Surfactants (anthropogenic substances that can change the surface tension of fluids or the interfacial tension between liquids, solids, and gases) are categorized as anionic, cationic, or nonionic, according to the electrical action of their molecular structure 1. Surfactants consist of a polar, water-soluble head group and an apolar hydrocarbon tail group having little solubility in water 2. They are omnipresent in industry, agriculture, medicine, and daily life, functioning as agents of dispersion, emulsification, solubilization, laundering, antiseptic, and other purposes 3. In recent years, surfactant production has increased markedly, and the majority of surfactants in China are directly discharged into water bodies, posing a threat to ecosystems and the environment.
Many studies have been performed on the toxicity of surfactants to organisms. Liwarska-Bizukojc et al. 4 researched the toxicities of three widely used anionic surfactants to aquatic organisms: Physa acuta Draparnaud, Artemia salina, and Raphidocelis subcapitata. Shcherbakova et al. 5 assessed the toxic effect of various surfactants on acetoclastic methanogens, and cationic surfactants were found to be the most toxic. Pavlic et al. 6 tested the toxicity of six anionic, two amphoteric, and one nonionic surfactant to freshwater green algae and marine diatoms.
Most of the toxicity research has emphasized single or pure toxicants. Aquatic organisms, however, are rarely exposed to only a single contaminant. Rather, they are typically exposed to mixtures of numerous man-made chemicals with varying constituents in varying concentrations and concentration ratios 7. The toxicity of single pollutants is always different from that of their combinations. Put simply, joint effects have additivity, synergism, and antagonism. Thus it is necessary to investigate the rules and mechanisms of the combined actions of various contaminants, as well as the discrepancies between effects of combinations and effects of the single contaminants.
Due to low production cost and ideal properties, sodium dodecyl benzene sulfonate (SDBS) as an anionic surfactant has wide applications in many fields and is the most common surfactant used in domestic detergents. Synthetic detergents are major pollutants of the aquatic environment 8. Cetyl trimethyl ammonium chloride (CTAC), a widely used cationic surfactant, has many industrial applications, such as in medicine, leather production, flocculation of water treatment, antiseptics, and others; it is released into natural environments in effluents. The overuse and massive discharge of the two surfactants constitute a potential menace to aquatic life.
In contrast to other organisms, algae—primary producers in water bodies and significant members of the biological chain—can reflect comprehensive effects on the aquatic ecosystem, making them indicators of water contamination 9. Microalgae can generally be considered as particles whose stability is due to the surface charge (which is electronegative at a pH of 2.5–11.5) 10. When microalgae are treated with CTAC, charge neutralization and particle bridging take place. Anionic SDBS does not exert much influence if used alone 11.
Dunaliella bardawil is a unicellular green alga of great halotolerance lacking a rigid polysaccharide wall. Instead, the algae are natural protoplasts enclosed by an elastic plasma membrane 12. This is also the only green algae without a cell wall; it is instead surrounded by an elastic cytomembrane. Thus variation in osmotic pressure accordingly results in changes of cell morphology and size. As with Dunaliella salina13, D. bardawil has been shown to accumulate massive amounts of β-carotene when grown under defined growth conditions 14.
To the the authors' knowledge, studies on the environmental toxicology and bioeffects of the two typical surfactants SDBS and CTAC have not been reported. The present study explored the toxicity of the two representative surfactants as well as that of their combination at different levels on D. bardawil. Cell growth, cellular morphology, β-carotene accumulation, and responses of both superoxide dismutase (SOD) and catalase (CAT) to pollutants were investigated.
MATERIALS AND METHODS
Chemicals and equipment
Sodium dodecyl benzene sulfonate (molecular wt: 348.48; purity ≥ 90.0%) and CTAC (molecular wt: 320.00; purity ≥ 98%) were purchased from Tianjin Fuchen Chemical Reagents. All the reagents used in the present study were of analytic grade. The centrifuge used was an Eppendorf 5804R.
Strain 435 (UTEX 200) of D. bardawil was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. Cells of D. bardawil were cultivated in defined medium 15 containing 1.5 M NaCl (which was sterilized at 121°C for 30 min before inoculation) at 26°C and 144 µmol photons m−2 s−1 provided by cool-white fluorescent lamps, under a 14:10-h light:dark cycle with shaking at 96 rpm.
Cells of D. bardawil were harvested at the log phase or late log phase. Algal pellets and equal volumes of fresh culture medium of 80 ml were added to previously sterilized conical flasks of 250 ml volume with each pollutant at various concentrations. The experiments included three groups: the SDBS group at concentrations of 200, 550, 900, 1,350, 1,800, and 2,400 mg/L; the CTAC group at concentrations of 0.4, 0.7, 1.0, 1.3, 2.8, and 3.5 mg/L; and the joint toxicity group at different levels as shown in Table 1. An algal culture with no pollutants served as control. Algal absorbance was determined at 630 nm at the same time of each day over a 10-d period, and morphological observations were subsequently made.
The standard curve for β-carotene was obtained with different optical density (OD453) values against the corresponding concentrations of the β-carotene/acetone standard solutions. The regression equation is y = 0.2839x + 0.0099, r2 = 0.9976, where y is the β-carotene content (in mg/L) and x is the OD453 value.
β-Carotene from D. bardawil cultures was extracted as described previously 16, 17. A cell pellet of 2 ml of algal culture harvested by centrifugation at 82 g for 10 min was dissolved in acetone, followed by agitation until all algal cells turned white, indicating that pigments were completely extracted. Saponification of chlorophyll and neutral fat contained in pigment mixtures was performed in 60% (w/v) ethanol/KOH (9:1) for 20 min at 4°C, avoiding illumination. The mixture was separated into two segments. Supernatant containing β-carotene was removed into volumetric flasks of 10 ml, and acetone was added for a constant volume. Finally, the OD453 value was measured and converted into β-carotene concentration based on the regression equation.
Using centrifugation at 4629 g for 10 min at room temperature, we harvested 10 ml of D. bardawil cells treated with surfactants in cultures for 48 h. After removal of the supernatant fluid, algal pellets were transferred to a 2-ml centrifuge tube and then resuspended with 1 ml of sodium phosphate buffer (50 mM, pH 7.8) and 0.02 g polyvinylpyrrolidone. The suspension was centrifuged at 12,000 g for 20 min at 4°C to crumble the cells. The supernatant was an enzyme extract and was maintained at −20°C for activity analysis.
SOD activity analysis
Activity of SOD was analyzed according to the method of Constantine and Stanley 18, with some modifications. The reaction mixtures of sample groups contained 1 ml of methionine (39 mM), 50 µl of enzyme extract, 1 ml of nitroblue tetrazolium (377 µM), and 1 ml of riboflavin (24 µM). In two control groups, 50 µl of sodium phosphate buffer was used instead of enzyme extract. After homogenizing, the mixtures, excluding one control group placed in total darkness, were illuminated by a fluorescent lamp (72 µmol photons m−2 s−1) for 20 min, and then the absorbance was determined at 560 nm. Nitroblue tetrazolium was reduced to blue formazan by O2−, which has a strong absorbance at 560 nm. However, the presence of SOD inhibits this reaction 19. One unit of SOD activity was defined as the amount of enzyme that caused a 50% inhibition of nitroblue tetrazolium reduction. The activity of SOD was calculated using the following equation
where SODA is SOD activity, ACK is the absorbance of illuminated control, AE is the absorbance of sample, V is the total volume of sample (ml), VT is the enzyme volume used in reaction (ml), and W is sample fresh weight (g).
CAT activity analysis
The activity of CAT was analyzed according to the method of Hao 20, with some modifications. A combination of 1 ml of enzyme extract and 0.5 ml of sodium phosphate buffer (50 mM, pH 7.8) were added to three test tubes. One of them was boiled to denature the enzyme and used as blank. The other two test tubes were preheated at 25°C, and then 2 ml of 0.1 M H2O2 was added in sequence and timed instantly. Changes in absorbance were taken to be proportional to the breakdown of H2O221. The absorbance was determined for 4 min at 240 nm by an ultraviolet spectrophotometer and read every 1 min. One CAT unit was described as the amount of enzyme whereby the A240 reduction was 0.1 in 1 min. The activity of CAT was calculated using Equation 2
where CATA is CAT activity, AS0 is the absorbance of control with denatured enzyme, AS1 and AS2 are the absorbance of tested reacting systems, VT is the total volume of extracted enzyme (ml), Vs is the volume of added enzyme (ml), W is sample fresh weight (g), and t is the reaction time (min).
Evaluation of joint toxicity
The joint toxicity of SDBS and CTAC was evaluated by the additive index method 22. Growth and inhibition rate of algae were calculated by Equations 3 and 4, respectively
where i represents mean growth rate; Nt and N0 are cell numbers at time t and t0, respectively; IR% is percentage of inhibition efficiency; and i(b) and i(tox) are the mean growth rates of the control group and the experimental group, respectively.
The curves of inhibition rate against concentrations of surfactants were plotted, and linear regression was used. Median inhibitory concentration (IC50) values were obtained by regression equations. The toxic summation of combined compound (S) was calculated using Equation 5
where A and B are median effective concentration (IC50) values of SDBS and CTAC alone, respectively, and Am and Bm are IC50 values of mixtures.
The additive index (AI) was obtained as follows: If S > 1.0, AI = –S + 1, and if S ≤ 1.0, AI = 1/S – 1.
Joint toxicity was assessed as follows: The joint effect is additive when AI = 0, antagonistic when AI < 0, and synergistic when AI > 0.
Each result shown is the mean of three replicated studies. The IC50 values for both cell growth and carotene content were calculated by the linear interpolation method. One-way analysis of variance of the data was performed using the program SPSS-13, and significance was determined at a 95 or 99% confidence limit.
Cell growth of D. bardawil
Figure 1 shows a decline in cell growth of D. bardawil subjected to surfactant treatment after 1 to 2 d and then a general increase. Lethal effects were seen in algae treated with SDBS at 2,400 mg/L, CTAC at 3.5 mg/L, and binary complex at level 6. Under treatment with SDBS at 2,400 mg/L, cell numbers constantly decreased until day 10. Cell numbers of algae treated by CTAC at 3.5 mg/L declined until day 3 and then reached a stationary stage, as shown in Figure 1B. Figure 1C shows that joint toxicity decreased algal growth until day 6, followed by a stationary stage. The algae treated with SDBS at 550, 900, 1,350 and 1,800 mg/L, all had similar growth curves (Fig. 1A). The curves of algae at CTAC concentrations of 1.0, 1.3, and 2.8 mg/L also had similar growth trends (Fig. 1B). The cell numbers of algae treated with a the mixture of 200 mg/L SDBS and 0.4 mg/L CTAC were above control at 7 to 10 d, showing a hormesis effect due to the antagonism between SDBS and CTAC. The cell numbers of algae treated with a mixture at level 5 were obviously above those shown in Figure 1A and B correspondingly. In addition, cell numbers of algae treated with 1,350 mg/L SDBS and 1.3 mg/L CTAC combined were greater than those treated with either surfactant alone, illustrating attenuated toxicity. Algae exposed to the binary system at level 3 grew better than those exposed to CTAC at 0.4 mg/L.
Analysis of joint toxicity effect
An IC50 based on cell number data was calculated. When SDBS acted alone, IC5010 d = A = 2,044 ± 637.3 mg/L; when CTAC acted alone, IC5010 d = B = 2.8 ± 1.49 mg/L; as SDBS concentration in binary mixtures, IC5010 d = Am = 2,305 ± 891.2 mg/L; and as CTAC in binary mixtures, IC5010 d = Bm = 3.3 ± 1.86 mg/L.
The AI value (−24.73, 0.159) indicated a probable antagonistic effect existing between SDBS and CTAC. Also, it was inferred from the magnitude of the EC50 that CTAC is more hazardous to D. bardawil than SDBS.
The IC50 for carotene content was also calculated. When SDBS acted alone, IC5010 d = 624.44 ± 278.82 mg/L; and in the binary system, IC5010 d = 787.3 ± 171.16 mg/L. When CTAC (D) acted alone, IC5010 d = D = 0.46 ± 0.034 mg/L; and in a mixture, IC5010 d= 0.99 ± 0.050 mg/L. Based on these data, we calculated AI = (−4.10, −1.67) < 0, indicating an antagonistic effect. It also could be concluded that CTAC was more hazardous than SDBS.
Observation of cell morphology
Compared with normal cellular morphology, the number of broken cells rose with the increase in surfactant concentration, as shown in Figure 2, which illustrates the toxic effects of surfactants on cell growth. When observed under an optical microscope, many of the algae treated with surfactants were less vigorous than control and even remained still. Cells exposed to toxicants appeared in various shapes, including globular, elliptical, and some special shapes such as those shown in Figure 2C1. If the culture was gently shaken, an agglomeration phenomenon could be more commonly observed in cultures containing CTAC than in those containing SDBS and their mixture, as shown in Figure 2B.
Response of β-carotene accumulation
After 1 to 2 d of surfactant treatment, growth of D. bardawil declined, as shown in Figure 3. Algal growth rates affected by SDBS at 550, 900, and 1,800 mg/L (Fig. 3A); affected by CTAC at 1.0 and 2.8 mg/L (Fig. 3B); and treated by a binary mixture at levels 3 and 5 (Fig. 3C) had quite similar curves, increasing until the day 3 but decreasing on day 4 and then advancing again. Sodium dodecyl benzene sulfonate at 2,400 mg/L, CTAC at 3.5 mg/L, and the binary complex at level 6 had lethal effects on algae, making absorbance unmeasurable at day 7 (Fig. 3A), day 3 (Fig. 3B), and day 4 (Fig. 3C). The curve of level 6 in Figure 3C is clearly seen to be closer to the curve of the control than are the curves of both SDBS at 2,400 mg/L and CTAC at 3.5 mg/L, indicating that algae with mixed surfactants at level 6 were less inhibited.
At the other concentrations, surfactants also inhibited carotene synthesis. The curve of algae affected by 1,800 mg/L SDBS was closer to that of cells treated with 2,400 mg/L SDBS in comparison with the curves of level 5 and 6 in Figure 3C. We inferred that SDBS alone at 1,800 mg/L was more hazardous to algae than it was in the mixture. By comparison, the curves of cells exposed to 1.0 and 1.3 mg/L CTAC alone were closer to each other than the curves of level 5 and 6 shown in Figure 3C, which also proved that the joint toxicity of the mixture was mitigated. These results were consistent with the results from the joint toxicity analysis. Statistical analysis showed significant variations between treatments (p < 0.05), and the correlation between β-carotene concentration and surfactant concentration was significantly negative (p < 0.05).
Figure 4 shows that SDBS, CTAC, and their mixture all had effects on SOD activity. From Figure 4A, it can be seen that SDBS alone increased enzyme activity at concentrations of 550, 900, 1,350, and 1,800 mg/L (and did not do so at 2,400 mg/L) and reached the maximum value at 1,350 mg/L. The combined action of SDBS and CTAC elevated SOD activity at concentrations 550, 900, and 1,350 mg/L and decreased it at 200 and 2,400 mg/L. Both of the two groups reached their maximum value at 1,350 mg/L. Moreover, SOD activity in the joint toxicity group increased more than that in the SDBS group at concentrations of 550, 900, and 1,350 mg/L but had a sharper decrease at concentrations of 1,800 and 2,400 mg/L. The sharper decrease indicated the complicated mechanism of surfactants influencing SOD activity.
Figure 4B shows a similar trend of SOD activity influenced by CTAC alone and in a mixture. The difference is that SOD activity in algae exposed to CTAC was greater than that in the binary system at all concentrations except 1.3 mg/L, for which both groups had the maximum value. Due to a hormesis effect with mixtures at level 1, this SOD activity is lower than those in algae at both 200 mg/L SDBS and 0.4 mg/L CTAC. By comparing Figure 4A and B, we concluded that the mixed surfactants at levels 2, 3, and 4 were more deleterious to algae than SDBS at 550, 900, and 1,800 mg/L but less harmful than CTAC at 0.7, 1.0, and 1.3 mg/L.
Figure 5A shows that CAT activity in the SDBS group increased at concentrations of 900 and 1,350 mg/L (lower than values in the joint toxicity group) and decreased conspicuously at 1,800 and 2,400 mg/L. In the binary system, CAT activity was increased at concentrations of 550, 900, and 1,350 mg/L and was reduced at 200, 1,800, and 2,400 mg/L. In both groups, CAT activity reached its peak value at 1,350 mg/L.
Figure 5B shows that CAT activity in D. bardawil exposed to CTAC at all concentrations was higher than that of algae treated with combinations of CTAC and SDBS. With CTAC alone, CAT activity increased at concentrations 0.4, 0.7, 1.0, and 1.3 mg/L and declined at 2.8 and 3.5 mg/L. In the binary mixtures, CAT activity was elevated at 0.4, 0.7, 1.0, and 1.3 mg/L and declined at 0.4, 2.8, and 3.5 mg/L. As with the results of SOD activity, we also concluded that the mixed surfactants at levels 2, 3, and 4 were more deleterious to algae than SDBS at 550, 900, and 1,800 mg/L but less harmful than CTAC at 0.7, 1.0, and 1.3 mg/L. Figure 5A and B shows that for CTAC, the mixed surfactants also produced a sharper decline in both CAT and SOD activity (shown in Figure 4B).
The potential impact of a surfactant is related to several factors, such as toxicity, concentration and persistence, the period of exposure, and the stability and toxicity of any metabolites 23. In terms of the relationship between toxicity and structure, results of the present study demonstrated a positive relationship between alkyl chain length and toxicity within a surfactant class, while N-containing amines and quaternary ammonium compounds had the greatest toxicity between classes 24. Our investigation also substantiated that the toxicity of CTAC is greater than that of SDBS.
Although the mechanisms of surfactant toxicity may vary with different species 25 and different categories of surfactants 24, it is generally accepted that such toxicity affects the structure and integrity of algal lipid membranes, binding with or denaturing membrane proteins 26, thus causing increased membrane permeability and leakage of compounds significant for cell viability like ions, amino acids, and others 27–30. In the present study, it was observed that the number and extent of broken cells rose with increasing toxic concentration. When the concentration was low, however, the hormesis phenomenon was observed. Hormesis is a dose–response relationship that is characterized by a low dose stimulation and a high dose inhibition 31. Sodium dodecyl benzene sulfonate and CTAC are anionic and cationic surfactants, respectively. In the present study, an antagonistic effect was observed when the two surfactants were used together. This could be explained by their distinct polarities. The opposite charges may lead to a reaction between them when they exist simultaneously, which may lessen their binding action to cellular membranes and thus mitigate toxicity. However, the new ionic bonds created may also affect the algal membrane in a different way from that of a single surfactant. The lowered toxicity of mixed compounds even could stimulate the growth of algae and produce the phenomenon of hormesis. Dunoliella bardawil (IC5010d = 2.8 ± 1.49 mg/L) is less sensitive to CTAC than the other green algae Selenastrum capricornutum (EC50 = 780 µg/L) 32 and Chlorella vulgaris (EC50 96h = 145 ± 13.35 µg/L) 33. Dunaliella has a higher tolerance to salinity stresses 34, which may be confirmed by the present results. For both SDBS and CTAC, the IC50 for carotene content is lower than that for cell growth, indicating that carotene accumulation is not entirely consistent with cell growth.
Once inside the cell, surfactants may affect thylakoid organization and chlorophyll synthesis (with consequent impairment of photosynthetic capacity) and may interact with bioactive macromolecules 35, 36.
From another aspect, in the presence of surfactants or other pollutants, some Dunaliella species often generate reactive oxygen species, including superoxide (·O), singlet oxygen (1O2), and hydrogen peroxide (H2O2) in the chloroplast under osmotic stress 37, which can seriously disrupt normal metabolism through oxidative damage to membrane lipids, proteins, pigments, and nucleic acids 38. For self-protection, algae developed an enzymatic system as a response to such impairments. The dominant enzymes are SOD, CAT, and peroxidase. Superoxide dismutase is capable of transferring active ·O to molecular oxygen and H2O2. The H2O2 produced is scavenged by CAT and a variety of peroxidase 39. Wu Jifa et al. 40 studied the toxicity of SDBS to Lateolabrax japonicus. The results indicated that CAT activity remained at a significantly high level (p < 0.05) over 18 d of exposure. It was shown in the present study that both SOD and CAT activity is promoted at certain concentrations, presumably to repair damage. For example, SOD activity of algae exposed to SDBS was significantly increased with increasing concentrations of SOD ranging from 550 to 1,800 mg/L. In regard to binary surfactants, both SOD and CAT activity showed a sharper decrease at high concentrations compared with one surfactant alone. We deduce that the hydrocarbon chains of mixtures possess more affinities to enzymes and that the binding action changed enzymatic structures, resulting in denatured enzyme.
In conclusion, the toxicity of CTAC is greater than that of SDBS and the joint effect of both together showed antagonistic effects, judged by AI value data (−4.10, −1.67) < 0 obtained from carotene content. Both cellular growth and β-carotene response underwent a general reduction at the initial 2 d and then increased again except for algae at the maximum concentrations tested. Algae cultivated at level 6 of the binary system showed hormesis due to the mitigated toxicity. Lethal effects on D. bardawil were found after treatment with SDBS at 2,400 mg/L, CTAC at 3.5 mg/L, and joint surfactants at level 6. Both SOD and CAT activity showed similar associations with varied concentrations of surfactants. Superoxide dismutase was significantly promoted by 550 to 1,800 mg/L SDBS, 0.7 to 1.3 mg/L CTAC, and mixtures at levels 2 to 4. Catalase was clearly promoted by 900 mg/L SDBS, 0.4 to 1.3 mg/L CTAC, and mixtures at levels 2 to 4.
This project was supported by the National Natural Foundation of China (grant 31171631) and Science and Technology Programs of Guangdong Province (grant 2011B031200005).