001R83100 Water Quality Criteria For The Evaluation Of Aquatic Life and Its Uses Ammonia Final Draft 193 1983 NEPIS online LAI 20060921 hardcopy single page tiff ammonia liter fish nh3 toxicity trout rainbow concentrations reported lc50 water acute species concentration ammonium effect effects excretion table hour image: ------- image: ------- WATER QUALITY CRITERIA FOR THE PROTECTION OF AQUATIC LIFE AND ITS USES AMMONIA Final Draft 20 January 1983 Prepared By U.S. Environmental Protection Agency Office of Research and Development Environmental Research Laboratory Duluth, Minnesota US Frv'r^r>?v^nt?! Protection Agency image: ------- TABLE OF CONTENTS Page Introduction 1 Acute Toxicity to Animals 7 Chronic Toxicity to Animals 42 Toxicity to Plants 59 Bioaccumulat ion 64 Other Data 65 Unused Data 76 Summary 80 National Criteria 84 Examples of Site-Specific Criteria 87 Tables 91 1 . Acute values for ammonia 91 2. Chronic values for ammonia 110 3. Species and family mean acute values (corrected for pH) for ammonia . . . . 114 4. Plant values for ammonia 117 5. Other data for ammonia 119 References 132 U.S. Envlronrnentc! Trctc^'cn Agenc li image: ------- INTRODUCTION* In aqueous ammonia solutions, un-ionized ammonia exists in equilibrium with the ammonium ion and the hydroxide ion. The equation expressing this equilibrium can be written as: ^3(8) + nH2°U) * NH3'nH2°(aq) * m*+ + OH~ + (n-OH2°U)* As indicated in this equation, the dissolved ammonia molecule exists in hydrated form. The dissolved un-ionized ammonia is represented for convenience simply as NHj. The ionized form is represented as NH^+. The term total ammonia refers to the sum of these; i.e., NH3 + NH^ . The toxicity of aqueous ammonia solutions to aquatic organisms is primarily attributable to the NH-j Species, with the NH^"*" species being relatively less toxic (Chipraan 1934; Wuhrmann et al. 1947; Wuhrmann and Woker 1948; Tabata 1962; Armstrong et al. 1978; Thurston et al. 1981c). It is, therefore, important to know the concentration of NH-j in any aqueous ammonia solution in order to determine what concentrations of total ammonia are toxic to aquatic life. The concentration of NH^ is dependent on a number of factors in addition to total ammonia concentration (Emerson et al. 1975; Thurston et al. 1979). Most important among these are pH and temperature; the concentration of NH-j increases with increasing pH and with increasing temperature. The ionic strength is another important influence on this equilibrium. There * The reader is referred to the Guidelines for Deriving Numerical National Water Quality Criteria for the Protection of Aquatic Life and Its Uses (5 July 1983) (U.S. Environmental Protection Agency 1983 ) in order to understand better the following text, tables, and calculations. 1 image: ------- is a decrease in tht; percentage of un-Ionized ammonia as the limit.: strength increases in hard water or in dilute saline solution. In most natural freshwater systems the reduction of percent NHg attributable to dissolved solids is negligible. In saline or very hard waters there will be small but measurable decreases in the percent NHo- Current analytical methods do not permit measurement of NH3 and NH^ separately. A number of analytical methods are available for direct determination of total ammonia concentrations in aqueous solutions. Once total ammonia is measured, and the pH and temperature of the solution determined, the fraction of total ammonia present as NH3 can be calculated based on the ammonia-water equilibrium. A review of analytical methods for ammonia in aqueous solution has been prepared by Richards (1981). Emerson et al. (1975) carried out a critical evaluation of the literature data on the ammonia-water equilibrium system and published calculations of values of pKa at different temperatures and of percent NH-j in ammonia solutions of zero salinity as a function of pH and temperature. The following table, reproduced from Emerson et al. (1975), provides values for percent NHg at one-degree temperature intervals from 0 to 30 C, and pH intervals of 0.5 pH unit from pH 6.0 to 10.0. An expanded version of this percent NH^ table is provided in Thurston et al. (1979), which provides tabulated values of the NH-j fraction, expressed as percentage of total ammonia, at temperature intervals of 0.2 degree from 0.0 to 40.0 C, and pH intervals of 0.01 pH unit from pH 5.00 to 12.00. For salt water, reports by Whitfield (1974) and Skarheim (1973) provide calculations of NHg as a function of pH, temperature, and salinity. image: ------- Percent NH-j in aqueous ammonia solutions tor 0-30 C and pH b-10. Temp. (C) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 6.0 .00827 .00899 .00977 .0106 .0115 .0125 .0136 .0147 .0159 .0172 .0186 .0201 .0218 .0235 .0254 .0274 .0295 .0318 .0343 .0369 .0397 .0427 .0459 .0493 .0530 .0569 .0610 .0654 .0701 .0752 .0805 6.5 .0261 .0284 .0309 .0336 .0364 .0395 .0429 .0464 .0503 .0544 .0589 .0637 .0688 .0743 .0802 .0865 .0933 .101 .108 .117 .125 .135 .145 .156 .167 .180 .193 .207 .221 .237 .254 7.0 .0826 .0898 .0977 .106 .115 .125 .135 .147 .159 .172 .186 .201 .217 .235 .253 .273 .294 .317 .342 .368 .396 .425 .457 .491 .527 .566 .607 .651 .697 .747 .799 7.5 .261 .284 .308 .335 .363 .394 .427 .462 .501 .542 .586 .633 .684 .738 .796 .859 .925 .996 1.07 1.15 1.24 1.33 1.43 1.54 1.65 1.77 1.89 2.03 2.17 2.32 2.48 pH 8.0 .820 .891 .968 1.05 1.14 1.23 1.34 1.45 1.57 1.69 1.83 1.97 2.13 2.30 2.48 2.67 2.87 3.08 3.31 3.56 3.82 4.10 4.39 4.70 5.03 5.38 5.75 6.15 6.56 7.00 7.46 8.5 2.55 2.77 3.00 3.25 3.52 3.80 4.11 4.44 4.79 5.16 5.56 5.99 6.44 6.92 7.43 7.97 8.54 9.14 9.78 10.5 11.2 11.9 12.7 13.5 14.4 15.3 16.2 17.2 18.2 19.2 20.3 9.0 7.64 8.25 8.90 9.60 10.3 11.1 11.9 12.8 13.7 14.7 15.7 16.8 17.9 19.0 20.2 21.5 22.8 24.1 25.5 27.0 28.4 29.9 31.5 33.0 34.6 36.3 37.9 39.6 41.2 42.9 44.6 9.5 20.7 22.1 23.6 25.1 26.7 28.3 30.0 31.7 33.5 35.3 37.1 38.9 40.8 42.6 44.5 46.4 48.3 50.2 52.0 53.9 55.7- 57.5 59.2 60.9 62.6 64.3 65.9 67.4 68.9 70.4 71.8 10.0 45.3 47.3 49.4 51.5 53.5 55.6 57.6 59.5 61.4 63.3 65.1 66.8 68.5 70.2 71.7 73.3 74.7 76.1 77.4 78.7 79.9 81.0 82.1 83.2 84.1 85.1 85.9 86.8 87.5 88.3 89.0 [From Emerson et al. 1975; reproduced with permission from the Journal of the Fisheries Research Board of Canada.] image: ------- Concentrations of ammonia have been reported in the .-U|u.-ic u~ toxu'ltv literature as a variety of different forms, such as NH3» NH^, N^-N, NH^Cl, and others. The use in a literature article of the terras NHg, NH-j-N, or ammonia-nitrogen may not necessarily mean un-ionized ammonia, but may be the author's way of expressing total ammonia. The use of the term NH-j in this document always means un-ionized ammonia, and NH^N means un-ionized ammonia-nitrogen. Throughout the following, all quantitative ammonia data have been expressed in terms of un-ionized ammonia, as mg/liter Nt^, for ease in discussion and comparison. Authors' ammonia concentration values are given as reported if authors provided data expressed as rag/liter NHg- If authors reported only total ammonia values, or used concentration units other than mg/liter, these were used with the reported pH and temperature values to calculate mg/liter un-ionized NH3« For calculations of NH3 in fresh water the table of Thurston et al. (1979) was used. For calculations in salt water the table of Skarheira (1973) was used. This Criterion Document was prepared in accordance with U.S. Environmental Protection Agency guidelines for the derivation of national water quality criteria (U.S. Environmental Protection Agency 1982a). The literature cited herein was obtained from a search of the literature through October 1982; data from primary references only were used. Of the literature cited in this document, a significant number of papers provided insufficient pH and temperature data to enable calculation of NH3 concentrations; such papers were relegated to the section on "Unused Data" unless they provided useful qualitative or descriptive information. In some instances information missing in published papers on experimental conditions image: ------- ,was obtained through correspondence with authors; data obtained in this manner are so Indicated by footnotes. Compounds used in the ammonia toxicity testing summarized here and their formulas and Chemical Abstracts Services (CAS) Registry Numbers are given below: Compound Formula CAS No. Ammonia NH3 7664417 Ammonium acetate ^^02^02 631618 Ammonium bicarbonate NH^Hct^ 1066337 Ammonium carbonate (NH^^COg 506876 Ammonium chloride NH^Cl 12125029 Ammonium hydrogen phosphate (NH^HPO^ 7783280 Ammonium hydroxide NH4OH (NH3'H20) 1336216 Ammonium sulfate (NHSQ 7783202 Papers stating use of other sources of ammonia were included if the source (e.g., excreted NH^ from fish) was deemed satisfactory. Papers using complex chemicals (e.g., ammonium ferricyanide, dec ylt rime thylammonium bromide) were not used. Finally, papers on ammonium compounds (e.g., NH^F, having anions that either might be themselves toxic or that would preclude calculation of NH3 concentration from the aqueous ammonia equilibrium relationship were not used. A number of review articles or books dealing with ammonia as an aquatic pollutant are available. Water quality criteria for ammonia have been recommended in some of these. Liebmann (1960), McKee and Wolf (1963), Epler (1971), Becker and Thatcher (1973), Tsai (1975), Hampson (1976), Steffens (1976), Colt and Armstrong (1979), and Armstrong (1979) have published summaries of ammonia toxicity. Literature reviews including factors affecting ammonia toxicity and physiological effects of ammonia toxicity to aquatic, organisms have been published by Lloyd (1961b), Lloyd and Herbert 5 image: ------- (1962), Warren (1962), Visek (1968), Lloyd and Swift (1976), and Kinne (1976). Literature reviews of ammonia toxicity information relating to criteria recommendations have been published by U.S. Federal Water Pollution Control Administration (1968), European Inland Fisheries Advisory Commission (1970), National Academy of Sciences and National Academy of Engineering (1973), Willingham (1976), U.S. Environmental Protection Agency (1977, 1980), National Research Council (1979), Willinghara et al. (1979), and Alabaster and Lloyd (1980). image: ------- ACUTE TOXICITY TO ANIMALS 1 i Freshwater Invertebrates Acute toxicity of ammonia to freshwater invertebrate species has been much less studied than that to fishes. The preponderance of available invertebrate data is comprised of studies with arthropods, primarily crustaceans and insects. LC50 and EC50 data are summarized in Table 1 for 12 species representing nine families. The acute toxicity of ammonia to Daphnia magna (Table 1) has been studied by several investigators, with reported 48-hour LC50 values ranging from 0.53 to 4.94 mg/liter NH3 (Parkhurst et al. 1979, 1981; Reinbold and Pescitelli 1982a; Russo et al., in prep.). Exposures (48 hours) of I), magna to NlfyCl in dilution water from two different sources were conducted by Russo et al. (in prep.). LC50 values (Table 1) ranged from 2.4 to 2.8 mg/liter NH3 in water of pH 7.95 to 8.15 and hardness 192 to 202 rag/liter CaC03, and from 0.53 to 0.90 mg/liter NH3 in water of pH 7.4 to 7.5 and hardness 42 to 48 mg/liter CaC03- On an acute (48-hour LC50) basis, in dilution water from the same source, Ceriodaphnia acanthina, Simocephalus vetulus, and IK magna all exhibited similar sensitivities (Table 1) to ammonia (Mount 1982; Russo et al., in prep.). The 48-hour LC50 value (Table 1) of 1.16 mg/liter NH3 reported by DeGraeve et al. (1980) for Daphnia pulicaria falls within the range of values reported for IK magna. Anderson (1948) reported a threshold toxicity value (Table 5) for £. magna of 2.4 to 3.6 mg/liter NH3 in Lake Erie water. Threshold concentration was taken to mean the highest concentration that would just fail to immobilize the test animals under prolonged exposure (Anderson 1944). A minimum lethal concentration of 0.55 rag/liter NH3 was reported for _D. magna by Malacca (1966), and a 24-hour I C50 value of 1.50 7 image: ------- rag/liter NH3 was reported by Gyore and Olah (1980) for Moina rectirostris (Table 5). Buikema et al. (1974) reported an EC50 (Table 5) for NH3 toxicity to a bdelloid rotifer, Philodina acuticornis, to be 2.9-9.1 mg/liter NH3 (calculated using reported pH values of 7.4 to 7.9). Tests of ammonia toxicity to a flatworm, Dendrocoelum lacteum (Procotyla fluviatilis), and tubificid worm, Tubifex tubifex, yielded LC50 values (Table 1) of 1.4 and 2.7 mg/liter NH3, respectively (Stammer 1953). Thurston et al. (in prep., a) conducted 26 flow-through toxicity tests with three mayfly, two stonefly, one caddisfly, and one isopod species; all tests were conducted with water of similar chemical composition. Ninety-six-hour LC50 values ranged from 1.8 to 5.9 mg/liter NH3 (Table 1). Results also indicated that a 96-hour test is not long enough to determine the acutely lethal effects of ammonia to the species tested, inasmuch as an asymptotic LC50 is not obtained within 96 hours. Percent survival data (Table 5) were reported for some mayfly, stonefly, and caddisfly tests in which LC50 values were not obtained; 60 to 100 percent survival occurred at test concentrations ranging from 1.5 to 7.5 mg/liter NH3- Gall (1980) tested NH^Ci with Ephemerella sp. near excrucians. Organisms were exposed to ammonia for 24 hours, followed by 72 hours in ammonia-free water; mortality observations were made at the end of the overall 96-hour period. An EC50 value (Table 5) of 4.7 mg/liter NH3 was obtained. Ammonia toxicity tests conducted using dilution water from the Blue River in Colorado resulted in no mortalities of either scud (Gammarus lac us tr is) or D^. majgjia after 96 hours' exposure to 0.08 ing/liter NH3« In a second test using river water buffered with sodium bicarbonate, 13 percent mortal icy occurred with scud at several concentrations tested, including the 8 image: ------- highest and lowest of 0.~"* And O.I? mg/Htov \'H^; *ov*n ami 1'» ;*«M .^ u mortality occurrtvi with r>- '-c-a^'.ia at the s'ilie* ct =>l. 1981). Five freshwater mussel species (Amblema _p. plicata, Anodonta imbecillis, Corbicula manilensis, Cyrtonaias t amp i c o en si s, and Toxqlasma texasensis) were exposed for 165 hours (Table 5) to a concentration of 0.32 rag/liter NH^J T_. texasensis was most tolerant to ammonia, and A. j>. plicata was most sensitive (Home and Mc.Tntosh 1979). During the ammonia tolerance tests, the more tolerant species generally had their shells tightly shut, whereas the least tolerant species continued siphoning or had their mantles exposed. Acute exposures of the freshwater crayfish (Orconectes nais) to NH^Ci gave LC50 values (Table 1) of 3.15 and 3.82 mg/liter NH3 (Evans 1979; Hazel et al. 1979). An LC50 value (Table 1) of 8.00 mg/liter was reported for the beetle (Stenelmis sexlineata) by Hazel et al. (1979). Freshwater Fishes Acute toxicity tests with freshwater fish species have been conducted with 23 different species from nine families; 96-hour LC50 values are summarized in Table 1. The acute toxicity of ammonia to rainbow trout (Salmo gairdneri) has been studied by many investigators, with reported 96-hour LC50 values ranging from 0.16 to 1.1 mg/liter NH3 (Table 1). Thurston and Russo (in press) conducted 71 toxicity tests with rainbow tiout ranging in size from sac fry (<0.1 g) to 4-year-old adults (2.6 kg), in wai er of uniform chemical composition. LC50 values (Table 1) ranged from O.lo to 1.1 mg/liter NH-j for 96-hour exposures. Fish susceptibility to NH-j decreased with increasing weight over the range 0.06-2.0 g, but gradually increased above that weight range. LC50 values for 12- and 35-day 9 image: ------- exposures (Table 5) were not greatly different from 96-hour values. No statistically significant differences in results were observed when different ammonium salts [NH^Cl, NH^HCO-j, (NH^HPO^, (NH^SO^l were used as the toxicants. Grindley (1946) also reported observing no appreciable difference in toxicity between toxicant solutions of NH^Cl and (NH^)9So^ with rainbow trout tests (Table 5). LC50 values (Table 1) ranging from 0.16 to 1.0 mg/liter NH3 for 96-hour exposures of rainbow trout to ammonia were reported by Calamari et al. (1977, 1981), Broderius and Smith (1979), Holt and Malcolm (1979), DeGraeve et al. (1980), and Reinbold and Pescitelli (1982b). Ball (1967) reported an asymptotic (five-day) LC50 value of 0.50 mg/liter NH3• Acute exposures to ammonia of rainbow trout of life stages ranging from one to 345 days' post-fertilization (325 days post-hatch) were conducted by Calamari et al. (1977, 1981). They reported a tenfold increase in the speed of intoKication processes between the embryonic and free larval stages; embryos and fingerlings (about one year old) were found to be less sensitive than the other life stages studied. LCSO values ranging from 0.49 to 0.70 mg/liter NH3 for 3-, 24-, and 48-hour exposures (Table 5) were reported by Herbert (1961, 1962), Herbert and Shurben (1964, 1965), and Herbert and Vandyke (1964). Rainbow trout (826 days old) subjected to 29.6 mg/liter NH-j reacted rapidly and strongly, overturned within two to three hours, and died within four hours (Corti 1951) (Table 5). Rainbow trout embryos and alevins were reported (Rice and Stokes 1975) to tolerate 3.58 mg/liter NH3 during 24-hour exposures; suscep- tibility increased during yolk absorption, with the 24-hour LCSO for 85-day-old fry being 0.068 mg/liter NH3 (Table 5). Nehring (1962-63) reported survival times of 1.3 and 3.0 hours at concenttations of 4.1 and 0.7 10 image: ------- ,mg/liter NH^, respectively (Table 5). Danecker (1964) reported survival times of S to 60 minutes at 0.4 to 4.0 rag/liter NHy, respectively, with <0.2 m,f*/l.1ter given as ,1 no-mortality concentration (Table 5). Allan et al. (1958) reported a median survival time of 1000 minutes at 0.18 mg/liter NH3 (Table 5). An acute value of 0.2 mg/liter NH3 attributed to Liebmann (1960) has been widely cited, in the EPA "Red Book" (U.S. Environmental Protection Agency 1977) and elsewhere, as being the lowest lethal concentration reported for salmonids. It is worthwhile to mention here a clarification and correction that was published in the American Fisheries Society's "Red Book Review" (Willingham et al. 1979): The research reported by Liebmann (1960) was that of Wuhrmann and Woker (1948); recomputation of the Wuhrmann and Woker data, using more accurate aqueous ammonia equilibrium tables, indicates an effect level of approximately 0.32 mg/liter NH3, not 0.2 mg/liter NH3 as cited by Liebmann. A 96-hour LC50 value of 0.44 mg/liter NH3 was reported for rainbow trout In a test conducted using dilution water from the Blue River in Colorado (Miller et al. 1981). Pitts (1980) conducted toxicity tests using ammonium chloride and river water. Tests were conducted with rainbow trout, and LC50 values ranged from 0.2 to 0.9 mg/liter NH3 for 96-hour exposures at temperatures of 10 and 15 C. Although acute toxicity studies with salmonids have been conducted preponderantly with rainbow trout, some data are also available for a few other salmonid species. Thurston et al. (1978) investigated the toxicity of a.timoriia to cutthroat trout (Salmo dark!), and reported 96-hour LC50 values of 0.52 to 0.80 rag/liter NH3 (Table 1). Thurston and Russo (1981) reported a 96-hoi,r LC50 value of 0.76 rag/liter NH3 for golden trout (Salmo il image: ------- aguabonlta) (Table 1). Taylor (1973) subjected brown trout (Salmo trutta) to 0.15 rag/liter NH3 for 18 hours, resulting in 36 percent mortality (Table 5); when returned to ammonia-free water, the test fish recovered after nearly 24 hours. No mortalities occurred during a 96-hour exposure at 0.090 mg/liter NH3, although fish would not feed. Woker and Wuhrmann (1950) reported 0.8 mg/liter NH-J was not acutely toxic to brown trout (Table 5). A 96-hour LC50 value of 0.47 mg/liter NII3 was reported for brown trout tested using dilution water from the Blue River in Colorado (Miller et al. 1981). Phillips (1950) reported that brook trout (Salvelinus fontinalis) evidenced distress within 1.75 hours at a concentration of 3.25 mg/liter NH3 and within 2.5 hours at 5.5 mg/liter (Table 5). Toxicity tests (Tables 1, 5) on (NH^^SO^ with pink salmon (Oncorhynchus gorbuscha) at different stages of early life stage development (Rice and Bailey 1980) showed that late alevins near swim-up stage were the most sensitive (96-hour LC50 = 0.083 mg/liter NHg), and eyed embryos were the most tolerant, surviving 96 hours at >1.5 mg/liter NH3. Buckley (1978) reported a 96-hour LC50 value of 0.55 mg/liter NH3 for fingerling coho salmon, Oncorhynchus kisutch (Table 1). Herbert and Shurben (1965) reported a 24-hour LC50 value (Table 5) of 0.28 mg/liter NH3 for Atlantic salmon (Salmo s_alar) . A comparison of relative susceptibilities of salmon smolts and yearling rainbow trout to 24-hour exposures to NH^Cl showed that the salmon were appreciably more susceptible than the trout in fresh water (Ministry of Technology, U.K. 1963). Data are available on the acute toxicity of ammonia to a variety of non-salmonid fish species. Thurston et al. (in press, a) studied the toxicity of ammonia to fathead minnows (Pimephales promelas) of sizes ranging from 0.1 to 2.3 g; LC50 values from 29 tests ranged from 0.75 to 3.4 mg/liter NH3 (Taoie 1). Toxictty was not dependent upon test tish size or source. 12 image: ------- '•LC50 values ranging from 0.73 to 1.7 rag/liter NH^ (Tables 1,5) for fathead minnows were also reported by Sparks (1975), DeGraeve et al. (1980), and Reinbold and Pescitelli (1982b). Toxicity tests with fathead minnows using ammonium chloride and river water yielded 96-hour LC50 values ranging from 0.6 to 2.4 tng/liter Nt^J fathead minnows exposed to 0.12 mg/liter NH3 in river water for 28 days incurred no mortalities (Pitts 1980). LC50 values (Table 1) for white sucker (Catostomus commersoni) exposed to ammonium chloride solutions for 96 hours (Reinbold and Pescitelli 1982c) were 1.40 and 1.35 mg/liter NH3• Reported LC50 values (Table 1) for 96-hour exposures of bluegill (Lepomis macrochirus) ranged from 0.26 to 4.60 mg/liter NH3 (Emery and Welch 1969; Lubinski et al. 1974; Roseboom and Richey 1977; Reinbold and Pescitelli 1982b; Smith and Roush, in prep.). LC50 values (Table 1) of 0.7 to 1.8 mg/liter NH3 for smallmouth bass (Micropterus dolomieui) and 1.0 to 1.7 mg/liter NH-j for largemouth bass (Micropterus saltnoides) were reported by Broderius et al. (in prep.) and Roseboom and Richey (1977), respectively, for 96-hour exposures. Sparks (1975) reported 48-hour LC50 values (in parentheses, as rag/liter ^3) for bluegill (2.30) and channel catfish (2.92), Dowden and Bennett (1965) reported a 24-hour LC50 value for goldfish (Carassius auratus) (7.2), and Chipman (1934) reported lethal threshold values of 0.97 to 3.8 mg/liter NH3 for goldfish (Table 5). Turnbull et al. (1954) reported a 48-hour LC50 value for bluegill to be within the range 0.024 to 0.093 mg/liter NH3 (Table 5); during the exposure they observed that the fish exhibited a lack of perception to avoid objects. Reported 96-hour LC50 values (Table 1) for channel catfish (Ictalurus pane, tat us) ranged from 1.8 to 4.2 mg/liter NH3 (Colt and Tchobanoglous 1976; Koseboora and Rirhey 1977; Reinbold and Pescitelli L982d). Vaughn and 13 image: ------- Simco (1977) reported a 48-hour LC50 for channel catfish of 1.24 to 1.96 mg/liter NH-j, and Knepp and Arkin (1973) reported one-week LC50 values of 0.97 to 2.0 mg/liter NH3 (Table 5). From studies with bluegill, channel catfish, and largemouth bass, Roseboom and Richey (1977) reported that bluegill susceptibility was dependent upon fish weight, with 0.07-g fish being slightly more sensitive than either 0.22- or 0.65-g fish; size had little effect upon channel catfish or bass susceptibility. LC50 values (Table 1) were determined with two species of field- collected fishes indigenous to Kansas streams, orangethroat darter (Etheostoma spectabile ) and red shiner (Notrqpis lutrensis) (Hazel et al. 1979); 96-hour LC50 values were 0.90 and 1.07 mg/liter NH3 for darter and 2.83 for shiner. Commercially obtained largeraouth bass, channel catfish, and bluegill (18 fish of each species) were also exposed for 96 hours to a concentration of 0.21 mg/liter NH3, resulting in zero mortality for bluegill and channel catfish and one mortality (6 percent) among the largeraouth bass tested. LC50 values (Table 1) ranging from 0.48 to 3.2 mg/liter NH3 for NH4C2H3°2' (NH4)2C03, NH4Cl, NH4OH, and (NH4)2S04 in 96-hour exposures of mosquitofish (Gambusia affinis) in waters with suspended solids ranging from <25 to 1400 mg/liter were reported by Wallen et al. (1957). Susceptibility of mosquitofish to ammonia was studied by Hemens (1966) who reported a 17-hour LC50 value of 1.3 mg/liter NH3 (Table 5); he also observed that male fish were more susceptible than females. Powers (1920) reported the relative susceptibilities of three fish species to ammonium chloride to be (most sensitive to least sensitive): straw-colored minnow (Notropis b 1 ennius) > bluntnose minnow (Pimephale s no ta tusj > goldfish. 14 image: ------- Rubin and Elmaraghy (1976, 1977) tested guppy (Poecilla reticulata) fry and reported 96-hour LC50 values (Table 1) averaging 1.50 mg/liter NH3; mature guppy males were more tolerant, with 100 percent survival for 96 hours at concentrations of 0.17 to 1.58 mg/liter NH3« LC50 values (Table 1) of 0.15 and 0.20 mg/liter NH3 at pH 6.0, and of 0.52 and 2.13 mg/liter NH3 at pTI 8.0, were reported by Stevenson (1977) for white perch (Morone americana). LC50 values (96 hours) of 1.20 and 1.62 mg/liter NH3 for spotfln shiner (Notr o pis sp1lopterus), and of 1.20 mg/liter NHj for golden shiner (No t emi gonus cryso1eucas), were reported by Rosage et al. (1979) and Baird et al. (1979), respectively (Table 1). Jude (1973), Reinbold and Pescitelli (1982a), and McCormick et al. (in prep.) reported 96-hour LC50 values ranging from 0.6 to 2.1 mg/liter NH3 for green sunfish (Lepomis cyanellus) (Table 1). Pumpkinseed sunfish (Lepomi8 gibbosus) were tested by Jude (1973) and Thurston (1981), with reported 96-hour LC50 values ranging from 0.14 to 0.86 mg/liter NH3 • Mottled sculpin (Cottus bairdi) were tested by Thurston and Russo (1981), yielding a 96-hour LC50 of 1.39 mg/liter NH3 (Table 1). Ball (1967) determined an asymptotic (six-day) LC50 value (Table 5) of 0.44 mg/liter NH3 for rudd (Scardinius erythrophthalmus). He compared the asymptotic LC50 values for this species against that obtained within two days for rainbow trout. Although the trout had proven to be more sensitive to ammonia than had rudd during the first day of the tests, the asymptotic LC50 for both species showed little difference. Rao et al. (1975) reported a 96-hour LC50 for carp (Cyprinus carpio) of 1.1 mg/liter NH3 (Table 1). Carp exposed to 0.24 mg/liter NH3 exhibited no adverse effects in 18 hours (Vamos 1963). Exposure to 0.67 mg/liter NH3 caused gasping and equilibrium disturbance in 18 min, frenetic swimming activity in 25 min, then sinking to the tank bottom after 60 min; after 75 min the fish were placed in ammonia-free water and all revived. Similar 15 image: ------- effects were observed at a concentration of 0.52 rag/liter NHj (Table 5). Pre-treating fish orally with 12.5 rag Suprastin (N-dimethyl-arainoethyl-N-p- chlorobenzyl-o-aminopyridin hydrochlor) , a chemical which reduces cell membrane permeability, somewhat reduced the toxic effect of ammonia. A lethal concentration (Table 5) for carp was reported to be 7.5 rag/liter NHg (Kempinska 1968). Acute exposures (Table 5) to ammonium sulfate of bitterling (Rhodeus sericeus) and carp were conducted by Malacca (1966), who determined minimum lethal concentrations (i.e., after such exposure, fish placed in ammonia-free water were unable to recover) of 0.76 mg/liter NH3 for bitterling and 1.4 mg/liter NH3 for carp. Nehring (1962-63) reported survival times of carp to be 2.4 and 6.0 hours at NH3 concentrations of 9.7 and 2.1 mg/liter N^, respectively (Table 5). Danecker (1964) reported survival time for tench (Tinea tinea) to be 20 to 24 hours at 2.5 mg/liter NH-} (Table 5). In a 24-hour exposure of creek chub (Semotilus atromaculatus) to NlfyOH solution (Gillette et al. 1952), the "critical range" below which all test fish lived and above which all died was reported to be 0.26 to 1.2 mg/liter NH3 (Table 5). In static exposures lasting 9 to 24 hours, with gradual increases in NH-j Content, lethal concentrations (Table 5) were determined for oscar (Astronutus ocellatus) (Magalhaes Bastos 1954); mortalities occurred at 0.50 rag/liter MH-j (4 percent) to 1.8 mg/liter (100 percent). Tests on oscar of two different sizes (average weights 1.6 g for "small" fish and 22.5 g for "medium" fish) showed no difference in susceptibility related to fish size. A 72-hour LC50 value (Table 5) of 2.85 mg/liter NH^ was reported by Redner and Stickney (1979) for blue tilapia (Tilapia aurea) . Factors Affecting Acute Toxicity of Ammonia There are a number of factors that can affect the toxicity of ammonia to aquatic organisms. These factors include effects of dissolved oxygen 16 image: ------- .concentration, temperature, pH, previous acclimation to ammonia, fluctuating or intermittent exposures, carbon dioxide concentration, salinity, and presence of other toxicants. Almost all studies of factors affecting ammonia toxicity have been carried out using only acute exposures. (a) Dissolved Oxygen A decrease in dissolved oxygen concentration in the water can increase ammonia toxicity. Vamos and Tasnadi (1967) observed mortalities in carp ponds at ammonia concentrations lower than would normally be lethal, and attributed this to periodic low concentrations of oxygen. Based on research in warmwater (20-22 C) fish ponds, Sel^si and Vamos (1976) projected a "lethal line" relating acute ammonia toxicity and dissolved oxygen, below which carp died. The line ran between 0.2 mg/liter Nltj at 5 mg/liter dissolved oxygen and 1.2 mg/liter NH3 at 10 mg/liter dissolved oxygen. Thurston et al. (in press, a) compared the acute toxicity of ammonia to fathead minnows at reduced and normal dissolved oxygen concentrations; seven 96-hour tests were conducted within the range 2.6 to 4.9 mg/liter dissolved oxygen, and three between 8.7 and 8.9 mg/liter. There was a slight positive trend between 96-hour LC50 and dissolved oxygen, although it was not shown to be statistically significant. Alabaster et al. (1979) tested Atlantic salmon smolts in both fresh water and 30 percent salt water at 9.6-9.5 and 3.5-3.1 mg/liter dissolved oxygen. The reported 24-hour LC50 values at the higher oxygen concentrations were about twice that at the lower. Several studies have been reported on rainbow trout. Allan (1955) reported that below 0.12 mg/liter NH3 and at about 30 percent oxygen saturation, the median survival time was greater than 24 hours, but at the same Concentration with oxygen saturation below 30 percent, the median 17 image: ------- survival time was less than 24 hours. Downing and Merkens (1955) tested fingerling rainbow trout at three different concentrations of NH-j at five different levels of dissolved oxygen. They reported, in tests lasting up to 17 hours, that decreasing the oxygen from 8.5 to 1.5 mg/liter shortened the periods of survival at all ammonia concentrations, and that a decrease in survival time produced by a given decrease in oxygen was greatest in the lowest concentration of NH^. Merkens and Downing (1957), in tests which lasted up to 13 days, also reported that the effect of low concentrations of dissolved oxygen on the survival of rainbow trout was more pronounced at low concentrations of NH3' Lloyd (1961a) found NHo to be up to 2.5 times more toxic when dissolved oxygen concentration was reduced from 100 to about 40 percent saturation. Danecker (1964) reported that the toxicity of ammonia increased rapidly when the oxygen concentration decreased below two-thirds of the saturation value. Thurston et al. (1981b) conducted 15 96-hour acute toxicity tests with rainbow trout over the dissolved oxygen range 2.6 to 8.6 mg/liter. They reported a positive linear correlation between 96-hour LC50 and dissolved oxygen over the entire range tested. Herbert (1956) reported on rainbow trout mortalities in a channel receiving sewage discharge containing 0.05 to 0.06 mg/liter NH^. They found that at 25-35 percent dissolved oxygen saturation more than 50 percent of the fish died within 24 hours, compared with 50 percent mortality of test fish in the laboratory at 15 percent dissolved oxygen saturation. The difference was attributed to unfavorable water conditions below the sewage outflow, including ammonia, which increased the sensitivity of the fish to the lack of oxygen. 18 image: ------- There is a reduction in fish blood oxygen-carrying capacity following ammonia exposure (Brockway 1950; Danecker 1964; Reichenbach-Klinke 1967; Korting I969a,b; Waluga and Flis 1971). Hypoxia would further exacerbate problems of oxygen delivery and could lead to the early demise of the fish. (b) Temperature Information in the literature on the effects of temperature on ammonia toxicity is varied. The concentration of NH3 increases with increasing temperature. Several researchers have reported an effect of temperature on the toxicity of the un-ionized ammonia species, independent of the effect of temperature on the aqueous ammonia equilibrium. Hazel et al. (1971) tested ammonia with striped bass (Morone saxatilis) and stickleback (Gas teros teus aculeatus) and found little difference in toxicity between 15 and 23 C in fresh water, although both fish species were slightly more resistant at the lower temperature; the influence of temperature on toxicity was less for striped bass than for sticklebacks. McCay and Vars (1931) reported that it took three times as long for brown bullheads (Ictalurus nebulosus) to succumb to the toxicity of ammonia in water at 10-13 C than at 26 C. The pH of the tested water was not reported; however, within the probable range tested (pH 7-8), the percent NH-j at the higher test temperature is approximately three times that at the mean lower temperature. Powers (1920) reported the toxicity of ammonium chloride to goldfish, bluntnose minnow, and straw-colored minnow to be greater at high temperatures than at low; however, in that study also no consideration was given to the increase in relative concentration of NH-j as temperature increased. Thurston et al. (in press, a) reported that the acute toxicity of NH^ to fathead minnows decreased with a rise in temperature over the range 12 to 19 image: ------- 22 C. Bluegill and fathead minnow were tested at low and high temperatures of 4.0 to 4.6 C and 23.9 to 25.2 C, respectively; rainbow trout were tested at 3.0 and 14.0 C (Reinbold and Pescitelli 1982b). All three species were more sensitive to un-ionized ammonia at the low temperatures, with toxicity being 1.5 to 5 times greater in the colder water; bluegill appeared to be the most sensitive of the three species to the effect of low temperature on ammonia toxicity. Colt and Tchobanoglous (1976) reported that the toxicity of NH3 to channel catfish decreased with increasing temperature over the range 22 to 30 C. LC50 values for bluegill, channel catfish, and largemouth bass at 28 to 30 C were approximately twice that at 22 C (Roseboom and Richey 1977). LC50 values for channel catfish tested in Iowa River water were 0.49 mg/liter NH3 at 2.5 C and 0.56 mg/liter at 5.1 C (Miller and UNLV-EPA 1982). An effluent containing ammonia as a principal toxic component showed a marked decrease in toxicity to channel catfish over the temperature range 4.6 to 21.3 C (Gary 1976). Herbert (1962) has reported that experiments with rainbow trout in his laboratory suggest that the effect of temperature on their susceptibility to NH-j toxicity is little if at all affected by temperature change; no details were provided. The Ministry of Technology, U.K. (1968), however, has reported that the toxicity of NH^ to rainbow trout was much greater at 5 C than at 18 C. Brown (1968) reported that the 48-hour LC50 for rainbow trout increased with an increase in temperature over the range 3 to 18 C; the reported Increase in tolerance between -12 to ~18 C was considerably less than that between ~3 to -12 C. Thurston and Russo (in press) reported a relationship between temperature and 96-hour LC50 for rainbow trout over the 20 image: ------- temperature range 12 to 19 C; ammonia toxicity decreased with increasing temperature. Lloyd and Orr (1969) investigated the effect of temperature over the range 10-20 C on urine flow rates of rainbow trout exposed to 0.30 mg/liter NH^, and found no apparent temperature effect on the total diuretic response of the fish, although the relative increase in urine production was less at higher temperatures. From a study of the behavioral response of bluegill to gradients of ammonia chloride it was hypothesized that low temperatures increased the sensitivity of bluegill and interfered with their ability either to detect ammonia after a certain period of exposure or to compensate behaviorally for physiological stress caused by ammonia gradients (Lubinski 1979; Lubinski et al. 1980). The European Inland Fisheries Advisory Commission (1970) has cautioned that at temperatures below 5 C the toxic effects of un-ionized ammonia may be greater than above 5 C. The basis for such a statement is not clearly documented in that report. Nevertheless, there is some merit to the argument that a decrease in temperature may increase the susceptibility of fish to un-ionized ammonia toxicity. It is important that this relationship be further studied. The available evidence that temperature, independent of its role in the aqueous ammonia equilibrium, affects the toxicity of NH3 to fishes argues for further consideration of the temperature/ammonia toxicity relationship. (c) PH Tha roxicity to fishes of aqueous solutions of ammonia and ammonium compounds has been attributed to the un-ionized (undissociated) ammonia present in the solution. Although there were observations in the early literature that ammonia toxicity was greater in alkaline solutions, the 21 image: ------- earliest reported thorough study of the pH dependence of ammonia toxicity was that of Chipman (1934). He concluded from experiments with goldfish, amphipods, and cladocerans that the toxicity was a function of pll and therefore of the concentration of undissociated ammonia in the solution. Wuhrmann et al. (1947) discussed the importance of differentiating between NH^ and NH^ when considering ammonia toxicity. They summarized some unpublished experimental data indicating a correlation between solution pH and ammonia toxicity to fish (indicated by persistent loss of balance). Wuhrmann and Woker (1948) reported on the experiments referred to in Wuhrmann et al. (1947); these were conducted using ammonium sulfate solutions at different pH values on rainbow trout. Either four or six fish were tested at each of nine ammonium sulfate concentrations. The authors concluded from the experimental results that NH^ was much more toxic than NH^+. Downing and Merkens (1955) tested rainbow trout at different concentrations of ammonia at both pH 7 and 8. They reported a consistency of results when ammonia concentration was measured as NH^- Tabata (1962) conducted 24-hour tests (Table 5) on ammonia toxicity to Daphnia (species not specified) and guppy at different pH values and calculated the relative toxicity of N^/NH^"*" to be 190 for guppy (i.e., NH3 190 times more toxic than NH^+) and 48 for Daphnia. From tests of the toxicity of ammonium chloride to juvenile coho salmon in flow-through bioassays within the pH range 7.0 to 8.5, the reported 96-hour LC50 for NH3 was approximately 60 percent less at pH 7.0 than at 8.5 (Robinson-Wilson and Seim 1975). Armstrong et al. (1973) tested the toxicity of ammonium chloride to larvae of prawn (Macrobrachium rosenbergii) in static six-day tests within the pH range 6.8 to 8.3; test solutions were renewed every 24 hours. They 22 image: ------- 'reported a 96-hour LC50 for NH3 at pH 6.83 which was approximately 70 percent less than that for pH 8.34. They concluded that the toxicity of ammonia was not due solely to the NH3 "olecule, that in solutions of different pH and equal NH3 concentrations survival was greatly reduced as NH^+ levels increased. Tomasso et al. (1980) tested the toxicity of ammonia at pH 7, 8, and 9 on channel catfish and reported that 24-hour NH-j LC50 values were significantly higher at pH 8 than at pH 7 or 9. Thurston et al. (1981c) tested the toxicity of ammonia to rainbow trout and to fathead minnows in 96-hour flow-through tests at different pH levels within the range 6.5 to 9.0. Results showed that the toxicity of ammonia, in terras of NH3> increased at lower pH values. They concluded that NH^ exerts some measure of toxictty, and/or that increased H* concentration increases the toxicity of NH3 • Acute (96-hour) exposures of green sunfish and smallmouth bass were conducted by McCorraick et al. (in prep.) and Broderius et al. (in prep.) at four different pH levels over the range 6.5 to 8.7. For both species, NH3 toxicity increased markedly with a decrease in pH, with LC50 values at the lowest pH tested (6.6 for sunfish, 6.5 for bass) being 3.6 (sunfish) and 2.6 (bass) times smaller than those at the highest pH tested (8.7). LC50 values found wltli rainbow trout for the ammoniacal portion (diammonium phosphate) of a chemical fire retardant at two different pH levels indicated greater NH3 toxicity at lower pH; the LC50 at pH 7.0 was 0.15 mg/liter NH3 and at pH R.O was 0.48 rag/liter NH3 (Blahra 1978). (d) Acclimation and Fluctuating Exposures The qviestion of whether fish can acquire an increased tolerance to ammonia by acclimation to low ammonia concentrations is an important one. If fish had an increased ammonia tolerance developed due to acclimation or 23 image: ------- conditioning to low ammonia levels, they would perhaps be able to survive what otherwise might be acutely lethal ammonia concentrations. Observations by McCay and Vars (1931) indicated that bullheads subjected to several successive exposures to ammonia, alternated with recovery In fresh water, acquired no immunity from the earlier exposures to the later ones. A greater number of researchers have reported that previous exposure of fishes to low concentrations of ammonia increases their resistance to lethal concentrations. Vamos (1963) conducted a single experiment in which carp which had been revived in fresh water for 12 hours after exposure to 0.67 or 0.52 mg/liter NHg for 75 min were placed in a solution containing 0.7 mg/liter NH3• The previously exposed fish exhibited symptoms of ammonia toxicity in 60-85 min, whereas control fish developed symptoms within 20 min. Redner and Stickney (1979) reported that blue tilapia acclimated for 35 days to 0.52 to 0.64 mg/liter NH-j Subsequently survived 48 hours at 4.1 mg/liter; the 48-hour LC50 for unacclimated fish was 2.9 mg/liter. Malacea (1968) studied the effect of acclimation of bitterling to ammonium sulfate solutions. A group of ten fish was held in an acclimation solution of 0.26 mg/liter NHg for 94 hours, after which the fish were exposed to a 5.1 mg/liter NH3 solution for 240 min; a control group of ten was treated identically, except their acclimation aquarium did not contain added (MH^^SO^* The ratio of the mean survival times of "adapted" vs. "unadapted" fish was 1.13; mean survival times for the adapted and unadapted fish were 78 and 88 minutes, respectively, indicating somewhat higher ammonia tolerance for adapted fish. Fromm (1970) measured urea excretion rates of rainbow trout initially acclimated to either 5 or 0.5 mg/liter NH3» then subjected to 3 mg/liter 24 image: ------- NH3' Fish previously exposed to 5 mg/liter NH3 excreted slightly less urea than those exposed to the lower concentration. Lloyd and Orr (1969) conducted acclimation experiments with rainbow trout and found that the rate of urine excretion increased with a rise in the concentration of un-ionized ammonia to which the fish were exposed. They presented some evidence for acclimation of rainbow trout to sublethal levels of ammonia, although these levels may be as low as 12 percent of the "lethal threshold concentration". Acclimation was retained for 24 hours, but was not retained after three days. They also suggested that environmental factors which affect the water balance of fish may also influence susceptibility to ammonia toxicity. Fromm (1970) acclimated goldfish to low (0.5 mg/liter) or high (5.0 or 25.0 rag/liter) ambient NH^ for periods of 20 to 56 days and found that urea excretion rate in subsequent 24-hour exposures to concentrations ranging from 0.08 to 2.37 rag/1iter was independent of the previous acclimation concentration or duration. Schulze-Wiehenbrauck (1976) subjected to lethal ammonia concentrations two groups of rainbow trout (56 g and 110 g) which had been held for at least three weeks at sublethal ammonia concentrations. In the experiment with 110-g fish, the sublethal acclimation concentrations were 0.007 (control), 0.131, and 0.167 mg/liter NH-jJ the fish from these three tanks were then subjected to concentrations of 0.45, 0.42, and 0.47 mg/liter NHg, respectively, for 8.5 hours. Fish from the two higher sublethal concentra- tions had 100 percent survival after 8.5 hours in the 0.42 and 0.47 mg/liter NH3 solutions, whereas fish ,from the 0.007 mg/liter NHj concentration had only 50 percent survival in 0.45 mg/liter NH^• In the experiment with 56-g fish, the acclimation concentrations were 0.004 mg/liter NH3 (control) and 0.159 nirf/liter NH35 these fish were placed in NH3 concentrations of 0.515 25 image: ------- and 0.523 mg/llter, respectively, for 10.25 hours. There was 100 percent survival of the acclimated fish, and 85 percent survival of the control fish. The results of these experiments thus showed an increase in resistance of trout to high ammonia levels after prior exposure to sublethal ammonia levels. Alabaster et al. (1979) determined 24-hour LC50 values of NH3 for Atlantic salmon smolts under reduced dissolved oxygen test conditions. Fish acclimated to ammonia before oxygen reduction evidenced LC50 values 38 and 79 percent higher than fish without prior ammonia acclimation. Brown et al. (1969) tested rainbow trout in static tests in which fish were moved back and forth between tanks in which the ammonia concentrations were 0.5 and 1.5 times a previously determined 48-hour LC50. If fish were transferred on an hourly basis, the median period of survival for the fluctuating exposure was reported to be the same as that for constant exposure (>700 rain). If the fish were transferred at two-hour intervals, the median survival time for the fluctuating exposure was reported to be less (370 min), indicating that the toxic effects from exposure to the fluctuating concentrations of ammonia was greater than those from exposure to the constant concentration. Thurston et al. (1981a) conducted acute toxicity tests on rainbow trout and cutthroat trout in which fish were exposed to short-term cyclic fluctuations of ammonia. Companion tests were also conducted in which test fish were subjected to ammonia at constant concentrations. Median lethal concentration (LC50) values in terms of both average and peak concentrations of ammonia for the fluctuating concentration tests were compared with LC50 values for the constant concentration tests. Based on comparisons of total dose exposure, results showed that fish were more tolerant of constant concentrations of ammonia than of fluctuating concentrations. Fish subjected 26 image: ------- to fluctuating concentrations of ammonia at levels below those iicuteiy toxic were subsequently better able to withstand exposure to higher fluctuating concentrations than fish not previously so acclimated. In static renewal exposures to ammonium chloride using river water as the dilution water, fathead minnows were reported (Pitts 1980) to survive for 28 days exposures fluctuating from 0.1 mg/liter NH3 for four days to 0.2 or 0.3 mg/liter Nil-} for three days. Four-day excursions above 0.1 mg/liter to concentrations of 0.42, 0.48, and 0.52 rag/liter resulted in 80 to 100 percent mortality in 28 days, as did four-day excursions to 0.73 mg/liter. No constant exposure tests were conducted simultaneously for comparative purposes; however, constant exposure tests conducted approximately a year earlier yielded LC50 values ranging from 0.6 to 2.4 mg/liter NHg • In summary, there is reasonable evidence that fishes with a history of prior exposure to some sublethal concentration of ammonia are better able to withstand an acutely lethal concentration, at least for some period of hours and possibly days. The relative concentration limits for both acclimation and subsequent acute response need better definition and a more complete explanation. Limited data on fluctuating exposures indicate that fish are more susceptible to fluctuating than to constant exposure to equivalent NH^ dose concentrations. Much more research is needed to examine further the effects of fluctuating and intermittent exposures under exposure regimes simulating actual field situations. (e) Carbon Dioxide An increase in carbon dioxide concentrations up to 30 rag/liter decreases total ammonia toKicity (Alabaster and Herbert 1954; Allan et al. 1958). C02 causes a decrease in pH, thereby decreasing the proportion of un-ionized ammonia in solution. Lloyd and Herbert (i960) found, however, 27 image: ------- that although total ammonia toxicity was reduced at elevated CC>2 levels, the inverse was true when considering un-ionized ammonia alone; more NH-j is required in low C(>2» high pH water to exert the same toxic effect as seen in fish in high C02> low pH water. The explanation presented by Lloyd and Herbert (1960) for the decreased toxicity of NH3 in low CC>2 water was that Cr>2 excretion across the gills would reduce pH, and therefore NH3 concentration, in water flowing over the gills. The basic flaw in Lloyd and Herbert's (1960) hypothesis has been discussed in Broderius et al. (1977). C02 will only form protons very slowly in water at the tested temperature. The uncatalyzed CC>2 hydration reaction has a half-time of seconds or even minutes (e.g., at pH 8: 25 seconds at 25 C, 300 seconds at 0 C (Kern I960)), and water does not remain in the opercular cavity for more than a few seconds, and at the surface of a gill lamella for about 0.5 to 1 second (Randall 1970; Cameron 1979). Thus the liberation of C02 will have little, if any, effect on water pH or, therefore, NH^ levels while the water body is in contact with the gills. Hence the liberation of C02 across the gills can have little, if any, effect on the NH3 gradient across the gills between water and blood. Szumski et al. (1982) hypothesized that in the course of its excretion C02 is converted in the gill epithelium to H+ and HCO^" which then pass directly Into the gill chamber where they cause an instantaneous pH reduction. Their interpretation of the published literature on fish respiratory physiology is questionable, and experimental evidence in support of their evaluation is required before it can be given serious consideration. 28 image: ------- . (f) Salinity Herbert and Shurben (1965) reported that the resistance of yearling rainbow trout to ammonium chloride increases with salinity up to levels of 30-40 percent seawater; above that level, resistance appears to decrease. Katz and Pierro (1967) tested fingerling coho salmon at salinity levels of 20 to 30 parts per thousand (57 to 86 percent saltwater) and found that toxicity of an ammonia-ammonium waste increased as salinity increased. These findings are in agreement at the levels tested with those of Herbert and Shurben (1965). Atlantic salmon were exposed to ammonium chloride solutions for 24 hours under both freshwater and 30 percent saltwater conditions; LC50 values (Table 5) were 0.15 and 0.3 rag/liter NH3, respectively, in the two different waters (Alabaster et al. 1979). As was discussed in Willingham et al. (1979), decreased NH3 toxicity with increased salinity may be partially explained, at least for low salinity levels, by the fact that there is a slight decrease in the NH^ fraction of total ammonia as ionic strength increases in dilute saline solutions (Thurston et al. 1979). At higher salinity levels, however, the toxicity to fishes of ammonia solutions must be attributable to some mechanism or mechanisms other than the changes in the NH^+/NH3 ratio. Further work is needed to confirm results already reported and to clarify the observed mitigating effect of total dissolved solids. (g) Presence of Other Chemicals The presence of other chemicals may have an effect on ammonia toxicity, and some experimental work has investigated this topic. Herbert and Vandyke (1964), testing rainbow trout, determined the 48-hour LC50 value for a solution of ammonium chloride and that for a solution of copper sulfate. They reported that a solution containing a mixture of one half of each of 29 image: ------- these LC50 concentrations was also the 48-hour LC50 for the two toxicants combined; i.e., the toxic response was simply additive. This information was also reported by the Ministry of Technology, U.K. (1964); it is not clear whether this was a separate study or the same study. Shemchuk (1971) measured copper uptake in two-year old carp from solutions of Cu(NH-j)^^ ; copper uptake in various fish tissues was reported, but no information was provided about toxicity. Vamos and Tasnadi (1967) applied cupric sulfate to a "carp pond" to reduce the concentration of free ammonia and reported that this measure proved successful to reduce the toxic effect of ammonia; few details were provided. Ministry of Technology, U.K. (1962, 1963) reported on the results of tests on rainbow trout in which 48-hour LC50 concentrations were determined for solutions of ammonium chloride, zinc sulfate and mixtures of these two salts. A fraction of each of those 48-hour LC50 concentrations, when combined in such a way that those fractions equalled unity, provided a mixture with a 48-hour LC50 concentration equal to that of either of the two toxicants alone. Results were similar for tests conducted in waters with alkalinities of 240 and 50 mg/liter CaC03. Herbert (1962) studied the toxicity to rainbow trout of ammonia-phenol mixtures. The mixtures contained fractions of the 48-hour LC50 concentrations of phenol and of ammonia; the combined fractions equaled unity. The toxicity of the combined fractions approximated the toxicity of either phenol or ammonia when tested separately but under test conditions of similar water chemical characteristics. The same information was reported by Ministry of Technology, U.K. (1961); it is not clear whether this was a separate study or the same study. 30 image: ------- Brown et al. (1969) conducted 48-hour tests on rainbow trout in mixtures of anraonia, sine, and phenol; the mixture coi\tain»d equal portions, by 48-hour LC50 concentration, of the three toxicants. They reported that each chemical nominally contributed equally to the toxicity. In a second series of three tests in which the mixture was adjusted to include approximately 75 percent of a 48-hour LC50 concentration of one toxicant and the balance split equally between the other two, they reported that the principal toxicant contributed about three-fourths of the toxicity. Broderius and Smith (1979), in 96-hour flow-through tests with rainbow trout, reported a synergistic effect for NH3 and HCN except at extremely low concentrations. Rubin and Elmaraghy (1976, 1977) estimated the individual and joint toxicities of ammonia and nitrate to guppy fry; the toxicities of the two in mixture were additive, except at very low ammonia-to-nitrate ratios. Tomasso et al. (1980) reported that elevated calcium levels increased the tolerance to ammonia of channel catfish. Derivation of Maximum Criterion for Fresh Water (a) pH Dependence of Acute Ammonia Toxicity The acute toxicity of total ammonia to aquatic organisms has been found to increase markedly with pH, with LC50s declining by as much as an order of magnitude per unit pll increase. Much of this variation can be accounted for by expressing toxicity on the basis of un-ionized ammonia concentration rather than total ammonia concentration, un-ionized ammonia being considered the principal source of toxicity. Although such a mode of expression substantially reduces the variation with pH, available data indicate that a residual dependence exists, with NH^ LCSOs increasing with increasing pH and the rate of increase declining as pH increases, apparently leveling off at high pH. 31 image: ------- The task at hand was to select a suitable mathematical expression to describe the pH dependence of LC50s, on an un-ionized ammonia basis, in the pH 6.S-9.0 range (this range being dictated by the water quality criterion for pH (U.S. Environmental Protection Agency 1977)) and to determine whether the available data support the use of a common (i.e., for all species) expression that has only a single species-dependent parameter. The data used (Figure 1) were for Daphnia sp., rainbow trout, fathead minnow, and coho salmon (Tabata 1962; Robinson-Wilson and Seim 1975; Thurston et al. 1981c). This includes all available sets for which, for a given species, the same investigators determined LC50s at a minimum of four pHs distributed over at least 1.5 pH units in the pH 6.5-9.0 range. Furthermore, a minimum of six data points (either by testing at more distinct pHs or by replication at some pHs) was required so that meaningful regression analysis was possible. Inspection of the data suggested that, for all data sets and for the pH range of concern, log(LCSO) versus pH has a slope that decreases with Increasing pH, nearing zero at the upper part of the range and perhaps approaching a limiting value at the lower part of the range. A suitable mathematical expression for such behavior is: XI L.C50 = v, / v~ H7 (1) I + 10X3(X2-pH) v ' where XI is the limiting LC50 at high pH as the slope approaches zero, X3 is the limiting slope of log(LCSO) vs. pH at low pH, X2 allows the intermediate portion between the limits to occur at a variable pH. This equation is not only consistent with the form of the data, but also with suggestions that the p!l dependence of ammonia toxicity is due to both 32 image: ------- 5.0 I i.o a 0.5 7.0 OAPHNIA 8.0 PH 9.0 0.5 I IO 0.2 o 10 So., 0-05L_i—^ RAINBOW TROUT 7.0 8.0 9.0 pH I.Or ^ 0.5 10 ^0.2 O.I O 0.05 2.0r 7.0 COHO SALMON 8.0 9.0 PH i.o 10 O> O in o 0.2 O.I FATHEAD MINNOW 7.0 8.0 9.O PH Figure 1. Acute NH3 toAicity at different pH values (data from Taoata 1962, Robinson-Wilson and Seim 1975, Thurston et al. 1981c). Dotted lines « regression based on individual data set; solid lines = regression based on pooled data sets. 33 image: ------- un-ionized and cationic ammonia being toxic, but with different potencies (Armstrong et al. 1978; Thurston et al. 1981c). Estimates for XI, X2, and X3 were obtained (Erickson 1982) for each of * the available data sets by least-squares, nonlinear regression, using log(LC50) as the dependent variable and pH as the independent variable. A logarithm transform was employed because the errors in LC50 estimates are largely proportional to LC50. The results of these analyses indicated X2 and X3 did not differ significantly (at the 0.10 confidence level) between data sets and were in fact quite similar (X2 » 7.3-7.7, X3 = 0.7-1.2). The data sets were therefore pooled for further regression analysis in which X2 and X3 were assumed to be the same for all sets. Deviations of this pooled analysis from the individual regressions (Figure 1) were not statistically significant and were, in fact, quite minor, the maximum deviation being less than 0.08 log units and the average being less than 0.03 log units. The individual regressions accounted for 89%, 96%, 99%, and 96% of the variance of log(LCSO) in the Daphnia, rainbow trout, coho salmon, and fathead minnow data sets, respectively. Respective percentages for the pooled analysis were 87%, 92%, 99%, and 92%. It was therefore concluded that the common values for X2 (7.32) and X3 (1.03) from the pooled analysis could be justifiably applied to these data sets. Equation 1 can then be interpreted as having a data set-dependent numerator, the limiting LC50 at high pH (XI), and a set-independent denominator, a multiplicative, pH correction factor (1 + 101>^3(7.32-pH)^. Four other d.-ita sets were available for which acute ammonia toxicity to a species was tested by the same investigators at three or more distinct pHs distributed over at Least 1.5 pH units in the pH 6.5-9.0 range. These sets 34 image: ------- • .were for Macrobrachium rosenbergii (a saltwater prawn), channel catfish, green sunfish, and sraallmouth bass (Armstrong et al. 1978; Tomasso et al. 1980; McCormick et al., in prep.; Broderius et al., in prep.). These data sets were not used in the previously-discussed analysis due to inadequate size, but rather were used to evaluate the applicability of the relationship derived above (Erickson 1982) . A line was fit to each set by taking the above common estimates for X2 and X3 and calculating XI as the geometric average of LC50 x (1 -I- 101 -03(7 -32-pH)) for the individual data (this minimizes the variance of the residuals between the observed log (LC50)s and the fitted line). Two of the sets (prawn, green sunfish) showed a marked pH dependence qualitatively identical with the previous discussion and had good P quantitative agreement with the lines so estimated (r = 99% and 83%, respectively). Of the other two sets, one (smallmouth bass) showed a marked increase of LCSOs with pH, but with a noticeably different shape in the pH range of Interest; the other set (channel catfish) showed no apparent pH trend at all. 2 Neither of the lines fitted to these two sets produced a significant r , but they also did not deviate enough from the data to reject statistically either the general mathematical model or the common estimates for X2 and X3. Furthermore, the residuals between the data of these two sets and the fitted lines were at most 0.3 log units (a factor of 2) and had standard deviations of only 0.13 log units (a factor of 1.5). Additionally, if the common estimates of X2 and X3 are used to correct an LC50 in these sets from the pH it was measured at to a pH of another datum in the set, the discrepancy between the two LC50s are lar^e (>0.2 log units) only if the pH difference is large (>1 unit) and one of the pHs is <7.5. image: ------- Available acute toxiclty data therefore suggest that the proposed relationship will perform well in most circumstances and will produce large errors only under restricted circumstances. Although the proposed relationship cannot be considered universally applicable, especially if residuals between fitted lines and data are not to exceed measurement error, the alternatives of using no pH relationship or of basing criteria only on species tested over a range of pHs are clearly less desirable. Deviations of some data sets from the proposed relationship are probably due to a combination of experimental error, species effects, and temperature effects. The relative contributions of each source are uncertain. It may be possible in the future to develop parameter estimates that better account for variations due to temperature and species, but the current data base will not support such an exercise. Of course, in site-specific applications, if evidence exists for significantly different pH relationships for species of importance to setting criteria, appropriate modifications should be considered. (b) Temperature Dependence of Acute Ammonia Toxicity Investigations of the temperature dependence of LCSOs on an un-ionized ammonia basis have produced conflicting results, varying from constancy with temperature to major increases with temperature (see earlier discussion). The relative contributions to this variability of species differences, experimental error, and the relationship of test temperatures to species temperature tolerance are uncertain and not definable from available data. Lacking a broadly-applicable description of the temperature dependence of ammonia toxicity, it will be assumed that, above 10 C, LC50s on an un-ionized ammonia basis are constant with temperature. Where LC50s do 36 image: ------- increase with temperature, this assumption will result in overprotection at temperatures above that at which data most important to the criterion were gathered and underprotection at lower temperatures. Because data for criteria were mostly collected in the 10-25 C range, large errors probably will not accumulate over this range, due to limited extrapolation from tested temperatures. Extension of such an assumption to low temperatures is questionable, however, because the paucity of the data in this range makes such a move a substantial extrapolation and because the temperature effects data presented earlier, unlike at higher temperature, consistently indicated increased sensitivity associated with low temperatures. At temperatures less than 10 C, it will be assumed that LC50s on a total ammonia basis are constant. Due to the effect of temperature on the pK of ammonia, from 10 C to 0 C this is equivalent to approximately a two-fold lowering of the LC50s on an un-ionized ammonia basis. Such a lowering is not inconsistent with temperature effects discussed earlier and constitutes a very simple algorithm. Of course, as for pll, where data for a species of importance to the setting of a criterion value contradicts the above assumptions regarding temperature, appropriate modifications should be made. (c) Application of pH and Temperature Relationships of Acute Ammonia Toxicity to Determination of Final Acute Values A Species Mean Acute Value (SMAV) Is the geometric average of the acute values (AVs), usually LC50s, available for a given species. A Family Mean Acute Value (FMAV) is the geometric average of the SMAVs available for a given family. A Final Acute Value (FAV) for a material is an estimate of the image: ------- FMAV at the 0.05 cumulative proportion in the cumulative distribution of FHAVs for all families tested for that material. These computations (see Guidelines) are not a subject of this discussion, but their application to pH and temperature dependent data is. The existence of pH and temperature dependence in AVs requires that they be corrected to a common pH and temperature basis before computing a FAV. After a FAV at this common pH and temperature is computed, it can be applied to other pHs and temperatures using the same equations used to correct the AVs. Since AVs on an un-ionized ammonia basis are assumed constant with temperature above 10 C, the "common" temperature can be the whole temperature range XIO C. Only AVs measured at <10 C must be temperature corrected, to 10 C, based on the assumption that AVs on a total ammonia basis are constant with temperature below 10 C. The temperature correction equation for AVs from <10 C is therefore: (1 + ioPKT-pH) 10 (1 + 10PK10-PH) T where AV-p is an AV measured at a temperature T <10 C, AV^Q is the estimated AV at temperatures >10 C, pH is the pH at which the AV was measured, pK-p is the pK of ammonia at the measurement temperature (=0.902 + ) , and pK^Q is the pK at 10 C (=9.73) (pK/temperature relationship ,- • ?7'-- _y from Emerson et al. 1975). After any necessary temperature corrections are made, Equation 1, with the common estimates for X2 and X3, can be applied for correction to a common pH. Equation 1 has a single species-dependent parameter, XI, the limiting AV at high pH, hereafter called LIMAV. This limiting value will be considered 38 image: ------- the common pH value. All available AV^gS will therefore be converted to LIMAVs by the relationship: LIMAV - AV1Q x (1 + io1.03(7.32-pH)) The limiting SMAV (LIMSMAV) for a species then can be computed as the geometric average of the LIMAVs for that species, and the limiting FMAV (1IMFMAV) can be computed as the geometric average of the LIMSMAVs for that family. The limiting FAV (LIMFAV) for un-ionized ammonia then can be computed from the LIMFMAVs available, by the same procedures used for computing FAVs from FMAVs for any material. A FAV on an un-ionized ammonia basis at a particular pH for temperatures XLO C can be computed as: _ LIMFAV _ FAV10 = l + 101.03(7.32-PH) <4> A FAV on an un-ionized ammonia basis at a temperature T < 10 C can be computed from the >10 C FAV at the same pH as follows: (I + l09.73-pH) (5) Application of these techniques to the data proceeded as follows. AVs from Table 1 were corrected for temperature (where necessary) and pH and averaged to obtain the LIMSMAVs and LIMFMAVs reported in Table 3. The fifth percentile was estimated, by the Guidelines method, to be 0.74 mg/liter NH3 • This number, however, exceeds the LIMSMAVs of important species (walleye and rainbow trout) and thus, by Guidelines procedures, should be decreased to the lower of these two LIMSMAVs. Additionally, the rainbow trout data in Table 1 indicate that sexually mature fish (M kg) are significantly more sensitive than the average of the tested fish. Since a species is not protected if such an important life stage is not protected, the LIMFAV was lowered to 0.30, the geometric average of the LIMAVs of rainbow trout in this size range. Thus, for temperatures >10 C: 39 image: ------- 0.30 3 (1 + 101.03(7.32-pII)) For temperatures <10 C: 0.30 (1 + 109-73-pH) FAVl " (1 + K)l-03(7.32-pH)) x (1 4 Available Information indicates that the acute response to ammonia is rapid. Therefore, a criterion based on the FAV cannot be treated as an average over any appreciable span of time, since such averaging implicitly allows significant excursions over the criterion value for an appreciable fraction of the averaging period and thus allows the occurrence of a time sequence of concentrations which would violate the intent of the criterion. Consequently, the criterion based on the FAV is a concentration that at no time should be exceeded. Saltwater Invertebrates Data on acute toxicity of ammonia to saltwater invertebrate species are very limited. LC50 values are summarized in Table 1 for five species representing five families. A 96-hour LC50 value (Table 1) of 1.5 mg/liter NH3 was reported (Linden et al. 1979) for the copepod, Nitocra spinipes. Lethal effects of NH^Ci on the quahog clam (Mercenaria mercenaria) and eastern oyster (Crassostrea virginica) were studied by Epifanio and Srna (1975) (Table 1). There was no observed difference in susceptibilities between juveniles and adults of the two species. Armstrong et al. (1978) conducted acute toxicity tests (6 days) on ammonium chloride using prawn larvae (Macrobrachium rosenbergii). LC50 results (Tables 1, 5) were highly pH-dependent. Acute toxicity of NH^Cl to penaeid shrimp was reported as a 48-hour composite LC50 value of 1.6 mg/liter NH^ for seven species pooled, including the resident species Penaeus setiferus (Wickins 1976). The acute 40 image: ------- ' toxicity of NH^Cl to the carldean prawn, >1. rosenbergii, was reported (Wicklns 1976) as LT50 values of 1700-560 minutes at concentrations of 1.74 to 3.41 mg/liter NH3 (Table 5). Hall et al. (1978) measured the acute toxicity of NH^Cl to grass shrimp (Palaemonetes pugio) (Table 5). Catedral and coworkers (1977a,b) investigated the effect of NH^Cl on survival and growth of Penaeus monodon; larvae had lower tolerance to ammonia compared with postlarvae. Brown (1974) reported a time to 50 percent mortality of 106 min for neraertine worm (Cerebratulus fuscus) at 2.3 mg/liter NH3 (Table 5). Effects of NH^Cl solutions on American lobster (Homarus americanus) were studied by Delistraty et al. (1977). Their tests were performed on fourth stage larvae which they believed to be the most sensitive life stage, or nearly so. They reported a 96-hour LC50 value (Table 1) of 2.2 mg/liter NH3 and an incipient LC50 (Table 5) of 1.7 mg/liter NH3. A "safe" concentration of 0.17 mg/liter NH3 was tentatively recommended. Saltwater Fishes Very few acute toxictty data are available for saltwater fish species. Holland et al. (1960) reported the critical level for chinook salmon (Oncorhynchus tshawytscha) to be between 0.04 and 0.11 mg/liter NH3 and for coho salmon to be 0.134 rag/liter NH3« A static test with coho salmon provided a 48-hour LC50 value (Table 5) of 0.50 mg/liter NH3 (Katz and Pierro 1967). Atlantic salmon smolts and yearling rainbow trout tested for 24 hours in 50 and 75 percent saltwater solutions exhibited similar sensitivities to ammonia (Ministry of Technology, U.K. 1963). 41 image: ------- CHRONIC TOXICITY TO ANIMALS The following discussion of chronic and partial chronic ammonia toxicity includes both data used in the derivation of the numerical 30-day average criterion (Table 2 data) and data that were not included in the criterion derivation, but that are important for an understanding of long-terra lethal and sublethal effects of ammonia on aquatic organisms (Table 5 data). Freshwater Invertebrates Few studies have been conducted on long-term exposure of freshwater invertebrates to ammonia, and life-cycle tests were conducted only for cladocerans. The lowest concentrations affecting reproduction in two life-cycle tests (Table 2) with ID. magna were 0.74 and 0.76 mg/liter NH-j (Russo et al., in prep.); a 28-day LC50 of 1.53 mg/liter NH^ was reported. In a chronic test (Table 2) conducted by Reinbold and Pescitelli (1982a), reproduction and growth of I), magna were affected at a concentration of 1.6 mg/liter NH^- A life-cycle test (Table 2) with £. acanthina (Mount 1982) showed effects on reproduction at a concentration of 0.463 mg/liter NH3• Two tests lasting 42 days were conducted by Anderson et al. (1978) on Nl^Cl with the fingernail clam, Musculium transversum (Table 5). Significant mortalities (67 and 72 percent) occurred in both tests at a concentration of 0.7 mg/liter NH^. In one of the experiments, significant reduction in growth was observed after 14 days of exposure to 0.41 mg/liter NH3• Sparks and Sandusky (1981) reported that fingernail clams exposed to 0.23 and 0.63 mg/liter NH3 incurred 36 and 23 percent mortality, respectively, In four weeks; after six weeks, 47 percent mortality occurred at 0.073 rig/liter NH^, and 83 percent mortality occurred at 0.23 and 0.63 42 image: ------- mg/liter NH-^- No growth at All occurred In all test chambers (conoontra- tions of 0.036 mg/liter NH3 and higher) other than the control after six weeks (Table 5). Two partial chronic tests, of 24- and 30-days' duration, were conducted by Thurston et al. (in prep., a) with the stonefly Pteronarcella badia (Table 5). Adult stonefly emergence was delayed with increasing ammonia concentra- tion, and little or no emergence occurred at concentrations exceeding 3.4 mg/liter NH-j. There was no significant relationship between food consumption rates of nymphs and concentrations up to 6.9 mg/liter NH-j • LC50 values for 24- and 30-day exposures were 1.45 and 4.57 mg/liter NH3, respectively. Freshwater Fishes A number of researchers have conducted long-term ammonia exposures to fishes, including complete life-cycle tests on rainbow trout and fathead minnows. Several '<1nds of endpoints have been studied, including effects on spawning and egg incubation, growth, survival, and tissues. The effects of prolonged exposure (up to 61 days) to ammonia of pink salmon early life stages was studied by Rice and Bailey (1930). Three series of exposures were carried out, beginning at selected times after hatching: for 21 days prior to completion of yolk absorption, for 40 days up to 21 days before yolk absorption, and for 61 days up to yolk absorption. All test fish were sampled for size when the controls had completed yolk absorption. NHg concentrations ranged from 0 (control) up to 0.004 mg/liter. For fry at the highest concentration of 0.004 mg/liter NH3 (Table 2), significant decreases in weight were observed for all three exposure groups. At a concentration of 0.0024 mg/liter NH3 (Table 2) the group of fry exposed for 40 and 61 dayu were significantly smaller, whereas a concentration of 0.0012 43 image: ------- mg/liter had no significant effect on growth. Effects were consistently more adverse for the 61-day-exposed fish. Thurston et al. (In press, b) tested rainbow trout in a laboratory study In which adult fish exposed for five months to concentrations of ammonia from 0.01 to 0.07 mg/llter NH^ Spawned of their own volition; baskets containing crushed rock served as the spawning substrate. There was no correlation between ammonia concentration and numbers of egg lots spawned, total numbers of eggs produced, or numbers of eggs subsequently hatched. Parental fish were exposed for 11 months, the first filial generation (F^) for four years, and the second filial generation (F2) for five months. Pathologic lesions were observed in both parental and F^ fish when ammonia concentra- tions reached and exceeded 0.04 mg/liter NH^ (Table 2). Measurements of blood ammonia concentrations in four-year-old F^ fish showed an increase when test water conditions reached or exceeded 0.04 mg/liter NH-j • Trout exposed for 52 months from day of hatching showed no dose/growth relationship ,it 10, 15, 21, and 5?. months. Burkhalter and Kaya (1977) tested ammonia at concentrations from 0.06 to 0.45 mg/liter NH-j On fertilised eggs image: ------- . after hatching, hypertrophy of secondary gill lamellae epithelium occurred at 0.23 rag/liter NH3, and karyolysis and karyorrhexis in the secondary gill lamellae were observed after 28 days at 0.34 mg/liter NH3 and higher. CalaraarL et al. (1977, 1981) exposed rainbow trout to ammonium chloride solutions for 72 days, beginning one day after fertilization and ending when fry were fed for 30 days. A 72-day LC50 of 0.056 mg/liter NH3 was calculated (Table 5); 23 percent mortality occurred at a concentration of 0.025 rag/liter NH3 (Table 2). Examination of 986 rainbow trout embryos at hatching stage after exposure to NH3 Concentrations of 0.010 to 0.193 mg/liter for 24 days showed an increase in macroscopic malformations with increasing ammonia concentration. Kinds of deformities observed were varying degree of curvature from median body axis, which in extreme cases produced a complete spiral shape, and various kinds of malformations in the head region with a number of cases of double heads. At the highest concentration tested, 0.193 mg/liter NH-j > 60 percent of the observed fish were malformed. Microscopic examination at hatching of 128 larvae from the same exposure showed abnormalities on the epidermis and pronephros that correlated with ammonia concentrations. The epidermis was thickened with an irregular arrangement of the various layers of cells and an increase in the number and dimensions of mucous cells. The pronephros showed widespread vacuolization of the tubule cells, together with a thickening of the wall. Increasing abnormalitLes were observed after exposure to concentrations over 0.025 mg/liter NH3 for epidermis and 0.063 mg/liter NH3 for pronephros. Broderius and Smith (1979) tested four-week-old rainbow trout fry for 30 days at concentrations of ammonia (reported grahically) ranging from -0.06 to 0.32 rag/liter NH3 (Table 5). Growth rate at -0.06 mg/liter NH3 was 45 image: ------- comparable to that of controls; above -0.10 mg/liter NH-j growth rate decreased, correlated with Increased NH3 concentration. The survival at 0.32 mg/liter NHg was reduced to 70 percent that of the controls. Schulze- Wiehenbrauck (1976) tested juvenile rainbow trout, approximately one-half-year-old but of different sizes, for periods of time from two to seven weeks, and at ammonia concentrations from 0.012 to 0.17 mg/liter NH3 • He concluded that 0.05 mg/liter NHj caused a slight decrease in growth during the first 14-day interval on nonacclimatized fish, but that decrease was completely compensated in the next growth interval; exposure to 0.13 mg/liter NH-j (apparently for 3 or A weeks) did not affect growth, food consumption, or food conversion. Smith (1972) and Smith and Piper (1975) reared young rainbow trout at three concentrations of ammonia (averaging 0.006, 0.012, and 0.017 mg/liter NH^) for a period of one year. There was no significant difference in fish growth reported among the three concentrations at four months. There was, however, a difference reported at 11 months; the fish at 0.012 and 0.017 mg/liter NH3 weighed 9 and 38 percent less than the fish at 0.006 mg/liter. Microscopic examination of tissues from fish exposed to the highest concentration, examined at 6, 9, and 12 months, showed severe pathologic changes in gill and liver tissues. Gills showed extensive proliferation of epithelium which resulted in severe fusion of gill lamellae which prevented normal respiration. Livers showed reduced glycogen storage and scattered areas of dead cells; these were more extensive as exposure titae increased. Ministry of Technology, U.K. (1968) reported on tests in which rainbow trout wore exposed for three months to concentrations of 0.069, 0.14, and 0.23 mg/liter NH3• The cumulative mortality of a control group (0.005 46 image: ------- tog/liter NTH^) was -2 percent. Cumulative .nortallty «t 0.069 and O.lt mg/liter NH-j was ~5 percent, and that at 0.28 mg/llter was -15 percent. Reichenbach-Klinke (1967) performed a series of one-week ammonia tests on 240 fishes of nine species (including rainbow trout, goldfish, northern pike (Esox lucius), carp, and tench) at concentrations of 0.1 to 0.4 mg/liter NH3• He observed swelling of and diminishing of the number of red blood cells, inflammations, and hyperplasia. Irreversible blood damage occurred in rainbow trout fry in ammonia concentrations above 0.27 mg/liter NH3 - He also noted that low NH^ concentrations inhibited the growth of young trout and lessened their resistance to disease. Smart (1976) exposed rainbow trout to 0.30 to 0.36 mg/liter NH3 (Table 5); 81 percent mortality occurred over the 36-day duration of the test, with most deaths occurring between days 14 and 21. Microscopic examination of the gills of exposed rainbow trout revealed some thickening of the lamellar epithelium and an increased mucous production. The most characteristic feature was a large proportion of swollen, rounded secondary lamellae; in these the pillar system was broken down and the epithelium enclosed a disorganized mass of pillar cells and erythrocytes. Gill hyperplasia was not a characteristic observation. Fronrn (1970) exposed rainbow trout to <0.0005 and 0.005 mg/liter NH3 for eight weeks. Subsequent examination of the gill lamellae of fish from the trace concentrations showed them to be long and slender with no significant pathology. Fish exposed to 0.005 mg/liter NH-j had shorter and thicker gill lamellae with bulbous ends; some consolidation of lamellae was noticed. Photomicrographs revealed that many filaments showed limited hyperplasia accompanied by the appearance of cells containing large vacuoles whose contents stained positive for protein. Other lamellae showed a 47 image: ------- definite hyperpLasi-i ot the epithelial layer, evidenced by an Increase Li Uie ' number of cell nuclei. Thurston et al. (1978) studied the toxicity of ammonia to cutthroat trout fry in flow-through tests which lasted up to 36 days (Table 5). Results of duplicate tests on 1.0-g fish both showed 29- and 36-day LC50 values of 0.56 mg/liter NT!-} • Duplicate tests on 3.3-g fish provided 29-day LC50 values of 0.37 and 0.34 mg/liter, slightly less than those of the 1.0-g fish. Tissues from heart, gastrointestinal tract, and thymus of cutthroat trout fry exposed to 0.34 mg/liter NH^ for 29 days were comparable to those of control fish. However, gills and kidneys of exposed fish showed degenerative changes. Gills showed hypertrophy of epithelium, some necrosis of epithelial cells, and separation of epithelium due to edema; kidneys showed mild hydropic degeneration and accumulation of hyaline droplets in renal tubule epithelium; reduced vacuolation was observed in livers- Samylin (1969) studied the effects of ammonium carbonate on the early stages of development of Atlantic salmon. The first set o£ experiments (temperature = 13 C) was conducted within the range 0.001 to >6.6 mg/liter NH-j beginning with the "formed embryo" stage; the experiment lasted 53 days. Accelerated hatching was observed with increasing (NH^^^G^ concentrations, but concentrations X).16 rag/liter NH^ were lethal in 12-36 hours to emerging larvae. Because (NH^^CO-j was used as the toxicant, the pll in the test aquaria Increased from 6.7 to 7.6 with increasing NH|j concentration. Growth inhibition was observed at 0.07 mg/liter NH-j (Table ?). Tissue disorders wore observed in eyes, brains, fins, and blood of MlanM^* salmon embryos and larvae exposed to concentrations from 0.16 to >6.6 mg/liter NH^, with increased degree of symptom at increased ammonia concentrations. Effects observed included erosion of membranes of the eyes 48 image: ------- and shedding of the crystalline lens, dilation of blood vessels in liver and brain, accumulation of blood in the occipital region and in intestines. Reaction to light and mechanical stimulation gradually disappeared with increased ammonia concentration, and the pulseheat slowed. Morphological differences in development between experimental and control larvae were observed from the tenth day of exposure, including a lag in yolk resorption, decrease in growth of the skin fold, and contraction of skin pigment cells causing the skin color to become paler than it was after hatching. At concentrations up to 0.07 rag/liter NHj no significant morphological differences were observed. A second series of experiments (temperature = 16.5 C) was carried out in the 0.001 to 0.32 rag/liter NHj concentration range, and began with larval salmon (Samylin 1969). Concentrations of 0.21 rag/liter NH3 and higher were lethal and caused weight loss in fry; 0.001 to 0.09 rag/liter NH^ caused a decrease in weight gain, although no differences in feeding activity, behavior, or development were observed in these concentrations compared to controls. Dissolved oxygen concentrations in this second series of experiments dropped as low as 3.5 mg/liter. Burrows (1964) tested fingerling chinook salmon for six weeks in outdoor raceways into which ammonium hydroxide was Introduced. Two experiments were conducted, one at 6.1 C and the other at 13.9 C, both at pH 7.8. In both cases fish were subsequently maintained in fresh water for an additional three weeks. A recalculation of Burrows' reported un-ionized ammonia concentrations, based on more recent aqueous ammonia equilibrium tables, indicates that the concentrations at 6.1 C were 0.003 to 0.006 mg/liter NH3> and at 13.9 C were 0.005 to 0.011 mg/liter 1^3. At both temperatures some fish at all ammonia concentrations showed excessive proliferation and clubbing of the gill filaments; the degree of proliferation 49 image: ------- was progressive for the first four weeks, after which no measurable increase was discernible. Examination of a sample of the fish tested at 6.1 C after three weeks in fresh water indicated no recovery had taken place from the extensive proliferation. In the experiment with larger fish at 13.9 C a marked recovery from hyperplasia was noted after the three-week fresh water exposure period. In the first experiment the proliferated areas had consolidated; in the second they had not. Burrows postulated that continuous ammonia exposure is a precursor of bacterial gill disease. Buckley et al. (1979) exposed duplicate groups (90 fish each) of hatchery-reared coho salmon for 91 days to "river-water" solutions of NH^Cl at concentrations of 0.019 to 0.33 mg/liter Nl^; these were compared with control groups reared at 0.002 mg/liter NI^- Hemoglobin content and hematocrit readings were reduced slightly, but significantly, at the highest concentration tested, and there was also a greater percentage of immature erythrocytes at the highest concentration. Blood ammonia and urea concentrations were not significantly different after 91 days, regardless of concentration of ammonia to which the fish were exposed. Rankin (1979) conducted ammonia tests with embryos of sockeye salmon (Oncorhynchus nerka) from fertilization to hatching. Total embryo mortality occurred at concentrations of 0.49 to 4.9 mg/liter NH-j; times to 50 percent mortality at these concentrations were 40 to 26 days. Mortality of the embryos exposed to 0.12 mg/liter NTl^ was 30 percent, and time to 50 percent mortality was 66 days. Two full life-cycle ammonia toxicity tests (291 and 337 days) were conducted with fathead minnows (Thurston et al., in prep., b). These tests began with newly hatched fry and were continued through their growth, image: ------- maturation and spawning stages; progeny were exposed from hatching through growth to 60 days of age. No statistically significant difference was observed based on spawning data (number of egg lots, egg lot size, egg lots per female, eggs per female per day) for any of the concentrations tested, up to 0.96 mg/liter NH3• However, there was a significant effect of ammonia on hatching success (Table 2), with the number of fry hatching decreasing as NH-j concentration increased to 0.187 mg/liter NH^ and higher; no effect on hatching success was observed at concentrations of 0.088 mg/liter and lower. Also, there was some indication that length of time for incubation from spawning to hatching increased with increasing NH3 Concentrations. No effect on fish growth was observed for either parental fish or progeny after 60 days' exposure and at exposure termination. Significant mortalities occurred among the parental generation at concentrations of 0.9 to 1.0 mg/liter NH3 after 30 and 60 days' exposure, and there were significant mortalities among progeny at concentrations of 0.2 to 0.4 mg/liter NH-j after 30 and 60 days' post-hatch exposure. Tissues from fathead minnows subjected to prolonged (up to 337 days) ammonia exposure were examined, including brain, heart, gills, kidneys, liver, and gastrointestinal tract (Smith 1981; Thurston et al., in prep., b). Growths, some massive, were observed on heads of several fish exposed to concentrations of 0.425 and 0.955 mg/liter NHg, and swollen darkened areas were observed on heads of several fish held at 0.216 and 0.228 mg/liter. Grossly and histologically the severity of the lesions, which varied from mild to severe, was positively correlated with ammonia concentration. Lesions appeared to be a connective tissue type that 51 image: ------- originated from the raeninx primativa covering the brain. At the higher ammonia concentrations proliferated tissue often completely surrounded the brain but was not observed around the spinal cord. Pathological changes were not observed in other tissues. An early-Hfe-stage test initiated at the blastula stage of embryo- genesis and extending through 39 days post-hatching was conducted with green sunfish by McCormick et al. (in prep.). Retardation of growth of green sunfish exposed from embryo through juvenile life stages was found at NHg concentrations of 0.489 mg/liter and higher, but not at 0.219 rag/liter and less (Table 2). In a long-term test on green sunfish, Jude (1973) reported that for treatments greater than 0.17 mg/liter NHg, mean fish weight increased less rapidly than controls after introduction of toxicant over the next four days. Thereafter, fish exposed to 0.26 and 0.35 mg/liter NHj grew at an increasing rate while fish exposed to 0.68 and 0.64 mg/liter NH^ remained the same for 12 days before greater increases in growth occurred. An early-life-stage test with bluegill from embryo through 30 days post-hatch was conducted on ammonia by Smith and Roush (in prep.). Significant retardation of growth due to ammonia exposure was observed at 0.136 mg/liter NH^; the no-observed-effect concentration was reported to be 0.063 ing/liter NH3 (Table 2). Broderius et al. (In prep.) conducted four simultaneous early-life-stage ammonia tests with smallmouth bass. These were carried out at four different pH levels, ranging from 6.6 to 8.7, to examine the effect of pH on partial chronic ammonia toxicity. Exposure to ammonium chloride solutions began with two- to three-day-old embryos and lasted for 32 days. The effect endpoint observed was growth, and ammonia was found to have a greater effect on growth at lower pH levels than at high. NH3 concentrations Cound to retard growth image: ------- (table 2) ranged from 0.05S8 mg/liter at pH 6.60 to 0.865 mg/Liter at pH 8.68. Early-life-stage tests (29-31 days' exposure) on ammonium chloride with channel catfish and white sucker were conducted by Reinbold and Pescitelli (1982a). No significant effect on percent hatch or larval survival was observed for channel catfish at concentrations as high as 0.583 rag/liter NH3 and for white sucker as high as 0.239 mg/liter NH3• Significant retardation of growth, however, occurred for channel catfish at concentrations of 0.392 mg/liter NHg and higher and for white sucker at 0.070 mg/liter NH3 and higher (Table 2). A delay in time to swim-up stage was also observed for both species at elevated (0.06 to 0.07 mg/liter Nl^) ammonia concentrations. Robinette (1976) cultured channel catfish fingerlings for periods of approximately one month at concentrations of 0.01 to 0.16 mg/liter NH3« Growth at 0.01 and 0.07 mg/liter NH3 was not significantly different from that of control fish; growth retardation at 0.15 and 0.16 mg/liter NH3 was statistically significant. Colt (1978) and Colt and Tchobanglous (1978) reported retardation of growth of juvenile channel catfish during a 31-day period of exposure to concentrations ranging from 0.058 to 1.2 mg/liter NH3« Growth rate was reduced by 50 percent at 0.63 mg/liter NH3, and no growth occurred at 1.2 mg/liter NH3» The authors hypothesized that growth may be inhibited by high concentrations of NH^"*" and low concentrations of Na+ in solution, and/or the NH^/Na4" ratio. Ammonia exposure for 30 to 40 days of goldfish and tench resulted in lesions .and diffuse necrosis of the caudal fin, causing it to degenerate progressively to the point of breaking off by degrees, ultimately leaving only a necrotized stump (Marchetti I960). 53 image: ------- Ver> li'-cle work has .n'eii ..uvu" to tiwost i «!:••; »• >vr ; >•» i • >•» .'. t ', v.- • .• .1. factors on chronic ammonia to image: ------- (1) At pH 7.7 and near 10 C, the threshold concentration for chronic toxicity of ammonia to rainbow trout has been estimated to be 0.031 mg/liter NH-j • Given the importance of this species, the FCV must be. at or below this number. The FCV at pH 7.7 and 10 C is therefore set to 0.031 mg/liter NH3• (2) Both the acute data and the chronic data of Broderius et al. (in prep.) indicate little or no increase of effect concentrations with pH above 7.7. The FCV is therefore set to 0.031 mg/liter for all pH >7.7 at 10 C. (3) At pH <7.7, the pooled salmonid chronic toxicity data (Table 2) show trends consistent with smallmouth bass chronic toxicity data (Broderius et al., in prep.) and the acute data. Thus, the pink salmon datum at pH 6.4 is not only important in its own right, but also likely lies close to where other trout and salmon would be at pH 6.4. The FCV will therefore be set to 0.0021 (the geometric average of the pink salmon data) at pH 6.4 and 4 C. Between pH 6.4 and pH 7.7, the log(FCV) will be assumed to vary linearly with pH, a trend which causes it to pass close to other salmonid data and which is similar to the trend in the acute toxicity pH relationship. (4) Temperature effects will be assumed to be the same as for acute toxicity. Based on this assumption, the pink salmon data point was corrected upward, to 0.0034 at 10 C, before computing the pH relationship between pH 6.4 and 7.7. (5) Therefore, for temperatures _>1G C and pH>7.7, FCV10 = 0.031 For temperatures MO C and pH <7.7, 0.031 100.74(7.7-pH) 55 image: ------- For temperatures <10 C, (1 + 109.73-PH) FCVT - FCVin x A. _ ~x i 1 -^ 1 OP^T P" i The criterion based on the FCV will be considered as a 30-day average. Provisions to limit excursions above this criterion for time intervals shorter than 30 days are believed to be unnecessary, given currently available data. (c) Acute/Chronic Ratios Although the ratios of acute effect concentrations to chronic effect concentrations were not used in the derivation of FCV above, they will be used in site-specific applications and therefore merit some consideration here. Acute/chronic ratios are available for ten species (Table 2). Because these ratios vary so widely (2.8-44), their dependence on species and physico-chemical factors should be evaluated so that they are properly applied. A clear trend exists in the species-dependence of the ratios. Of the ten species, the five most acutely sensitive have ratios >11 and the five least acutely sensitive have ratios <6. This has considerable significance because ratios are applied to FAVs which reflect species with high acute sensitivities. To produce an appropriate FCV therefore requires using a ratio appropriate to such species. The question thus arises as to the range of acute sensitivity from which ratios can be justifiably used. The five highest ratios here cover the LIMSMAV range <1.8. This range includes a large number of diverse families and the FAV is unlikely ever to exceed this value; thus there is little reason ever to consider the five lowest ratios. Within the LIMSMAV range <1.8, there is no clear trend of ratio with 56 image: ------- sensitivity; thus there is also no good reason to try to limit further this set of ratios based on proximity to the FAV. The sraallmouth bass data in Table 2 indicate that acute/chronic ratios may increase with decreasing pH. This is consistent with the comment earlier that the effect of pH on chronic toxicity in the 6.5-7.5 pH range is greater than the effect of pH on acute toxicity. The large ratio for pink salmon also suggests such a pH dependence of the ratio, if it is assumed all salmonids have similar chronic and acute sensitivities. The paucity of data makes firm conclusions impossible, but it is probably inappropriate to apply the pink salmon ratio (measured at pH 6.4) to the pH range (>7.5) at which other ratios were measured. Eliminating the pink salmon ratio from the five highest ratios leaves four ratios for diverse fish species (white sucker, fathead minnow, bluegill, rainbow trout) that were measured at similar pHs (7.7-8.3). These four ratios have a geometric average of 16 and have no apparent trends with pH, temperature, or species sensitivity. This average is therefore as appropriate as can be derived from the available data for application to FAVs derived in the pH >7.5 range (this range being based on the range in which the ratios were measured (7.7-8.3), with extrapolation to lower pH being modest because of the indications of effects on ratios of pHs <7.5, and extrapolation to higher pH being liberal because of the lack of significant effects of pH in this range). No recommendation is made here about appropriate ratios for lower pHs, except that they should probably be higher than 16 and will require further testing. Use of a constant ratio, of course, implicitly makes the assumption that the pH dependency of chronic toxicity is the same, on a relative scale, as for acute toxicity. Although this assumption was rejected earlier, the 57 image: ------- rejection was based on a broad pH range that extends below pH 7.5. For a limited pH range and/or one restricted to pHs above 7.5, this assumption should cause only minor errors as long as the ratios are, as advised above, derived from data within the range in question. An acute/chronic ratio should also be based on acute data consistent with the FAV it is applied to. Specifically, if sensitivity varies with organism size, the ratio should be modified based on this variation so that it reflects acute data from the same size range as the acute data on which the FAV Is based, or the original ratio should be applied to the value the FAV would have had if a sensitive size range had not been used. Saltwater Animals Little information is available on long-term effects of sublethal ammonia exposures on saltwater species, and no chronic data are available for any saltwater fish species. Three-week exposure (Wickins 1976) of P_. setiferus to NH^Cl yielded an EC50 value (Table 5), based on growth reduction, of 0.72 mg/liter ^3. A six-week test (Table 5) with M. rosenbergii resulted in reduction in growth to 60-70 percent that of controls for prawn exposed to concentrations above 0.12 mg/Hter NH3 • A "maximum acceptable level" was estimated to be 0.12 mg/liter NH3• Armstrong et al. (1978) conducted growth tests (Table 5) on NH^Cl using prawn larvae (M. rosenbergii). Retardation in growth was observed at sublethal concentrations (0.11 mg/liter NH-j at oH 6.83 and 0.63 mg/liter NH-j at pH 7.60), and this effect was greater at low pH. 58 image: ------- TOXICITY TO PLANTS Bacteria and Freshwater Plants Ammonia is known to play an important part in the nitrogen metabolism of aquatic plants. In the aquatic environment, nitrogen plays an important role in determining the composition of phytoplankton and vascular plant communi- ties and in some cases can act as a limiting nutrient in primary production. Ammonia can also be toxic at certain concentrations. Data concerning the toxicity of ammonia to freshwater vascular plants and phytoplankton are contained in Table 4. Few of the papers examined contained sufficient information to enable calculation of un-ionized ammonia concentrations, altough total ammonia solutions were more toxic at high than at low pH, indi- cating that toxicity was likely due primarily to NHj rather than NH^ . Some information on ammonia effects on bacteria is also included here. The bacterial species Escherichia coll and Bacillus subtills were found to be sensitive to NH^Cl (Deal et al. 1975); 1100 mg/liter NH3 killed 90 percent of an ]5. coli population in 78 minutes. _B. sub tills, an aerobic, spore-forming bacterium, was destroyed in less than two hours in 620 mg/liter NH-j • NH-} inhibition of the bacteria Nltrosomonas (that convert ammonium to nitrite) and the bacteria Nltrobacter (that convert nitrite to nitrate) was studied by Anthonisen et al. (1976) and Neufeld et al. (1980). NH3 inhibited the nitrification process at a concentration of 10 mg/liter (Neufeld et al. 1980). The NH-j concentrations that inhibited nitrosomonads (10 to 150 rag/liter) were greater than those that inhibited nitrobacters (0.1 to 1.0 mg/liter), and NH^, not NH^"1", was reported to be the inhibiting species (Anthonisen et al. 1976). Acclimation of the nitrifiers to NH3> temperature, and the number of active nitrifying organisms are factors that may aftect the inhibitors' concentrations of NH-j in a notification system. 59 image: ------- Langowska and Moskal (1974) investigated the inhibitory effects of 24-hour exposures to NH-j on pure cultures of ammonifying and denitrifying bacteria. Effects examined were based on ability of the bacteria to produce some specific metabolic processes, such as proteolysis, aramonification, denitrification, and nitrification. Ammonifying and denitrifying bacteria were most resistant to MR-}', proteolytic and nitrifying bacteria were the most sensitive. Concentrations ranging from 0.8 to 170 mg/liter NH-j did not adversely affect denitrifying and ammonifying bacteria; 220 mg/liter caused reduction of the examined metabolic processes. Proteolytic bacteria were unaffected at 0.8 nig/liter NH^, but were reduced to zero at 4.2 mg/liter; nitrifying bacteria were unaffected at 2.6 to 5.1 rag/liter and reduced to zero at 13 to 25 mg/liter. Experimental data concerning the toxicity of ammonia to freshwater phytoplankton are limited. Przytocka-Jusiak (1976) reported ammonia effects (Table 4) on growth of Chlorella vulgaris with 50 percent inhibition in five days at 2.4 mg/liter NH-j, and complete growth inhibition in five days at 5.5 rag/liter. The MR-} Concentration resulting in 50 percent survival of C_. vulgaris after five days was found to be 9.8 mg/liter NH^• In a separate study, Przytocka-Jusiak et al. (1977) were able to isolate a C. vulgaris strain with enhanced tolerance to elevated ammonia concentrations, by prolonged incubation of the alga in ammonium carbonate solutions. C_. vulgaris was reported to grow well in solutions containing 4.4 mg/liter NH-}, but growth was inhibited at 7.4 mg/liter (Matusiak 1976). Tolerance to elevated concentrations of NH-j Seemed to show a slight increase when other forms of nitrogen were available to the alga than when ammonia was the only form of nitrogen In the medium. The effects of ammonia on growth of the algal species Ochromonas sqciabii is was studied by Brettliauer (1978). He 60 image: ------- fpund that concentrations (assuming pH 6.5 and 30 C) of 0.6 mg/liter NH3 killed the organisms, and at 0.3 mg/liter development of the population was reduced. Concentrations of 0.06 to 0.15 mg/liter NHg had insignificant effect on growth, and 0.015 to 0.03 mg/liter enhanced growth. Effects of ammonia on four algal species (Table 4) were studied by Abeliovich and Azov (1976). Ammonia at concentrations over 2.5 mg/liter NH3 inhibited photosynthesis and growth of the algal species Scenedesmus obliquus and inhibited photosynthesis of the algae Chlorella pyrenoidosa, Anacystis nidulans, and Plectonema boryanum. Hosier (1978) reported that NH^ concentrations causing 50 percent reduction in oxygen production by the green alga Chlorella ellipsoidea and blue-green alga Anabaena sub cy1indrica were 16.0 x 10~8 and 251.0 x 10~8 Vig NH3-N/cell, respectively. The rate of photosynthesis in the blue-green alga _P. boryanum was observed to be stimulated by NH^+, but inhibited by NH3 (Solomonson 1969); the magnitude of these effects was dependent on the sodium-potassium composition of the suspending media. NH3 inhibition of photosynthesis was associated with a conversion of inorganic polyphosphate stored in the cells to orthophosphate. Champ et al. (1973) treated a central Texas pond with ammonia to a mean concentration of 25.6 mg/liter Nl^. A diverse population of dinoflagel- lates, diatoms, desmids, and blue-green algae were present before ammonia treatment. Twenty-four hours after treatment the mean number of phytoplankton cells/liter was reduced by 84 percent. By the end of two weeks (NH-j = 3.6 mg/liter) the original concentration of cells had been reduced by 95 percent. Much of the work concerning the response of freshwater vegetation to high ammonia concentrations is only descriptive or is a result of research exploring the possible use of ammonia as an aquatic herbicide. 61 image: ------- Champ et al. (1973) reported virtually complete eradication of rooted aquatic vegetation (water shield, Brasenia schreberi, and American lotus, Nelumbo sp.) in a central Texas pond within two weeks after treatment with anhydrous ammonia; NHg concentration was 25.6 rag/liter 24 hours after ammonia addition, and 3.6 rag/liter two weeks later. In experiments with Potamogeton lucens, Litav and Lehrer (1978) observed that ammonia, which forms a readily available nitrogen source for the plant, can be toxic when present at high concentrations, with ammonia causing appreciable injury to detached branches. Ammonia inhibition of growth of Eurasian watermilfoil (Myriophyllum spicatum) affected length and weight similarly and affected roots and shoots similarly (Stanley 1974). Litav and Agami (1976) studied changes in vegetation in two rivers subject to increased pollution from agricultural fertilizers, urban sewage, and industrial wastes, and attributed the changes in plant species composition primarily to ammonia and detergents. Agami et al. (1976) transplanted seven species of "clean water" macrophytes to various sections of river, and found that ammonia affected only Nymphaea caerulea. Use of high concentrations of ammonia to eradicate aquatic vegetation was described by Ramachandran (1960), Ramachandran et al. (1975), and Ramachandran and Ramaprnbhu (1976). Saltwater Plants Data concerning the toxicity of ammonia to saltwater phytoplankton are presented in Table 4. Ten species of estuarine benthic diatoms were cultured for ten days in synthetic media at a range of NH3 Concentrations from 0.024 to 1.2 mg/liter NH3 (Admlraal 1977). A concentration of 0.24 mg/liter NH-j retarded the growth of most of the tested species (Table 4) . Relative tolerance to ammonium sulfate of five species of chrysomunads was studied by 62 image: ------- Pinter and Provasoli (1963). Coccolithus huxleyi was most sensitive, and Pavlova gyrans and Hymenomonas sp. were most tolerant, with intermediate tolerance exhibited by Syracosphaera sp. and Ochrosphaera neapolitana. Shilo and Shilo (1953, 1955) reported that the euryhaline algae Prynmesium parvum was effectively controlled with applications of ammonium sulfate, which exerted a lytic effect. Laboratory and field tests showed that the concentration of ammonium sulfate necessary for cell lysis decreased with increasing pH, indicating that un-ionized ammonia and not the ammonium ion is responsible for the lytic activity of ammonium sulfate on P^. parvum. Effect of ammonia on the dinoflagellate Amphidinium carterae was studied by Byerrura and Benson (1975), who reported that added ammonium ion at concentrations found to stimulate the photosynthetic rate also caused the algae to release up to 60 percent of fixed CC>2 to the medium. Natarajan (1970) found that the concentrations of fertilizer plant effluent toxic to natural phytoplankton (predominantly diatoms) in Cook Inlet, Alaska, were between 0.1 percent (1.1 mg/liter NH^) and 1.0 percent (11 mg/liter NH-j). At 0.1 percent effluent concentration * C uptake was reduced only 10 percent, whereas at 1.0 percent effluent concentration a 24-33 percent reduction in the relative C uptake was observed. Effects of ammonium sulfate on growth and photosynthesis of three diatom and two dinoflagellate species were reported by Thomas et al. (1980), who concluded that increased ammonium concentrations found near southern California sewage outfalls would not be inhibiting to phytoplankton in the vicinity. Provasoli and McLaughlin (1963) reported that ammonium sulfate was toxic to some marine dinoflagellates only at concentrations far exceeding those in seawater. No data were found concerning the toxicity of ammonia to saltwater vegetal ion. 63 image: ------- BIOACCUMULATION No data are available concerning the accumulation of ammonia by aquatic organisms. 64 image: ------- OTHER DATA A number of investigators have studied effects of ammonia on behavior and various metabolic processes of exposed animals, or have conducted field studies. This research has dealt predominantly with freshwater fishes. Freshwater Invertebrates The effect of ammonia (Table 5) on the ciliary beating rate of clam gills was investigated by Anderson et al. (1978). Concentrations of 0.036 to 0.11 ing/liter NHj caused a reduction in ciliary beating rate of fingernail clams; the effect of these concentrations ranged from 50 percent reduction in beating rate to complete inhibition of cilia. Adult clams (>5 mm) were more sensitive than juveniles (<5 mm); adults were also slightly more sensitive than the unionid mussel (Elliptic complanata) and the Asiatic clam (C. manilensis). Shaw (1960) investigated effects of ammonium chloride on sodium influx in the freshwater crayfish, Astacus pallipes. Ammonia produced an inhibition of sodium influx; a concentration of 18 rag/liter NH^+ reduced the influx to about 20 percent of its normal value, and influx reduction was related to greater ammonia concentration. This effect was attributed to NH^ ions and not to any toxic effect exerted on the transporting cells by un-ionized ammonia. NH^"1" did not affect chloride influx nor the rate of sodium loss. Ammonia was added to a Kansas stream at a 24-hour average concentration of 1.4 mg/liter NH3, and a 24-hour drift net sampling was conducted (Liechti and Muggins 1980). No change in diel drift pattern was observed, but there was an increase in magnitude of drift, a shift in kinds of organisns present, and changes in benthic standing crop estimates; the ammonia concentration was concluded to be nonlethal. image: ------- Fre shwa te r Fi she s Herbert and Shurben (1963) Investigated the effect on susceptibility to ammonium chloride solutions of rainbow trout forced to swim continuously against water currents of different velocities prior to ammonia exposure. Forcing rainbow trout to swim for one to two days at 85 percent of the maximum velocity they could sustain increased their susceptibility only slightly, corresponding to a 20 to 30 percent reduction in the 24- or 48-hour LC50. The behavioral response of blacknose dace (Rhinichthys atratulus) to ammonium chloride solutions has been studied (Tsai and Fava 1975; Fava and Tsai 1976); the test fish did not avoid concentrations of 0.56 or 4.9 mg/liter NH^, nor did these concentrations cause significant changes in activity. Avoidance studies were conducted by Westlake and Lubinski (1976) with bluegill using ammonium chloride solutions. Bluegill detected concentrations of approximately 0.01 to 0.1 mg/liter NH3» and evidenced a decrease in general locomotor activity. No apparent avoidance of ammonia was observed, and there was some indication of an attraction. Behavioral responses of bluegill to a five-hour exposure to 0.040 mg/liter XH-j> although variable, were related to at least a small amount of physiological stress either at the gill or olfactory surfaces. At a concentration of 0.004 mg/liter NH^, bluegill evidenced slight temporary increases in both activity and turning behavior; no preference or avoidance was demonstrated, with responses seemingly exploratory (Lubinski et al. 1978, 1980). Wells (1915) investigated the avoidance behavior of bluegill to ammonium hydroxide solutions and reported that fishes did not avoid ammonia prior to being killed by it. In a study of the repelling ability of chemicals to green sunfish, Summerfelt and Lewis (1967) concluded that concentrations of ammcaia 66 image: ------- 'high enough to repel fish would ho rapidly tcit» .-»v>>tJtn, ,-i <-«.»>..-i f.-u->u i with threespine stickleback, solutions of ammonia concentration O.J/ mg/liter NH-j elicited a positive (attraction) response from the test fish (Jones 1948). Weltering et al. (1978), in tests with largemouth bass and mosquitofish, demonstrated that predator-prey interactions were sensitive to sublethal concentrations of NHj- Ammonia concentrations of 0.63 and 0.86 mg/liter NH3 decreased prey consumption and bass growth; bass were reported to be more sensitive than mosquitofish to NH3« The effect of ammonium chloride on consumption of juvenile chinook salmon by brook trout was studied by Hedtke and Norris (1980). At the lowest test concentration of 0.29 mg/liter NH3> trout consumption rates decreased as much as 65 percent. As ammonia concentration increased, however, consumption of prey increased and was double that of controls at the highest tested concentration of 0.76 mg/liter NH-j- Increased consumption rate was related to both increased NH-j concentration and increased prey density. The magnitude of the effect of ammonia was not the same at all prey densities, having a greater effect on consumption rate at high than at low prey densities. Mortalities were observed among prey salmon at the highest NH3 levels, and these were attributed to the combined effect of NH^ and stress from presence of the predator. Brook trout exhibited toxic effects due to NH-j • NH/^Cl and NH^HcOg Solutions were injected intraarterially into rainbow trout (Hillaby and Randall 1979). The same dose of each compound was required to kill fish, but there was a more rapid excretion of NH3 after NH4HC03 infusions, resulting in higher NH3 Concentrations in blood, than after NH^Cl infusions. Ammonium acetate solutions of different concern rations were injected intraperltoneally into three species of fishes 67 image: ------- (Wilson 1968; Wilson et al. 1969). LD50 values (mmoles/kg body weight) for channel catfish for one to four hours was 26.7 to 18.7, for goldfish for one hour were 29.3 and 29.6 in two separate tests, and for rainbow trout for one hour was 17.7. Goldfish was the most resistant species tested and rainbow trout the least resistant. Nehring (1964) compared toxicity of ammonia in the water to toxicity of ammonia administered orally and concluded that the threshold and lethal concentration values were considerably lower for ammonia in water than for ammonia administered orally. Acute symptoms of NH-j toxicity to brown trout sac fry and 12-day-old fry were described by Penaz (1965), who exposed fry to concentrations ranging from 0.08 to 50.0 mg/liter NH^- Symptoms caused by Ntt-^ exposures were: rapid spasm-like movements at concentrations of 2.0 mg/liter NH-j and higher within 16-17 minutes of exposure; after 40 minutes these symptoms were also observed at 0.4 mg/liter NH-j > After 2.5 hours these abnormal movements ceased, and at 10 hours heart activity was decreased and fish lost movement ability at the higher (>2.0 mg/liter N^) concentrations. Other symptoms included Inability to react to mechanical stimulation and disorders in rhythm of mouth movements culminating in the mouth's staying rigidly open. Thumann (1950), working with rainbow trout and brook (=brown?) trout, described observed symptoms of ammonia poisoning to fishes to be convulsions and frequent equilibrium and positional anomalies. Smart (1978) reported that exposure of rainbow trout lo an acutely lethal concentration of 0.73 mg/liter NH^ resulted in an increase in oxygen consumption, increase in ventilation volume, decrease in percent oxygen utilization, increase in respiratory frequency and amplitude (buccal pressure), decrease in dorsal aortic blood PQ? » increase in dorsal aortic blood pressure, and increase in mean heart rate. Physiological parameters 68 image: ------- not significantly affected by NH_-j exposure were "cough" race, dorsal aort li- blood pH, blood P50, erythrocyte count, heraatocrit, and hemoglobin concentration. Coho salmon exposed to concentrations ranging from 0.094 to 0.162 rag/liter Nll-$ (Souea and Meade 1977) exhibited hyperexcitability, hyperventilation, ataxia, and progressive acidemia; metheraoglobin concentrations in blood of exposed fish did not differ significantly from those of controls. Effects on trout (species not specified) blood with exposure to accumulated excreted NHg were investigated by Phillips et al. (1949) and were reported to include an increase in blood carbon dioxide content and a decrease in oxygen content. Arillo et al. (1979d) measured gill sialic acid content in rainbow trout exposed to NH^Ofl or NH^Cl solutions ranging from 0.05 to 0.5 mg/liter NH-j, and reported that increasing NH^ concentrations produced increasing gill sialic acid content. Elevated gill sialic acid levels were also produced by higher ammonium ion (NH^ ) concentrations at identical NH^ concentrations, and the authors concluded that NH^ was a stressor causing elevated sialic acid levels. Exposure of rainbow trout (14-cm length) for four hours to NH^Cl and NH^On solutions of concentrations ranging from 0.094 to 0.50 mg/ltter NH^ resulted in increased proteolytic activity and free amino acid levels in the fish livers, but no statistically significant change in fructose 1,6-biphosphatase enzyme activity (Arillo et al. 1978, 1979a). Renal renin activity was reported (Arillo et al. 1931b) to increase in rainbow trout exposed to concentrations of 0.048 to 0.61 rag/liter NH-j • A significant decrease in liver glycogen and increase in free glucose were observed in rainbow trout exposed to NH^Cl solutions for four hours at a concentration of 0.048 mg/liter NH3, and a decrease in total carbo- hydrates was observed at 0.12 mg/liter NH3 (Arillo et al. 1979b). For 69 image: ------- trout similarly treated with .umnonlum hydroxide, .-slgivif ti-aui. Uei-redsiea ii> glycogen and carbohydrates, and increase in glucose occurred at 0.097 mg/liter N!^. A statistically significant increase in rainbow trout liver concentrations of cyclic-31,5'-adenosine-raonophosphate (cAMP) was reported by Arillo et al. (1979c) to be induced by a four-hour exposure to elevated ammonia concentrations of 0.011 to 0.124 mg/liter NH3« Decreases in liver glycogen levels were also measured and were significantly different from controls only in the trout exposed to 0.048 mg/liter NHj, the highest exposure used for glycogen measurements. The authors concluded that cAMP measurements provided a very sensitive means of discerning fish stress even at very low toxicant concentrations, although quantitative measurement of stress intensity was not possible. Lysosoraal lability was also investigated as an indicator of stress in rainbow trout due to ammonia exposure (Arillo et al. 1980) , and was reported to increase significantly for fish subjected to concentrations of 0.048 to 0.61 mg/liter NHj. Exposure of rainbow trout for four to 48 hours to 0.024 to 0.61 mg/liter NH3 resulted in changes in various brain and liver metabolites; the magnitude of the changes was depen- dent on both exposure time and NH-j concentration (Arillo et al- 1981a) . Exposure of walking catfish (Clarias batrachus) to ammonia caused inhibition of fish brain cholinesterase and kidney peroxidase activity (Mukherjee and Bhattacharya 1974, 1975a). Plasma corticosteroid concentrations were measured (Tomasso et al. 1981) in channel catfish exposed to 1.1 mg/liter NH^ for 24 hours; corticosteroid levels increased initially, peaked after eight hours, then decreased. The overall increase was approximately tenfold over normal levels. Korting (I969b) reported that carp exposed to 1 mg/liter NH-j exhibited an increase in number of blood erythrocytes, reaching an initial maximum 70 image: ------- after several hours followed by a gradual decrease; after 50 hours the number was less than the average for non-exposed fish. Other blood changes from the ammonia exposure were: thickening of individual erythrocytes, reduction of osmotic resistance of erythrocytes, increase in concentrations of urea and lactic acid, and decrease in ATP concentration. Levi et al. (1974) reported that goldfish exposed for 24 hours to NH^Cl solutions exhibited increases in cerebral and blood concentrations of glutamine and in other amino acids, with changes most pronounced in the brain. Concentrations of free amino acids in livers showed only slight increases of a few amino acids, including glutamine, and the concentration of lysine decreased. No change in concentrations of free amino acids was observed in kidneys. Rainbow trout exposed to 0.33 mg/liter NH^ had significantly higher packed cell volumes; exposures to concentrations of 0.24 rag/liter NH^ and higher resulted in significantly raised blood glucose and plasma cortisol concentrations (Swift 1981). Diuretic response of rainbow trout exposed to concentrations of 0.09 to 0.45 mg/liter NH3 was studied by Lloyd and Orr (1969). After an initial lag period, urine production increased rapidly during exposure then returned to normal within a few hours after discontinuation of NH3 exposure. A no-obs^rved-effect concentration was reported to be 0.046 mg/liter NHo - Goldfish were exposed to solutions containing 1.0 to 1.9 mg/liter NH^ (Fromm 1970; Olson and Fromm 1971); onset of death was characterized by a gradual cessation of swimming movements and settling to the bottora of the tank. Some goldfish near death were returned to ammonia-free water in which they recovered to at least some degree. In similar experiments (Fromm 1970; Olson and Fromm 1971) rainbow trout were exposed to ambient total ammonia concentrations of 0.04 to 0.2 mg/litei NH3• There wad a decrease in total 71 image: ------- nitrogen excreted with increase in ambient NH-p and a concomitant decrease in the. NH^ portion of total nitrogen excreted; urea and protein nitrogen excretion rates showed no changes as ambient NH-j increased. Onset of death for trout was characterized by violent thrashing movements. Exposure of rainbow trout to solutions of NH^Ci for 24 hours (Froram and Gillette 1968; Fromm 1970) showed that an increase in ambient water NH3 concentration resulted in a corresponding increase in blood NH-j concentrations, and a decrease in total nitrogen and NH-j excretion. The decrease in NH^ excretion accounted for half or less of the total nitrogen excretion, depending on the water NH^ concentration, indicating that the reduction in NH3 excretion was to some extent compensated for by increased excretion of some other nitrogenous compound(s). Young fry (2-20 days old) of loach (Misgurunus anguilicaudatus) and carp were exposed for five to 70 hours to N-labeled ammonium chloride solutions at six concentrations from 0.002 to 0.064 mg/liter NH-j (ito 1976), and the proportion of ^N relative to total N in the fishes determined. Ammonia was shown to be directly absorbed by the fry; nitrogen conversion rate increased with increasing ammonia concentration and exposure time. Nitrogen conversion rates for carp fry decreased as fry age increased from 3 to 20 days. After 48 hours of exposure to 0.064 mg/liter NH-j followed by transfer to ammonia-free water, rapid excretion (15-20 percent) of the absorbed N occurred during the first hour in ammonia-free water. Excretion rate then slowed, with about 50 percent of the absorbed N being retained after 48 hours in ammonia-free water. Comparison of N absorption rates between live and sacrificed three-day-old carp fry showed one-third to one-half the uptake of N by dead fry compared with live, 12 image: ------- indicating that the uptake of ammonia from water by live fish occurs not only by simple membrane permeation but also by metabolic action. Flagg and Hinck (1978) reported that exposure to NH3 lowered the resistance of channel catfish to the pathogen Aeromonas hydrophila. In 17- and 28-day tests, increasing exposure concentrations from 0.02 to 0.04 rag/liter NH^ resulted in increasing numbers of bacteria in host livers. Schreckenbach et al. (1975) reported that ammonia in pond water leads to outbreaks of gill necrosis in carp, accompanied by an increase in ammonia concentration in serum of the fish. This is aggravated at elevated pH levels due to increasing inhibition of ammonia excretion at increasing pH levels, with ammonia excretion being almost totally blocked at pH values above 10.5. After investigating the possible role of parasites, bacteria, viruses, and other ultramicroscopic agents in causing gill necrosis, the authors concluded that pH-dependent intoxication or autointoxication with ammonia was the sole cause of the gill damage. Studies of the treatment and prophylaxis of gill necrosis using 28 different therapeutical preparations led to the conclusion that only those preparations that lowered the water pH level and/or ammonia concentrations resulted in an improvement in clinical symptoms. Increase in frequency of opercular rhythm in fishes was monitored as a means to measure fish response to sublethal concentrations of ammonia (Morgan 1976, 1977). Ammonia threshold detection concentration (Table 5) for largemouth bass was approximately 30 percent of the lethal concentration (LC50) for that species. Increases in largemouth bass opercular rhythms and activity were electronically monitored (Morgan 1978, 1979) to determine threshold effect ammonia concentrations (Table 5); for a 24-hour exposure the effect concentration for opercular rhythms was 0.028 mg/liter NH-j and for activity was 0.0055 mg/liter. Lubinski et al. (1974) observed that ammonia stress apparently caused bluegill to consume more oxygen. 73 image: ------- In field experiments in an Arizona mountain lake, mortalities of oa rainbow trout were attributed to high un-ionized ammonia concentrations and high pH levels; 20 to 100 percent of test fish died in 24 hours at NH^ concentrations of 0.109 to 0.225 mg/liter (Fisher and Ziebell 1980). Ammonia added to a Kansas stream at 24-hour average concentrations of 1.4 mg/liter NH3 resulted in fry of slender madtora (Notorus exllis), Notropis sp., and orangethroat darter being collected in large numbers in a 24-hour drift net sampling; these fishes are not normally found in drift net samples, and their presence was attributed to toxic effects of the ammonia (Liechti and Huggins 1980). Saltwater Invertebrates Sublethal toxicity of NH^Cl to the quahog clam and eastern oyster was studied by Epifanio and Srna (1975) who measured the effect of ammonia over 20 hours on the rate of removal of algae (Isochrysis galbana) from suspension (clearing rate) by the clams and oysters. Concentrations of 0.06 to 0.2 mg/liter MH3 affected clearing; no difference was observed between juveniles and adults. The effect of ammonia on the ciliary beating rate of the mussel Mytilus edulis was studied by Anderson et al. (1978). Concentrations of 0.097 to 0.12 mg/liter NH3 resulted in a reduction in ciliary beating rate from 50 percent to complete inhibition (Table 5). Exposure of unfertilized sea urchin (Lytechinus pictus) eggs to NH^Cl resulted in stimulation of the initial rate of protein synthesis, an event that normally follows fertilization (Winkler and Grainger 1978). NH^l exposure of unfertilized eggs of St rongylocent rot us purpuratus, L^. pictus, and Strongylocentrotus drobachiensis was reported (Paul et al. 1976; Johnson et al. 1976) to cause release of "fertilization acid", more rapidly and in greater amounts than after insemination. Activation of unfertilized L_. pictus eggs by NH/jCl exposure was also evidenced by an increase in 74 image: ------- Intracellular pH (Shen ami Stelnhardt 1978; Steinhardt and Mazia 1973). Ammonia treatment was also reported to activate phosphorylation of thymidirce and synthesis of histones in unfertilized eggs of the sea urchin S^. purpuratus (Nishioka 1976). Premature chromosome condensation was Induced by ammonia treatment of eggs of L. pictus and S. purpuratus (Epel et al. 1974; Wilt and Mazia 1974; Krystal and Poccia 1979). Ammonia treatment of S^. purpuratus and S. drobachiensis fertilized eggs resulted in absence of the normal uptake of calcium following insemination, but did not inhibit calcium uptake if ammonia treatment preceded insemination (Paul and Johnston 1978). The polychetous annelid (Nereis succinea), the channeled whelk (Busycon canalieulaturn), and the brackish water clam (Rangia cuneata) were subjected to ammonia concentrations of 0.85, 0.37, and 2.7 mg/liter NH3 and ammonia excretion measured (Mangura et al. 1978). The excretion of ammonia in these species was inhibited by non-lethal concentrations of ammonia; the authors concluded that ammonia crosses the excretory epithelium in the ionized form, and that the process is linked to the activity of the Na+ + K+ ATPases. When blue crab (Callinectes sapidus) were moved from water of 28 ppt salinity to water of 5 ppt, a doubling of ammonia excretion rate occurred; addition of excess NH^Ci to the low salinity water inhibited ammonia excretion and decreased net acid output (Mangura et al. 1976). The effect of gaseous NH3 on hemoglobin from blood of the common marine bloodworm (Glyeera dibrachiata) was examined (Sousa et al. 1977) in an attempt to determine whether there was competition between NH^ and oxygen in binding to hemoglobin; such an NH-j/02 relationship was not found. Saltwater Fishes Mo other data were found for saltwater fish species. image: ------- UNUSED DATA Many references cited In the References section were not used in the text or tables, for a variety of reasons. Listed below are unused references, with a brief explanation for their being relegated to this category. For those several cases where more than one reason applies to a given paper, it is listed only under the principal reason for its not being used. The following references were not used because the research they reported was conducted using aquatic organisms not resident in North America: Alderson (1979), Arizzi and Nicotra (1980), Brown and Currie (1973), Brownell (1980), Chin (1976), Currie et al. (1974) Dockal and Varecha (1967), D'Silva and Verlencar (1976), Giussani et al. (1976), Greenwood and Brown (1974), Grygierek et al. (1978), Inamura (1951), Nicotra and Arizzi (1980), Orzechowski (1974), Reddy and Menon (1979), Sadler (1981), Saha et al. (1956), Shaffi (1980b), Singh et al. (1967), Stroganov and Pozhitkov (1941), Thomas et al. (1976), Turoboyski (1960), Vailati (1979), Woker (1949), Wuhrmann (1952), Wuhrmann and Woker (1953), Wuhrmann and Woker (1955), Wuhrmann and Woker (1958), Yamagata and Niwa (1982). The following references were not used because insufficient water chemical composition data were provided to permit calculation of NH-j: Belding (1927), Binstock and Lecar (1969), Chu (1943), Danielewski (1979), Das (1980), Ellis (1937), Hepher (1959), Joy and Sathyanesan (1977), Kawamoto (1961), Mukherjee and Bhattacharya (1978), Oshima (1931), Oya et al. (1939), Patrick et al. (1968), Rao and Ragothaman (1978), Roberts (1975), Rushton (1921), Scidmore (1957), Shelford (1917), Shevtsova et al. (1979), Sigel et al. (1972), Southgate (1950), Wolf (1957a), Wolf (1957b), Zgurovskaya and Kustenko (1968). 76 image: ------- The following references were not used because they report or) published elsewhere which was cited in this Jiviuarut ! row t h«» .»thoi publicatlon(s): Burkhalter (1975), Colt (1974), Dept. of Environment, U.K. (1972), Herbert (1955), Hillaby (1978), Larmoyeux and Piper (1973), Ministry of Technology, U.K. (I960), Ministry of Technology, U.K. (1966), Rice (1971), Smart (1975), Wilson (1974). The following references were not used because they were foreign- language papers for which no translation was available, and no useful information could be obtained from the abstract: Desavelle and Hubault (1951), Fedorov and Smirnova (1978), Frahm (1975), Garcia-Romeu and Motais (1966), Guerra and Comodo (1972), Guseva (1937), Hubault (1955), Jocque and Persoone (1970), Kawamoto (1958), Korting (1976), Krauss (1937), Kuhn and Koecke (1956), Leclerc and Devlaminck (1950), Maraontova (1962), Oya et al. (1939), Pequignot and Moga (1975), Pora and Precup (1971), Revina (1964), Saeki (1965), Schaperclaus (1952), Scheuring and Leopoldseder (1934), Schreckenbach and Spangenberg (1978), Steinmann and Surbeck (1922a), Steinmann and Surbeck (1922b), Svobodova (1970), Svobodova and Groch (1971), Teulon and Simeon (1966), Truelle (1956), Varaos and Tasnadi (1962a), Vamos and Tasnadi (1962b), Vamos et al. (1974), Yasunaga (1976), Yoshihara and Abe (1955). The following references were not used because they relate more to ammonia metabolism in fishes, than to ammonia toxicity: Bartberger and Pierce (1976), Becker and Schmale (1978), Brett and 7,ala (1975), Cowey and Sargent (1979), Creach et al. (1969), Cvancara (1969a), Cvancara (1969b), De and Bhattacharya (1976), De Vooys (1968), De Vooys (1969), Driedzic and Hochachka (1978), Fauconneau and Luquet (1979), Fechter (1973), Fellows and Hird (1979a), Fellows and Hird (1979b), Flis (1968a), Flis (1963b), Florkin and Duchateau (1943), Forster and Goldstein (1966), Forster and Goldstein 77 image: ------- (I960), Fromm (1963), Cirard and Payan (1980), Goldstein and Forster (1961), Goldstein and Forster (1965), Goldstein et al. (1964), Gordon (1970), Gregory (1977), Grollman (1929), Guerin-Ancey (1976a), Guerin-Ancey (1976b), Guerin-Ancey (1976c), Guerin-Ancey (1976d), Hays et al. (1977), Hoar (1958), Muggins et al. (1969), Janicki and Lingis (1970), Katz (1979), Kaushik and Luquet (1977), Kloppick et al. (1967), Kutty (1978), Lawrence et al. (1957), /» Lum and Hatnraen (1964), Maetz (1973), Maetz and Garcia-Roraeu (1964), Makarewicz and Zydowo (1962), Mason (1979a), Mason (1979b), Matter (1966), McBean et al. (1966), McKhann and Tower (1961), Moore et al. (1963), Morii et al. (1978), Morii (1979), Morii et al. (1979), Mukherjee and Bhattacharya (1977), Nelson et al. (1977), Payan (1978), Payan and Maetz (1973), Payan and Matty (1975), Payan and Pic (1977), Pequin and Serfaty (1963), Pequin and Serfaty (1966), Pequin and Serfaty (1968), Pequin et al. (I969a), Pequin et al. (1969b), Raguse-Degener et al. (1980), Ray and Medda (1976), Read (1971), Rice and Stokes (1974), Rychley and Marina (1977), Savitz (1969), Savitz (1971), Savitz (1973), Savitz et al. (1977), Schooler et al. (1966), Smith (1929), Smith (1946), Smith and Thorpe (1976), Smith and Thorpe (1977), Storozhuk (1970), Sukumaran and Kutty (1977), Tandon and Chandra (1977), Thornburn and Matty (1963), Vellas and Serfaty (1974), Walton and Cowey (1977), Watts and Watts (1974), Webb and Brown (1976), Wood (1958), Wood and Caldwell (1978). The following references were not used because the material the authors used was a complex compound or had an anion that might in itself be toxic: Curtis et al. (1979), Johnson and Sanders (1977), Simonin and Pierron (1937), Vallejo-Freire et al. (1954). The following references were not used because they dealt with complex effluents or waste waters, of which ammonia was a primary component: Brown 78 image: ------- •'et al. (1970), Calamari and Marchetti (I1)?1*). <7upM et al. (t, Twan ami Cella (1979), Janicke and Liidemann (1967), Lloyd and Jordan (196J), Lloyd and Jordan (1964), Martens and Servizi (1976), Matthews and Myers (1976), Mihnea (1978), Nedwell (1973), Okaichi and Nishio (1976), Perna (1971), Rosenberg et al. (1967), Ruffier et al. (1981), Sahai and Singh (1977), Shaffi (1980a), Vamos (1962), Vamos and Tasnadi (1972), Ward et al. (1982). Three references consisted only of an abstract, providing insufficient information to warrant their use: Liebmann and Reichenbach-Klinke (1969), Mukherjee and Bhattacharya (1975b), and Redner et al. (1980). 79 image: ------- SUMMARY All concentrations used herein are expressed as un-ionized ammonia (Nil-}), because NH^, not the ammonium ion (NH^ ) has been demonstrated to be the principal toxic form of ammonia. The data used in deriving the criteria are predominantly from flow-through tests in which ammonia concentrations were measured. Ammonia was reported to be acutely toxic to freshwater organisms at concentrations (uncorrected for pH) ranging from 0.53 to 8.00 rag/liter NH-j for 12 Invertebrate species representing nine families and from 0.083 to 4.60 mg/liter NH3 for 23 fish species from nine families. Among fish species, reported 96-hour LC50 values ranged from 0.083 to 1.09 mg/liter for salmonids and from 0.14 to 4.60 mg/liter NH-j for non-salmonids. Reported data from chronic or partial chronic tests on ammonia with two freshwater invertebrate species, both daphnids, showed effects at concentra- tions (uncorrected for pH) ranging from 0.304 to 1.2 mg/liter Nt^i and with nine freshwater fish species, from five families, ranging from 0.0017 to 0.612 mg/liter NH3- Concentrations of ammonia acutely toxic to fishes may cause loss of equilibrium, hyperexcitability, increased breathing, cardiac output and oxygen uptake, and, in extreme cases, convulsions, coma and death. At lower concentrations ammonia has many effects on fishes including a reduction in hatching success, reduction in growth rate and morphological development, and pathologic changes in tissues of gills, livers, and kidneys. Several factors have been shown to modify acute NH3 toxicity in fresh water. Some factors alter the concentration of un-ionized ammonia in the water by affecting the aqueous ammonia equilibrium, and some factors affect the toxicity of un-ionized ammonia itself, either ameliorating or exacer- bating the effects of ammonia. Factors that have been shown to affect 80 image: ------- ammonia toxicity include dissolved oxygen concentration, temperature, pH, previous acclimation to ammonia, fluctuating or intermittent exposures, carbon dioxide concentration, salinity, and the presence of other toxicants. The most well-studied of these is pH; the acute toxicity of NH-j has been shown to increase as pH decreases. Sufficient data exist from toxicity tests conducted at different pH values to formulate a mathematical expression to describe pH-dependent acute NH^ toxicity. The very limited amount of data regarding effects of pH on chronic NH^ toxicity also indicate increasing NH-j toxicity with decreasing pH, but the data are insufficient to derive a broadly applicable toxicity/pH relationship. Data on temperature effects on acute NH^ toxicity are limited, and somewhat variable, but indications are that NH3 toxicity is greater at low (<10 C) temperatures. There is no information available regarding temperature effects on chronic NH3 toxicity. Examination of pH-corrected acute NH-j toxicity values among families of freshwater organisms tested showed that the most sensitive families are Percidae and Salraonidae, with walleye and rainbow trout being the most sensitive tested species in each of these families; invertebrates are generally more tolerant than fishes. Available chronic toxicity data for freshwater organisms show that the most sensitive families among those tested are Salmonidae and Catostomldae (suckers), with pink salmon and white sucker being the most sensitive tested species in these families. Limited data for invertebrates, mostly cladocerans and one insect species, indicate they are generally more tolerant than fishes; however, the fingernail clam appears to be as sensitive as salmonids. The range of acute/chronic ratios for ten species from six families was 2.8 to 44, and acute/chronic ratios were higher for the species found to be the most sensitive on -in acute (pH-corrected) 81 image: ------- basis. Available data indicate that differences in sensitivities between warm and cold water families of aquatic organisms are inadequate to warrant discrimination in the national ammonia criterion between bodies of water with "warm" and "cold" water fishes; rather, effects of organism sensitivities on the criterion are most appropriately handled by site-specific criteria derivation procedures. Data for concentrations of NH3 toxic to freshwater phytoplankton and vascular plants, although limited, indicate that freshwater plant species are appreciably more tolerant to NH^ than are invertebrates or fishes. The ammonia criterion appropriate for the protection of aquatic animals will therefore in all likelihood be sufficiently protective of plant life. Available acute and chronic data for ammonia with saltwater organisms are very limited, and insufficient to derive a saltwater criterion. A few saltwater invertebrate species have been tested, and the prawn Macrobrachium rosenbergii was the most sensitive. Acute toxicity of NH^ appears to be greater at low pH values, similar to findings in freshwater. Data for saltwater plant species are limited to diatoms, which appear to be more sensitive than the saltwater invertebrates for which data are available. Although a great deal of information has been published about ammonia toxicity to aquatic life, much of it provides little, if any, quantitative data. There are some key research needs that need to be addressed in order to provide a more complete assessment of ammonia toxicity. These are: (1) acute tests with saltwater fish species, and additional saltwater invertebrate species; (2) life-cycle and early-life-stage tests with representative freshwater and saltwater organisms from different families, with investigation of pH effects on chronic toxicity; (3) fluctuating or intermittent exposure tests for a variety of species anJ exposure patterns; 82 image: ------- (4) tests at cold water temperatures, both acute and chronic; (5) more hlstopathological and histochemical research with fishes, which would provide a rapid means of identifying and quantifying sublethal ammonia effects; (6) studies on effects of dissolved and suspended solids on acute and chronic toxicity. 83 image: ------- NATIONAL CRITERIA To protect freshwater aquatic life, the criteria for ammonia (in rag/liter un-ionized NH-j) are: (1) the concentration at all times should be less than or equal to the numerical value given by 0.15 x f(T)/f(pH), where f(pH) - 1 + 10l-03(7.32-pH) f(T) - I, T _> 10 C * 1 4- 109-73-pH pKT-pH ' T < 10 C (2) The average concentration over any 30 consecutive days should be less than or equal to 0.031 x f(T)/f(pH), where f(pH) = 1, pH _> 7.7 » 100.74(7.7-pH)> pH < 7.7 f(T) « 1, T >_ 10 C ! + iQ9.73-pH * 1 + iOPKT~pH • T < 10 C Criteria values for the pH range 6.5 to 9.0 and the temperature range 0 C to 30 C are provided in the following tables. Total ammonia concentrations equivalent to each NH-j criterion are also provided in these tables. Data available for saltwater species are insufficient to derive a criterion for saltwater. 84 image: ------- (1) Maximum allowed concentrations for ammonia.* pH 0 C 5 C 10 C 15 C 20 C 25 C 30 C Un-ionized Ammonia (rag/liter NHj) 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 0.008 0.014 0.021 0.030 0.040 0.049 0.056 0.061 0.065 0.068 0.071 31.9 29.5 25.7 20.8 15.5 10.6 6.84 4.22 2.54 1.53 0.94 0.013 0.021 0.032 0.046 0.061 0.074 0.084 0.091 0.096 0.100 0.104 31.9 29.5 25.7 20.8 15.5 10.6 6.84 4.22 2.54 1.53 0.94 0.019 0.031 0.048 0.069 0.091 0.110 0.125 0.135 0.141 0.145 0.147 Total Ammonia 31.9 29.5 25.7 20.8 15.5 10.6 6.84 4.22 2.54 1.53 0.94 0.019 0.031 0.048 0.069 0.091 0.110 0.125 0.135 0.141 0.145 0.147 (mg/liter 21.8 20.1 17.6 14.2 10.6 7.29 4.71 2.92 1.78 1.09 0.69 0.019 0.031 0.048 0.069 0.091 0.110 0.125 0.135 0.141 0.145 0.147 NH3) 15.0 13.9 12.1 9.84 7.34 5.06 3.28 2.05 1.27 0.80 0.52 0.019 0.031 0.048 0.069 0.091 0.110 0.125 0.135 0.141 0.145 0.147 10.5 9.69 8.48 6.89 5.15 3.56 2.33 1.47 0.93 0.60 0.41 0.019 0.031 0.048 0.069 0.091 0.110 0.125 0.135 0.141 0.145 0.147 7.41 6.86 6.00 4.88 3.66 2.55 1.68 1.08 0.70 0.47 0.33 * To convert these values to mg/liter N, multiply by 0.822. 85 image: ------- (2) 30-day average allowed concentrations for ammonia.* pH 0 C 5 C 10 C 15 C Un- ionized Ammonia (mg/liter 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 9.00 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 S.75 9.00 0.0018 0.0027 0.0042 0.0064 0.0098 0.0138 0.0139 0.0140 0.0142 0.0145 0.0150 6.82 5.87 5.06 4.36 3.77 2.99 1.70 0.97 0.56 0.33 0.20 0.0027 0.0041 0.0063 0.0096 0.0148 0.0208 0.0209 0.0210 0.0211 0.0214 0.0219 Total 6.82 5.87 5.06 4.36 3.77 2.99 1.70 0.97 0.56 0.33 0.20 0.0040 0.0061 0.0094 0.0144 0.0220 0.0310 0.0310 0.0310 0.0310 0.0310 0.0310 Ammonia 6.83 5.89 5.07 4.37 3.78 3.00 1.70 0.97 0.56 0.33 0.20 0.0040 0.0061 0.0094 0.0144 0.0220 0.0310 0.0310 0.0310 0.0310 0.0310 0.0310 (mg/liter NH3 4.65 4.01 3.45 2.98 2.58 2.05 1.17 0.67 0.39 0.23 0.14 20 C NH3) 0.0040 0.0061 0.0094 0.0144 0.0220 0.0310 0.0310 0.0310 0.0310 0.0310 0.0310 ) 3.21 2.76 2.38 2.06 1.78 1.42 0.81 0.47 0.28 0.17 0.11 25 C 0.0040 0.0061 0.0094 0.0144 0.0220 0.0310 0.0310 0.0310 0.0310 0.0310 0.0310 2.24 1.93 1.67 1.44 1.25 1.00 0.58 0.34 0.20 0.13 0.09 30 C 0.0040 0.0061 0.0094 0.0144 0.0220 0.0310 0.0310 0.0310 0.0310 0.0310 0.0310 1.58 1.37 1.18 1.02 0.89 0.72 0.42 0.25 0.15 0.10 0.07 * To convert these values to rag/liter N, multiply by 0.822. 86 image: ------- KXAMP1.KS OF SITR-SrKOU'lO CR National criteria are subject to modification, if appropriate, to reflect local conditions. One method provided in the Site-Specific Criteria Guidelines (U.S. Environmental Protection Agency 1982b) for such modification is to base certain calculations only on those species that occur in the body of water of interest. As an example of how site-specific criteria for ammonia may differ from the national criteria, calculations were performed for several sites. The sites were chosen on the basis of readily available information on the presence of fish and invertebrate species and on a reasonable diversity between sites. The sites were: (1) St. Louis Bay - mouth of the St. Louis River on Lake Superior at Duluth, Minnesota/Superior, Wisconsin (A. R. Carlson, pers. comra. 1982) (2) Straight River at Owatonna, Minnesota (A. R. Carlson, pers. comm. 1982) (3) Piceance Creek, Colorado (Goettl and Edde 1978; Gray and Ward 1978) (4) Poudre River at Fort Collins, Colorado (W. T. Willingham, pers. comm. 1982) (5) Colorado River at Utah/Colorado border (W. T. Willingham, pers. comm. 1983) The calculations here are limited to temperatures of 15 C and to a single pH typical of the site (see following table). Therefore, the criteria here should not be considered as final criteria for the sites because variation with pH and temperature was not explored. For each site, available surveys of species occurrence were used to identify which of the families tested for acute toxicity (Table 3) were image: ------- present. Minimum data set requirements for the diversity of families were met except where inappropriate to a site (U.S. Environmental Protection Agency 1982b). The national LIMFMAV (Table 3) was used for each family, even if it is based in part or whole on species not occurring at the site, unless LIMSMAVs were available for all species in the family at the site, in which case a site-specific LIMFMAV was computed that was based only on site species. The LIMFMAVs so developed for each site are listed in the following table. The Guidelines method for estimating the FAV as the fifth percentile of FMAVs was applied to the set of LIMFMAVs selected for each site. If the LIMFAVs so computed exceeded the LIMSMAV of an important species at a site, or of an important size class of an important species, the LIMFAV was lowered to the lowest such LIMSMAV. The FAVs at each site were then computed by adjusting the LIMFAVs to the site pHs, using the acute pH relationship already discussed. The maximum criterion at each site was set to one-half of the site FAV. In all cases, the site pH was >7,5, so an acute/chronic ratio of 16 was used. At sites 2, 4, and 5, this ratio was applied directly to the FAV to obtain an FCV. At sites 1 and 3, where the LIMFAV was lowered to reflect an age/size class, the ratio was applied to the value the FAV would have had if the LIMFAV was only lowered to LIMAVs averaged over all data for a species, not just an age/size class. If the resultant PCV at a site exceeded the average chronic value (measured at pH >7.5) of an important species present at the site, the FCV was lowered to this species' chronic value. The 30-day average concentration was set to the FCV. If rainbow trout are present at a site, the maximum allowable concentration is the s^uie as the national value. If raiubow trout are 88 image: ------- ab,sent, but percids or other salmonids are present, the maximum allowable concentration Is about twofold higher than the national number. This is not due to differences in general species sensitivities, but rather reflects unavailability of information on the sensitivity of different age/size classes for species other than rainbow trout; these higher numbers should therefore be treated with caution as perhaps being relatively underprotective compared to the national criterion. Only where both Percidae and Salmonidae are absent (site 5) is the maximum allowable concentration greatly higher, about fourfold, than the national number. Again, however, a large part of this difference may be due to the dearth of toxicity information on sensitive age/size classes. The 30-day average criterion shows even less variation among sites than the maximum criterion, due to the size class issue not affecting sites differently. If rainbow trout are present, the average criterion is near the national value. If rainbow trout are absent, but percids or other salmonids are present, the average criterion is only slightly (<20%) higher than the national value. Only if both Percidae and Salmonidae are absent from the site is the number substantially higher than the national value and the difference is little more than twofold. 89 image: ------- Site-specific aramoni.* criteria examples for t lv Site Number LIMFAVs: Elmidae Ephemerellidae Asellidae Actacidae Tubificidae Tctaluridae Perlodidae Poeciliidae Baetidae Percichthyidae Cyprinidae Daphnidae Cottidae Dendrocoelidae Catostomidae Centrarchidae Salraonidae Percidae pH Temperature (C) 1 a 6.22 3.86 3.46 3.03 2.87 2.70 a a a 1.92 1.84 1.65 a 1.57 1.49 0.90 0.82 7.6 15 2 8.30 6.22 a 3.46 3.03 2.87 a a 2.36 a 1.92 a a a 1.57 1.49 a 0.82 7.5 15 3 a 6.22 3.86 a 3.03 a 2.70 a 2.36 a 1.92 a 1.65 a 1.57 a 0.90 a 8.1 15 4 a a 3.86 3.46 3.03 2.87 a a 2.36 a 1.92 1.84 a a a 1.30b a 0.82 7.9 15 5 8.30 6.22 3.86 3.46 3.03 2.37 2.70 a 2.36 a 1.92 1.84 a a 1.57 1.49 a a 8.1 15 Maximum allowed concentration (ing/liter NH3): as un- ionized ammonia (as total ammonia) 30-day average concentrat (mg/liter NH3) : as un-ionized ammonia (as total ammonia) 0.10C (9.3) ion 0.028 (2.6) 0.21 (25) 0.026 (3.0) 0.13C (3.9) 0.03ld (0.9) 0.27 (13) 0.034 (1.6) 0.5S (18) 0.073 (2.2) (a) Family not present at site. (b) LIMFAV adjusted to reflect only site species. (c) LIMFAV lowered to L1MAV of adult rainbow trout. (d) FCV lowered to CV of rainbow trout. 90 image: ------- jO « 8 li *. * ox • tf °5 4- 18 t~ irlratlon L NH»I in o jO 4» ^ *•• ^» Ul « J i 5 I o |s - X — 3 E0> 1 *" 23 — 10 3 - — h- CM oo •" c 3 * K-, in i 00 CM 8 ^ •o o o o in ^ i £s t o 5 G -o I — CM O V 1C c •t- c 10 U ID 10 C C 10 £i Cladocer Cer i odap r- 2 * ID d> o in 3 „. 4 f^ 1O 2; o CO ON * CM O in O »-J y tn CJ X _,- 4-- (M O V ID C c" in 10 e Cladocer Daphn la g- fal c — - 10 4. 03 O in 3 or co * *o o o CM O\ O CO CM O in o ,£ to "„. 5 x: 4 — CM O V 10 c •> o c ro 10 c Cladocer Daphn la g- h. c ~ -~» 10 4. a> 8 Ul 3 ce ( O * in CO r— i- CM 0 in 0 s: to o X ^ i -o CM O V £ » a 10 E Cladocer Daphn la , g- h. c 3 ^ 10 . * 8 U) 3 K 1 co CM CM O 03 CM O in O „( s •I to 0 vr X _,_ I -o CM O V 10 c - a lo E Cladocer Daphn la §- h. c Z j a 4- e o t/) DC O c r- • in ^ in o o in O _,_f 2 to vr X _c 4- — CM O V <0 c c Q c? 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in 0 c E 3 'JJ] O 10 ».h !-. O IO O C_ E a T c 0) 8 i- E 3 '^ U 10 •>.b E^ ° 10 O (- (0 a. s CT fe c e in O u £ ^J ^ 8 ^i c O * L- (0 O CL -S. 130 image: ------- f— co r- r*- cj* -— r-- • 00 — O> _ |~- (0 — (O C7\ — 4- O 4- CD L CD . U — •>. CD 8 g 10 a. k4- in in — — N *. e — — 4- • 1_ 10 d> 10 OC -2 -n a ^ § £i 4- 1 <-> C -J K^i -tf- K°\ £) O a N^ vo ro in r~ tn y g . ... c B o o o — o 3~ c +- c: ro O O L. •*• 4- .C UJ 3 * O O O -o O in m in i? L S B S s — SZ -0 -C . s £ in c L) in 4- 3 in 1 •j- CL 0 c O m ^ a. B ^ oo 00 ao j E * 0 1, 1 g o\ in 0 O oo o — in f> L • CM (N CN •— ~- 9- X *- 4? 3 -o — 4? a. 3 ^ •° SI -o O CN ~— •/ O TI .... i j in oJ r^ co co co j . 153 in 9 * i_ 1 3 0 — C § en 10 • •" ._ in 8 §a> T> 3 — E — 3 — — — — — 610 O SO 0 U 0 *r ^f xj- •**• n > .0 .c £XXXX — ^ t"1 O^ Z z ^ ic I . * H) 4- m in o 3 0 X -C I. — — CT -D jC C O CO O U5 4- 4- z a. o> ID 10 > - - k. C m in O) — z L L. I O O - c 10 c JT in O x +- 4- L. — 2 3304- 4- 3 CD SE 3 TJ 4- O IO 35 10 in~33 l_L- l_ — O CT C. U) j *• •*- S 3 a o -* E » ^ — 3 a. m L -in »— 60) w C 3 jr — 4- I o -c O u 0) 10 E U 10 £ c « — c 8L. (/) O L I/I 10 >- •» jD £ Cp 3 in ^r — CO i/) O 4- k. L. U *u 1/110 inio OO 0 IOO 10— ^16 -CO a i_ig LID oo oc i/i in 334-5- o o a -Q u 10 ID — O -C o u 4- m 4- a> o 3 o: cr uJ uj < ID -O U TD CD K% in •— c 0 t s *• 10 4- 10 CD 3 4- HJ k t 4? ^ C 10 X a. •*- ^ c O 4- (D O C 3 g __ 10 C O I/I o. • <« c o As s <4^ v K1 X 2 in 5 •£ (O § ^_ **- _ •^ 10 3 O tr ^z 131 image: ------- REFERENCES Abeliovich, A. and Y. Azov. 1976. Toxicity of ammonia to algae in sewage oxidation ponds. Appl. Environ. Microbiol. 31(6): 801-806. Admiraal, W. 1977. Tolerance of estuarine benthic diatoms to high concentra- tions of ammonia, nitrite ion, nitrate ion and orthophosphate. Mar. Biol. 43(4): 307-315. Agami, M., M. Litav, and Y. Waisel. 1976. 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