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JCL: T6G: RF ,XF:Conoco Chemicals Compan A Division of Conoco Inc. 15990 North Barker's Landing Road P.O. Box 19029 Houston. TX 77224 January 30, 1984 Mr. Bob Townsend Kleen-Brite Labs 100 Fair Street Brockport, NY 14420 Dear Mr. Townsend: Per your request, enclosed is information on the aquatic toxicity of Linear Alkyl Benzene Sulfonates. No applicable information could be found on sulfonic acids. Conoco Chemicals has done no aquatic testing on these products. The enclosed articles are from open scientific literature. Please contact us if you have questions on the enclosed. Sincerely, Thomas G. Grumbles, C.I.H. Director, Industrial Hygiene ajo Enclosure cc G. V. Curran Wv 00oH640 HUMAN SAFETY AND ENVIRONMENTAL ASPECTS OF MAJOR SURFACTANTS A Report To The SOAP AND DETERGENT ASSOCIATION May 31, 1977 h CLs Arthur D Lttle Inc OOOOl TABLE OF CONTENTS (continued) Page Acute Irritation - Skin - Ocular 137 1 38 Subacute Toxicity - Oral 138 Percutaneous 140 Chronic Toxicity - Oral 140 Acute, Subacute and Chronic Toxicity - S'jimary 141 Carcinogenicity and Co-Carcinogenicity 142 Hutagenicity 143 Reproduction Studies Teratooenesls 144 145 Carcinogenicity, Mutagenicity and Teratogenicity - Suntnary 150 Pharmacology - Absorption and Metabolism 151 - Hematological Effects 152 Glucose Tolerance 153 - Oye Uptake 154 Pharmacology - Stfmary 154 B. Human Studies 154 Skin Irritation Skin Sensitization Pharmacology Human Studies - Suimary 154 156 i57 158 C. Epidemiology 158 Accidental Exposure Occupational Exposure 158 159 Bibliography 160 10 Arthur Dljttlc Inc TABLE OF CONTEXTS (continued) (continued) D. Metabolic Pathways of Biodegradation E. Sumary Bibliography Page B4 89 90 Environmental Safety A. Aquatic Toxicity 1. Acute Toxicity a. Methodology b. Intact LAS Structure-Activity Relationships c. Acute Toxicity to Fish - Intact LAS d. Acute Toxicity to Fish-Biodegraded LAS e. MBAS-Related Acute Toxicity to Fish in Sewage Effluents f. Acute Toxicity to Invertebrates 2. Chronic Toxicity 3. Effects of Environmental Conditions on Toxicity 4. Interactions with Other Chemicals B. Effects of LAS on Higher Plants C. Effects on Birds and Wildlife D. Mode of Action E. Aquatic Toxicity and Aquatic Safety Blbllography Human Safety A. Animal Studies Acute Toxicity - Oral - Percutaneous - Systemic Exposure 98 98 99 99 100 103 108 112 IK 118 119 122 124 125 125 128 130 134 134 134 137 137 9 Arthur l) ijitle Inc vvv 0000126*2 TABLE OF CONTENTS (continued) Page III. Biodegradation A. Laboratory Test Systems Used In LAS Studies 51 1. Oxygen Uptake -- Biochemical Oxygen Demand (BOD) 51 2. COg Evolution 53 3. Enrichment Cultures 54 4. Die-Away Tests 55 a. River Water Test 55 b. Fortified and Inoculated Waters 56 c. Shake Culture Test 56 d. British STCSD (Standing Technical Committee on Synthetic Detergents) Test 57 e. Swiss EAWAG (Eidgenossische Anstalt fur Wasser und Gewasserschutz) Test 59 f. Bunch-Chambers Test 59 5. Simulated Treatment Processes 60 a. Activated Sludge 60 b. Trickling Filters 66 c. Anaerobic Systems 67 6. Soil 69 7. Influence of Test System Variables 70 a. Media 70 b. Inoculum 73 c. Temperature 75 d. Surfactant Concentration 76 e. Reference Compounds for Test Validity 77 f. Analytical Test Methods T8 B. Field Tests 78 C. Effects of Chemical Structure 82 VVV 000i2643 8 Arthur D Little Inc TABLE OF CONTENTS Sunjp8'.8 I. Introduction A. Chemical Characterization of LAS 1. Primary Product - LAS 2. Secondary Products 3 Inorganic Sulfate Bibliography Page 1 II 12 13 1 16 18 II. Environmental Levels A. Analytical Methods 1. Physical Methods 2. Specific Chemical Techniques a. Methylene Blue Active Substances(MBAS) b. Other Methods 3. Physicochemical Analyses B. Water Quality Standards 1. National Regulations 2. State and Local Regulations C. LAS Levels in Natural WaterBodies 1. Pathways for LAS irto the Environment a. Sewage Treatment Plant b. Septic Tank Systems 2. MBAS Concentrations in the Environment a. Surface Waters - Streams andRivers b. Estuaries 0. Summary Bibliography 19 19 20 21 21 23 24 27 27 29 30 31 31 34 3S 35 41 44 46 7 Arthur Dljltle Inc VVV 000012644 e,.y of Ga^narus was reduced at the lowest LAS concentration employed - -- -r;/1). The possible toxicity of detergent components other than LAS rf.. rat considered. The effect of 30 days' exposure of 4 species of fresh water fish to the jr* formulation described by Arthur (1 970) above was examined by McKim et^ . [1975 ). Statistically significant reductions in the 30-day standing crop rated (Table 1-1 ). Tnese few studies which only approach the problem of the possible chronic wuity of LAS to aquatic organisms indicate that long-term exposure may re- in toxic effects because of the increased sensitivity of early developatil stages; e.g., sac-fry and larvae, as compared to adult organisms. Howw, before any definitive evaluation rr* the chronic effects of LAS can be titrated, further work is necessary usinc^ test samples containing well charfttenzed LAS as the only surfactant and on typical environmental degradation tuples. Moreover* further chronic studies with fish and organisms of lower levels would also be required. 3- Effects of Environmental Conditions on Toxicity toxicity of a chemical in an aquatic environment, natural or experiK is dependent on physical, chemical and biological conditions. Differ'P*cies of fish exhibit various degrees of tolerance to toxic pollutants or. temperature, water hardness, dissolved oxygen and heavy metals. j j j vw 000012645 Arthur D Little !nc 2.___Ch_ronjc Toxicity Oner of ere first reports of LAS chronic toxicity to fish was by Sardach a\. (1965) wno found that LAS at concentrations of 0.5 mg/1 for 24 days re. i*ed ir oarage to the chamoreceptors of the taste buds of yellow bullheads -;j;ur-jj_s nata 1 is). Tne study of Pickering and Thatcher (1 970) on the effects :t .AS (SjA iRteri.ii Reference Sample, LAS Lot No. 1-1, LAS-60.8: ) to fish rer-iins to the present the only effort to examine the chronic toxicity of LAS. T'v. examined a nunoer of responses with the fathead minnow (Pir.ephales prerfl^s) in continuous flow systems including 5-week growth, egg production, hatchjtil'ty and fry survival. Five-week growth, egg production >nd hatchability .ere not affected by mean LAS concentrations up to 2.7 mg/1. In agreement with otrer studies, the fry were more sensitive than other stages with deaths occurr'r.g at levels of LAS of 0.63 mg/1 or above, and the greatest sensitivity at 7 :a 14 days. T:>e authors noted thtt even during the 96-hour TLm tests that 80 to 90! of the LAS as measured by MBAS was lost. For chronic studies, difficulty encountered because of the increasing efficiency of biodegradation even trcj;h a dilution device was used to feed LAS. Standard deviations of MBAS alues in 7-day composite samples amounted to 25% of the MBAS values. Arthur (1970) studied the effects of a detergent containing LAS (14*,), alcohol ethoxylate (2.3;:), sodium soap (2.5") and inorganic salts. The re mits were reported in terms of LAS concentrations alone for the invertebrate orqanisms which were the amphipod (Garrmarus pseudolimnaeus) and 2 species snails (Campeloma decisum and Physa integra). The organisms were exposed acutely followed by a 6-week exposure of survivors. Survival of Physa was Effected at LAS concentrations up to 4.4 mg/1, whereas Gammarus was affected Ci.4 ng/i and Campeloma at between 1.9 and 4.4 mg/1. Survival of and F2 VVV Q0001Z&*6 118 Arthur Dluttiu. Inc. found the 96-hour IC^q to larvae of the mayfly (Isonychia sp.) to be -3 -g/i (Litchfield-Wilcoxon confidence limits, 4,23-6.72) for a well defined ,,-3le of LAS (C10-13.2a. C^-32.7%, C^-37.9^, C13-13.2S, C14-3.0>. Hendricks e_t aj_. (1974) found that a high molecular weight LAS (C.|3, *,,.362) was more toxic to the snail (Goniobasis sp.) than a low molecular ,*ight LAS {Ci ^ g , MW-342). The respective 24 hr LC5q values were 19.4 ;i*.6-33.9) and 92 mg/1. Although algae are not aquatic fauna, tieir critical role as the lowest troohic level of the aquatic food chain makes their discussion appropriate it this point. Hal1 (1973) has examined the effects of surfactants on phytojlmkton and finds that results from toxicity assays provide useful data for redaction of aquatic environmental safety. For the 3 species examined, Ulenastrum caoricornutum. Microcystis aeruginosa and Navicula seminulum, :*e 5-day minimum a 1gistatic concentrations of LAS were 1000 mg/1, 50 mg/1 d 50 mg/1, respectively. Thus, for invertebrates, toxicities of LAS vary widely due in some fasure to the protective morphological characteristics (exoskeletons, closure frcranisms) of many organisms. These traits allow organisms in the adult stage *4 resist exposure to toxicants from any source. On the other hand, early *velopmental forms of many bivalves, crustaceans and lower forms are susceptible to LAS at concentrations found toxic for sac-fry of fish. In some cases, effects of LAS were noted on larval stages at concentrations as low as nq/l. VVV 0000126^7 117 Arthur DLjttle Inc At an LAS concentration of 5 rng/1 for 6 hours, siphon retraction was com pletely abolished in the cockle, while the same exposure resulted in only a slight reduction in this response in the clam. Among the crustaceans, the dimming ability of larval stages of the spider crab and barnacle were re duced severely (100-fold) by LAS at a concentration of 10 mg/1. In a subsequent examination of the mussel (Mytilus edul is) by Granmo (1972), fertilization and early developmental stages were inhibited at concen trations as low as 0.05 mg/1 and larval growth was depressed at an LAS concen tration of 0.1 mg/1. As part of the chronic study on the effects of an LAS-containing deter ment (LAS-14.OS, alcohol ethoxylate-2.3%, sodium soap-2.5%) on 3 invertebrate species, Arthur (1970) reports 96-hour Tl_m values for the amphipod Gaimarus pseudolimnaeus and for the snails Physa inteqra and Camoelona decisum of 7, 9 and 27 mg/1, respectively, based on the LAS content of the detergent. The possible toxicity of other components of the detergent was not considered. Aoffett and Grosch (1967) have reported that LAS induces "gross developmental abnormalities" in larvae of 5 genera of marine invertebrates at 1 to 3 mg/1 IAS. The genera studied were Arbacia (sea urchin), Asterias (starfish), jpicul- (sponge), Jhaatopteris (annelid) and Holqula (tunicate). The exact ature of the gross abnormalities was not described. In another report by same authors (1968), brine shrimp (Artemia sp.) exhibited a 50% leth Hty at 22 hours following an 8-hour exposure to 5 mg/1 LAS. Dolan et al_. I l 1 1 VVV 000012648 116 Arthur D Little Inc sj5nificantly and the percentage survival and growth of larvae decreased sig-tficantly at 1.0 and 0.5 mq/1 , respectively. Swedmark et al_. (1971), in a broad-ranging study of marine organisms* jve studied the effects of LAS (uncharacterized) on a number of marine bi valves and crustaceans. The LC^g walues at 6 to 8C are shown in Table 1-H. Otner than the cockle and scallop adults of the species examined were markedly rore sensitive. These data parallel the findings in fish with respect to in creased sensitivity of early developmental stages. TABLE 1-H ACUTE TOXICITY OF LAS TO MARINE SIVALVES AND CRUSTACEANS Species______________ Mussel (Mytilus edulis) Clams (Mya arenaria) Cockle (Cardium edule) Scallop (Pecten maximus) Decapod (Leander adspersus) Decapod (Leander sguilla) Hermit crab (Eupaqurus bernhardus) Spider crab (Hyas areneus), adult stage I zoea larvae Shore crab (Carcinus maenus) Barnacle (Balanus balanoides), adult stage II naupluis larvae Swedmark et aU (1971) 115 96-Hour LC^ (mq/1) >100 70 15 <5 SO >100 >100 >100 9 >100 50 3 VVV 000013649 Arthur D Little inc amounts of K3AS alone do not appear to exert any adverse effects various fresh water fish. Rainbow trout, golden orfe, goldfish, bream, tench, roach, perch, carp, raffe, pace, chib, pike, rudd, gudgeon, stoneloech, spired loach ana bullhead fish have been reported to survive, grow and breed in two small, artificially created lakes (Colworth Lakes). The naln source of water in the lakes is sewage effluent which, due to the nature of the site, contains high MBAS levels (3 mg/1)* higher than normal BOD and a large amount of total dissolved solids (Unilever Ltd., unpublished data). An additional study indicating that toxicity to aquatic organisms of sewage effluents cannoc readily be attributed to LAS was carried out by Calabrese and David (1967) with oysters (Crassostrea virqinica). Although rot a fish study, the results are appropriately considered in this discussion. Effluents from treatment of sewage without ano ,/1th biodegraded LAS (5 mg/1) had approximately the same toxicities to oysters with respect to survival of larvae, development of eggs and increase in length of larvae. Thus, biodegraded LAS did not contribute to the toxicity of the sewage effluents. f. Acute Toxicity to Invertebrates Daphnia. a commonly tested invertebrate, showed no effect from exposure to LAS (44.731 LAS) at concentrations less than 1 mg/1. The 24 hr LC^g for this species was 3.46 (2.31-5.22) mg/1 (Shell Chemical Company, unpublished data). The effects of LAS (supplied by SDA, 60.8% LAS) on the oyster (Crassostrea *j_rqinica) have been studied by Calabrese and Davis (1967). At concentrations of LAS greater than 0.025 mg/1, the development of fertile eggs was reduced VVV 000012^50 114 Arthur D Little Inc r$velt et il* (1971), in a study of San Francisco Bay, determined toxicity to . ,rir.e (yoiden shiner, Notemigur.cus chrysoleucas) related to sewage effluents and found significant reductions in MBAS levels after biolo- sewage treatment and concomitant reductions in toxicity. Toxicity #.*ritutable to MBAS in this study was difficult to separate from the overall siicity of sewage effluents. There was a significant difference in concen- ntions of MBAS in effluents from primary and ether more extensive treatment, ^xesses. Mean MBAS values in effluents from 4 primary treatment plants in f* San Francisco Bay area were 10.9, 5.0, 7.2 and 7.3 mg/1. For activated plants, the average was 1.1 mg/1, occasionally reaching levels of 6.7- ?.Jpg/l. It was determined from a mathematical model that M8A5 and ammonia ttroqen were significantly correlated with toxicity of primary effluents. ever, the direct addition of LAS to primary effluents had little effect on its toxicity lending further support to the view that MBAS levels are not con clusive measurements related to acute toxicities of primary effluents. In a study at the Elm Farm Sewage Treatment Plant, which employs an actited sludge treatment process, it was found that MBAS levels contributed by US were generally reduced by greater than 95*. Testina of this effluent for toxicity in fathead minnows (Pimephales promelas) resulted in complete wnrival (Renn, 1974). In an effluent solution of only 36% MBAS removal, all f the tested individuals died. In this instance, MBAS concentrations were ^'5 PPr (Colgate-Palmolive Company, unpublished data). 0oooizb51 113 Arthur DLuttle Inc / The most fish-toxic components of a LAS are also the ones most rapidly biodegradable. The biodegradation, even a partial one, reduces greatly the toxicity of the surface-active agent. Different LAS isomers with different initial fish toxicity tend to be reduced and to become equal upon biodegradation. The value of the LC^q of any LAS tends to increase consider ably with the progress of biodegradation. e. MBAS-Related Acute Toxicity to Fish in Sewage Effluents The data summarized above clearly show that LAS is rapidly degraded in laboratory simulations of the activated sludge sewage treatment process. The biodegradation of LAS results in a 10- to 100-fold reduction in acute toxi city to fish. In the actual environment, the situation is considerably more complex because of wide variations in treatment of waste waters and in the ex treme diversity of effluents reaching natural water bodies with respect to amount and characteristics of materials other than LAS. Thus, the toxicity to aquatic organisms of sewage effluents and waters containing these effluents cannot be readily attributed to LAS even though these waters contain MBAS. The problems surrourdirg the use of MBAS as an analytical tool for LAS in natural waterways, especially those which receive sewage effluents, have been considered above (Section II.B). J 1 1 1 1 00001*^ 112 Arthur D Little: Inc TABLE 1-G nUE TOXICITY OF LAS AND PRESUMPTIVE BIODEGRADATION INTERMEDIATES Intact LAS*Cjj Sulfophenylundecanoic acid, aisodiun salt (mixed isomers, 6tnrough 10-phenyl) 3-(Sulfophenyl)butyric acid, disodium salt A-(Sulfophenyl)valeric acid, disodium salt 48-Hour LCrq (mq/1) Daphnia magna Pimephales promelas 5.7 + 0.6 16.0 208 + 85 ^6,000 ^5,000 76.6 + 12.4 %.10,000 ^10,000 Kimerle and Swisher, 1977 vw 000012653 111 Arthur D bale Inc HLAS was 0.72 mg/1. After biodegradation to 25% of the initial surfactant concentration (MBAS), 96-hour LC^g for the bluegill was increased to 1.64 ng/1 and to 2.3-7.2 mg/1 with 92% degradation. Undiluted biodegraded HLAS resulted In LC5Q values ranging from 4.6 to less than 4.6 mg/1 for 24 and 48 hours. At 50% and 90% biodegradation levels, 24-hour LC^g values were 5.0 and greater than 5.0 mg/1. A test using intact LLAS gave a 3.89 mg/1 LC^q in 96 hours for bluegills. This LAS product also showed decreased toxicities for bio degradation products, with LC^g of 10.3 mg/1 MBAS. Kimerle and Swisher (1977 ) obtained evidence that toxicity of a cormiercial LAS preparation (ci2"^14^ t0 DaPhn^a maqna decreased from an LC^g of 3 mg/1 for the parent product to a level of 6 mg/1 for a partially (50%) de graded product. Further biodegradation resulting in 80 to 90% removal of the initial concentration of MBAS produced LCgg values of 20 to 35 mg/1. More over. they showed that certain presumptive LAS biodegradation intermediates have little or almost no toxicity to either Oaphnia maqna or fathead minnow (Pimephales promelas) (Table 1-G ). The preparation of a set of computations designed to predict the acute fish toxicity of complex mixtures of LAS from a knowledge of their molecular composition has led Divo (1974) to several conclusions with respect to the relationship of biodegradation of LAS to toxicity. These conclusions agree with the data from the few studies performed to date: VVV 000012654 110 Arthur D Little Inc In the first systematic study of this problem, Swisher et al_. (1964) examined the effect of biodegradation on toxicity to bluegill (Lepomis r^crochirus) fingerlings. They found that addition to continuous flow activated sludge units of as much as 100 mg/1 C^- or C^-LAS resulted in effluents that did not exhibit any lethal toxicity. MBAS concentrations in the test tanks ranged from 0.1 to 0.9 mg/1. Toxicity tests of effluents from acclimated, as well as unacclimated sludge yielded toxic levels of LAS at 1 to 2 mg/1. A minimally altered LAS, mixed isomers of sulfophenylundecanoic acid disodium salt, gave a 96-hour TLm of 75 mg/1 indicating that even a single oxidative alteration of the alkyl chain of LAS is sufficient to markedly reduce toxicity. Borstlap (1967) found that the acute toxicity (minimum lethal concen tration) for guppies (Lebistes reticulatus) decreased markedly from 5 mg/1 to >1000 mg/1 with the biodegradation of the commercial LAS product D0BS-C300 (sulfonate of Dobane C-300 n). Similar sharp reductions in toxicity of other commercial LAS products following their biodegradation have been reported for guppies (Poecilia reticulatus) and harlequins (Rosboral spp.) (Shell Research Ltd., London, unpublished data) as well as for rainbow trout (Salmo qairdnerii) (Unilever Ltd., unpublished data). Cairns and Dickson (1973) have reported on a series of toxicity tests **'th intact and biodegraded LAS on bluegills and snails using high (HLAS) low (LLAS) molecular weight products. The 96-hour LC^q value for intact 00001*^ 109 Arthur D Little Inc flow assays at 6-8*^, 96-hour LCg0 values were 1.0, 1.5 and between 1.0 and 5.0 mg/1 LAS, respectively. Tests conducted at 15-17C gave 96-hour LC^g values of less tnan 1.0 mg/1 for cod and plaice. As for fresh water fish, early developmental stages were more sensitive than adults. Concentrations of LAS of 0.1 and 0.3 mg/1 significantly reduced survival time of cod and plaice, respectively, in the stages from hatching to yolk absorption. Sublethal responses such as impaired swimming activity and breathing rate as well as reduced opercular movement were observed in cod after exposure to 0.5 mg/1 LAS for 24 hours. In contrast, flounder were more resistant, exhi biting normal swinming behavior after 21 days in 0.5 mg/1 LAS. Considering the available data on the acute toxicity of intact LAS to fish, the LC50 values for fingerlings and adults of a number of fresh water and marine species range from 1.0 to 10.0 mg/1. For those fresh water and marine species that have been examined, early developmental stages (e.g., sac-fry) are more sensitive to the acute toxic effects of LAS. Acute effects of LAS on sub-lethal manifestations of toxicity (swimming, breathing rate, opercular movement) occur at concentrations at or slightly below the LC^q values for the two species that have been examined for these responses. d. Acute Toxicity to Fish-Biodegraded LAS Although intact molecules of LAS are readily biodegraded in waste waters and waste water treatment plants as well as in natural waterways, there is a paucity of reliable information dealing with toxicity of degraded LAS to aquatic organisms. VVV 0000X2656 108 Arthur D Little ln< C/o- Clf- TABLET-? EFFECTS OF LAS ON BLUEGILL (LEPOMIS MACROCHIRUS) Development Staqe Fingerling Sac-Fry 5-day 1 -day Newly hatched fry Fertilized eggs* Fertilization Sperm TLm (mq/1) 3.80 (24 hr) 3.4 (24 hr) 2.3 (6 day) >5.6 (24 hr) Median Response (mq/1) 3.7-4.0 (hatching) 10 5.4-5.7 (active sw ming - gyration) *Eogs burst at LAS concentrations >4.0 mg/T. Hokanson and Smith, T97T VVV 000012657 107 Arthur D Little inc '-AS necessary to reduce swimming activity to zero in 6 hours in the test system used by Karchetti were 4.7 and 3.2 rg/1, respectively. In this study, the lethal effects and suoacute effects on locomotor activity appeared to be related. Hokanson and Smith (1971) studied the toxicity of a well defined LAS sample (90S active; C1Q-16 , C,-, Z: , . . C12-29.5S, C^-IS*. >C14-2.52) in Mississippi River water to various developmental stages of the bluegill (Lepomis macrochirus) ranging from sperm and unfertilized egg to fingerlings. They found that the feeding sac-fry were most sensitive to LAS: eggs and fingerlings exhibited intermediate sensitivity; egg fertilization was the least sensitive developmental step (Table 1-F ). lubinski et aj_. (1974) also examined the toxicity of LAS to the bluegill (Lepomis macrochirus1 in a continuous flow bioassay and found a 96-hour LC.q value of 6.5 mg/1. They also developed a concept of aquatic toxicity based on fractions of the 96-hour LC^q values of each of the identified toxi cants in the Illinois River. They determined that the Illinois River water is not normally toxic to bluegills and that the major source of potential toxicity for fish would probably come from armonia and cyanide, with LAS, copper, fluoride and zinc also contributing fractional toxicity. In the only extensive study of marine fishes and their responses to LAS, Swedmark et al. (1971) investigated 3 species; i.e., cod (Gadus morrhua L.), founder (Pleuronectes flesus L.) and plaice (P. platessa L.). In continuous I i WV 000012658 106 Arthur D Little Ir* TABLE 1-C ACUTE TOXICITY OF LAS TO FRESH WATER FISH Species --*>n atherinoiaes n nacrochirus whales oromelas *mi5 cornutUS rilurus melas Common Name Emerald shiner Bluegi11 Fathead minnow Common shiner Black bullhead TLm [mq/1 3.0 4.0 4.2 4.9 6.4 95% Confidence Limits (mq/1) 2.96-3.56 3.70-4:30 Not given 4.58-5.18 6.08-6.68 u*.er and Santer, 1 967. VVV 000012659 105 Arthur D Little Inc 3 several species of fresh water fish (Table 1-E ). The results Indicated ;-3t "sufficient difference in sensitivity to LAS exists among species of i$h to warrant attention to this factor when assessing the potential hazard f LAS to aquatic populations." Pickering (1966) has investigated the effects of the same type of LAS reparation used by Thatcher and Santer (1967 ) on eggs of the fathead minnow pjrepnales promelas) in a continuous-flow bioassay. The results expressed 9-day TLm values for survival of hatched fry ranged from 2.3 to 2.6 mg/1 :n 4 replicate tests. The 1-day TLm value was 3.6 mg/1, with the threshold :f mortality at 0.9 mg/1. The results with this species indicate that the egg and fry stages are more sensitive to LAS than are adults. Dooley (1968) also examined the acute toxicity of this LAS sample ob tained from the SDA on mosquito minnows (Gambusia affinis). At an LAS con centration of 0.12 (1000 mg/1), the survival time for males was 9 minutes and for females was 17 minutes. As the concentrations of LAS were reduced, Survival times increased until the populations exhibited 72-hour survival a level of 0.00078% (7.8 mg/1). The gills of fish killed by LAS were Imaged showing a mattc-u condition, occasional blood masses and loss of gill *cosa cells. I I 1 T I In addition to determining 6-hour LC^q values of 8.4 and 7 mg/1 in gold- f,stl (Carassius auratus L.) with C^- and Cj^-IAS products, respectively, ,'*rchetti (1968) examined swimming activity. The concentrations of and WV 000012660 104 Arthur D bttie Ire ;en extensively studied for their aquatic toxicity. Divo (1974) found in cased toxicity with an increase in chain lengths among a series of tetra'S with chains from 10 to 13 carbon units. Kimerle and Swisher (1977 ) ifirm this trend with 48-hour LC^q values in fathead minnow (Pirrephales ^elas) of 86.1, 21.5 and 5.3 rr,g/l for C.jq, and dial kyl tetral in jane sulfonate mixtures, respectively. However, these side products are ;nsiderat>ly less toxic than LAS isomers of comparable chain length. c. Acute Toxicity to Fish - Intact LAS In considering the reported data on the acute toxicity to fish of LAS -j LAS-containing detergents, a number of factors should be weighed in the nination of experimental results, especially in relation to the use of *se data to set aquatic safety standards. Abel (1974) has reviewed in de:' 1 the problems surrounding the assessment of toxicity of synthetic deter-*ts to fish and aquatic invertebrates. In addition to wide variations in 'jerimental protocols with respect to water temperature and chemistry and '^sure patterns (static vs. continuous flow; water volume to organism mass v-io), the lack of adequate chemical characterization of the LAS samples '"`led and the wide range of susceptibilities among different aquatic species ';se genuine difficulties for the comparative evaluation of acute toxicity dies. In a series of continuous flow-through bioassays, Thatcher and Santer ^7) determined acute toxicities of an LAS preparation (SDA Interim Ref**rce Sample: LAS Lot No. 1-1), 60.8" surfactant and 36.1% sodium sulfate. 103 Arthur D Little Inc / i iAri % 1 -o_ ACUTE toxic 1 t r or in tact las,. EFFECT OT CHAIN l EKtiTH LAS 48-Hour LC50 (mg/1)^ Homoloques Pimephales promelas C10 cll C,2 C13 CH C16 o rvj C18 43.0 16.0 4.7 0.4 0.4 LC50 (mg/1)Z Carassius auratus 61.0 22.5 8.5 3.3 - LC50 (mg/1)23 Lebistes reticulatus 50 5 1 1 15 LC50 tmg/1)4 5 96-Hour LC50 (1 Idus melanotus Lepoxis r'ACroc 16.6 6.5 2.6 0.57 0.26 0.68 21.2-47.5 11.6 1.18-6.5 1.11 0.25-0.42 0.087 0.38 1. Kimerle and Swisher, 1977. 2. Gafa, 1974. 3. Borstlap, 1967. 4. Hirsch, 1963. 5. Procter and Gamble Company* unpublished data. LC5q values of individual LAS homologues dependent on phenyl group position. Lower LC^q values correspond to LAS with higher proportions of 2-phenyl isomers. < < < o o o o M M O' O' Nj I 1 m r- J r - 4 r- i Arthur D Little 8. 599-612 (1979) Archives O' Environmental Contamination and Toxicology- Acute Toxicity Studies of SurfactaiUS to Daphnia magna and Daphnia pulex jr _.o. Alan W. Maki and William E. Bishop The Procter & Gamble Company U.S.A., (vorydale Technical Center, Cincinnati. Ohio 4S2I7 Abstract. Several experiments were designed to examine the acute toxicity of surfactants to Daphnia. Specific tests were designed to develop compari sons between existing acute toxicity data for fish and similar data for Daph nia, and to provide data on the effects of various environmental factors on resultant toxicity of surfactants to Daphnia. Amtr toxicity data for a series of homologous linear alkyl benzene sulfonates (LAS) demonstrate increases of up to one order of magnitude in toxicity for each increase of two alkyl carbons. LC 50's obtained with Daphnia magna are similar to those obtained with bluegills, L. Macrockirus. Comparative tests with D. magna and D. pulex indicate no statis tical differences in 48-hr LC 50 values for three anionic and two nonionic surfactants. A 50 mg/L concentration of suspended, naturally-occurring kaolin significantly reduced the toxicity of longer chain length LAS homologs and had no effect on nonionic surfactant toxicity. In tests with variable hardness concentrations, the acute toxicity of LAS to D. magna is a combined function of both culture and test water hardness. The toxicity of a nonionic surfactant to D. magna was higher in soft water and was not affected by culture water hardness levels. Unlike previously published data for fish, the results of acute toxicity tests with D. magna cultures previously exposed to 0.4 mg/L LAS for periods up to seven generations indicated no significant difference in LAS susceptibility compared to simultaneously tested unexposed controls. The use of laboratory toxicity tests with aquatic species serves the important function of predicting the effects on aquatic life of chemical substances poten tially released to surface water environments. As such, toxicity data for aquatic species is integral to the development of aquatic safety assessments for new or expanded-use chemicals and for the establishment of relevant water quality criteria. Traditionally, acute tests have been done with representative fish species since they are the most visible and economically significant component of most aquatic communities. Recently, as the utility of acute toxicity data for aquatic safety programs has been realized and the number of chemicals requir- 0090-4341/79/0008-0599 $02.80 1979 Springer-Veriag New York Inc. 600 A. W. Maki and W. E. Bishop ing the submission of acute data in support of use or registration has increased, the demand for such data has produced increased interest in the use of invertebrate species for the development of needed toxicity information. Representa tives of the genus Daphnia have emerged as some of the more important test organisms and as such they typify the advantages of employing macroinverte* brates for development of toxicity data: ease of laboratory culture and mainte nance in a strictly defined media, small culture space and water requirements when compared to fish, ease of handling and counting, small test volume re quirements, significantly shorter life cycles, and greater disease resistance. This program was designed to examine, the application of Daphnia acute toxicity data to aquatic safety programs for the evaluation of effects of residual concentrations of surfactants potentially reaching surface water communities. Specific objectives were to: 1. Examine the effects of changes in surfactant chemical structure on the biological activity as measured by acute toxicity to Daphnia, and com pare effect concentrations with those published for fish species. 2. Test the relative susceptibility of D. magna and D, pulex to surfactant toxicity. 3. Examine the effects of environmental variables such as presence of suspended solids, variable water hardness, and previous surfactant ex posures or acclimation on the acute toxicity of several classes of surfac tants. Materials and Methods Test Materials and Analyses The test materials employed in these investigations included both anionic and nonionic surfactants. The identifications and chemical descriptions are '_i:ed in Table >. Since the surfactants were of varying degrees of activity, a determination of percent active material was performed on each material immediately prior to toxicity test initiation. A standard curve for each anionic homolog was obtained utilizing the analysis for methylene blue active substances {Arner. Pub. Health Assoc. 1971). Table I. Chemical characterization of the test surfactants. Generic names are used throughout the text Generic Name C,0 LAS through C,, LAS Linear alkyl ethoxylates Ci^AEi through C,4AE, Structure CHa - (CH*)X " CHj i V SO,Na* CHj - (OH*), - <C,H,0)*H Chemical Characterization Anionics, alkyl chain length range: CI0-C,S. molecular weight range: 341-453 Nonionics X - 13 Y - 1 to 9 Toxicity of Surfactants to Daphnia Table 2. Dilution water quality for alt Daphnia toxicify tests with surfactants Parameter Concentration <mg/L) Hardness pH Dissolved oxygen Nitrate Nitrite Copper Iron Lead Sodium Zinc 120 mg/L as CaC03 7.4 - 0.2 8.5 - 9.5 <0.05 <0.05 <0.001 <0.05 <0.01 M.6 <0.001 601 Water samples were taken from each test concentration at the termination of the 48-hr expo sure period, preserved with \7c formaldehyde, analyzed by the MBAS procedure.^Tid compared with the standard curve for confirmation of expected nominal test concentrations. Results for the oomonic surfactants are based on nominal concentrations since accurate analytical methodologies for quantification of dilute aqueous concentrations of these materials are not available. Test Procedure The methods used for culture procedures and acute toxicity tests followed the guidelines estab lished by the USEPA <EPA-660/3-75-009. 1975). The quality of the carbon-filtered well water used as dilution water for all tests is listed in Table 2. All tests were carried out at a constant temperature of 21 a l`C under a 16-hr illumination period. Prior to a test, adult Daphnia. sorted by size, were isolated in separate aquaria and the young produced overnight were tested the following day. In this manner, known age individuals. 24-hr old or less, were used to initiate all tests. The test containers were 250 ml Pyrex beakers with a total solution volume of 200 ml. The beakers were cleaned and sterilized after each test. Prior to use. the beakers were rinsed in hot tap water, brushed in 957c reagent grade alcohol, and rinsed in deionized water and allowed to dry. All tests were of 48-hr duration and all concentrations were done in triplicate. Fi\t Daphniu were added with a glass pipet to each beaker at the test initiation. Mortality was recorded after 24 and 48-hr intervals. The individuals were not fed during the test. Results were analyzed using a computerized probit analysis program providing for calculation of LC 50 values and associated 957c confidence intervals (Finney 1971). Deviations from this standard method were employed for tests incorporating kaolin day (Georgia origin, mean panicle size 4ji). For these tests, 50 mg L kaolin was added to a 4 L volume of dilution water and vigorously mixed. Five 1-L beakers were then filled with 600 ml of this suspension, placed on magnetic stirrers, and the required amounts of test surfactant added to achieve the desired test dilutions. The solutions were then vigorously mixed for 30 min and the three 200 ml replicates of each test concentration were decanted into the test containers. For tests designed to determine the effects of prior exposure or acclimation to sublethal concentrations of the anionic surfactant Cn, LAS. D. rnaii/ui was cultured in a 0 4 mg L LAS concentration for periods of 24-hr (short-term exposures) up to seven continuous generations (long-term exposures) prior to comparative toxicity testing w ith unexposed indiv idu.il>. During the long-term acclimation period, the 0.4 mg L LAS concentrations in the culture chambers were replaced three times weekly to minimize degradation of the LAS. All tests were run simultaneously with unexposed individuals to examine (he significance of the prior exposure on acute toxicity of LAS. JL 602 too 0- 10.0- A. W. Maki and W. E. Bishop s J] 95% CONFIDENCE LIMITS </ lCcn Imgtf! 10- 0.1 48 HR. ice5Uft PAPHNIA 96 HR LC50 BIUEGUL ~T~ i `no '12 '14 ^16 '18 '20 LAS ALKYL CHAIN LENGTH Fig. I. The effect of variations in the alkyl carbon chain length of linear alkyl benzene sulfonate on resultant acute toxicity to Daphnia magna. Results and Discussion Structure: Activity Correlations The effect of an increase in the alkyl carbon chain length of the anionic surfac tant, LAS, on resulting acute toxicity is summarized in Figure 1. An increase in r --48 HR. LC5Q AEj AE^ AEg AEg AEjQ AE^ C - ETHOXYtATE distribution 14 Fig. 2. The effect of variations in the ethoxylace distribution of nonionic surfactants on the resul tant acute toxicity to Daphnia magtta. toxicity of approximately one order of magnitude is demonstrated for each additional two carbons between Ci0 and C,8. with a subsequent slight decrease at C,,. The acute toxicity measured as 48-hr LC 50 values drops consistently from 29.5 mg/L at C,0 to 0.11 mg/L at Ct6. demonstrating a slight but statisti cally insignificant increase to 0,12 mg L at C,*. Also plotted in Figure l are the bluegill 96-hr LC 50 values for the same homologous series of anionic surfactants (A.D. Little 1977). These data demon strate a similar relationship between increased toxicity and increasing alkyl r r vvy 00001266 <04 A. W. Maki and W. E. Bishop carbon chain length. Acute toxicity values for bluegill and Daphnia indicate that anionic surfactant toxicity data obtained for representatives of one trophic level may be extended to species of different trophic status within aquatic communities. Similar Daphnia toxicity tests completed with nonionic, linear alcohol ethoxylate surfactants with a constant alkyl carbon chain length of CM and an ethoxylate distribution ranging from AEt to AE,, indicate that increasing ethox ylate distribution causes a decrease in acute toxicity to Daphnia (Figure 2). The 48-hr LC 50 value for CuAEi is 0.83 mg/L, while the corresponding value for C14AE is 10.1 mg/L, demonstrating greater than one order of magnitude de crease in toxicity over this increased ethoxylate composition (Table 3). Consistent changes in biological activity, measured as acute toxicity to Daphnia, resulted from variation in chemical composition within these homol ogous series of anionic and nonionic surfactants. These relationships demon strate that it may be possible to predict subsequent toxicity of formulation changes for new materials by knowing precise chemical structure and basic toxicity data for representatives of the homologous series. Thus, individuals attempting to design safety assessment programs for new chemicals or indi viduals attempting to establish water quality criteria have much to gain from an examination of data for similarly structured chemical substances. Species Comparison Testing Five surfactants representing three anionics and two nonionics were utilized during comparative acute toxicity tests with D. magna and D. palex. All tests with a particular material were run simultaneously under similar conditions with young daphnids 24-hr old in an attempt to control any extraneous variables Table 3. Acute toxicity of homologous series of surfactants to Daphnia magna Anionic Surfactants 48 hr LC50 (ppm) 9S% Confidence limits (ppm) C,, LAS C,, LAS C,, LAS C,, LAS CM las C,, LAS C,, LAS Nonionic Surfactants 29.55 21.15 5.88 2.63 0.68 0.11 0.12 48 hr LC50 (ppm) 27.94 - 31.05 18.49 - 22,25 5.24 - 6.49 2.37 - 2.85 0.58 - 0.77 0.068 - 0.126 0.074 - 0.154 95% Confidence Limits (ppm) C,AE, c,ae5 C,,AE: Ct<AE, C,,AE,, CI4AE,, 0.83 1.53 0.73 1.76 4.17 10.07 0.73 - 0.91 1.18 - 1.97 0.64 - 0.81 1.43 - 2.03 3.05 - 6.02 9.46 - 10.66 Wy 000012668 Toxicity of Surfactants to Daphnia 605 /^''Tble 4. Results of 48 hr acute toxicity tests with surfactants and two species of Daphnia 48 hr LC50 (m/L) Test Material C,, LAS Cl4 LAS C, LAS C|*AE1 C|AE* Daphnia magna 6.84 (5.29 - 8.46) 0.80 (0.69 - 0.91) 0.20 (0.!? - 0.22) 0.14 (0.11 - 0.16) 0.24 (0.20 - 0.27) Daphnia pulex 8.62 (7.28 - 9.91) 0.59 (0.51 - 0.69) 0.15 (0.13 - 0.17) 0.10 (0.07 - 0.12) 0.21 (0.18 - 0.23) otherwise influencing directional susceptibility of the two test species. Results of these comparative tests with anionics ranging from Cl2 to C,s LAS, and the nonionics C14EAt and CMAE4, indicate that no statistically significant differ ence can be shown between the 48-hr LC 50 values for the two species (Table 4). Slight differences existing between the two species are most likely due to the sample size--15 individuals from three replicates of each test concentration. These differences would most likely be normalized with a higher number of replicates. Representatives of the genus Daphnia have become some of the more important test organisms for aquatic safety evaluation programs, due to the Native ease of laboratory culture and short generation times of approximately ^ -ght days. Traditionally, the most popular Daphnia test species has been D. magna; however, recently, questions have arisen over the most appropriate Daphnia species for testing. Daphnia magna has a somewhat limited geograph ical distribution, being typically found in small, hard water lakes (Brooks 1957). It has been argued that the more cosmopolitan species, D. pulex, is a more valid test organism for water quality demonstrations (Buikema et al. 1976). Such a conclusion, however, is not warranted by the available data. Using LC 50 values as a measure of acute toxicity, D. magna and D. pulex have been reported to exhibit virtually identical sensitivities to a wide variety of organic and inorganic compounds (Canton and Adema 1978; Winner and Farrell 1976; Buikema et al. 1976). The similarity of response for D. magna and Z>, pulex demonstrated in this investigation indicates that both species are equally sensi tive to anionic and nonionic surfactants. Effects of Suspended Solids on Surfactant Toxicity Numerous investigators have examined the effects of various physical and chemical environmental factors on the toxicity of surfactants to aquatic life. These studies have demonstrated that acute toxicity thresholds are generally r vw 000012669 606 A. W. Maki and W. E. Bishop observed at lower concentrations of nonionic surfactants (Swedmark et ai. 1971) and anionic surfactants (Hokanson and Smith 1971) when test tempera ture is increased. Similar studies examining the effects of variable concentra tions of dissolved oxygen (Herbert et al. 1957; Hokanson and Smith 1971) and salinity concentrations (Eisler 1965) on the acute toxicity of surfactants have shown effect levels to be significantly lower at reduced oxygen concentrations and when tested at high and low salinity extremes. Little information, however, concerning the interactions of suspended solids with surfactant toxicity is cur rently available. Since natural surface waters typically contain measurable concentrations of suspended inorganic sediments and intact surfactants are known to interact with these solids, it is reasonable to assume that toxicity tests completed with the presence of suspended sediments would closely model the chemical/physical complex likely to exist in surface waters to which unde graded surfactants may have been discharged. The results of the present tests with Daphnia magna demonstrate variable toxicity, with interactive effects between the 50 mg/L suspension of kaolin clay, the chemical class of surfactant tested, and individual alkyl chain lengths (Table 5). For the anionic surfactants tested, the 48-hr LC 50 values were observed at significantly higher concentrations of both the C14and C18 homologs, but no significant difference was observed with CM LAS (Figure 3). The acute toxicity of the nonionic surfactants tested varied as a function of alkyl carbon chain length; however, there was no effect on the 48-hr LC 50 values when kaolin clay was present (Table 5). The results of these tests with surfactants demonstrate that the presence of suspensions of purified, naturally occurring kaolin clay, significantly alters the Table 5. Effects of a 50 mg L suspension of kaolin clay on the acute toxicity of a homologous H series of anionic surfactants to Daphnia magna 48 hr LC50 and Associated 959c Confidence Limits (ppm) l Anionic Surfactants Without Kaolin With Kaolin f C,, LAS 19.3 (14.3 - 23.5) 13.0 (5.1 - 17.4) C14 LAS 1.0 (0.94 - 1.1) 1.4 (1.2 - 1.5) C,, LAS 0.09 (0.07 -- 0.11) 0.18 (0.12 - 0.24) Nonionic Surfactants c,*ae3 C^AE, Cj,AEj young adults 1.9 (1.0 - 2.6) 0.12 (0.08 - 0.16) 5.0 - 20.0 >80.0 1.7 (1.1 - 2.3) 0.12 (0.05 - 0.18) <5.0 >80.0 \0000126TM. Toxicity of Surfactants to Daphnia 607 Fig. 3. The influence of suspensions of naturally-occurring Kaolin clay on the acute toxicity of linear alkyl benzene sulfonate to Duphnia magna. observed acute toxicity threshold and LC 50 concentration. The proposed mechanism by which toxicity is reduced is believed to result from absorption of the surfactant on the clay and subsequent settling of this suspension in the test container, with resultant loss of active surfactant from solution. The degree of adsorption and subsequent observed loss of toxicity is dependent on the chemi cal class and specific composition of the surfactant. In general, the longer alky l VVV 000012671 60S A. W. Maki and W. E. Bishop chain length homologs of anionic surfactants demonstrate significant reduc tions in toxicity in the presence of kaolin, while nonionic surfactants represent ing a wide range of alkyl chain lengths appear unaffected by the suspended solids additions. These results lead to the conclusion that the addition of sus pended solids to a toxicity test chamber can be important to testing designed to determine acceptable or nonhazardous concentrations of surfactants to aquatic life, but that the decision to employ solids during the testing procedure should be predicated on the demonstrated ability of the material in question to strongly adsorb to natural solids. In these instances, the addition of known solids is advisable to more closely simulate the physical/chemical form of test material existing in the environment. jEffects of Hardness Ions Water hardness affects the toxicity of many chemicals in solution and the toxicity of surfactants has been variously reported to increase, decrease, or be unaffected by water hardness. (Abel 1974). Henderson et al. (1959) found nonionic toxicity to be unaffected by water hardness, but reported the anionic sodium alkyl sulphate to be more toxic in soft water. Hokanson and Smith (1971) reported the toxicity of LAS to be significantly greater in waters of 290 tng/L CaC03 than in waters of 15 mg'L CaC03. The toxicity and bioconcentration of the anionic surfactant, sodium lauryl sulphate, were directly proportional to the hardness of the test solution and to the hardness of the water in which the fish were acclimated. (Tovell et al. 1974). Toxicity and bioconcentration were related to the concentration of divalent c cations (Ca*2 of Mg*2) in the test solution. The toxicity and bioconcentration of a nonionic surfactant, CJ2 alkyl ethoxylate. were inversely proportional to the test water hardness, and not related to the cation composition of the test solu tion or to the hardness of the water in which the fish were acclimated (Tovell et al. 1975). The acute toxicity of LAS to D. magna is a function of both culture and Table 6. Effects of culture and test water hardness on the acute toxicity of an anionic and nonionic surfactant on Daphnia magna Culture Water Test Water 48 hr LC50 (95% Confidence Interval!0 Type Hardness* Type Hardness C,,., LAS Neodol 45-7 Soft Soft Moderately hard Moderately hard Hard Hard Very hard Very hard 50 50 125 125 225 225 350 3 JO Soft Very hard Soft Very hard Soft Very hard Soft Very hard 2J 350 25 350 25 350 25 350 7.1 (5.4-16.9)* 3.5 (2.9-4.1) 4.2 (3.2-6.0) 4.0 (3.4-4.8) 2.0 (.5-2.9) 3.2 (2.6-3.8) 1.8 (.6-2.4) 4.0 (3.1-J.0) .36 (.28-.45) .65 (.52-.79) .56 (.52- 74) .62 (.48-.77) .36 (.28-.451 .88 (.64-1.90) .36 (.28- 451 .90 (.72-1.301 oi|/L as CaCO: * m^/L. based on nominal concentration* I VVV 000012672 Toxicity of Surfactants to Daphnia 10 8- 64 20 LAS 609 HARD(325-350ppm) CULTURE 1 Acclimation) WATER HARONESS-mg/Zas CaC03 Fig. 4. The influence of variations in culture and dilution water hardness on the acute toxicity of an anionic and nonionic surfactant to Daphnia magna. test water hardness (Table 6). In hard water the toxicity in LAS is independent of culture conditions. In soft water, however, the toxicity of LAS is directly related to culture water hardness levels. That is, in soft water LAS is signifi cantly more toxic to Daphnia cultured at high hardness levels (figure 4). This apparent discrepancy may indicate a significant additional physiological stress induced by testing high hardness acclimated Daphnia in low hardness test solutions. By allowing a sufficient time for acclimation to low hardness, a significant reduction of LAS toxicity in soft water may be affected (Table 6). The nonionic surfactant, Neodol1 45-7, was more toxic to Daphnia in soft water (Table 6). The compound has a mixed alkyl chain length of linear CH and CIS and an average of seven moles of ethylene oxide per mole of alcohol. Furthermore, the sensitivity of Daphnia to Neodol was not conditioned by the* * Neodol is a registered trademark of the Shell Chemical Company 610 A. W. Maki and W. E. Bishop Table 7. Acute toxicity of C,, LAS to acclimated and unacclimated D. magna Acclimated* Unacclimated Acclimation Period 48 hr LC50b (95% Confidence Interval) 48 hr LC50b (95% Confidence Interval) 24 hr 24 hr 5 generations 7 generations 2.4 (2.1 - 2.7) 2.8 (2.1 - 3.3) 3.2 (2.0 - 4.0) 2.6 (1.8 - 3.3) 2.9 (2.5 - 3.2) 2.5 (1.7 - 3.1) 4.3 (3.7 - 4.9) 3.0 (2.0 - 3.8) Nominal LAS concentration = .4 mg/L b mg/L hardness of the water in which the organisms were cultured (Figure 4), The results of these Investigations underscore The importance of documenting the quality of test dilution waters and the culture conditions of aquatic species used in toxicity evaluation programs. Effects of Prior Surfactant Exposures Existing data indicate that fish are able to acclimate to surfactant concentra tions. A 30-day exposure of bluegill, L. macrochirus. to 3 mg/L ABS (alkyl benzene sulphonate) produced a 48-hr LC 50 63% higher than that of unaccli mated control fish tested simultaneously fLemke and Mount 1965). Hokanson and Smith (1971) also found that acclimation of bluegill to 0.5 mg/L LAS in creased lethal threshold concentrations by 50 to 400% that of unexposed con trols at several levels of oxygen saturation. If aquatic life is able to acclimate to previous surfactant exposure with no effects on survival or reproductive pa rameters, this capability could have significance to natural surface w'ater com munities existing in receiving waters continuously exposed to low background concentrations of the surfactant, indicating that results of laboratory toxicity tests may yield unnecessarily restrictive values when projected to these real world populations. To determine if this acclimation effect also occurs in D. magna. cultures were exposed to C,,.a LAS at a residual concentration of 0.4 mg/L. selected as approximately 1/10 the 48-hr LC 50 concentration, for up to seven generations (approximatiey 14 weeks). Subsequent tests with unexposed controls indicate no significant difference in the acute toxicity of Ctl,,, LAS to acclimated or unacclimated cultures (Table 7), leading to the conclusion that previous expo sure of D. magna to concentrations of LAS as high as 1/10 the 48-hr LC 50 docs not alter the sensitivity of the organisms to acute effects. Caution should be used, therefore, in extrapolating results of acclimation reported for fish, to aquatic life in general. VVV 000012674 Toxicity of Surfactants to Daphnia 611 Conclusions 1. Data for a series of linear alkyl benzene sulfonates and a series of linear alkyl ethoxylates show regular and predictable effects of variation in chemi cal structure on subsequent acute toxicity to D. magna. 2. Comparative toxicity testing with D. magna and D. ptilex and several anionic and nonionic surfactants indicated that both species have statisti cally similar 48-hr LC 50 values and that both species are equally valid test organisms for investigators attempting to develop water quality information. 3. Acute toxicity tests withD. magna and two surfactant types carried out in solutions with and without suspended kaolin clay, indicate that observed loss of toxicity is dependent on the chemical class and specific composition of the surfactant. While nonionics were generally unaffected, the Cu and Ci,, alkyl chain lengths of LAS have reduced toxicity in the presence of kaolin. A. In hard water, the acute toxicity of LAS to D. magna is independent of culture conditions, while in soft water toxicity is directly related to culture water hardness. The acute toxicity of a nonionic surfactant was greater in soft water and was not affected by culture conditions. 5. Acute toxicity tests with D. magna cultures previously exposed to 0.4 mg/L LAS for periods of up to seven generations indicated no significant differ ence in LAS susceptibility compared to simultaneously tested unexposed controls. References Abel. P.D: Toxicity of synthetic detergents to fish and aquatic invertebrates. J. Fish Biol. 6. 279 (1974). A. D. Linte, Inc: Human safety and environmental aspects of major surfactants. 1 American Public Health Association: Standard methods for the examination of w ater ar.d w astewa- ter. 13 ed. APHA, AWWA, Water Pollution Control Federation. 1015 13th St.. N. W., Washington. D. C. (1971). Brooks, J. L: The systematics of North American Daphnia. Mem. Conn. Acad. Arts Sci. 13, (1957). Buikema, A. L.. D. R. Lee, and J. Cairns. Jr.: A screening bioassay using Duphru: pulex for refinery wastes discharged into freshwater. J. Testing and Evaluation. 4, 119 i 19"6>. Canton. J. H.. and D. M. M. Adema: Reproducibility of short-term and reproduction toxicity experiments with Daphnia magna and comparison of the sensitivity of Dapfuua /mrgna with Daphnia puiex and Daphnia cucullaia in short-term experiments. Hvdrobiologta. 59, 135 (1978). Eisier, R: Some effects of synthetic detergents on estuarine fish. Trans. Am. Fish. Sod. 94, 26 (1965). U.5. Environmental Protection Agency: Methods for acute toxicity tests with fish, rrucroinvertebrates, and amphibians. EPA-660i'3-75-009 Washington, D.C. (1975). Finney. D. J: Probit analysis. London:Cambridge University Press. 11971), Henderson, C., Q. H. Pickering, and I. M. Cohen: The toxicity of synthetic detergents and soaps to fish. Sew. Ind. Wastes 31, 295 (1959). Herbert, D. W. M., G. H. J. Elkins. H. T. Mann, and J. Hemens: Toxicity of synthetic detergents to rainbow trout. Water Waste Treat. J. 6, 394 (19571. Hokanson. J. E. F , and L. L. Smith; Some factors influencing the toxicity of LAS to the bluegill. Trans. Am. Fish. Soc. 100, l (1971). Prioritization of Chemicals According to the Degree of Hazard in the Aquatic Environment by Dean R. Branson* / 4 Chtmirak designated as "priority pollutants" or "toxics" have received special attention recently because the discharge of these compounds into public water is to be restricted to the maximum possible with linle regard to water quality or economics. The selection or many of the 129 priority cemicais was not based on an objective scientific assessment of the exposure and effect data. In fact, for some compounds, including ctnaphthene and 4-cMorophenyl-phenyl ether, the necessary data for listing were non-existent. As an alternative to arbitrarily listing or delisting chemicals for the purpose of prioity control, (his paper gi^iv promising scientific approach to selecting priority chemicals based on the principles of hazard as.evstnent for chemicals in the aquatic environment. According a the hypothesis, the highest priority cb micals are those with the least margin of safety, defined as the gap between the no-observable-effect ciM'crnlralions and the amhienl exposure concentrations. The no-observable-effec t concen rations are based on the results of chronic or sensitive life stage tests with aquatic organisms and the acceptable daily intake rate for fish eates. The ambient exposure concentrations are levels either measured in fish and water, or roughly estimated from a simple nomogram that requires only t*u of the following three factors: environmental release rate, ratio of dissipation to biocoacenlration potential, or ambient residues in fish. The chemicals studied to illustrate this approach to prioritizing chemicals based on hazard assessment are: polvehlurmated biphenvls, di-2-ethylhexyl phthalate, U--r dkyflwwai write--aod pentachloro- phenol.' ' -------- ---------------------VVV 000012677 Introduction 17* principles of hazard assessment used in this paper tre those developed at a recent workshop in Pellston. Michigan (/). At this workshop, represen tatives of government, industries, and universities reached a consensus that hazard in water could be assessed only after both effect and exposure of the chemical substance had been taken into account, ^ure 1 illustrates the relationship. In general, the margin between the no-effect concentration and the expected expov.ire concentration in water is the statement of hazard or margin of safety. Estimates of these two concentrations are made in a sequential fashion, and the error limits on these two estimates irrow as the amount of information about biological ifects and exposures increases. It is important to `Environmental Socnces Kcsc.-rch. The Dow Chemical Com pany, MiJli/iJ. Michigan 4KS40. February 1980 recognize that the essentiaJ environmental informa tion can be estimated even from preliminary screen ing studies but the error limits on these estimates are usually quite large. To illustrate the use of hazard assessment techniques in prescreening priority chemicals, no emphasis on error limits was made. This concept of hazard assessment is fairly new in the field of aquatic toxicology. Another new concept in our field is regulation of those specific chemicals which represent the greatest cause for concern for pollution in the aquatic environment. The 1977 amendments to the Clean Water Act contained a list of 65 categories of substances, which was later de* fined in terms of 129 specific priority chemicals (2). These chemicals are to receive the maximum possi ble discharge control in effluents. This paper suggests combining these two concepts into an ob jective selection criterion for prescreening priority pollutants. Such selection criteria are needed, since Congress provided a process for adding and sub- 133 s N \ Tier I Tier >1 Refining Estimate* | Tier ill Figure ]. Principle of sequential hazard assessment of chemicals in the aquatic environment. Large gaps between the no observable adverse effect concentration and the exposure concentration indicate a low degree of potential hazard and vice versa. tracting chemicals from the list of priority chemicals. If the hypothesis is correct that principles of hazard assessment can he employed as a prescreen for selecting priority chemicals, then it may be possible to distinguish between priority chemicals and those chemicals whiih more appropriately should be clas sified as chemicals with less potential to cause harm to man or the environment. The discharge of chemi cals that might be removed from the list will also be regulated but to the extent that water quality is pro tected rather than to the maximum possible extent with little regard to economics. Case Study Chemicals Four chemicals were selected to illustrate the principles of hazard assessment--two with fairly high environmental release rates, di-2-ethyihexyl phthalate (DEHP) and linear alkyibenzene sulfonate (LAS), and two other chemicals with moderately high environmental release rates, polychlorinated biphenyls (PCB), and pentachlorophenol (PCP). The environmental release rate, according to a National Science Foundation (NSF) workshop (J), is the maximum amount of material which may enter public receiving water and is calculated from the amount produced minus the amount consumed, de stroyed, or contained. The estimates of environ mental release rates in the NSF workshop were based on 1972 production records. For DEHP and LAS, the maximum environmental release rate in 1972 was 100 million to 500 million Ib/yr, and for PCB and PCP 10 million to 50 million Ib/yr. The distinction between production rates and environmental release rates is important, because rarely is all of the chemi cal produced released to the aquatic environment. In order to assess the hazard of chemicals in the aquatic environment, certain environmental values must be measured or estimated. Table 1 shows the environmental information forthe case study chemi cals. This information includes the maximum con centrations of the case study chemicals which should not cause an observed adverse effect to fish-eaters, including man, i.e., acceptable daily intake DI) values combined with fish residue data. Thi_ values were derived from long-term feeding su s 134 Environmental Health Perspectives VVV 000012678 E-- : Tahl* I. Lxsenlial information for assessing Ihe hazard of chemicals in tu.` aquatic ;.irunmrnt. Hjj.i: d to Ha.iarj to fish caters Chemical A inherit exposure Conee rUralion in water. me.1. (ppm** NOEC nig. 1. (pr-mP Safety margin' Ambient concentration in fish. (ppm)* ADI residues lor fish eaters4 Safety margins' LAS PCP DEHP PCB <0.05 <0.00003? -0.00:5 0.000010 0.5 -0.005 0 003 0.O05 >10 150 -12 500 0.5 0.01 -10 >5 ??0 1500 9 900 ISO 18 <5 <1 'Samples from areas outside the mixing rone of an effluent; large lakes, rivers, and estuarie*. concentration of chemical in true solution. NOHC -- no observable adverse effect concentration in chronic or sensitive life-stage tests. 'Safety margin = no observable adverse etTeci concentration/ambient exposure concentration. VVV 000012679 aKish residue = acceptable daily intake (ADI) *zg/g/day x 60 kg fish eater/0.2 kg fish eaten per day. in rodents and assumed a safety factor of 100. For the case study chemicals, the following ADI values were used: DEHP. 0.6 mg kg-day (4); LAS, --2.5 mg/kgday 15); PCB, --0.016 mg/kg-day (6); PCP, 0.03 mg/ kg-day (7). It is appropriate to use long-term ex posure data since the hazard assessment of the case .--study chemicals was based on ambient concen- ations of the chemicals. If all the information in Table I is available, hazard assessment is an easy task, involving merely dividing the no-et'fect concentration by the exposure con centration, but rarely, if ever, is all the information available to everyone's satisfaction. Typically, the data least available or reliable are the ambient ex posure concentrations. As an alternative to direct measurements of the exposure concentration, it seemed reasonable that rough estimates of exposure could be made for the purpose of prioritizing chemi cals, by examining the quantities released to the aquatic environment, the resulting residues in fish, the bioconcentration potential, and the degradation potential. Correlation Between Release Rates, Persistence/Accumulation, and Fish Residues It was assumed in reviewing the available infor mation for this paper that in the future more and better data will be available on the ambient concen trations of chemicals in fish tissue. It was further assumed that these concentrations were a direct function of the persisience/accumulation properties d environmental release rates. The PCB residues in Table 1 of 5 ppm were for the irs 1972 and 1975 and were increasing with time t6). The linear aikylbenzene sulfonate residues in fish are estimated to be less than 0.5 ppm based on limited measurements of the concentrations in water and bioconcentralion factors of approximately ten (8). Pentachlorophenol measurements are about 0.01 ppm and declining (D. Stallings, personal communi cation, 1979). The DEHP residues are not well documented. Characteristically, they are measured as by GC-MS M!e 149, which is the common mass ion for all phthahte esters. The limited monitoring data which are available suggest that the ambient levels in tish tissue of DEHP are at least 10 ppm (9). The third part of this correlation deals with the persistence/accumulation. A composite of these properties was represented as a ratio of dissipation and bioconcentration potential; ratio DIS/BCF. Dis sipation was assumed to be the sum of both the evaporative loss of the chemical from water and the rate of photochemical, hydrolytical, or biological degradation (Table 2). Since quantitative measure ments of the rate of degradation were either not readily available or comparable, relative rates were assigned, where 100 was the maximum rate and a minimum rate of 1 was chosen for substances like metal ions which generally do not degrade orevaporatefrom water. For LAS, a relative degradation rate of 100 was assigned based on the first-order rate constant for COa evolution, 0.10/day as reported by Larson (10). For PCP, a relative degradation rate of 50 was assigned based on 100% disappearance in various soils after 4 hr to 30 days (11). For DEHP, a relative rate of 10 was assigned based on 50% of the parent compound remaining in a hydrosoil test after 14 days (72). For PCP. a relative degradation rate of 1 was assigned based on the second-order rate con stant for 14COa evolution from l4C-[2,5,2'-tri- chlorobiphenyl] in activated sludge, 0.009 g/g-hr (13). In the future, rate constants should replace these arbitrarily assigned relative rates. The bioconcentration factors used to calculate the ratio of dissipation to bioconcentration potential were all measured values (Table 3) and relative dissi pation rates were obtained from Table 2. What can he seen by this ratio of dissipation to bioconcentra lion potential is that the greater the dissipation and February 1980 135 Table 2. Dissipation of cliemiuls from staler. Chemical LA 5 PCP DEHP PCB' Water solubility, mg/l.* > 10,000 8.000 6 0.00025 H\.ipor.itio:i L k < 100 <0.1 <0.1 l 4 Degradation bm!' photo l.'dat.'. e rate)6 l iH> 50 10 I Relative dissipation rate DIS) 100 50 11 5 *rH 7 5 biuO is maximum value combining both evaporation and degradation rate*. Metal ion dissipation has minimum value of I, 'Representative PCB isomer: 2,S,2'-tnchlorobiphenyl 113). Table 3. Ratio of dissipation to bioconcentretion potential of chemicals in aquatic environment. Chemical Relative Dissipation IDISP Factor (BCF) Ratio DIS/BCF L\S PCP DEHP PCB 100 10* 10 50 200* 0.25 11 800* 0.014 5 50,000* 0.0001 tOO is maximum value combining both evaporation and degra dation rates. Metal ion dissipation was a minimum value of 1. 6Daca of Comotto et al. (<5). 'Data of Dow Chemical Co. (//). tfDaca of Branson (15). Data of Nisbet (6). the lower the bioconcentration potential, the larger the value of the ratio. Chemicals like LAS receive a large number, DIS/BCF - 10, because of high degradation and low bioconcentration potential; chemicals like PCB's with high bioconcentration factors and low dissipation potential receive very small numbers, DIS/BCF = 0.0002. The nomogram for predicting the unknown ambient exposures (Fig. 2) illustrates an empirical relationship between fish residues, environmental release rates, and.the ratio of dissipation to bioconcentration potential. Recog nizing that the straight lines plotted on this graph are only made from two points each, the absolute posi tion of the line is tentative at best. It seems reason able, however, to assume that higher environmental release rates would result in a parallel line higher on this graph and lower release rates would result in a lower line. The graph does serve to illustrate the h>pothesis that if any two of the three pieces of information are known, the third piece can br~e.sti- mated. For example, if the DIS/BCF ratio a chemical is 0.001 and its release rate is about 107 Ib/yr, then the predicted ambient residues in fish are about 1 ppm, but if the environmental release rate is about 10H Ib/yr then the ambient residues in fish would be about 100 ppm. Also, given that fish resi due data (Cf) are available, measured or estimated from Figure 2, the ambient exposure concentration in water (CV) can be estimated by dividing by the bioconcentration factor: C,, -- C/BCF. The shaded 136 Ratio OIS/BCF Relative Peniitwice/Accumulation: Early Warning Zone <0-1 Figure 2. Preliminary nomogram for predicting the unknown ambient exposures. The ambient concen tration of a chemical in water, C,,. can be roughly estimated from the ambient concentration in fish. Cf, and the bioconcentration factor at steady state, BCF. i.e., C* * C//BCF. Environmental Health Perspectives VVV 000012680 area illustrates qualitatively that chemicals with high environmental release rates and/or persistence/ accumulation properties will end up in this zone. Most of the chemicals, on the other hand, will end up outside this zone, since these curves were drawn with chemicals with known tlsh residues and fairly high environmental release rates. Correlations of environmental properties of chemicals derived from large data bases confirm that most of the chemicals are outside this zone of con cern. Vieth and Konasewich (personal communica tions, 1979) have shown that only about iwo-lhirds of the 2,100 chemicals manufactured or used around the Great Lakes have octanol-water partition coeffi cients suggestive of low concern for fish residues. Also Bailey (personal communications, 1979), indi cated approximately two-thirds of 600 chemicals screened for biodegradability in a simple biochemi cal oxygen demand test yielded 30% or more BOD in 20days, indicating a low degree of persistence in the aquatic environment. Both of these properties, ac cumulation and persistence, appear to be related to Jpw water solubility (Table 2). In general, the en- onmental fate of the majority of chemicals cur.ently manufactured and used by society is charac terized by a low degree of persistence and accumu lation and will, therefore, be of low' hazard because the ambient exposure concentrations are low. Hazard Assessment Figure 3 compares the margin between the no ef fect concentrations and the ambient exposure con centrations for the case study chemicals. A recip rocal of hazard was used to show hazard by the height of the bar graph, i.e., narrow margins of safety yield tall bar graphs and vice versa. The PCB ex ample illustrates a case in which residues in fish would cause concern to fish eaters but the ambient levels of PCBs in water would cause little or no concern to aquatic life. On the other hand, DHHP illustrates a case in which the aquatic organisms could be at risk but not the fish eaters. Both of these materials fall across an arbitrary line where the mar gin between effect and exposure is equal to or less than five. A safety margin of five appears to be a reasonable criterion for distinguishing between chemicals that should and should not be classified as "priority.'' Neither LAS nor PCP appears to be rea sonably classified as a priority chemical. The safety margin concept was also employed by Klapow and Lewis (N) in selecting water quality standards in the marine environment. More impor tantly, the safety margin of the typical standard was less than a factor of five above the ambient exposure concentration, e.g., for copper, ambient 0.002 mg/1., standard 0.005 mg/1., margin of safety 2.5; and for 1 t Httard of Ambient Concentration in Water to Aquatic Organisms Hurd of Ambient Conoantrttion in Fith to Fr*#i Eater* *Sefety Margin, - go Obwfyebfr Adrene Effect Concentration Ambient Exposure Concentration Priority Chemical* ere thoe* with Safety Margin* Let* Than Five (1/Hazard - 0.7) According to thh Working Hypothesis VVV '/vuux cool Figure i. Safety margins ofchemicals in the aquatic environment. The numerical values associated with each chemical are only meant to illustrate the concept of prioritization of chemicals according to safety margins, i.e.. degree of hazard. Confidence limits and difference in geographical sites for ambient measurements should be considered before using these values for more than illustrating the concept. February 1980 137 zinc. ambient 0.00't :ng/L. standard 0.02 mg/!., mar gin ol *;;vt'ety 1.5. Therefore the margin of sat'ctv factor of live used iteie to select priority chemicals is conservative. Research Needs The basic premise for suggesting areas of research to improve hazard assessments of any chemical in the aquatic environment for any decision-making values is that exposure and effect concentrations must be available for both and that the biggest re search need is estimating exposure concentrations. The following areas of research are very important. It is necessary to develop and validate test methods for measuring rates of degradation, evap oration. and bioconcentration of chemicals in water. These test methods must accommodate chemicals with low water solubility (less than 1 ppm), and the results should be reported as measured rate con stants. With more experience in generating rate con stant data, it is hoped that some preliminary esti mates of their value can be based on structure through various regression correlations. The lack of reliable information on the amount of chemicals released to the environment is the single largest source of error in environmental hazard as sessment (Baughman, personal communications, 197$). The approach used by the 1975 NSF Work shop seems promising and should be updated. It takes the use patterns of industrial chemicals into account. There is a glaring lack of field studies to show that laboratory data correctly forecast the fate and effects of chemicals in the real world. Predictive models need to be validated for several different aquatic environments based on time-concentration data with adequate material balance accountability. It would be valuable to add more data points (case study chemicals) to Figure 2, as data are generated from more and better analysis of fish and water sam ples, DIS/BCF, ratios and environmental release rates. These data should be used in prioritizing and deprioritizing chemicals. Conclusions Principles of hazard assessment can be used as a prescreen in selecting priority chemicals. These principles involve estimates or measurements of both the exposure and the no effect concentrations. In addition to narrow safety margins, chemicals should not be classified as priority chemicals unless there is evidence of (a) potential to cause significant human toxicity including carcinogenicity, muta genicity and teratogenicity; or (b) analytical de tection at tovlcologic.illy Munilic.int in five or more control!,ib!r point sources Rough estimates ot ami sent exposure lions can be made if err. ir-mimer,:..! ; eleas- i ,:te ..n the ratio of dissipation iDlS) to btoeonce:::: a''o potential (PCI i are know n. Also, the ratio i)IS BC: can be used as an early warning index for chemical or used for predicting fish residues or enx ironment.: release rates. Under the principles of environmental ha/urJ a:sessment, both PCB and DEHP would be candidate for a list of priority chemicals. Neither LAS or PC. would be priority chemicals. REFERENCES 1. Cairns. J.. Dickson. K. L.. and Marki. A. W., Eds. Estimat ing the Hazard of Chemical Substances to Aquatic Lift (ASTM STP 657). American Society for Testing and Mate rials, Philadelphia, 1978. 2. EPA Recommended List of 129 Pnonty Pollutants. Com pound Name, Legislative History of the Clean Water Act Appendix V; Attachment N'o. 2. April 27. 1977, pp 404,405. 3. National Science Foundation. Final Report of NSF Work shop Panel to Select Organic Compounds Hazardous to thi Environment. October 1975. 4. EPA Proposed Water Quality Criteria for Phihalate Esters Unpublished. 5. Little, A. D., Inc. Human Safety and Environment: " nect> of .Major Surfactants, A report to the Soap and gem Association. May 31, 1977. 6. Nisbel, I.C.T. Criteria Document for PC'Bs. EPA Report 44<V9-76-021 (1976). 7. National Academy of Sciences. NAS Finds No Hard Evi dence of Carcinogenic Compounds in Drinking Water. Drinking Water News. pp. 3. 4 (June 8, 1977). 8. Comotto. R. M., Kimerle, R. A., and Swisher, R. R. Hiocon- centranon potential and metabolism of 1ine.tr a'kylbenzene sulfonate by daphids and fathead minnows. In: (ASTM STP 667) 1967, American Society for Testing and Materials. Philadelphia, p. 232. 9. Ciam, C. S.. Chan. H. S., Neff. G. S., and Atlas. E. L. PhthaJate ester plasticizers: a new class of marine pollutant. Science 199: 419 (1978). 10. Larson, R. J. The role of biodegradation kinetics in predicting environmental fate. Paper presented at a workshop on Dio- transformation and Fate of Chemicals in the Aquatic Envi ronment. University of Michigan Biological Station, Pellston, Mich., August 21, 1979. 11. Dow Chemical Co. Dow Response to EPA-RPAR on Pen* lachlorophenol. Midland. Michigan 48640, February 1979. 12. Johnson, B. T.. and Lulves, W. Biodegradation of di-n-butyl phthalate and di-2-cthylhexyl phthalute in fresh water hydro soil. J. Fish Res. Bd. Can. 32: 333 (1975). 13. Branson, D. R. A new capacitor fluid -- a case study in product stewardship. In: Aquatic Toxicology and Hazard Evaluation (ASTM STP 634), F. L. Mayer and J. L. Hamelink. Eds.. American Society for Texting and Materials, Philadelphia. 1977, p. 44. 14. Klapow. L. A., and Lewis. R. H. Analysis of toxicity data for California marine water quality standards. J Water Poll. Control Fed. 51: 2054 (1979). 15. Branson. D. R.. Blau. G. E.. and Mayer. F. L Biocc rra- tion kinetics ofdi-2-e thy Ihe xy I phthalate in fathead mi. Paper presented at Association of Oificial Anal Chemists, 91st Annual Meeting, paper 176. October I. .a. 1977. 138 Environmental Health Perspectives YVV 000012682 To- ^ ,j, y Osce .Time- WHILE YOU WERE OUT CulASUsi* r(Lcst^*~S of- ^?>/ Phono- Area Code </5 Number 3 lrd~d Extension TELEPHONED CALLED TD SEE YOU " PLEASE CALL WILL CALL AGAIN - WANTS TO SEE YOU URGENT RETURNED YOUR CALL Message_&4rfr ^/rusvux^cf 7/0 - A 37 - 5 35^ A- %J GrlU^r^^f" S# 5 *> 7 (L550 Operator fr2v*-'_ y. AMPAD EFFICIENCY 23-000 50 SHT. PAD 23-001 250 SHT. DISPENSER BOX /***- try- s&^ m C /Cfee* - /3a//o ^r/s /iW /^/Z'' S/yr*/U 3*0 cfkrt3 a)Y /y/'ZO 3 *3<**tA