Interpretation of Data from Nonstandard Studies: The Fate of Octamethylcyclotetrasiloxane in a SedimentlWater Microcosm System

David Kent*

Technology Sciences Group, Inc., Washington, DC. Paul Fackler and Deborah Hartley

Springborn Laboratories, Inc., Wareham, MA

James Hobson

Technology Sciences Group, Inc., Washington, DC

As part of a TSCA Section 4 Consent Order agreement, the biodegradation of the organosilicone octamethylcyclotetrasiloxane (OMCTS) was investigated using an updated version of the Bourquin Eco-Core design. This study illustrates the concept that caution must be exercised in all data interpretation but especially when nonstandardized studies are used in the regulatory decision- making process. Because of OMCTS’ unique combination of high volatility and low water solubility, modifications to the test procedures were necessary, as were a series of supplemental studies conducted to aid in the interpretation of the original study results. While the correct interpretation of the data was achieved, the use of supplemental studies highlights the importance of professional judgment in conducting testing for regulatory purposes. Researchers should not hesitate to question the appropriateness of standard and nonstandard study designs as related to the specific physico- chemical properties of the test material being investigated (e.g., Henry’s Law constant). In addition, investigators should be prepared to conduct supplemental studies and analyses as needed to ensure the accurate interpretation of study results. Misinterpretation of data can have serious ramifications in a regulatory context, whether it may result in keeping an environmentally sound product off the market, in misdirection of limited financial and personnel resources away from more serious environmental concerns, or in placing unwarranted limitations on a given chemical or chemical product. 0 1996 by John Wiley & Sons, Inc.

I NTRODUCTI 0 N

The behavior of a chemical introduced into the environ- ment is a function both of its physicochemical proper- ties and the processes that control persistence, mobil-

ity, and accumulation. Although a chemical may be degraded by abiotic mechanisms (e.g., chemical oxida- tion, hydrolysis, and photodegradation), the decompo- sition of organic chemicals is very often mediated by biological processes, e.g., microbial metabolism. How- ever, while biodegradation is one of the most important -

determinants of the environmental fate of many organic

difficult to measure for aquatic ecosystems. Standard-

* To whom correspondence should be addressed at Technology Sciences Group, 1101 17th Street, NW, Suite 500, Washington, chemicals, it is often poorly understood and commonly DC 20036.

Environmental Toxicology and Water Quality: An International Journal, Vol. 11 (1996) 145-149 0 1996 by John Wiley & Sons, Inc. CCC 1053-47251961021 45-05

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146 KENT ET AL.

ized techniques to quantify biolysis of anthropogenic organic chemicals are limited.

One technique that is available for measuring the biodegradation of organic chemicals in the environ- ment includes the use of core chambers, first developed and tested at the U.S. Environmental Protection Agency laboratory in Gulf Breeze, Florida, as part of the Eco-Core Program (Bourquin, 1975; Bourquin et al., 1977; Bourquin et al., 1979; Spain and Van Veld, 1983; Spain et al., 1980, 1984). Originally used with salt marsh sediment systems, these core chambers acted as laboratory microcosms to simulate natural conditions for studying the biodegradation of test materials in cer- tain lentic environments. The method was designed for organic compounds with moderate or greater water solubility and moderate to low volatility.

Recently, a modified and updated version of the eco- core technique has been used in the study of cumene with reasonably good results (Williams et al., 1993). Cumene is a volatile organic compound that is also moderately soluble. Although this method of determin- ing biodegradation was applied to this volatile com- pound successfully, the method must be utilized with care, as specific properties of some compounds may lead to misinterpretation of the resultant data.

As an illustration of this, the method was used to study the biodegradation of an oligomeric silicone, oc- tamethylcyclotetrasiloxane (OMCTS). OMCTS is used predominantly as a site-limited intermediate in the pro- duction of polymeric silicones (polydimethylsiloxanes, PDMS), and to a more limited extent in personal care consumer products such as antiperspirants and skin and hair care products (Syracuse Research Corpora- tion, 1985; Versar, 1986). OMCTS is characterized by arelatively unique combination of high volatility (vapor pressure = 1.0 mm Hg at 25°C; Versar, 1986), a high Henry’s Law constant (> 17; Ann Arbor Technical Ser- vices, 1990), and low water solubility (74 pg/L in fresh- water; Springborn Laboratories, 1989a,b). OMCTS also has a relatively high octanol/water partition coef- ficient (log KO, = 5.1; Versar, 1986). This combination of properties make OMCTS both a highly volatile and a highly sorptive compound, suggesting that it could potentially distribute to the atmosphere as well as be adsorbed onto sediment particles (Mueller et al., 1995). As a result of the perceived potential for widespread environmental use and dispersion and a concern for accumulation in aquatic sediments, OMCTS was the subject of a TSCA Section 4 Consent Order (CO) [U.S. Environmental Protection Agency (EPA), 19861. Un- der this CO, a comprehensive aquatic toxicity and environmental fate testing program was sponsored by the Silicones Environmental Health and Safety Council. These study results are published elsewhere (Kent et al., 1994; Fackler et al., 1995; Hamelink et

al., 1995; Hobson, 1995; Hobson and Silberhorn, 1995; Sousa et al., 1995). The current paper describes only the results and interpretation of the biodegradation study.

Figure 1 depicts the basic test apparatus, Whereas previous designs (e.g., Williams et al., 1993) used intact sediment cores in their test systems, the microcosms for this study consisted of natural sediment and water collected from an uncontaminated pond, which was passed through a 2.0 mm stainless sieve to assure uni- formity and to remove coarse debris.? Radiolabeled OMCTS was added directly to the aqueous phase of each individual test system and the systems were moni- tored for 56 days. The rate and extent of mineraliza- tion and disappearance of the radiolabeled material was determined by passing C0,-free air through the core chambers and trapping exhaust gases on a vola- tile organic trap and an alkaline CO, trap. Sterile controls were included in the test to examine the po- tential for abiotic biodegradation. In addition, micro- bial biomass, as determined by standard plate count techniques, was evaluated at test start and near the end of the study to determine the possible toxicity or enhancement effects of the test substance on micro- bial viability.

An important modification to Bourquin’s procedure involves the pattern of aeration. Since the test materials evaluated by Bourquin and co-workers were of rela- tively low volatility (e.g., methyl parathion), they were able to maintain a continuous aeration of the test ves- sels throughout the study period. Due to the high vola- tility of OMCTS, the test vessels in the current study were aerated intermittently, for 10 min periods, once every three days. This variation was adapted from a study of cumene (Williams et al., 1993), and strikes a balance between providing adequate aerobic condi- tions for maintenance of a healthy microbial commu- nity while providing sufficient residence time to allow biodegradation to occur.

RESULTS AND DISCUSSION

Based on the overall results of the study, it was con- cluded that no biodegradation of OMCTS occurred in the test system. The majority of the added 14C-OMCTS was volatilized and collected on the Tenax trap. High Performance Liquid Chromatography -Radiometric De- tection (HPLC-RAM) analyses showed that less than 5% of the total dose remained in the water by 28 days. Similarly, only a small amount (maximum of 6.7%)

7 Requested by U.S. EPA as an attempt at uniformity for good laboratory practices (GLP) goals.

INTERPRETATION OF DATA 147

Inlet Tube for Aeration Outlet Port

Drierite Ascarite BaloH)2 Cannister Cartridge Solution Scintillation

0 M KOH Rubber Septum for

Water Sampling

Fig. 1. SedimenUwater microcosm

partitioned to the sediments; these values also were less than 5% by day 28 of the study.$

Variable small amounts (<LO% of the total 14C ap- plied) were, however, observed in the KOH traps. These results might suggest that some “biodegrada- tion” was occurring in several of the microcosm cham- bers. However, the sterile control chambers showed similar degradation that, as evidenced by a lack of microbial populations in the sterile controls, indicates the apparent “biodegradation” was not related, in fact, to biological processes. Because OMCTS is known to be susceptible to alkaline hydrolysis (Cecil Frye, per- sonal communication), this “degradation” was be- lieved to be the result of KOH contamination of some Tenax traps and possibly a few microcosm chambers. During the study there was some evidence of backflow in the form of condensation observed in the tubing connecting the Tenax traps to the KOH traps originally believed to be condensed water vapor that had purged from the microcosm chamber. However, because the results of the chemical analyses suggested some form of degradation had occurred, the possibility that some of the KOH had backflowed into the Tenax trap was considered. Furthermore, even though closed glass stopcocks were located on the inlet and outlet ports of the microcosm chambers, alkali may have also reached some of the chambers themseives inadvertently. This is evidenced by a large proportion of dosed 14C remain-

* Since the intent of this paper is to discuss the issues of data interpretation rather than specific study results, only limited data are included here. Comprehensive results of the biodegradation study from which this paper is derived can be found in Springborn Labora- tories (1991) .

ing in the day 28 and 56 sterile control microcosm chambers. HPLC-RAM analysis confirmed that this I4C was not OMCTS.

Analyses of the KOH traps indicated that only a small proportion of the originally applied 14C was trapped in the KOH (<lo%). If backflow did occur, and some of the KOH was pushed back into the Tenax trap, then some of the I4C found in the KOH trap could have originated from adsorbed 14C-OMCTS in the Tenax trap. Because the percentages of dose recovered from the KOH trap were small in all instances, and did not show a consistent pattern of formation, CO, production probably did not occur.

If the study had been terminated at this point, as would be the norm with regulatory testing, the apparent ambiguity of some of the results could have made inter- pretation of the data equivocal. Regulatory reviewers would likely have used a worst-case interpretation. However, because the importance of professional judg- ment was explicitly recognized in the CO, the investiga- tory team chose to conduct a series of three supplemen- tal experiments. The goal of these additional experiments was to reduce the uncertainty associated with the test results, and to establish definitively that any apparent “degradation” was related to chemical degradation from KOH contamination and not biologi- cal processes. In the first experiment, 30 pL of the I4C- OMCTS stock solution (0.100 mg/mL) was dosed into a microcosm chamber containing 82 mL of NANOpure water. A Tenax trap was affixed to the chamber and the system purged for a period of 30 min. After purging, a solution of 1.OM KOH was drawn into the Tenax trap using a syringe, and the trap stored at room tem- perature overnight. The following day the trap was

148 KENT ET AL.

eluted with 10 mL methanol, and the highly basic ex- tract (pH 11) neutralized with HCl before analysis by reverse-phase HPLC-RAM. This analysis demon- strated that the adsorbed OMCTS had completely de- graded to the same product observed during the defini- tive %day study.

In the second experiment, after purging OMCTS from the water in the first experiment, an additional 20 pL of the 14C-OMCTS stock solution (0.100 mg/ mL) was added to the microcosm chamber. Five drops of 1.OM KOH was added and the solution was allowed to sit overnight. The following day the solution was neutralized with concentrated HC1 and subjected to reverse phase HPLC-RAM. The results again indicated that the OMCTS had completely degraded to the ob- served product.

In the third experiment, the solution that had been neutralized in the second experiment was purged with nitrogen gas for a period of 2 h. Aliquots of the solution were removed every 30 min for total I4C determina- tions. The results of this experiment proved that the observed product was nonvolatile, as the dpm/mL of this solution did not measurably decrease (approxi- mately 3300 dpm/mL) during the 2 h purge.

This series of supplemental experiments show that the chemical degradate observed could be formed on the volatile organic trap and in water in the presence of KOH, but was not sufficiently volatile to migrate in the air from water to the Tenax trap. Taken together with the presence of degradation product in the sterile controls, these studies demonstrate that the product observed was not the result of biodegradation in the microcosm chambers. Rather, the data supports the argument that the most likely source of the degradation product is the alkaline hydrolysis of OMCTS from backflow of KOH into the Tenax trap. The apparent backflow of materials, and the need to perform a series of supplemental studies to fully evaluate the results, illustrates the challenge of conducting this type of non- guideline study on such a difficult substance.

In general, this enclosed water/sediment system method appears to be a plausible means of determining biodegradation of some organic compounds. However, a compound such as OMCTS, characterized by the unique combination of high volatility and extremely low water solubility, illustrates some of the potential difficulties associated with the interpretation of experi- mental results. In the Consent Order agreement, EPA recognized the challenges of working with certain com- pounds and included language that acknowledged “good professional judgement will be applied in deter- mining the scientific adequacy and validity of the test results . . .” and that it might be necessary to modify the CO “if successful completion of some tests ap- pear[ed] not feasible scientifically” (U.S. EPA, 1989,

p. 820). Inclusion of such language in a consent order has been atypical, but reflects EPA’s growing recogni- tion of the importance of proper interpretation of re- sults, especially for technically difficult nonstandard- ized studies. The prudent use of professional judgment contributed to the production of scientifically defensi- ble results in this study, and in the overall OMCTS assessment program.

This study was part of a series of studies performed on OMCTS sponsored by the Silicones Environmental Health and Safety Council, then known as the Silicones Health Council. Participating member companies were as follows: Dow Corning Corporation; General Electric Silicones; Bayer Corporation (formerly Miles, Inc.); Rhone-Poulenc, Inc.; Shin-Etsu Silicones of America, Inc.; OSi Specialties, Inc. ; and Wacker Silicones Corporation. Comments on the original manuscript by three anonymous reviewers greatly enhanced the presentation of this information and are sincerely ap- preciated.

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