SELECTIVE MONITORING TO ASSESS THE BIOAVAILABILITY OF ORGANIC CHEMICALS IN AQUATIC SYSTEMS S. W. Morrall, C. A. Smith, W. M. Begley, D. J. Versteeg and J. M. Rawlings Human and Environmental Safety Division, The Procter & Gamble Co., PO Box 538707, Cincinnati, OH 45253 The risk a chemical poses to the aquatic environment is a function of concentration, toxicity, and bioavailability. The concentration of a chemical in the environment represents a maximum dose that is potentially available to an organism. Bioavailability describes the fraction of this potential dose that the organism actually encounters. Bioavailability is determined to an extent by physical properties, such as aqueous solubility, vapor pressure, and partitioning. However, the entire system; biota, environmental compartment composition, and physico-chemical properties are intimately related and their interaction ultimately defines the actual dose or bioavailability of a chemical. We can do a reasonable job of estimating both toxicity, via laboratory testing, and environmental concentration, by direct measurement and modeling. However, accurate assessment of the bioavailability of organic compounds in aquatic systems has remained elusive due to the complexity and variability inherent to the environment. In an aquatic system, organic compounds can be in molecular solution, associated with dissolved organic carbon (DOC), sorbed to colloids, suspended particulates, and sediment. The chemical is available to different organisms to varying degrees, depending upon what form it is. For example partitioning from a dissolved phase to sediment will reduce bioavailability to many fish, but increase availability to organisms that “graze” on periphyton attached to the sediment. Additional variables that impact effects to biological organisms and ultimately risk to the ecosystem, include the physical properties and chemical composition of the aquatic system, route of chemical exposure to organisms, and form of ionizable molecules. Integration of all these factors Preprints of Extended Abstracts Vol. 41 No. 1 668 ELEGANT ANALYTICAL CHEMISTRY APPLIED TO ENVIRONMENTAL PROBLEMS — A PRACTICAL SYMPOSIUM Organized by V. Turoski, S.D. Richardson and J. Plude Symposia Papers Presented Before the Division of Environmental Chemistry American Chemical Society San Diego, CA April 1-5, 2001 is needed to understand the bioavailability of organic chemicals in aquatic systems. This complexity can be reduced in controlled laboratory model systems and with integrated field sampling techniques. However, one should not forget that bioavailability and environmental effects are a product of biology and chemistry, and are not fully described by either discipline alone. Aquatic Toxicity In conventional laboratory testing of toxicity to aquatic organisms care is taken to deliver a constant freely soluble aqueous concentration of test chemical to the organism. Bioavailability is eliminated from the equation and aqueous concentration is equivalent to exposure. Toxicity endpoints are expressed in terms of the aqueous exposure, as opposed to the internal dose or body burden (tissue concentration). One rationale for this design is that the tests are used to identify potential hazard. This hazard is placed into context by an environmental risk assessment, which is expressed as a ratio of an environmental concentration divided by a no observable effect level (PEC/PNEC). This approach builds conservatism into the risk assessment, because bioavailability is not accounted for in the environment (PEC) and designed out of the lab toxicity test (which determines the PNEC). The actual dose of chemical that reaches the site of toxic action in the organism is unknown. This is reflected in the myriad of quantitative structure activity relationships (QSAR) based primarily on descriptors of water to organism partitioning (such as log P or Ko/w) that have been developed for aquatic toxicity. The compounds of interest in this research are primarily ingredients formulated into consumer products that are disposed “down the drain” during routine use. Surfactants, fragrance materials, disinfecting actives, and small water soluble polymers are among the compounds considered. A commonality of these “down the drain” chemicals is they are introduced into the aquatic environment via sewage and waste water treatment plant (WWTP) effluents. Consequently, they are bound to colloidal and dissolved organic matter upon dilution into surface waters. While desorption can occur with dilution, the predominate environmental form is associated with organic material and can exhibit significantly less bioavailability than the molecular solutions employed in laboratory testing. Body Burden and Bioconcentration Factor (BCF) An alternative approach, that is gaining acceptance, is to express the toxicity endpoint in terms of tissue concentration (body burden), rather than aqueous concentration. This enables a relatively direct comparison between controlled laboratory test data and a complex dynamic environment. In simple terms, the body burden measured in organisms collected from the environment is divided by the tissue based toxicity value determined in the lab. This ratio, how much chemical is in the organism versus the amount known to cause an effect, provides a reasonable estimation of environmental risk across the variability of environmental and biological parameters mentioned previously. The “mussel watch” program implemented this past decade is one example of using feral organisms as biological samplers to monitor chemical body burden. In Preprints of Extended Abstracts Vol. 41 No. 1 669 practice, collection and sacrifice of organisms from the environment is not always feasible nor desirable. There are several approaches that enable practical application of body burden based risk assessment. We are pursuing a combination of placing (caging) laboratory test organisms in the environment, making laboratory tests more relevant (similar) to environmental conditions, and selective sampling to determine bioavailable concentrations in the field. Bioconcentration factor (BCF), the ratio of a chemical’s concentration in an aquatic organism versus the concentration in surrounding water at steady state, is the key metric for linking laboratory data to field reality. Estimation of Bioavailability using Solid Phase Micro Extraction (SPME) As the name implies, SPME extracts a very small amount of analyte, which makes it useful for measuring partitioning in a complex system without perturbing the system. Several researchers have employed SPME to estimate the bioavailability of hydrophobic compounds in aquatic systems. In these cases, the SPME fiber is allowed to equilibrate with the system and is used as a surrogate for the biological organism. Our approach differs from this “biomimetic” sampling by utilizing SPME as an analytical tool to estimate the freely soluble fraction, effectively linking lab test data with complex environmental systems. In order to accomplish this, the sampling time is strictly controlled to prevent saturation of the fiber and minimize fouling by DOC, which can alter the adsorption characteristics and selectivity of the fiber. Analyte associated with DOC is also extracted by the fiber (probably via adsorption of the DOC:analyte complex) at longer extraction times. Consequently, sampling is typically completed within 2 minutes, which provides sufficient quantities for HPLC/MS analyses of most compounds. The standard laboratory toxicity test water is a reference for assessing relative differences observed by SPME measurements in natural waters. Distilled water is not a good reference solvent, as ionic strength, hardness, and pH can effect adsorption, particularly ionizable and surface active molecules. The experiment is summarized in Figures 1. The analytical response of linear dodecyl benzene sulfonate (C 12 LAS, a common surfactant) versus concentration in HQ distilled water, tap water, and river water. The LAS response (adsorption) is greatly reduced in the HQ water, where there is no hardness and very low ionic strength. However, response is the same for the tap and river waters, indicating that LAS has minimal association with DOC or suspended river water solids. This conclusion was confirmed in experiments where up to 10 mg/L standard Suwanee River humic acid (ISHS, Minneapolis) had no effect on SPME response in a 1 mg/L LAS solution. Toxicity testing in both lab and river water is consisitent with these data, showing little difference between the two. Preprints of Extended Abstracts Vol. 41 No. 1 670 Figure 1 SPME CALIBRATION STUDY USING C12LAS WITH HIGH QUALITY, TAP AND RIVER WATERS LAS Concentration (mg/L) 0 20 40 60 80 100 120 Peak Area 0 1e+5 2e+5 3e+5 4e+5 5e+5 6e+5 HQ Water Tap Water River Water FILE: SPME\CALCOMP.JNB In contrast, the addition of 10 mg/L humic acid significantly reduced the SPME response for short chain (C8,10) cationic surfactant in a test water. [Figure 2] The difference in SPME response for 1mg/L surfactant is approximately twofold, which is consistent with observed toxicity being ameliorated 50% in actual river water. In practice, SPME is a useful tool for measuring partitioning in well controlled laboratory systems. However, we have found that under field conditions significant changes in SPME fiber properties occur with multiple exposures and they are best limited to single use applications. Preprints of Extended Abstracts Vol. 41 No. 1 671 Figure 2 SPME resonse to C8,10 cationic surfactant with (lower) and without (upper) humic acid. Concentration of Cationic Surfactant (ug/L) 0 200 400 600 800 1000 1200 1400 1600 1800 Area Ratio 0.0 0.5 1.0 1.5 2.0 Hard Water w/ Humic Acid Hard Water linear regression 95% CI Biomimetic Sampling A variety of sampling devices have been used to identify pollutants with the greatest potential for bioaccumulation. Among these are hydrophobic solid phase extraction media and semi-permeable membrane devices (SPMD). In each case the principle of the experiment is similar, the device is placed in the field, allowed to reach a steady state with the surrounding water, then removed for analyses of sorbed components. Hydrophobic compounds with low Henry’s Law Constants (HLC) are typical of what is identified in these experiments. Biological processes, such as ingestion (or other routes of exposure), metabolism, and elimination, are not accounted for in these sampling schemes. Physical partitioning from an aqueous to a hydrophobic phase is the dominant mechanism determining what is identified. Integration of Sampling Approaches in Field Experiments A combination of water column, SPME, passive SPE, caged organisms, and feral fish samples were collected downstream from trickling filter (TF) waste water treatment plants (WWTP) located on small streams in Southwest Ohio and Central Kentucky. While activated sludge is the dominant WWTP, the TF sites were chosen because it is a less effective process and effluents contain sufficient levels of chemical to measure in field studies. Similar measurements were conducted during clean water laboratory toxicity tests. Bioconcentration factors are used to relate lab tests to field conditions, and to identify appropriate sampling strategies for estimating bioavailability of specific Preprints of Extended Abstracts Vol. 41 No. 1 672 chemicals. This is a work in progress with the ultimate goal of developing lab based toxicity and exposure test systems that will accurately assess the risk new chemicals pose to aquatic environments. Selected References Aquatic Toxicity QSAR 1. Morrall, S. W.; et al(1997) Chapter 21 in “Quantitative Structure-Activity Relationships in Environmental Sciences-VII”, Schuurmann, G. and Chen, F. eds., 299-314. 2. Dyer, S. D. et al (2000) Environmental Toxicology and Chemistry, Vol 19, No 3, pp 608-616. 3. Roberts D. W. (1991) Sci Tot Environ 109/110:557-568. SPME and Biomimetic Extraction 1. Boyd-Bland, A. A. and Pawliszyn, J. B. (1996) Anal. Chem, 68 1521-1529 2. Mayer, P., (2000), “Partitioning Based Approaches to Study Exposure and Effects of Hydrophobic Organic Substrates” Dissertation Utrecht Unieversity, Utrecht , The Netherlands. 3. Vaes, W. H. J. et al (1996) Anal. Chem 68, 4463-4467 Preprints of Extended Abstracts Vol. 41 No. 1 673 . 1 668 ELEGANT ANALYTICAL CHEMISTRY APPLIED TO ENVIRONMENTAL PROBLEMS — A PRACTICAL SYMPOSIUM Organized by V. Turoski, S.D. Richardson and J. Plude Symposia. from a dissolved phase to sediment will reduce bioavailability to many fish, but increase availability to organisms that “graze” on periphyton attached to