Document baKo981nXZQvYEp4GwRKvKzrZ

EPA-660/3-74-013 September 1974 THEORETICAL MODEL AND SOLUBILITY CHARACTERISTICS OF AROCLOl( 1254 IN WATER: Problems Associated With Low-Solubility Compounds In Aquatic Toxicity Tests by W. Pater Schoor Gulf Breeze Environmental Research Laobrstory National Environmental Research Center Gulf Breeze, Florida 32561 . Program Element 1EA077 ROAP/Task. No. 10AKC/18 National Environmental Research Center Office of Research and Development U.S. Environmental Protection Agency Corvallis, Oregon 97330 MOMS 066732 ABSTRACT A theoretical model of the behavior of substances having low watersolubility is presented and discussed with respect to aqueous bioassay. Ultracentrifugal techniques were used in an attempt to study size distribu tions of Aroclor 1254 aggregates in aqueous emulsions. Results Indicate strong adsorption from emulsion by surfaces and a water-solubility at 20C of less than Q.lug/t in distilled water and approximately 40X of that value in water containing 30 g/i NaCl. Implications with regard to aqueous bioasaey are discussed. This report wsa submitted in fulfillment of Program Element 1EA077, RQAP/Task No. 10AKC/18 by the Gulf Breeze Environmental Research Laboratory under the sponsorship of the Environmental Protection Agency. Work was com pleted as of September* 1974. ii *0NS 066 ?33 CONTENTS Section! I CONCLUSIONS II RECOMMENDATIONS III INTRODUCTION IV THEORY V MODEL VI EXPERIMENTS WITH AROCLOR 1254 VII RESULTS VIII DISCUSSION IX REVERENCES Page 1 2 3 5 10 13 15 27 30 *ans 66?34 Hi TABLES No. 1. Effect of storage tine on amount of Aroclor 1254 remaining in the water phase. Pa 17 2. Isomer distribution of Aroclor 1254 type II emulsion after standing for various periods of time in 31 glass bottle. 18 3. Adsorption of Aroclor 1254 type II emulsion on Polyallomer centrifuge tubes on standing. 21 4. Adsorption of Aroclor 1254 on stainless steel centrifuge tubes as a function of time and concentration. 21 5. Adsorption of Aroclor 1254 on stainless steel centrifuge tubes. ' 24 6. Centrifugation of Aroclor 1254 in water of varying aalinitiaa 24 at 69*000 x g (max.). 7. Distribution of isomers of Aroclor 1254 type 11 emulsion oh standing in stainless steel centrifuge tubes. 25 8. Distribution of Isomers in the absorbed fraction of Aroclor 1254 type III emulsion on standing in stainless steel centrifuge tubee. 26 Iv MGNS 066735 ACKNOWLEDGMENTS The author thanks Maaars. D. Lamb and W. Burgess for assistance with the analytical work and Dr. Ralph Birdwhlstell, Dean, School of Chemistry, University of West Florida, for reviewing the manuscript. ArodoifS* 1254 It a registered trademark of the Monsanto Company, St. Louis, Missouri. MOWS 064736 Section 1 CONCLUSIONS An extrapolation from the theory presented suggests that the use o "carriers" be continued with caution, because of two independent effects that may be present. One effect can most simply be described as an alteration of the aggregate-solvent interactions by "carriers" forming transltlon-llke links between aggregates and solvent molecules. In such a fashion, solute aggregates are surrounded by "carrier" molecules, thus enhancing the ability of the aggregate to remain in a stable emulsion by permitting greater solute-solvent interaction. This can be illustrated graphically in Fig. 1 by enlarging region "B" over a greater range of aggregate sizes since some aggregates previously belonging to regions "A" and "C" now become more stabilized. It may also be visualized by flattening the two curves in Fig. 2, thereby extending their region of overlap. Thus, when added with a "carrier", more of an insoluble compound may be Introduced into a stable water emulsion. The other effect may be due to possible interference with the uptake of a test compound by an organism. Any such uptake must by neces sity be* preceded by an adsorption to a surface of the organism such as the gills in a fish. If at this time the "carrier" molecules, which are located at the surface of the aggregate, affect the actual process of adsorption in any way, there will be a resultant change in the rate of transfer of the compound into the organism. If the rate of uptake is related to toxicity, there will be a concomitant change in toxicity. 1 MONS 066737 Section II RECOMMENDATIONS This study shows* both theoretically and experimentally, that In so far as physical Interactions are concerned, emulsions differing in degree of dispersion and stability can be formed, depending on the method of preparation and subsequent treatment. Consequently, the following questions should be answered before conducting bloassays in disperse aqueous systems: (a) What are the solubility characteristics of the compound under Investigation? . (b) To what extent ere these characteristics related to field conditions? (c) How can the solubility characteristics and field Conditions be best simulated in the laboratory? Such Information would undoubtably result In more precise data on acute toxicity as well as long-term effects regarding aqueous bloassay of water-insoluble test compounds. 2 MONS 066738 Section HI INTRODUCTION Laboratory experiments designed to determine the effects of chemicals on aquatic organisms require that the tests be conducted under conditions which reproduce those present in nature as closely as possible. In order to accomplish this in a precise and scientific fashion, the physical state of a compound in an aqueous dispersion must be known. Convenience, time and other factors have in the past often led to the use of techniques in the laboratory which do not take into consideration that the solubility characteristics of a compound may possibly affect the toxicity, necessitating extrapolation from an apparent toxicity established in the laboratory to an expected toxicity under field conditions. In many Instances, the practice of using extra polation in scientific investigations is necessary and has proven to be a valuable tool when certain conditions cannot be met. However, the range through which the extrapolation is carried out must be chosen with great care, because without sufficient experimental and theoretical justification, a resulting extrapolation in this light may well prove to be unrealistic. Since natural water conditions represent a multi component system, any attempt to quantitatively understand it must be preceded by a study of the system under ideal conditions. While the knowledge thus gained may or may not be of consequence in direct appli cation, it, nevertheless, provides a more precise scientific basis for choosing valid limits for extrapolation. The physical state of a compound in water is not a simple and straightforward phenomenon, even given the Idealized conditions of a 3 HONS 066739 two-component system - a single solute and a single solvent. A definable system should, however, be the -starting point of any investigation aimed to scientifically arrive at data which lead to a quantitative understanding of the behavior of a compound in water. With this data a more precise attempt can be made to extrapolate from a system employed in the laboratory to the obviously much more complex system present in natural waters. The purpose of this work is to provide a working theory on the behavior of substances of low water solubility and to teat this theory by investigating the solubility characteristics of Aroclor 1254. HONS 066740 Section IV THEORY To explain and predict the characteristics of water-insoluble sub stances at low concentrations* an attempt is made here to redefine the basic principles underlying a disperse system. No attempts have been made to Include in the definition the somewhat obsolete and often vague definitions of emulsions, suspensions, colloids, etc. The characteristics ascribed to each becoming readily apparent as the theoretical treatment of the proposed model continues. In this paper, an Ideal or true solution Is defined as a solute dis persed In a solvent so that any single molecule of solute is surrounded by enough solvent molecules to Insure that at any instant all soluts mole cules are distributed statistically equidistant, assuming a dilution at which interactions between solute molecules become negligible. The ideal solution, under the conditions described, is represented by the presence of single solute molecules. Solute aggregates consisting of two or more molecules may represent a deviation from the ideal solu tion because, at least theoretically, these aggregates could consist of any number of molecules whose behavior would not necessarily coincide with that of a single molecule. For each solute and a single solvent, there la assumed to exist amongst all aggregates a maximally stable aggregate which, due to its nature, remains statistically equidistant from all other aggregates for at least a certain period of time. The stability of this aggregate depends solely on the molecularly char acterized interactions at the solute-isolvent interphase and on tem perature. 0*6?+i 5 By definition, a single solute molecule In a disperse system possesses e certain sphere of influence, the nature of which governs the fete of the solvent molecules that surround it, which In turn effects the behavior of the solute molecule, and thus determines the characteristics of the solute molecule In the system. While precise Information is lack ing, it Is known, nevertheless, that the range of effect of a solute molecule may extend through several layers of surrounding solvent molecules. This means, of course, an orderly alignment Involving either oppositely charged polar regions or non-polar regions on the solute and the solvent molecules. If this interaction between solute and solvent molecules is of signlflcance, the above defined ideal solution can be visualised, pro vided also that there is no competition among the solvent molecules belong ing to respective spheres of influence of two separate solute molecules. The complexity of the situation is increased in cases where the interactions between solute end solvent molecules (solute-solvent inter actions) become lees pronounced, end, as a result, the interactions between solute end solute molecules (solute-solute interactions) become more pro nounced. This implies that the sphere of influence around the solute molecule 1s dimlnlshsd with respect to ths solvent molecules which ars . now no longer attracted to the seme degree. As two or more solute mole cules start to form aggregates, the factor of size of aggregates versus their stability In a solvent becomes of utmost importance. A generalized illustration of the size distribution of aggregates that one might expect to find in a suspension Is shown in Fig. 1. 6 HONS 066742 MOWS 0 6 6 7 4 3 : INCREASE IN AGGREGATE DIAMETER -------- ` Figure 1. Theoretical relative stability of different sizes of aggregates in an emulsion during a given time interval. Region "A" describes an area in which Che aggregates are too snail to exist independently because interactions in the sphere of influence at that point are such that solute-solute Interactions, which have now become aggregate-aggregate interactions, are more pronounced Chan the aggregate-solvent interactions. Therefore, these aggregates are expected to coalesce, moving them into region "B", which describes a range of.aggregate sizes of maximum stability. The aggregate-aggregate interactions in this range are weaker than in region "A" for that size of aggregate. Region "C" described aggregates which are too heavy to remain In suspension for a given period of time and will settle out or break into smaller, more stable aggregates. The exact shape of this curve and especially that of region "B", depends on how tightly the sol vent is held within the sphere of Influence of the solute aggregate, which is a function of the molecular interactions between solute and solvent. The distribution of different aggregate sizes in terms of molecularly characterized interactions is shown in Fig. 2. The actual equilibrium reaction taking place is described in a simplified manner at the top of the figure. The two curves relate the hypothetical strength of interactions of solute-solvent (aggregate-solvent) type and solute-solute (aggregateaggregate) type to aggregate size. The region where the curves cross corresponds to a distribution of aggregate sizes of maximum stability. MQMS 066744 8 KMMIMIUM RETWEEN SINGLE MOLECULE (A) AND AGGREGATES () AND (C) A B C 5 *4 990 SNOW Figure 2. Theoretical strength of interaction between solute and solvent Section V . , MODEL Aroclor 1254 was chosen as a model compound because It has been extensively used In bioassay at this laboratory (Duke et al., 1970; Ninmo et al., 1971a; Nimmo et al., 1971b; Hansen et al., 1971; Lowe ct al., 1972; Walsh, 1972; Cooley et al., 1972). . One approach to estimate quantitatively the solubility of Aroclor 1254 in water and the behavior of its aggregates is to use ultracentrifugal analysis* This technique permits the selective removal of particles of a certain size. For a spherical particle having a density of (p) and a radius of (r) the molecular weight (M.W.) is represented by; M.W. - 4/3irr3 NQ . 1 where NQ is Avogadro a Humber. . (1) Two opposing forces (f) which determine the fate of a particle in solution: sedimentation 3 f - 4/3irr (p-p0)g, and (2) buoyancy f - 6irrn (3) where (pQ) is the density of the solvent, (g) is gravity, and (n) is the viscosity of the solvent. To remove a small particle from an emulsion at a reasonable rate, a force larger than gravity must be applied. Using the ultracentrifuge, (g) 2 ' . in equation (2) is replaced with (w x), the angular velocity of the centrifuge rotor (<n) times the distance of travel (x) of the emulsified particle.1 1 The equations used are normally found in any textbook on physical chemistry, and. their reproduction here is intended merely for the convenience of the reader. 10 HONS 066746 The rat* of sedimentation during centrifugation la daacrlbad by: dx _ 2r2(p-p0)oj2x dt 9n (4) where (t) la time In seconds to resell equilibrium. Integration yields: _ 2r2(p-p0)u2t In X2 - In xj 9n Th* radlua of a spherical particle Is then given by: (5) where 9n(ln xj-ln x2) 1/2 r 2(p-p0)w2t w 0.10472 (rpm)rotor n g/cm/sac P g/cm2 x cm t aec (6) . ' ' Knowing the radius ot s particle or assuming a radius, the time necessary to remove the particle from an emulsion Is given by: 9n(ln x2-ln xi) 2(p-p0)r2w2 (7) 11 HGNS 064)747 The following are particle size Units calculated using equation (6) for given centrifugation tines, with n - 8.94 x 10-3 g/aec/cn, x1 - 4.7 cn, Xj." 15.3 cm, P - 0o - 0.508 g/cm3 at Z5.000 rpn. Tine (hra) Radius of particle (nm) ` 1 16.3 2 11.5 3 9.3 4 8.1 6 6.6 a 5.7 The following are particle size limits calculated using equation (6) for given centrifugation times, with q - 8.94 x 10"3 g/sec/cn, x^ - 6.00 cm, Xj * 10.73 cm, p - pQ- 0.508 g/cm3 at 45,000 rpm. Time (hrs) Radius of particle (nm) 1 7.6 2 5.4 3 4.4 4 3.8 (208,000 g/mole1! 636 molecules) 6 3.1 8 2.7 12 2.2 (40,000 g/mole1; 124 molecules) ^Average molecular weight Aroclor 1254 - 327 g/mole (Hutzinger et al., (1972). 12 KOhS 066748 Section VI EXPERIMENTS WITH AROCLOR 1254 Wide-mouth Jars, 30 cm high and 14 cm wide, were used to produce 31 of Aroclor 1254 emulsion per batch. Mechanical considerations concerning the proper physical agitation of Aroclor 1254 and water made it necessary to use 250 ml of Aroclor 1254 in the jar to submerge the blades of the stirrer. Agitation for 0.5 hr at 60C and 1,800 rpm produced a cloudy emulsion which was allowed to settle for 48 hra, when the range of concentration was found to be 1-20 mg/1 and the emulsion became almost clear. This emulsion is referred to as type-1. A second homogenization was carried out by transferring to a jar identical to the one used previously volumes of type-I emulsion to produce emulsions of 10-300 yg/i, end stirring 1 hr at 25C and 1,800 rpm. This emulsion is referred to as type-II. Type-Ill emulsions were prepared by taking an appropriate volume of type-I emulsion* adding it to a stainless steel blender jar to make a total volume of 500 ml, and homogenizing at high speed for 5 min* All centrifugations were performed in a Beckman Model L3-50 ultra centrifuge at 20C using SW 50.1 and SW 25.2 rotors. The extraction procedure was that of Schoor (1973), with modifica tions of the ratio of water to hexane. Evaporation was carried out by placing the hexane extracts in a water bath at 35C and allowing a gentle stream of air to blow across. This method was found superior to dis tillation in percentage recovery and time Involved. When the extract volumes had ta be reduced to less than 10 ml, dried, pre-purlfied nitrogen was used instead of air. 13 M0NS 066749 A Hewlett-Packard Model 5700 gas chromatograph with a linear electron-capture detector (3Ni) was uaed for quantitative determina tion of the Aroclor 1254. The linearity of this detector eliminated uae of different standarda at each attenuation or reduction In volume of the sample, either being very time consuming and subject to errors. An OV-101 column (2X OV-101 on Cas Chrom Q, 100-120 mesh) vae operated mt 195C with the detector at 300C and the argon-methane (10:1) carrier gas at a flow rate of 25ml/mln. Except where noted, quantitation was performed by comparing total peak heights of ssmple and standard. To determine the amount of Aroclor 1254 adsorbed on valla of the 34 ml stainless centrifuge tubes, the water phase was decanted and any adhering droplets removed with e disposable plpet. Since acetone Injacted with the sample was detrimental to the chromatographic column, a sonic probe and hexane were uaed for removal of Aroclor 1254 from the valla of the tubes. This was necessary because the thin layer of water remaining on the wells shielded the Aroclor 1254 and prevented It from being desorbed into the hexane phase. SonifiestIon emulsified the water at the boundary layer, thus allowing the hexane to contact the adsorbed Aroclor 1254. HOMS 066750 Section VII RESULTS A typical chromatogram of an Aroclor 1254 standard In hexane (A) and a haxana extract of a type-II emulsion (B) Is shown In Fig. 3. Soms of the 11 peaks indicated are multiple peaks. Only peaks 1-7 were used to calculate the "total" peak height on which all quantita tions were based. Peaks 8-11 were excluded, because they were often too small to permit accurate calculations. The effect of storage time on Aroclor 1254 emulsions of type-I and type-II la shown In Table 1. There is a fairly rapid Initial decrease In Aroclor 1254 In all cases and it appears that a plateau la reached at around 7 ug/l. This should not be Interpreted to mean that solubility Is approached at that point, only that perhaps a stable emulsion Is reached at that point. The haxana extract of type-II emulsion (chromatogram B) Indicates a relative reduction In peak height for the early eluting peaks. This phenomenon is better described by the results shown In Table 2. For comparison peak 7 was arbitrarily assigned a relative value of 100Z. The results indicate that on standing a type-II emulsion shows a reduc tion of the Individual peaks, with the early eluting components, or less chlorinated biphenyls (Zitko, 1970), being reduced much more than tha late eluting ones. The degree of reduction depends somewhat on the preparation and Initial concentration of Individual type-II emulsions (Table 2). Type-Ill emulsions of comparable "total" concentration show a relative distribution of the isomers Identical to that of the standard. 15 MOMS 066251 HONS 0 6 6 7 5 2 Tblt 1. EFFECT OF STORAGE TIME ON AMOUNT OF AROCLOR 1254 REMAINING IN THE HATER PHASE Tin* (days) 0 2 5 6 8 9 13 15 19 20 21 23 26 28 33 34 . 41 . . 43 yg/t Aroclor 1254 Type I Type II 2300 301 115 113 112 97 502 483 87 . 78 428 355 350 280 50.2 286 23.6 11.3 123 . 98.5 54.7 48.1 44.5 6.7 7.1 6.5 7.7 7.4 15.5 6.8 *ONS 066753 17 Table 2. ISOMER DISTRIBUTION OF AROCLOR 1254 TYPE II EMULSION AFTER STANDING FOR VARIOUS PERIODS OF TIME IN 31 GLASS BOTTLE Tin* <d*y) Total cone. (lig/i)1 1 X Peak Height^ 2 Peak Numbers 23 4 5 6 7 2 9 13 19 20 21 41 21 . 33 38 286 123 98.5 54.7. 58.1 44.3 15.5 13.4 3.6 1.6 76 93 95 95 98 104 100 79 78 89 94 98 99 100 79 79 93 98 99 96 100 72 75 85 93 91 95 100 64 70 80 89 92 94 100 61 65 82 90 99 93 100 37 41 56 70 77 -88 100 16 27 44 55 76 87 100 12 21 39 45 64 82 100 9 10 31 46 63 100 (3i4 ppm) (41) (80) (87) (100) ^Calculation* are baaed on the relative height of peak 7 (aee below) 2 Peak nuabera are shown on the chronatogran in Fig. 1. 18 MOMS 066754 `I*-" The distribution of Isomers in a hexane extract of the gill tissue of a pink shrimp (Penaeus duorarua) exposed to 2.5 Ug/t Aroclor 1254 for 20 days is shown in parentheses at the bottom of Table 2. Because peaks 2, 4 and 7 showed obvious contamination peak 6 was assigned the arbltrsry, relative 100Z value. The "total" concentration of 3.4 mg/kg was based on the total height of peaks 1, 3, S and 6, and on the wet weight of gill tissue (blotted to remove adhering water). Filtration of type-I emulsion through 450 nm (0.45m) Mllllpore* filters revealed obstructed passage of Aroclor 1254 aggregates smaller than 450 nm. Starting with a 1 mg/l emulsion and changing filters after each filtration, less than 0.01 vg/t of the material remained In the water after ,15 passages. Since aggregates In the starting emulsion were most likely smeller than 450 nm (calculations using equation 1 lead to roughly 10^ times the average molecular weight of Aroclor 1254), the Aroclor 1254 must have been adsorbed on the filter. This was also evidenced by the fact that the filter paper turned slightly transparent after the first passags during which about 95Z of the material was removed from the emulsion. The first centrifugation experiments were carried out by centri fuging 180 ml of 42 ug/l Aroclor 1254 type-II emulsion in 60 ml polyacstste centrifuge tubee for 60 min at i.07,000 x g (max.). MGNS 066755 19 At an 85X total recovery the following distribution was found: Acetone extract of tubes 66Z Hexane rinses of tubes 18X Top 50 ml water phase 5% Bottom 10 ml water phase 11% The low recovery (85Z) was probably due to Incomplete extraction of the tubes in spite of refluxing with acetone. Polyallomer centrifuge tubes were tried next. When 180 ml of 286 ug/Jt type-II emulsion were centrifuged in 60 ml Polyallomer tubes for 60 min. at 107,000 x g (max.) the following distribution was found: Acetone extract of tubes Hexane rlnsea of tubes -22Z Top 25 ml water phase 5Z Bottom 35 ml water phase .6Z These percentages were based on the total amount of starting material, i.e., assuming 100Z recovery Instead of the 85% in the case of the polyacetate tubes. Extraction of the Polyallomer tubes by reflux ing with acetone produced too many interfering peaks on the chroma togram, making complete recovery calculations impossible. Direct adsorption on Polyallomer tubes was achieved by permitting type-II emulsions to sit undisturbed in the tubes. Table 3 shows the outcome for two different concentrations. To permit recovery and study of the material adsorbed on surfaces, 34 ml stainless steel centrifuge tubes were used for static, tests, MOfcS 066756 20 Teble 3. ADSORPTION OF AROCLOR 1254 TYPE II EMULSION ON POLYALLOHER CENTRIFUGE TUBES ON STANDING Tine (hrs) 0 3 72 0 1 3 Aroclor 1254 (ug/l) In water phase 125 86 3.3 45 35 27 Tlble 4. ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES AS A FUNCTION OF TIME AND CONCENTRATION Tine (hrs) Total (Mg) (Mg/1) Aroclor 1254 tvoe II emulsion Water S.. S. tube (ug) (wg/t) (Mg) X adsorbed 0.5 ' 1 3.83 . , J.8J 113 HJ 2 3.83 .113 16 3.83 113 1 0.48 14 2 . 0.06 2 3.63 107 3. J1 97 3.20 94 3.14 . 92 0.35 10 0.03 1 1 Stainless steel centrifuge Cubes. 21 0.18 0.30 0.33 0.51 0.08 0.02 * * 5 9 13 16 23 67 HONS 06675? ss well as for ultrecentrlfugal analysis. Table 4 shows the ^mounts of Aroclor 1254 adsorbed on the wall of a stainless steel cenTrlfuge tube in relation to starting concentration and time. The amounts adsorbed from the 14 pg/1 and 2 ug/t emulsions were greater than that adsorbed from the 113 yg/1 emulsion during the same time period. It should be pointed out that 0.100 pg of Aroclor 1254 adsorbed as a mononolecular layer per tube represents about IX of the minimum area available. The calculated inside area of a stainless steel centrifuge 2 '. tube was 60.8 cm . This area must be considered minimum because the surface was assumed to be ideally smooth, which certainly is not the case. However, for the approximations involved, this figure was used. 2 A simple calculation using equation (1) yields 0.613 nm for the cross-sectional surface area of an average Aroclor 1254 molecule using the average molecular weight of 327 (Hutzlnger et; al., 1972), and 3 p 1.505 g/cm (W. B. Fapageorge, Monsanto Company, St. Louis, Missouri, personal communication). Utilizing a molecular model with the phenyl groups at right angles to each other and bond length (Pauling, 1940) as the basis for calculations, a cross-sectional area 22 of 0.643 nm for the fully chlorinated and 0.356 nm for the unchlori nated or biphenyl molecule was obtained. Values falling between are not linearly related to amount of chlorination. Using 0.613 nm2 as an approximate, average cross-sectional area, 0.100 pg of Aroclor 1254 2 occupies 1.13 cm in the form of a monomolecular layer. Thiar corresponds to approximately 3 ug/2. in a 34 ml stainless steel centrifugl tube. 22 HONS 066758 It can be seen that even at 50% adsorption from a 3 yg/i emulsion only about 1% (maximum) of the available surface area Is occupied, and surface saturation was not a factor. fi The amounts of Aroclor 1254 in the form of emulsions of type-II and type-III adsorbed on the vails of the stainless steel centrifuge tubes are shown in Table 5. There is a difference In adsorption of the two different types of emulsion in the absence of NaCl. At least for type-III emulsions, the Introduction of 30 g/l NaCl appears to have no effect on the amount of Aroclor 1254 adsorbed. However, centrifugation reveals a difference In the size of the aggregates formed in the presence of NaCl, as shown in Table 6. In comparison with an Aroclor 1254 standard, the relative distri bution of the isomers in emulsions of type-II and III is quite different, as shown in Tables 7 and 8. However, in all cases the adsorbed Aroclor 1254 had a higher percentage of early eluting (gas chromatography) laoaers than did that which remained in solution. t } 23 M0NS 066 759 Table 5. ADSORPTION OF AROCLOR 1254 ON STAINLESS STEEL CENTRIFUGE TUBES TiaX ((hi.) Type II Emulsion 0 g/1 NaCL US Aroclor 1254^ adsorbed i Type III Emulsions . 30 g/t NaCl 0 g/I NaCl 0.5 1.0 2.0 : 4.0 19 22 0.19 0.30 0.33 0.62 0.09 0.10 0.14 0.19 0.39 0.10 0.14 0.45 ^Data adjusted to A.00 pg total starting amount. Table 6. CENTRIFUGATION OF AROCLOR 1254 IN WATER OF VARYING SALINITIES AT 69,,000 x g (MAX.) . pg/i Aroclor 1254 remaining in water phase Tima (hra) g/t NaCl 0 15 30 0.5 ; 1,9 2.0 13.9 12.5 7.2 7.1 6.6 4.6 'Started with 50 ug/1 Type 111 emulsion. 24 6.0 4.9 2.9 : _i____ MQNS 066760 Table 7. DISTRIBUTION OF ISOMERS OF AROCLOR 1254 TYPE II EMULSION ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES l Storage U*y) Hrs In tube ug/l i2 2 Peak height.1' 2 Peak number 3 4 56 1 0 310 water 93 90 98 99 98 100 100 5 0 115 water 53 71 73 91 98 98 100 2 97 water phase 49 67 69 83 100 100 100 2 12 adsorbed 96 106 103 127 119 100 100 s 0 112 water 51 67 71 82 96 97 100 2 . 102 water phase 48 66 68 79 98 98 100 2 8.0 adsorbed 69 82 85 104 107 100 100 13 0 97 water 47 64 68 81 97 98 100 2 86 water phase 43 59 66 78 92 96 100 2 6.1 adsorbed 47 68 77 94 101 98 100 ^Compared to standard Aroclor 1254 (Fig. 1). Calculations are based on the relative heights of peak 7. 2 `Peak numbers are shown on the chromatogram in Fig. 1. HONS 066761 Tabl* 8. DISTRIBUTION OF ISOMERS IN THE ABSORBED FRACTION OF'AROCLOR 1254 TYPE III EMULSION ON STANDING IN STAINLESS STEEL CENTRIFUGE TUBES NaCl (g/D hrs in water phase adsorbed tube <ug/D (Mg) 1 X Peak heights* 2 Peak number 2 345 6 02 47.4 0.122 149 127 135 130 98 100 30 1 46.9 0.075 144 121 129 129 105 100 0 22 39.7 0.190 139 118 113 122 127 100 ^Compared to standard Arodor 1254 (Fig. 1). Calculations are based on the relative heights of peak 6. 2Peak numbers are shown on the chromatogram in Fig. 1. hqns 6676i 26 Section XX DISCUSSION The original Intent for conducting the work described was to find tha absolute solubility of Aroclor 1254 in fresh and salt water. This, unfortunately, waa not completely accomplished to any accurate degree, because a series of significant problems occurred at the beginning of the centrifugation experiments. Recovery of Atoclor 1254 after centrifugation vae low and, hence, led to the discovery that adsorption occurred on the walls of the polyacetate centrifuge tubes as well ae on Velysllomer and etalnless ateel centrifuge tubea. Ultimately, only the stainless steel centrifuge tubes were used In the adsorption and ultracentrlfugal studies. The apparent disappearance of early eluting Isomers, such as shown la Table 2, has been observed by others. It was found to occur In the eggs of the double-crested cormorant and regarded as possibly due to metabolic breakdown (Huczlnger et al., 1972), Similar behavior In tha carcasses of bobwhlte quail after exposure to Aroclor 1254 was observed and believed to be becauee of Isomeric transformations (Begley and Cromartle, 1973). Application of Aroclor 1254 to different types of soil showed a reduced recovery of the early eluting, lower chlorinated biphenyls (Iwsta et al., 1973), and It was postulated that this may have been due to evaporation from the soil. My studies did not substantiate the Observations by Zitko' (1970) that when Aroclor 1254 emulsions are centri fuged the dissolved fraction Is richer In the lower chlorinated biphenyls than la the original preparation. However, the difference could be due to the method of the preparation of his emulsion, which was similar to my type-III emulsion. In both type-II and type-ill emulsions the distribution 27 MGNS 066763 of isomers in the water phase shows a loss of the lower chlorinated biphenyls on standing (Tables 7 and 8). This loss was accounted for in all cases by adsorption on the stainless steel centrifuge tubas, the "lost" lower chlorinated biphenyls always being found in the adsorbed fraction. Thus, at least from water emulsions of Aroclor 1254, loss of the lower chlorinated biphenyls is due to their relatively greater affinity for surfaces. The published values for solubility of Aroclor 1254 In fresh and salt watar of 2-3 ag/l and 1-1.5 mg/l, respectively (21tko, 1970), appear much too high. A conservatively high estimate based on ay ultra centrifugal experlaants indicates the average solubility of the leomere to be leas than 0.1 pg/i for fresh watar and approximately 0.04 pg/i (calculated from Table 6) in water containing 30 g/i NaCl. It is extremely difficult. In my opinion, to obtain an absolute value for the true solubility of the average molecular weight isomer of Aroclor 1254. The problem lias in the fact that at low concentrations, long centrifugation times (In exceae of 12 hra at 243,000 x g (max.) theoretically are necessary to eliminate aggregates from the emulsion. At the low concentrations necessary to ellminatt undesirable stirring back after completion of the centrifugation (Bowman at al., I960), adsorption on the walls of the stainless steel centrifuge tubes (67X at 2 pg/l for 2 hrs, Table 4) makes it all but impossible to employ ultracentrifugation for extended periods of time. It appears that at least in the case of type-111 emulsions the adsorp1tlon from water emulsions containing 0 and 30 g/i NaCl was the same (Table 5), although the rate of sedimentation was quite different. The 28 06676, explanation for this lies in the fact that the size of the Arodor 1254 aggregate is nuch larger in the presence of salt and, vhlle this Is not apparent at 1 i g, the larger aggregates are renoved more quickly from the salt-containing emulsion during ultracentrlfugstlon. This agrees very veil with my hypothesis that a larger aggregate is more etable under the given conditions and in the presence of salt, which is conducive to greater solute-solute (aggregate-aggregate) interaction. MONS 066265 29 Section X REFERENCES Begley* C. E., and E. Cromartie. Elimination Pattern of Aroclor 1254 Components In the Bobwhite. J. Chromatogr. Sci. 25s219-226, 1973. Bowman, M. C., F. Acree, Jr., and M. K. Corbett. Solubility of Carbon-14 DDT in Water. J. Agric. Food Cham. 8(5):406-408, Sept. 1960. Colley, N. R., J. M. Keltner, Jr., and J. Forester. Mlreac and Aroclor 1254 : Effect On and Accumulation by Tetrahymena pyriformia Strain W. J. Protozool. 19(4):636-638, 1972. Duke, T. W., J. I*. Love, and A. J. Wilson, Jr. A Polychlorinated Biphenyl (Aroclor 1254) in the Water, Sediment, and Biota of Escambia Bay, Florida. Bull. Environ. Contam. Toxicol. _5 (2) .*171-180, 1970. Hansen, D. J., P. R. Parrish, J. I. Lowe, A. J. Wilson, Jr., and P. D. Wilson. Chronic Toxicity, Uptake and Retention of Aroclor 125^in Two Estuarine Fishes. Bull. Environ. Contam. Toxicol. j(2): 113-119, 1971. Hutzlnger, 0., S. Safe, and V. Zltko. Polychlorinated Biphenyls. Analabs Res. Notes. 12(2):1-11, July 1972. Iwata, Y., W. E. Westlake, and F. A Gunther. Varying Persistence of Polychlorinated Biphenyle in Six California Soils Under Laboratory Conditions. Bull. Environ. Contam. Toxicol. 2(6):204-211, 1973. Lowe, J. X., P. R. Parrish, J. M. Patrick, Jr.,^and J. Forester. Effects of the Polychlorinated Biphenyl ArodorS) 1254 on the American Oyster (Crassostrea virglnica). Mar. Biol. 17(3):209-214, Dec. 1972. Nlrnmo, D. R., R. R. Blackman, A. J. Wilson, Jr., and J. Forestar. Toxicity and Distribution of ArocloirPl254 in the Pink Shrimp (Penaeus duorarum). Mar. Biol, . li(3):191-197, Nov. 1971(a). Nlmmo, D. R., P. D. Wilson, R. R. Blackman, and A. J. Wilson, Jr. Polychlorinated Biphenyl Absorbed from Sediments by Fiddler Crabe and Pink Shrimp'. Nature 231:50-52. May 1971(b). Pauling, L.. Nature of the Chemical Bond. Ithaca, Cornell University Press, 1940. 164 p. . Schoor, W. P. In Vivo Binding of p,p'-DDE to Human Serum Proteins. Bull. Environ. Contam. Toxicol. 2(2)^70-74, 1973. Walsh, G. E. Insecticides, Herbicides and Polychlorlrated Biphenyls in Estuaries. J. Wash. Acad. Sci. 62^(2) :122-139, 1972. Zltko, V. Polychlorinated Biphenyls Solubilized In Water by Nonionic Surfactants for Studies of Toxicity to Aquatic Animals. Bull. Environ. Contam. Toxicol. 5(3):219-226, 1970. 30 HONS 066766  l.l'HMIMNC KPA 660/3-74-013 TECHNICAL REPORT DATA tl'lrau rrnd mi thr rcrcrte beforr 1* | 4. II1U ANO'JUUMfUt .. Theoretical modal and aolubllity characteristic! of Arodof*'1254 in water: Problems associated with low- solubility compounds in aquatic toxicity tests. r. authorisi ' W. Peter Schoor, Ph.D. . ! PERFORMING one 'NIZATION NAM* AND AOORfBS U. S. Environmental Protection Agency Gulf Bretts Environmental Research Laboratory Sabine Island Culf Breese, Florida 52561 17. (PONSORINO AGENCY NAME ANO AOOAiSB 3- RECIPIENT*! ACCSSSiOPPNO. ^ 6. REPOST OAT* September 1974 S. PSRP ORM1NO ORGANIZATION COD* B. PfRPORMlNO ORGANIZATION RBPORT NO. o. HooAaUIlIWUT WO. '"" 1 EA077 / 10AKC / 018 11. CONTRACT/ORANT NO. " " 13. TYPI OP RIPORT ANO PSRIOO COVIRSO Final 14. SPONSORING AQINCV COOI IS SUPPLIMtNTARV NOT*! tA AMTAAdV A theoretical model of the behavior of substances having low water-solubility Is presented and discussed with respect to aqueous bloassay. Ultxacsntrlfugal techniques were used in an attempt to study size distributions of Aroclor^l254 aggregates In aqueous emulsions. . Results indicate strong adsorption from emulsion by surfsees sod a water-solubility at 20C of less than 0.1 lihl in distilled water and approximately 40X of that value in water containing 30 *g/i NaCl. Implications with regard to aqueous bloassay are discussed. 1,7. KEY WOROS AND DOCUMENT ANALYSIS it. DESCRIPTOR* b.>OBNTIPIRS/OPSN SNOBO TERMS e. COSATI 1*14/Group Solubility AroclojFl254 Theoretical Modal, Water Aquatic Toxicity Tests Low-Solubi.llty Compounds Emulsion t, bii^miu-VioNSTArlMiNr Rslssst to public Adsorption . c IB. SECURITY CLASS {Tttit Hcportf 30. SECURITY CLASS fTMt f*gtl Unclsssifltd 31. NO. OP PAOtt' 37.PRICI ' ' ' PA P** >})) (* >J