product were then aseptically added in each flask. The sterility of surfactant solutions was previously confirmed. A 250 mL was taken from each flask to measure the concentrations of active matter of surfactant in the simulative wastewater. The bottles with remaining 250 mL of wastewater were then aerobically incubated for 24 h at 30 ± 0.5 °C in a water-bath with shaker (70 rpm) and rate of 1 L per minute of filtered air was provided. Bottles which served as negative control contained no surfactant. For each surfactant a preliminary range-finding test covering several orders of magnitude in surfactant concentrations was carried out in order to determine the concentrations range for each surfactant used in final test. Tested concentrations ranged from 0 to (in g L-1): 25 SI M B e Ir a ^ 'iŠ § ^ z (S O iì e .> S3 3 ro « -O s o o o ^ 13 13 << << J m << << J < >y CD T3 .Si a ö o -9 sä « s ss ^ c o C IN ro ^ m U ss ^ C o C IN ro ^ m U o •■o c^ o •■o ci co o ••o ITj cf << c^ 13 o :3 m m Ö o q (S 3 -a U ,<2 J Z + O 12 3 « ro O C ^^ 13 S ^^ s p cd C + « ia -iS s ^ -a o lU Ji OJOOoO ^^ ^^ g ^ ^ o Qp^p^ o in ci co Zi 00 iN iN iN iN iN iN iN co IN iN O co co iN IN co co c^ CD 00 ck ^^ m 3471 >1.50 X 10-2 > 3471 > 1.5 X 10-2 - Texapon 8.2 1.8 5.96 X 10-6 175.6 5.81 X 10-4 24.39 Cumolsulfonat 15000 > 72.6 >3.27x10-4 > 72.6 > 3.27 X 10-4 - Servamine 4.3 2.8 8.00 X 10-6 6.18 1.76 X 10-5 6.98 Oxidet 3.1 1.0 3.89 X 10-6 2.8 1.09 X 10-5 9.68 Arylpon 4.1 1.9 4.28 X 10-6 4.4 9.91 X 10-6 12.20 Empilan 630.0 570 1.13 X 10-3 > 4524 > 8.96 X 10-3 9.52 Ethomeen 1.8 1.1 3.14 X 10-6 6.1 1.74 X 10-5 33.33 Genapol 15000 > 14340 1.13 X 10-3 > 14340 > 1.13 X 10-3 - values of 0.15 mg L-1 and 1.8 mg L-1, respectively. Straight chain alkyl sulphate (AS) Texapon was the second most toxic anionic surfactant, while Lutensit (AS with branched chain) showed no toxic effect. Branched chain secondary alkane sulphonate (SAS) Hostapur and alkyl benzene sulphonate (ABS) Cumolsulfonat, also with branched alkyl chain, proved to be harmfull to A. junii. Some toxicity data obtained from the literature are listed in Table 3. For some surfactants literature data could not be found so the presented toxicity data were obtained from the valid material safety data sheet provided by the manufacturer. Table 3: Toxicity data for various types of surfactants. Surfa- Species EC 50 References ctant (mg L-1) SAS Pseudomonas putida > 1.0 Data sheet* LAS Daphnia magna 8.1 17 Dunaliella sp. 3.5 37 AS Gloeocapsa sp. 50 38 Vibrio fischeri 2.6 39 Acinetobacter junii 2.25 40 ABS Daphnia magna > 100 Data sheet* EOP Bacteria > 1.0 Data sheet* QAC Pseudomonas putida 6.0 23 Vibrio fischeri 0.5 Acinetobacter junii 0.12 41 AO Photobacterium phosphoreum 2. 4 42 Daphnia magna 6. 8 AE Ceriodaphnia dubia 0. 39 2 5. 36 EA Daphnia magna 0. 17 Nitrifying bacteria 64. 0 Data sheet* AE+EA Bacteria > 1. 0 Data sheet* *The data was retrieved from the valid material safety data sheet provided by the manufacturer. Since the anionic surfactants are the most popularly used type of surfactant, their toxicity towards aquatic organisms was widely researched. The chronic toxicity of anionic surfactants occurs at concentrations usually greater than 0.1 mg L-1.12 Hennes-Morgan and Oude13 found the ranges of toxicity values for the LAS, alkyl ethoxy sulphates and AS on Daphnia magna to be broad and largely overlapping. The EC50 values were ranging roughly between 1-15 mg L-1 of these anionic surfactants. However, the LAS tended to be the most toxic followed by the alkyl ethoxy sulphates that were either more toxic than or as toxic as the AS surfactants. Some studies indicated that molecular lipophilicity exerts a considerable influence on the toxicity of anionic surfactants,14 and the effect depends on both the character of the polar head group and the length of apolar alkyl chain.15 It seems that in our study the toxicity of anionic surfactants did not depend on the type of active group; the surfactants that share the same sulphonate active group were ranging from highly toxic to non-toxic to A. junii. To compare the toxicity of different surfactants regarding the chain length, the EC50 value should be expressed in mol L-1 (Table 2) to demonstrate the toxicity as a function of number of surfactant molecules. The order of toxicities of anionic surfactants were as follows: Kutriacid C10_13 (12 rich) > Texapon C12 > Solfodac C10-13 (12 rich) > Hostapur C13-17. Based on these results it seems that the toxicity of anionic surfactants decreases with increase in alkyl chain length. However, these conclusions cannot be assuredly verified from current experimental data. The exact distribution of alkyl chain length homologues of the tested surfactants could not be established so their contribution to the observed toxicity could not be evaluated. Although Kotani et al.^^ stated that the the C18 AS compounds caused human skin cell injury whereas C10 and C16 compounds caused more severe membrane destruction and protein denaturation, most literature data report that the toxicity of anionic surfactants increases with increasing lipophilicity of the molecule.17 Holman and Macek18 found the toxicity of LAS homologues to aquatic organisms was increasing with increase in chain length and/or molecular weight. In a quantitative structure-activity relationship study Versteeg et al.^"9 found that toxicity of anionic surfactants to aquatic organisms increases with increasing length of the alkyl chain. However, this trend was not observed when the commercial products that contained LAS of different alkyl chain lengths were tested.2 Hall et evaluated the acute toxicities of 17 industrial surfactants having a wide range of physicochemical characteristics and reported that the base structure of the surfactants (aromatic or aliphatic, branched or linear) were not a factors controlling toxicity. Surfactants containing branched alkyl chain were non-toxic (Lutensit) or considerably less toxic (Cumolsul-fonat, Hostapur) than straight chain anionics. Common perception is that anionic surfactants with branched alkyl chain are poorly degradable and therefore environmentally unacceptable. However, recent studies showed that alkyl sulphates with either a small number of methyl branches along the hydrophobic chain21 or 2-alkyl branc-hes22 were as rapidly and completely biodegraded as their linear counterparts. In our study branched chain anionics proved to be more environmental friendly than their linear chain counterparts. 3. 2. Cationic Surfactants Servamine belongs to the quaternary ammonium compound (QAC) type of cationic surfactant. The Servami-ne proved to be toxic to A. junii with EC50 value of 2.8 mg L-1 (Table 2), which is similar to EC50 values for the most toxic representatives of other classes of tested surfactants. The QACs are known to be toxic in low concentrations to Gram-positive bacteria.23 Augustin et al.24 hypot- hesized that QACs can disarrange the biological wastewater treatment process. The prevalence of QAC-resistant bacteria was elevated in effluents of sewage treatment plants receiving QACs.25 In previous study from our laboratory26 the QAC with 12 carbon atoms in alkyl chain acted less toxic against phosphate-accumulating bacterium A. junii than the QAC with 16 carbon atoms in alkyl chain. Garcia et al.27 carried out the acute toxicity tests on D. magna and Photobacterium phosphoreum for six catio-nic surfactants: the EC50 on D. magna ranged from 0.13 to 0.38 mg L-1 whereas EC50 on P. phosphoreum ranged from 0.5 to 0.63 mg L-1. They did not observe the incremental difference in toxicity between homologues of different chain length. Since Servamine was the only representative of QAC in this study, and it contains imidazole ring instead of long alkyl chain, no relations between the chain length and toxicity of cationic surfactants could be concluded. 3. 3. Amphoteric Surfactants In this study one amphoteric surfactant, Oxidet, belonging to amine oxide (AO) group was tested. The pure C14 AO component of Oxidet proved to be toxic to A. junii with EC50 of 1.0 mg L-1 (Table 2). The AO has been shown to have anti-microbial activity (Table 3) that increases with increasing chain length, exhibiting a cut-off effect at chain lengths of approximately C14.28 Garcia et al.^'9 showed that the toxicity to Daphnia sp. increased when increasing the alkyl chain length. The relations between the chain length and toxicity of AO against A. junii could not be established in this study since Oxidet was the only representative of this particular type of surfactant. 3. 4. Nonionic Surfactants The Ethomeen, an ethoxylated amine (EA) was the most toxic nonionic surfactant with EC5q of 1.1 mg L-1 (Table 2). Empilan, a member of alcohol ethoxylate (AE) group and Genapol, ethylene oxide/propylene oxide polymer, showed no toxic effect whilst Arylpon, a mixture of 60% EA and 15% (AE), proved to be toxic with EC50 of 1.9 mg L-1. Since AE type of surfactant proved to be non-toxic to A. junii, it seems that the toxicity of Arlypon mixture comes from 60% of present EA. Lewis30 found that surfactants in mixture had antagonistic, additive and syner-gistic interactions on toxicity to aquatic organisms. In our case it seems that the toxicity of surfactant mixture is defined by the most toxic surfactant present in the mixture. The literature indicates that nonionic surfactants are in general more toxic to aquatic organisms than anionic surfactants.13 Such generalization could not be considered in this study since the toxicity of nonionic and anionic surfactants were wide ranged and overlapping. We observed that the toxicity of nonionic surfactants increases with increase in chain length; C16-18 Arylpon was considerably more toxic than C11-14 (13 rjch) Empilan. The most toxic nonionic Ethomeen is a tallow alkyl amine, meaning it is a mixture of alkyl amines with many different chain lengths ranging from C14 to C19, with C18 being abundant. We also observed that the Empilan with 8 ethoxylated groups (EO) was considerably less toxic than Ethomeen with 5 EO. Still, no conclusions could be drawn from these observations since the different nature of hydrophilic groups in tested surfactants does not allow us to establish the effect of alkyl chain length or the number of ethoxylated groups on toxicity. Feijtal and Van de Plassche31 stated that there was no relationship between the carbon chain length or the number of ethoxylated groups (EO) and the toxicity for AE surfactants. Warne et al? found that the AE containing eight EO was notably more toxic than the AE containing three EO but could not state with any degree of confidence that there was a trend of increased toxicity with increased ethoxylation. In contrary, Wong et al.32 stated that toxicity of AEs tended to increase with increasing alkyl chain length and decreasing number of EO groups. The branched alkyl chain of Empilan might be responsible for its lack of toxicity which was confirmed by Madsen et al.33 observation of neglible aquatic toxicity for branched nonionic surfactants. The biodegradation of surfactants was not expected with pure culture of A. junii since usually a consortium of bacteria is required for successful biodegradation.34 The Genapol seemed to be biodegraded by A. junii since the number of bacteria in the reactors with Genapol after 24 h incubation was up to four times greater when compared to control reactor with no Genapol added. However, since the surfactant was not the sole carbon source in the growth media, the increase in cell number could simply be due to the lack of antibacterial activity of the surfactant, or for some miscellaneous reason. No literature data about the biodegradation of this substance could be found. The material safety data sheet information states the biodegradation of > 47% which was the lowest among all the surfactants used in the study. The increase in number of bacterial cells after the 24 h of incubation when compared to control was not observed for other two non toxic surfactants Lutensit and Cumolsulfonat. The number of cells was actually slightly decreasing; however, the EC50 was not obtained at 15 g L-1 of commercial product so we classified them as non-toxic, but not possibly biodegradable by A. junii. It should be noted that toxicity values of commercial surfactants against A. junii reported in this study are probably the upper limits. The adsorption of surfactants to insensitive organisms, dead cells in flocs and inorganic matter in the wastewater treatment plant should protect viable susceptible bacteria from the toxic effect of surfactants. Traina et al.^^ found that dissolved humic substances reduced the aqueous bioavailability of LAS and Cano et al.36 found that organic carbon in sediments reduced the toxicity of ABS to Hyalella azteca. 4. Conclusions The results showed that the toxicities of nonionic and anionic surfactants to phosphate-accumulating bacterium A. junii were wide ranged and overlapping. When all of the tested surfactants were compared there could be no generalization in toxicity regarding the class or the alkyl chain length of the surfactant. One observed feature is that surfactants with branched alkyl chain had least toxic or no toxic effect to A. junii. Our experiments proved that the most abundantly used types of surfactants (LAS, AS and QAC) are also the most toxic ones to A. junii. Ever expanding detergent and cosmetics industry will assuredly give rise to concentrations of these commercially manufactured surfactants in sewage effluents. Thus, it is important to investigate the influence of these abundantly present chemicals on phosphate-accumulating bacteria inhabiting the activated sludge in order to prevent or understand the possible failures of EBPR systems. 5. Acknowledgments This research was supported by The Ministry of Science, Education and Sports of the Republic of Croatia (project no. 119-1191155-1203). We would like to thank Mr. Bozo Pranjic from Croatian detergent producing company Labud, Zagreb, for providing us with the surfactant samples. 6. References 1. G. G. Ying, Environ. Int. 2006, 32, 417-431. 2. M. St. J. Warne, A. D. Schifko, Ecotoxicol. Environ. Saf. 1999, 44, 196-206. 3. M. S. Holt, J. Waters, M. H. I. Comber, R. Armitage, G. Morris, C. Newberry, Water. Res. 1995, 29, 2063-2071. 4. G. W. Fuhs, M. Chen, Microb. Ecol. 1975,2, 119-138. 5. M. Sidat, F. Bux, H. C. Kasan, Water SA 1999, 25, 175-179. 6. J. Hrenovic, D. Tibljas, H. Büyükgüngör, Y. Orhan, Food. Technol. Biotechnol. 2003, 41, 331-338. 7. APHA, AWWA and WEF, Standard methods for the examination of water and wastewater, 21st Edition, Port City Press, Baltimore, 2005. 8. G. F. Longman, The analysis of detergents and detergent products, Wiley-Interscience, London, 1975. 9. A. V. Few, R. H. Ottewill, J. Colloid Sci. 1956, 11, 34. 10. M. E. Turney, D. W. Cannell, J. Am. Oil Chem. 1965, 42, 544-546. 11. 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Utsunomiya, T. Watanuki, K. Matsushita, M. Nishina, I. Tomita, Chemosphere 1997, 35, 2479-2490. 38. S. R. D. Tozum-Calgan, N. Z. Atay-Guneyman, J. Environ. Sci. Health Part A. 1994, 29, 355-370. 39. L. Mariani, D. Pascale, O. Faraponova, A. Tornambe, A. Sarni, S. Giuliani, G. Ruggiero, F. Onorati, E. Magaletti, Environ. Toxicol. 2006, 21, 373-379. 40. J. Hrenovic, T. Ivankovic, Cent. Eur. J. Biol. 2007, 2, 405414. 41. J. Hrenovic, T. Ivankovic, L. Sekovanic, M. Rozic, Cent. Eur. J. Biol. 2008, 3, 143-148. 42. M. T. Garcia, E. Campos, I. Ribosa, Chemosphere 2007, 69, 1574-1578. Povzetek Testirali smo vpliv strupenosti dvanajstih vrst različnih (anionskih, kationskih, amfoternih in neionskih) komercialnih surfaktantov na kulturo Acinetobacter junii. Sedem je bilo strupenih (EC50 vrednosti med 1 in 10 mg L-1), medtem ko smo jih pet opredelili kot nestrupene (EC50 višja od 100 mg L-1). Najbolj strupen surfaktant je bil Kutriacid (EC50 = 1.4 mg L-1). Strupenost anionskih surfaktantov je naraščala s krajšanjem alkilne verige, vendar ni bila odvisna od vrste aktivne skupine, medtem ko je strupenost neionskih surfaktantov naraščala z dolžino verige. Iz primerjave vseh testiranih surfaktantov ni bilo možni dobiti splošnih zaključkov o vplivu vrste ali dolžine verige na strupenost, ugotovili smo le, da bolj razvejana veriga povzroča manjšo strupenost ali pa ni strupena za A. junii.