Introduction
Perfluorooctane sulfonic acid (PFOS, C8F17SO3H), a perfluorinated compound, is a synthetic material. PFOS possesses superior heat resistance, chemical resistance, water repellency, oil repellency, rub resistance, and surface activity; hence, it is widely utilized in fire blowing agents, in coating agents for carpets, clothing, paper, and leather, in commercial stain preventing agents, in metal cleaners, and in aircraft hydraulic fluids. While PFOS is highly valued as a chemical agent for industrial and consumer uses, it is not easily degraded and is known to remain in the environment and biological materials for long periods of time [1,2].
Cerebellum tests using rats have shown that PFOS can induce neurotoxicity, and it can also cause reproductive toxicity in pregnant rats as indicated by a decrease in the survival rate of newborns. A recent study reported that the average blood PFOS concentration for Koreans was 6.06 ng/mL [3].
PFOS was registered as a persistent organic pollutant (POP) at the 4th Stockholm Convention on POPs held at Geneva, Switzerland, in May 2009. In South Korea (hereafter Korea), PFOS was designated as a POP by amending the enforcement decree of the POPs Control Act in April 11, 2011, and consequently, its manufacturing, export, and utilization have been severely restricted.
The 3M company voluntarily stopped PFOS production in 2002 and began developing alternatives such as perfluorobutane sulfonate with a reduced number of carbon chains; but they have not yet succeeded in developing a substance that can completely replace PFOS to date (UNEP/POPS/POPRC.6/Add.3/Rev.1, 2011.10.14). In Korea, Shin’s research team at Changwon National University (Department of Chemistry) are developing PFOS alternatives with a decreased or increased number of carbon atoms and a decreased number of fluorine atoms [4]. The objective of this study was to evaluate the biodegradation potential of five species four PFOS alternatives that were developed at Changwon National University. It is hoped that this research will aid in the identification of potentially useful PFOS alternatives.
Materials and Methods
Test Materials
This study used microorganisms that were collected from 15 sampling points at city sewage treatment plants, industrial waste water treatment plants, rivers, lakes, and the sea throughout Korea. The microorganisms were cultured at the good laboratory practice (GLP) culture room of the Korea Environment Corporation for more than 1 month prior to use in experiments.
PFOS alternatives that have been synthesized by Shin’s laboratory at Changwon National University include C8F17KO3S (molecular weight [MW], 538.22, PFOS in a form of potassium salt), C10H8F13KO3S (MW, 494.31), C8H8F9KO3S (MW, 394.30), C6H8F5KO3S (MW, 294.28), and C5H8F3KO3S (MW, 244.27); all of these chemicals were used in this study.
Biodegradation tests were performed according to the Organization for Economic Cooperation and Development TG 301C protocol using basic culture medium prepared at the GLP laboratory of the Korea Environment Corporation. An OxiTop Control system (Control 100, WTW, Munich, Germany) was used in the experiments.
Biodegradation Test
A total of 12 biochemical oxygen demand (BOD) bottles were used in the biodegradation tests. In bottles 1 through 5, the five test materials (C8F17KO3S, C10H8F13KO3S, C8H8F9KO3S, C6H8F5KO3S, C5H8F3KO3S) were added 0.015 g each, and 150 mL of deionized water (DW) was injected. In bottles 6 through 10, the five test materials were added 0.015 g each, and then 1.8 mL basic culture medium, 131.53 mL DW, and 16.67 mL activated sludge were injected. To investigate the condition of the activated sludge, 0.015 g of a control material (aniline, C6H7N) was added in bottle 11, and then 1.8 mL basic culture medium, 131.53 mL DW, and 16.67 mL activated sludge were injected. Bottle 12 contained 1.8 mL basic culture medium, 131.53 mL DW, and 16.67 mL activated sludge, and it was used for comparison purposes with bottles 6 through 10. The experiment was conducted in an incubator for 28 days under aerobic conditions at 24.9 to 25.1°C and pH 6.70 to 7.92.
Biodegradation Rate Calculation
The biodegradation rate (%) was calculated as follows:
Test material:
Control material:
BOD (mg/L) of test materials (or control materials) that is measured by a BOD curve.
Theoretical oxygen demand (ThOD, mg/L) that is the total amount of oxgen required to oxidise test meterials (or control material) completely.
CCHHClClNNNaNaOOPPSS
Separation and Identification of Degrading Microorganisms
Mineral medium prepared in the biodegradation laboratory at Chonnam National University were used as the separation medium, and PFOS alternatives used in the test were the only carbon source. As for the microorganism separation, the growth of microorganisms was visibly confirmed after culturing at 35°C in incubator under aerobic conditions for 24 hours. These microorganisms were further subcultured five times to achieve pure separations. The separated microorganisms were identified by 16S rRNA sequencing analyses.
Results
Biodegradation rates of aniline (i.e., the control material in bottle 11) were 60.5% and 72.1% on days 7 and 28, respectively, thus confirming that the activated sludge (microorganisms) used in this study are behaving normally. The BOD values for bottle 6 with C8F17KO3S, PFOS in a form of potassium salt reached 116.1 mg/L on day 7, 129.3 mg/L on day 14, and 139.6 mg/L on day 28 (Figure 1A). On the contrary, the BOD values for bottle 12 without test materials reached 120.8 mg/L on day 7, 137.8 mg/L on day 14, and 150.9 mg/L on day 28. The BOD values for bottle 7 with C10H8F13KO3S reached 126.0 mg/L on day 7, 151.0 mg/L on day 14, and 164.6 mg/L on day 28 (Figure 1B). The BOD values for bottle 8 with C8H8F9KO3S reached 128.3 mg/L on day 7, 150.0 mg/L on day 14, and 170.6 mg/L on day 28 (Figure 1C). The BOD values for bottle 9 with C6H8F5KO3S reached 122.5 mg/L on day 7, 141.0 mg/L on day 14, and 154.5 mg/L on day 28 (Figure 1D). The BOD values for bottle 10 with C5H8F3KO3S reached 119.3 mg/L on day 7, 133.8 mg/L on day 14, and 144.2 mg/L on day 28 (Figure 1E).
After calculating the biodegradation rates, we found that bottle 6 with C8F17KO3S, which is the PFOS in a form of potassium salt, and bottle 10 with C5H8F3KO3S had negative values, thus indicating that biodegradation did not occur at all for 28 days. Biodegradation rates of bottle 7 with C10H8F13KO3S were confirmed at 6.8% on day 7, 17.1% on day 14, and 17.7% on day 28. Biodegradation rates of bottle 8 with C8H8F9KO3S were 9.3% on day 7, 15.1% on day 14, and 24.3% on day 28. Biodegradation rates of bottle 9 with C6H8F5KO3S were 2.0% on day 7, 3.7% on day 14, and 4.1% on day 28. Considering that the biodegradation rate for highly degradable materials is greater than 60% in general, we cannot conclude that all four PFOS alternatives used in the present study were well degraded. However, the biodegrading rate of C8H8F9KO3S was confirmed as the most superior (Table 1).
For three PFOS alternatives (bottle 7 with C10H8F13KO3S, bottle 8 with C8H8F9KO3S, and bottle 9 with C6H8F5KO3S) that were confirmed to undergo biodegration, the degrading microorganisms were separated and subjected to sequencing analyses. Samples from the C10H8F13KO3S treatment showed greater than 99% of high homology with Serratia marcescens, Acinetobacter spp., and Pseudomonas geniculata. Samples from the C8H8F9KO3S treatment had more than 99% of high homology with Bacillus spp., Serratia marcescens, and Chryseobacterium spp. Samples from the C6H8F5KO3S treatment had more than 99% of high homology with Bacillus cereus and Serratia marcescens (Table 2).
Discussion
C8F17KO3S, which is the PFOS in a form of potassium salt, as well as potential PFOS alternative C5H8F3KO3S with a decreased number of carbon and fluorine atoms did not undergo biodegradation at all. While C8H8F9KO3S, which has the same number of carbons as the parent compound but a reduced number of fluorines, showed the highest biodegradation rate followed by C10H8F13KO3S and C6H8F5KO3S with 6 carbon atoms and 5 fluorine atoms also exhibited susceptibility to biodegradation, but at a markedly low rate. Together, these results suggest that it may be it advantageous to develop PFOS alternatives with 8 carbons, the same as PFOS, but a reduced number of fluorines, as such chemicals are more susceptible to biodegradation than the parent compound.
Three microorganisms were confirmed to degrade C8H8F9KO3S and C10H8F13KO3S, which showed a relatively high biodegradation rate. Two microorganisms were identified during the biodegradation of C6H8F5KO3S, which exhibited a relatively low biodegradation rate. Such results follow the general principle that there are more species of microorganisms that can act as degradators for chemicals with a higher biodegradation rate.
Commercialization of PFOS alternatives should proceed with an overall consideration of ecotoxicity, human toxicity, functionalities, and economic feasibility. It is therefore necessary to perform additional studies regarding this matter.
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