Bioassays--procedures and results

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Water Environment Federation is collaborating with JSTOR to digitize, preserve and extend access to Journal (Water Pollution Control Federation). http://www.jstor.org Pollution _Water et al}5 exposed Gammarus pulex to a high-level exposure of the fungicide Thiram for 1 hour, subsequently placed test or ganisms in freshwater, and compared mortality observed in organisms exposed to freshly prepared solutions to organisms Bioassays? and procedures results 48- Bioassay procedures to describe, evaluate, and predict the potential hazard of toxic materials to organisms and ecosys tems, and the health-related aspects of polluted waters con tinue to receive widespread attention. Symposium proceedings and books pertinent to toxic substances management and test have procedures as have appeared, several literature reviews pertinent to specific pollutants. The proceedings of the fifth annual ASTM symposium on aquatic toxicology presented a collection of papers directed to research needs in aquatic tox hazard and icology new assessment, tox in aquatic concepts icology, biological availability and sediment toxicity, and haz ard assessment water and environment sessment chemical the propounded that state-of-the-art in the the fate of chemicals cause between on affects adverse as hazard that concept the relationships requires understanding concentrations A criteria.1 quality to modeling overview pertinent aquatic aquatic life and environmental exposure.2 This book featured defini tions of chemical, physical, and biological processes that de termine chemical fate; modeling of physical and chemical fate water processes, microbial quality, and transformation as a tool and modeling degradation; in assessing chemical an including in pollution dustry Specific control, toxic pollutants including petroleum copper,6 zinc,7 account lead,8 were of the work presented to aquatic in a new organisms and specific petroleum cadmium,9 chlorine were on aquatic in reviewed hydrocarbons,5 haloge cluded aquatic species was tested. However, different samples examined,14 of one and acute test compound may result inmarked toxicity differences, and all compounds were biodegradable by more than 80% within 4 weeks. Bluzat June 1983 for adjuncts and adsorption solubility, de sorption phenomena, partition coefficient, volatility, hydro factors reactivity. Physicochemical lysis, and photochemical influencing bioconcentration of organic solutes were examined suggesting a direct proportional relationship between biocon centration The factors and can correlation octanol-water be extended coefficients.17 partition to give a simple relationship between bioconcentration factors (BCF) and aqueous solubil ities. The relationships between physicochemical properties of 21 substituted phenols and their toxicity and accumulation in guppies were determined, and related to lipophilicity defined as log P from 1-octanol/water the to APKa and system, value.18 Results indicated that if LC50 values were corrected for ion ization using an empirically formulated relation between tox icity and pH, the resulting regression could be used to predict toxicity for the pH range of 6 to 8.When corrected for ion ization, log BCF of eight phenols was highly correlated with log P, but not with APKa. Koch19 discussed structure-activity way of as 10 chlorophenols to predict vironmental the and a molecular toxic by values toxicity en of on Based chemicals. exper et edulis and organic chemicals, Geyer that water suggested expressed acute approximate pollutants iments with Mytilus al.20 and solubility water n-octanol par tition coefficients were useful screening tests for organic chem ical bioaccumulation. Data interpretation for bioassay research often requires a statistical approach. Calamari et al.21 applied a mathematical model of accumulation and elimination kinetics of Cr, Cd, and Ni in rainbow trout at levels approximating water quality criteria. The between estimation statistical single and sorption toxicity differed up to a factor of 1000 for the different organ isms such by useful and mixture metal release are described model. Based used differences parameter and assumed that by a reversible on PCB uptake reaction as a exposures experiments with plankton, amathematical model was developed to predict PCB accumulation and determine the roles of feeding and Various chemical, physical, and biological phenomena may alter chemical structure and therefore the fate and toxicity of certain compounds in natural and laboratory toxicity test sys tems. The biod?gradation of five surfactants in relation to their seven water pressure, vapor uptake ecosystems.13 DESIGN, INTERPRETATION, STRATEGY to demonstrated often prep in the toxicity studies and environmental fate predictions. A labo ratory method for testing the volatility of compounds from aqueous solution was described16 and the results discussed within the context of environmental behavior of chemicals. Key properties with regard to ultimate environmental fate in two-compartment toxicity are data after respectively, connectivity index (MCI) as a novel descriptor of molecular structure in a quantitative comparison with partition and bio logical properties of a molecule. The MCI was proposed as a nated hydrocarbons,11 oil well drilling fluids,12 and acidic de position As suspensions. hours 96 45%, correlation textbook.4 residuals,10 old 96-hour physico-chemical and and bio the water of 71 haz ard. Cairns et al? provided methods, advantages, difficulties, in water pol and future possibilities of biological monitoring lution condensed into six categories including: early-warning systems; receiving system functional methods, relationships, based on com and indexes; receiving system methodology munity structure; toxicity testing; preference and avoidance studies; and future needs within the field as a whole. Funda mentals of freshwater pollution and its effects on living or ganisms, and 48 suspension, decreased Toxicity studies, A. F. Maciorowski, L. W. Little, L. F. Raynor, R. C. Sims, J. L. Sims same to the exposed aration. on uptake under various An conditions.22 allometric for pesticide bioaccumulation was also reported using size body frequency distribution and appropriate allometric model relationships to permit ticide bioaccumulation qualitative cept.23 The terns and those in the a more operational approach to pes than the traditional trophic level con similarity literature between from experimental laboratory and field pat ex relations describing periments suggested that empirical contaminant flux as functions of body weight form the basis for predicting contaminant body burdens in natural systems. 801 Water Pollution_ Jensen et al.24 employed a bioenergetic model to simulate PCB uptake by five fish species, which suggested that differences in PCB residues between and among species from different en are vironments to related and metabolic growth, Pollutants size, differences, of parameters. occur rarely exposure rate and in the environment, singly was of the mixtures near concentration and addition, chemical regression for mixture ture. A /-test was developed t values such that insignificant values support antag additivity, significant negatively and positively values support significant synergism. support onism, metal Heavy was antagonism also for reported was in the presence decreased Correlations between from fects actual of Zn.28 laboratory toxicity test results and ef environmental exposure consider requires ation of various biotic and abiotic factors. An Exposure Anal ysis Modeling System (EXAMS) designed for rapid evaluation of synthetic organic chemical behavior in aquatic ecosystems, combined loadings, transport, and transformation into differ ential equations using the law of mass conservation to allow of computation environmental expected concentration, fate, and persistence of chemicals.29 The Exposure Commitment Method for pollutant exposure evaluation was described as a time-independent to exposure approach measured evaluation as the concentration and duration of the presence of a pollutant in an environmental medium.30 The commitment method was said to be a convenient procedure for comparing contributions to intake and exposure from various pathways and in express ing source-receptor to assess criteria Frische relationships. environmental behavior et al.31 of chemicals examined with respect to selection and preliminary quantification. Factors of prime importance included the quantity entering the environ ment, accumulation, mobility, and persistence, or direct in direct toxic effects. These factors were quantified using pro duction volumes, physicochemical data, results of degradation and accumulation and experiments, toxicity values. Quanti fied criteria for "well-known" organic chemicals were evalu ated using appropriate weighing factors demonstrating that persistence and were accumulation most mutage important; nicity and carcinogenicity had to be strongly weighted; and mobility, although indispensible for estimating chemical dis tribution and was persistence, not included it can because have beneficial as well as adverse effects. A screening procedure to evaluate environmental behavior of chemicals by comparative evaluation of various parameters, termed the Ecotoxicological accumulation, and bound residues The ment used system An studies. ing of dissolved was described and may and egg an to activate used develop monitor a gasometer termed supersaturation be larval for continuous device and gases trout rainbow inexpensive alarm system and, therefore, protect fish from hyperbaric or hypobaric gas MICROORGANISMS of the acti vated sludge test; and photomineralization by irradiation with UV light.32The cumulative data are subsequently ranked and aspects of water quality laboratory techniques, including bioassay A compendium included various and of microbiological A bioassay tests.35 screening method on based measurement of light output changes from luminescent bac teria was described as a practical and reliable method for tox icity monitoring.36 Busch37 described a system and test pro cedure to determine relative toxicity of water and effluent sam ples based on the biochemical oxygen demand (BOD) bottle bacterial test system. A procedure using oxygen uptake by bacteria in the presence of a pollutant was said to detect toxic responses within 40 minutes.38 Parker39 developed a microbial bioassay procedure that used active biomass and metabolic activities in terms of ATP and oxgyen uptake that allows com parison of the microbial response to toxins with the response of a standard as gested reference a surrogate This compound. parameter was procedure for analysis organic sug priority pollutants. The application of four bacterial screening proce dures to assess changes in the toxicity of chemicals inmixtures was examined, resulting in the conclusion that the screening tests had individual sensitivity patterns and that it seems un wise to use a single test for assessing the presence of toxicants in water or effluents.40 Trevors41 examined sensitivity differ ences between respiration, growth inhibition of active cells, and percentage mortality or resting cells using Pseudomonas to pentachlorophenol, exposed fluorescens and a was growth more sensitive toxicity indicator than respiration. Bacterial assays have been used to evaluate the potential impact of various substances to microbial species and com munities. Studies on the effect of water soluble fractions from emulsified, degraded, and artificially weathered crude oil dem onstrated that microbial uptake of glutamate was inhibited by emulsified oils, but not by oils that had been altered by bio or weathering.42 and recovery degradation tional responses munities biomass, et Sayler of using 20 estimates of microbial and over activity a between variation determine cluded that multiple covery al.43 15-month responses from population are com density, Multivariate period. sites. contaminated required func analysis were used to functional measures environmental evaluated microbial sediment analysis of variance and discriminant munity Profile Analysis, included consideration of bioaccumulation retention and potential with algae and fish tests; mammalian elimination with rat tests; information of biodegradability, metabolism, Dosing and monitoring methods in aquatic organism tox icity tests are often challenging, and Amelung33 described an apparatus for continuously saturating water with dissolved iron procedures two mollusk species. Sublethal concentrations of Se were found to decrease Cd-induced mortality in the snail Lymnea stagnalis by 50%,27 while the amount of Hg accumulated by the mussel Mytilus edulis the assessment. the mix and components to complete compared pressures.34 concentrations near 0.1 of their LC50 value contributed to mixture toxicity. Schaeffer et al.26 used multiple regression analysis and the assumption of response additivity to obtain coefficients results salts. quan tification of joint toxicity remains of interest. The acute me dian lethal concentration of equitoxic mixtures of 8 and 24 toxicants was determined in a series of experiments and results expressed by means of aMixture Toxicity Index.25 The toxicity properties, and test correlated with respective physicochemical to evaluate the and contamination, It was of microbial effect certain con com and re phys iological traitsmay not reflect population and biomass estimates of community response. The effects of PCBs on nitrification were examined using laboratory assays and it was determined that >10^g/L PCB inhibited nitrification in water from an unpolluted reservoir, but inhibition was not observed in pure 802 Journal WPCF, Volume 55, Number 6 Pollution _Water or cultures a contaminated from in water res nutrient-rich ervoir.44Mahaffey et al.45 evaluated Kepone toxicity to mixed population of estuarine organisms and demonstrated that un der anaerobic conditions, it reduced the number of colony forming units but had no effect on the number of organisms. The effects of carbaryl on four bacterial indicator species of fecal pollution and pathogenic bacteria were examined in pure culture and variously combined mixed cultures. Mixed cul tures provided a better estimate of environmental effects of chemicals than cultures.46 pure Trevors et al.41 used a Pseudomonas fluorescens assay to demonstrate that toxicity results were affected by the sequence of exposure to pentachlorophenol (PCP) and 2,3,4,5-tetra chlorphenol (TCP) including both the concentration used and addition. Cell suspensions the sequence of chlorophenol treated with PCP and subsequently removed from exposure were not affected by a second PCP dose; if the second dose was TCP the test species was sensitive to TCP. The toxicity of inorganic tin to estuarine sediment microbial populations was found to b? influenced by a number of chemical factors.48 The use of gelatin and silica gel as a gelling agent decreased toxicity, cysteine had no apparent effect on toxicity, serine or 3-hydroxyflorone enhanced toxicity, while humic acids re duced toxicity. Itwas concluded that toxicity levels determined in the laboratory should be extrapolated to the environment with caution. Bacterial growth is often used as a measure of productivity and eight methods of assessing growth rates were compared by Christian et al.49 Linear and exponential growth rate con stants were from computed cell and ATP concentrations, cell division frequency, and RNA synthesis as measured by [3H] adenine uptake. Estimates of in situ bacterial productivity and growth were found to vary with themethods used and the assumptions regarding bacterial growth state. A bioassay method for determining the concen tration of assimilable organic carbon (AOC) in drinking water distribution systems used growth of fluorescent pseudomonads as a function of AOC to examine water concentration.50 samples from various The method water was treatment used stages that AOC increased if and piping material, demonstrating water was ozonated or placed in contact with polyvinyl chlo ride pipes. Gordon et al.51 assessed calorimetry as a technique and organic nu for studying marine microbial metabolism trient waters. in marine found concentrations Microcalori and was that the wide range at measurable a constant metabolism of glucose submicromolar efficiency concentrations was indicated over studied. concentrations of organic compound biodegradability has Determination obvious implications for environmental toxicology. The reli ability of the river die-away (RDA) test for establishing bio degradability of chemicals was assessed for reproducibility and RDA test results were not reproducible for di-2-ethylhexyl phthalate and phtalic acid in replicated tests with Missouri River water.52 It was suggested that RDA test biod?gradation measurements are too variable and too dependent on labo ratory treatment of samples to be applied directly to aquatic environments. Means and Anderson53 compared five different methods formeasuring biod?gradation (BOD, shake flask, C02 evolution, activated sludge, and the Gledhill test) using five June 1983 metab rapidly Several used investigators bac than other microorganisms teria in bioassays. Babich and Stotzky54 examined the influence of abiotic environmental factors on nickel toxicity to mycelial growth rates of several filamentous fungi (Achyla, Saprolegnia, Cunninghamella, Aspergillus). Nickel toxicity was eliminated with increasing pH and reduced in the presence of chlorophyll and humic acids; addition of Mg and Mn reduced Ni toxicity was which not affected by K, Na, or Fe Ca, re A additions. liable, accurate technique for identifying and analyzing sol vent-pesticide interactions in bioassays was described that uses the fungi talotia and sp., acetone, Interaction Sclerotina ultimum, Pythium any and Pes homeocarpa, the fungicides with responses and metalaxyl set of bioassay given captan.55 parameters were dependent on both the acetone and fungicide concentra tions, but the method compensates for discrepancies and in the most dicates A bioassays. bioassay use test parameters,to suitable technique the using in subsequent Tetra protozoan hymena pyriformis was described that uses the rate of change of oxygen over uptake, a 10-minute as the period, endpoint.56 The procedure was not sufficiently sensitive to detect some chemicals at drinking water limits, but may be useful for in dustrial waste monitoring. The toxicity of the carbonate in secticide was cartap examined a Paramecium using primau relia assay providing a 24-hour LC50 of 2.5 mg/L.57 ASSAYS WITH ALGAE AND OTHER PLANTS New to algal approaches investigators. were assays et a/.58 Domotor An communities. phytoplankton several by proposed that demonstrated autoradiog thermal stress in raphy facilitated detection of species-specific automated mon biological itoring system based on changes in natural fluorescence of algae was designed and evaluated.59 Comparison of cell counts, in vivo fluorescence, as surement indicators phaeophytin of toxicity and a, a mea chlorophyll to Selenastrum capricor nutum showed that cell count data alone failed to indicate toxic adequately metric measurements of heat production from glucose by Vib rio alginolyticus demonstrated that glucose metabolism by this bacterium were compounds bio-volumes, changes, density Certain compounds. organic olized in all tests, while others degraded relatively rapidly in some and slowly or not at all in others. Inconsistencies were evaluated relative to the physical, chemical, and biological con ditions inherent to each test. curate and stress in vivo while indication.60 rapid fluorescence According an gave et to Sellner ac al.,61 natural fluorescence can be enhanced by adding DCMU, sug gesting that the almost instantaneous response of alga fluo rescence to stress may a provide time-con for replacement suming algal assay procedures. Testing of volatile substances is difficult in the Algal Assay Procedure Bottle Test and a atile test a constant that maintains modification in culture substances of concentration containers was vol devised.62 Algal assays continue to be used extensively in determining the limiting nutrient in waters and assessing eutrophication of Results potential. such studies were reported for Balaton Lake (Hungary),63 English lakes,64Hastings Lake (Canada),65 the Rideau River (Canada),66 Lake Michigan,67 deep ocean Lake (Canada),69 and Wolderwijd waters,68 Memphremagog Lake (Netherlands).70 Young et al11 used Scenedesmus bioas says waters. to evaluate The phosphorus problem of poor availability growth in municipal of many marine waste algae in 803 Water Pollution_ and it was artificial seawaters was examined selenium that Selenastrum demonstrated was sources natural from be a problem.72 may deficiency suggested that Visser and Couture73 capricornutum growth inwater influenced the nature by amount and of the dissolved organics present. As a rapid response bioassay for nutrient additions under field conditions, Stephens and Shultz74 suggested measuring the ATP concentration of peri strips. phyton in using a single bioassay method The difficulties mine status nutrient a water of vestigators. Several bioassays phosphorus demand were were stressed for determining in and nitrogen a combination and compared, to deter several by of techniques allowed useful conclusions about nutrient status, but no single technique could be considered conclusive.75,76 Chiaudani and Vighi77 preferred amultistep approach to iden tify limiting nutrients. Lean et al.18,19 described the compli cated interrelationships between light and nitrogen and phos phorus uptake and suggested that taken alone, results from 14C reason One be misleading. bioassays was from the presence 14C methods could and solutions eliminate used containers such of for trace metals and in the assay, were contamination erratic results in shock to procedures ob Yallop81 reported.80 tained results that cast doubt on the validity of the light-dark bottle method of determining primary productivity. Because of the strong influence of "light history" on algal cell oxygen it was uptake, that suggested oxygen measurements uptake be Imaoka made in the presence of dichlorophenyl-dimethylurea. et a/.82 used red tide organism algal assays to demonstrate that removal phosphorus control tive was wastewater from municipal an effec method. Algae continue to be widely used in toxicity testing. Tests with various substances were reported including: snowmelt,83 leachate from Mount St. Helens ash,84 chlorine,85 pulp and paper coal wastewaters,86 hexa- ammonia,91 tachloropheol,94 and and and liquids polychlorinated humic oils,87"89 detergents,90 biphenyls,92,93 et al.,96 Walsh materials.95 pen com pared the sensitivity of Selenastrum capricornutum and Skel and to etonema costatum to that of Daphnia andMysidopsis 10 wastewaters, and found to be more algae sensitive than crustaceans. An apparent synergistic effect between the pesticides feni trothion and aminocarb and the solvent used in their for mulation was observed in bioassays with green algae and Daphnia.91 Einer et a/.98 distinguished between effects on algal communities of aminocarb itself and those caused by other components of the commercial formulation Matacil. Selen astrum assays capricornutum were to used assess treatment efficiency in treatability studies of atrazine and dinoseb man ufacturing wastewaters.99 Veber et al100 suggested algae could be used to remove atrazine residues from polluted waters. Stratton and Corke101 found permethrin degradation products to be more toxic than permethrin to green and blue-green algae, and permethrin and itsmetabolites interacted differently in different combinations. In situ bioassays conducted in 60 L clear plastic bags were used by Hoffman et al.102 to evaluate effectiveness of algicides in pond water. The order of effec tiveness was simzine > diuron > copper > ethanolamine > copper citrate. The impact of herbicides on phytoplankton used in controlling aquatic macrophytes revealed that effects of 2-propenal varied for such species at different tempera tures.103 Saroja and Bose104 found chlorophyll content to be the most sensitive indicator of methyl parathion toxicity to Chlorella. Effects of organochlorine insecticides on algae and other were microorganisms reviewed.105 Jouany et al.m studied the toxicity of hexavalent chromium (Cr6*) to the green alga Chlorella vulgaris and the invertebrate Daphnia magna, both individually and inmixed culture using 24- to 96-hour and 28-day Mixed bioassays. culture results were substantially different than expected from single species experiments in that presence of Chlorella reduced Cr toxicity to Daphnia. Many investigations dealt with metal toxicity to several Five species. algal as well genera, algal a natural as phytoplankton assemblage were used in determining effects of a mixture of 10 heavy metals using primary productivity re and duction other used Foster108 responses.107 200 isolates, representing 87 species of green algae, in sensitivity tests with Cu, Pb, Zn, and Cd, and found that isolates were generally resistant tometals normally present in their habitats. In related research,109 of species algae in metal-polluted present rivers seem to be determined by the degree of metal pollution. Fisher110 observed that conditions in a polluted estuary selected for heavy term metal in marine tolerance to evaluate study the algal As diatoms. species the Great Lakes, toxicity studies of heavy metals size algal were fractions of a long trends in part composition on various conducted.111 The influence of Co, Ni, Cu, and Cd on Anacystis nidulans was to assist examined in response of blue-green interpretation algae atmining sites resulting inmutants tolerant to high metal levels.112Of interest were synergistic and antagonistic effects noted with different combinations of metals. Interactions be tween Cd, Pb, and Ni in metal mixtures to three freshwater green were algae as were investigated,113 those and of Se, Hg, Cu in tests with Dunaliella.114 Petersen115 addressed the ap plicability of metal speciation in studies of the joint action of Cu on Zn and model Scenedesmus quadricauda and a proposed to account for observed biological effects as a function of metal Several speciation. studies on mecha concentrated nisms of metal toxicity to algae, including surface adsorption of Zn,116 structure/toxicity relation of inorganic and organic tin compounds;117 the relationship of Hg chemical structure accumulation capacities118 and metal toxicity effects of pH wtih Chlorella"9 Generation time, maximum cell density, and chlorophyll content are commonly employed in assessing pollutant impact on For algae. the marine diatom Thalassiosira rotula, chain length was a useful indicator because the chains tended to break apart at toxic Cd and Ni levels.120Usefulness of algae as biological metal monitors in the aquatic environment was investigated by Seeliger and Cordazzo,121 who concluded that Enteromorpha was an effective Cu and Hg monitor, and by who Burdon-Jones,122 brown obtained results unsatisfactory as a monitor Padina algae for with the 10 metals. The role of plants is becoming more common in toxicity, tests. Onion root tips were biomonitoring, and mutagenicity used to study biological effects of Cu in drinking water,123 and the aquatic plant Elodea was used in research on biological effects of Cu and methyl-mercury.124-126 Lemna demonstrated pentachlorophenol.127 monitoring stream Test results with its similarity to animals in responses to for To facilitate use of macrophytes water quality, Haslam128 developed a pol lution index method. The potential of aquatic plants as in dicators of metal pollution was demonstrated in Dutch,129 804 Journal WPCF, Volume 55, Number 6 _Water western and U. Europe,130 S. waters.131 toxicology assays, the Arabidopsis for tests in situ soil- of and For short-term genetic plant seems to be suitable water-borne Italian pollutants.132 researchers suggested that a root tip assay with the bean Vicia as a screening test and reliable, inexpensive on assess effects of pollutants seagrass. is sensitive, faba for mutagens.133 To Walsh et al.134 devised a flow-through system containing whole plants or leaves of Thalassia testudinum. Plant genetic and assays cytogenetic and programs, with use to protocols, respect research recommended been have in screening discussed,135 environmental impact on exploited populations, Jensen and Marshall144 examined the applicability of the surplus produc tion model commonly used with fish to laboratory populations of D. pulex exposed to chronic radiation stress. The model was found to describe the observed relationship between equilib rium Use as a rapid detection D. pulex cladocerans, specifically the most represent commonly and magna, Daphnia freshwater employed invertebrate bioassay test species. A revised standard bioassay procedure for determining acutely toxic thresholds of soluble substances has been described, including culture techniques and test initiation and operation.139 The test is designed to determine the percentage of free swimming organisms at test termination, LC50 values are obtained by interpolation, and confidence limits are estimated using probability tables. An interlaboratory comparison of the D. magna survival/life cycle test was performed using silver and endosulphan as challenge for procedure im toxicants, resulting in recommendations of insol interference the examined provements.140 M?ller141 uble particles in the Organization for Economic Cooperation and Development (OECD) D. magna reproduction toxicity test protocol using various concentrations of silica gel, alu minum oxide, dextrane, and cellulose particles and concluded that the test is of limited value for insoluble test materials. Bowman et al.142 espoused conduct o? Daphnia or Hyalella bioassays of dislodgeable pesticide residues from foliage as an alternative to chemical analysis for the determination of safe worker reentry into crop fields treated with pesticides. Filtra tion rates and phototactic behavior of D. magna have been proposed as indexes of chronic Cu stress following comparison of these response with the more survivorship, number of juvenile molts, and maturity, neonate body used commonly length.143 of responses age at reproductive The two former re sponses and neonate body length were reduced at Cu concen trations that did not reduce longevity or reproduction, while juvenile brood molt size number, responses June 1983 age were at reproductive to Cu erratic maturity, and mean exposure. To assess as exposure, for tetraurelea gen detecting in marine for health system use The hazards.145 of organisms for the detection of carcinogens was environments evaluated, benzo(a)pyrene using equivalent activity in the Ames test.146A host-mediated assay for the detection of mutagenic compounds was also developed mussels using in clean grown into transferred of mussel activity exceeded a value taneous frequency. and polluted on extract the however, dependent, Freshwater radiation of level the of Paramecium sponges as model genic Invertebrates and fish are widely used in bioassays to de termine various lethal, physiological, pathological, and behav ioral effects to the species under study. In this section emphasis literature. For spe is placed on procedural and methodological cific pollutant effects on marine and freshwater invertebrates and fish, the reader is referred to other titles contained in this annual literature review issue ("Bioaccumulation and Toxicity of Heavy Metals and Related Trace Elements," "Effects of Pollution on Freshwater Invertebrates," "Effects of Pollution on Freshwater Fish," and "Estuarine and Marine Pollution"). and otoxicity and cytoxicity of oil shale process water was discussed mussels INVERTEBRATES AND FISH size population well as the relationship between field and population size. Several invertebrate bioassays have been devised to examine the potential effects of chemicals to human health and marine pollution. while other studies136137 have indicated that cultivated barley chromosomes are easily identified, making barley a suitable species for induction studies of chromosome abberations. A screening technique based on differences between antibiotics in the production mechanism of and mutagens/carcinogens plastid-free Euglena mutants was used for rapid determination of mutagenic activity.138 Pollution induced The and yeast clean and muta direct was bacteria five the times dose never frequency mutagenic to about corresponding areas polluted water.147 spon Benoit et al.148 evaluated the 32-day fathead minnow early life stage toxicity test in determining estimated maximum ac on effects Toxic four of concentrations toxicant ceptable chemicals. early untested organic stages developmental dem onstrated that larval growth and survival were the most sen sitive toxicity indicators while embryo hatching and larval deformities were the least sensitive. The tests produced good replication and are capable of providing a rapid means to de water velop quality criteria when to predict used long-term chronic toxicity. Acute toxicity test procedures with fathead minnows and coal synfuel blends were examined indicating that: static test conditions were characterized by declines of dissolved oxygen (DO) and phenolics over 96-hours; renewal of exposure solutions at 12- or 24-hour intervals retarded, but did not prevent such declines; flow-through test conditions (9 volumetric turnovers daily) provided suitably stable DO and phenolic conditions.149 A novel evaluation of the relationship between body contaminants and bone development was em striped bass from the ployed to examine young-of-the-year Hudson River and other East Coast locations.150 Mechanical properties of striped bass vertebrae were subjected to analysis for strength, stiffness, toughness, and rupture. Fish from sites contaminated with PCB, DDT, DDD, DDE, and chlordane displayed vertebrae with the least strength, stiffness, toughness, and rupture under lowest force, compared to hatchery reared fish. Bresch151 described a test that considered xenobiotic ac tion on zebrafish reproduction. Successive reproduction cycles were investigated with the numbers of eggs released as the primary test response. This reproduction test, in conjunction with an embryo-larval test, may be capable of replacing full life-cycle tests and provide more information than a single embryo-larval test. Fish gill ventilation and coughing frequency remains an area of interest because of its potential as a rapid screening test. Carlson152 characterized ventilatory characteristics of bluegill sunfish by using non-contact sensing electrodes in conjunction with buccal pressure changes of cannulated fish to characterize definitive features of gill purge biopotential recordings from free-swimming fish. Cairns et al.153 developed a 96-hour tox 805 Water Pollution_ icity test using ventilation variable. sponse frequency of steelhead as the re Ventilation were frequencies from buccal and opercular activity. Ventilatory responses were detected at a Cu concentration of 144 ??g/L as compared to a "safe" concentration of 444 to 819 /?g/L determined in chronic exposure of embryos and juveniles, indicating that the test was ventilatory at least as sensitive as chronic tests. toxicity Thompson et al.154 demonstrated the applicability of time-se ries intervention analysis to determine the ventilatory reactions of bluegill sunfish exposed to sublethal Cu concentrations in fluctuating intermittent exposures. Tests have also been developed to determine the presence and of effects mutagens, carcinogens, and teratogens, geno toxins on fish species. Laboratory and field studies of aquatic animals were reviewed indicating that fish have metabolic to mammals similar pathways an dussumieri, for disposition of car certain screening 20 fish species, Boleopthalmus cinogens.155 After edible mud was skipper, as a cytoge studied netic model for in vivo mutagen detection.156 Utility of the killifish Nothobranchius rachowi in a sister chromatid ex as was the use of sister chromatid was test described157 change exchange frequency in flatfish exposed to carcinogens.158 The suitability of a micronucleus test for genotoxic compound de tection also using reported, eastern the and mudminnow an represents Umbrea was pygmea to alternative chromosome clearance time, and parameters, regimes dosing for crude oil;160 induction of hepatic microsomal cytochrome P450 and aldrin epoxidase in the sculpin Leptocottus armatus to petroleum exposed refinery and concentrations adenylate and wastewater;161 energy adenylate the use of as in charge dicators of hypoxic stress in estuarine fish.162 COMPARATIVE TESTING, MICROCOSMS, AND IN SITU TESTS realization that toxic chemicals may affect human health, as well as plants and animals in the environment has increased the concept of integrated assessment strategies to evaluate chemicals. and Brusick Young163164 prepared two manuals related to Level 1 biological testing assessment and data format. The first consisted of a bioassay sensitivity lit erature review for validating test systems including the Ames test, rodent toxicity, in vitromammalian clonal toxicity assays, and consideration of the role of aquatic ecological and terres trial assays ecological assessment. in environmental The sec ond manual outlined the rationale and proposed methods for 1 health effects and ecological effects bioas Level performing says including methods for sampling and testing gases, sus pended particulate matter, liquids, and solids. Specific assays for determining toxicity and mutagenicity using a variety of test systems and responses hazards were described. fish, terrestrial insect, to assess In addition aquatic potential environmental to tests for mammalian, crustacean, algal, terrestrial plant, and microbial toxicity, consideration of quality control for toxicity studies was discussed. Short- and long-term toxicity studies with Cd were con ducted with freshwater organisms from different trophic levels was et al.166 and fishes, amphibians, examined determined to be of coal-liquor effects 0.37 /?g Cd2+/L. water soluble (WSF) on growth and survival of two freshwater fraction a daphnid, midges, reproduction, a green and and of growth, Suppression alga. was survival at concentrations observed of 0.02 to 0.42% WSF for the invertebrates, with algal populations being the least sensitive of the species tested. The acute toxic of responses mined for pairs tested simultaneously lauryl sulfate, cadmium, species sodium were and deter methano myl.167 One species in each test was that recommended by the U. S. Environmental Protection Agency (EPA), the other a closely related species. Species pairs consisted of estuarine algal phytoplankters, mysid shrimp, copepods, and fish. For each toxicant, the species pairs yielded similar lethal or effective concentrations. were Mysids most to cadmium sensitive and methanomyl, with algae being most sensitive to sodium lauryl sulfate. Payne168 examined metabolism of oil spill surfactant compounds rainbow by a crustacean trout, Cancer irroratus, and the mollusk Chlamys islandicus demonstrating that each had the capacity for enzymatic hydrolysis of the complex fatty ester acid mixtures. Comparative aquatic lation of environmental microcosm systems evaluation organism tests but microcosm often and water, (sediment, realistic conditions. exposure in assess aids more allow simu Three-phase for gas) evaluating substances under a variety of ecological conditions were de scribed by Porcella et al.169Response variables were related to functional ecosystem general anaerobic piration, processes res (photosynthesis, carbon degradation, and cycling, aquatic chemistry). A compartmentalized flow-through model ecosys tem for studies of transport and degradation of pollutant has also been described.170171 Microcosm-related mathematical models related to persistence were analyzed by Gard.172 Several authors used microcosms to study effects of oil and oil-related constitutents including effects of short-term expo sure of crude oil and dispersants on benthic community me crude tabolism,173 The crust?cea, algae, "no-toxic-effect-level" Dauble ments, aberration tests.159Other physiological tests designed to indi cate stress included: induction of mixed function studies (MFD) activity of the American eel Anguilla rostrata as related to exposure bacteria, including using a variety of lethal and sublethal endpoints.165 The lowest enumerated water-sediment oil on simulated natural lakes gas using the water-soluble microcosms;174 laboratory fraction of a crude oil liquefaction product on laboratory pond microcosms;175 and no. 2 fuel oil on natural phytoplankton Microcosm assemblages.176 studies were also to de conducted termine the significance of interfaces in the distribution and metabolism of di-2-ethylhexyl phthalate in a static laboratory Interface ecosystem.177 cumulations at the occurred adsorption occurred in organisms Dickson interfaces. inhabiting and/or feeding et al.178 used microcosms three-phase to study the aquatic fate and effect of benz(a)anthracene, had no acute effect on aquatic structure munity and as measured organisms function. remained benz(a)anthracene ac and rapidly After in the sediment 60 with which by com most days, no evidence of metabolism. Atrazine toxicity to submerged vascular plants was investigated by Correll and Wu179 using simulated estu arine microcosms. Macrobenthic estuarine de communities veloped in sand-filled aquaria in the laboratory and field were exposed fects.180 to chlorpyrifos Molluscan larvae to assess colonizing community laboratory structure aquaria ef were sensitive at 0.1 Mg/L, but later developmental stages charac terizing field aquaria were not sensitive at 5.9 Mg/L-Arthropod abundance was diminished significantly at the former concen 806 Journal WPCF, Volume 55, Number 6 Pollution _Water in tration mostat for studies trace metal trace of latter concentration to overcome metals culture from non-teflon only the high Strachan et al.m developed a teflon che contamination The reservoirs. at and aquaria laboratory in field communities. and medium were components of problems vessels and pH glass dissolved oxygen probes and short pieces of silicone rubber tubing that necessarily passed through the peristaltic pump. A study of the biogeochemical fate and toxicity of Hg using controlled experimental ecosystems showed strong influence Hg affinities for organic matter in chemical speciation, trans and port, cosm bacteria, containing demonstrated gochaetes early effects The toxicity.182 algae, that of heterotrophic stages of Cu on an were responses succession and rotifers, protozoa, stress and oli in greater con nutrient high centrations.183 The impact of Cd on freshwater bacterial com munities in chemostats approximating natural river conditions resulted in measurable responses including bacterial produc tivity, accumulation by free bacteria, Cd budget in continuous and Cd culture, from removal the water and Capone phase.184 Carpenter185 developed a perfusion method for assaying nitro genase activity in unmanipulated marine sediments and found more precise results than those obtained from conventional sealed-flask assays. Laboratory were microcosms to study used the effects of temperature and salinity,186 and salinity and or ganic nutrient concentration,187 on the survival and growth of Vibrio which cholerae, that suggested member tochthonous of the estuarine this is an species microbial au community. Microbial decomposition of the aquatic macrophyte Car ex sp. was investigated in two microcosm studies, which suggested that pH reduction would significantly reduce rates of litter decomposition.188189 Nutrient enrichment increased ATP lev els associated with microbiota on fresh or partially degraded ATP The litter. increase when occurred were microcosms in cubated in light, but not in the dark, suggesting that nutrient enrichment primarily stimulated the photosynthetic compo nent of the detrital community. Several reports dealt with the use of artificial streams for assessing toxicant effects in lotie ecosystems. Sanders190 sug gested design criteria for artificial stream facilities and dis cussed the strength and limitations of this approach. Specific applications of artificial stream research included an assess ment of aufwuchs food quality from artificial streams dosed with separately hydrate, and Cu, chlorine, organic content and dextrose.191 of aufwuchs carbo Protein, samples were altered by taxonomic or physiological condition of the community. Hansen and Garton192 evaluated the effects of diflubenzuron on a complex laboratory stream community demonstrating that insect fauna suffered direct toxjc effects at 1.0 vg/L, but algal and fungal flora were only mildly affected at the same concentration. The effects of oil and gas drilling fluids on bio mass and community structure of marine benthic microbial and communities invertebrate seawater were examined assess that colonize using biochemical sands in running methodology to impact.193 In contrast to microcosm studies perimentally more natural conditions test methods provide an opportunity designed to simulate ex in the laboratory, in situ to examine toxic effects directly in the field. The dispersion, persistence, and biological effects of cypermethrin were evaluated following its application in a pond, which demonstrated that the insecticide was sorbed onto suspended solids. This permitted survival offish, although June 1983 insects was crust?cea and observed.194 Ap trichlorphenol and 1 mg/L penta plication of 5 mg/L into chlorophenol duplicate compartments of a natural ex perimental pond demonstrated thatDaphnia populations were entirely eliminated after 8 days for the former and 3 days for the latter.195Both chemicals resulted in decreased autotrophic increased populations, phytoplankton and microor flagellate ganism populations, and significant decreases in dissolved ox ygen from balance shifts of autotrophic and heterotrophic pop ulations. et al.196 Tsushimoto reported that dioxin was reduced to 30% of its original concentration following pond applica tion, but most of its metabolites were detected in the water micro aquatic of mortality and column aquatic of an Treatment vegetation. ar outdoor tificial pond with 15 fig/L permethrin indicated rapid loss of the compound from water and while readily absorbed by duck were residues weed, not persistent.197 The fates long-term of three organochlorine pesticides were examined by dosing small experimental ponds.198 Although chemically different, the re sidual behavior of the compounds followed a similar pattern resulting in high initial concentrations in biota, and a slow buildup and decline of sediment concentration. Three years after application of 50 /xg/L of radiolabeled compounds, 14C residues of 0.1 mg/kg were detected in sediments and certain insects even though no detectable symptoms of poisoning were observed over the investigation period. Hildebrand et al.199 determined the effect of the herbicide Roundup on rainbow trout in field experiments following operational application of 2.2 kg/ha, and 10X and 100X this field dose. Results indicated that no mortality McLachlan was at any observed and Hartz200 of dosed surface treatment the the supralittoral levels. zone of an open sandy beach with crude oil, (weathered and fresh oil), fresh oil mixed with dispersions, and weathered oil at the water, and monitored subsequently Meiofauna meiofauna. was reduced 1month after dosing in all cases, but returned to normal in all cases by 5 months except for the site dosed with fresh oil and dispersant. McGreer201 investigated factors balthica on a affecting distribution of the bivalve Macoma mudflat receiving wastewater effluent, and demonstrated that and chlorinated effluent was not toxic in lab oratory tests, which was confirmed by performing 7-day in situ bioassays. The effect of experimental acidification (pH 4) on drift density in a mountain freshwater macroinvertebrate stream demonstrated that drift increased for the first 5 days, but no significant differences were observed between drift en tering and leaving the acidified reach over longer periods.202 unchlorinated HEALTH EFFECTS ASSAYS Test development, and evaluation, validation of short-term bioassays for mutagenicity and cellular toxicity received in tensive investigation. A test approach using a battery of bioas says with careful selection of test systems similar to target or ganisms represents the current testing trend. Statistical analysis and interpretation of results of testing have also received at tention. Evaluating, monitoring, and controlling the quality of complex toxicological research studies, particularly those dealing with toxic hazards of chemicals, were reviewed.203 Kraybill204 listed six specific factors that should be addressed in research studies designed to elucidate exposure/response relationships and risk analysis including; routes of exposure; 807 Water Pollution_ media; integrated exposure; fluctuations in contaminant levels; factors controlling absorption; and fluctuating exposure levels. The mutagenic and carcinogenic evaluation of environmental pollutants, and several methodological problems including a critical evaluation of the Ames test, were discussed at the Eu test ropean Environmental Mutagen Society.205 Mutagenicity ing, bioassay procedures, and analytical techniques for de trace tecting amounts of in environ substances carcinogenic on aquatic species.208 Several tiered-testing scenarios were developed to screen chemicals that are potentially hazardous to public health.209 A systems for analysis approach and ing test was presented. The development of short-term, eukaryotic or assays tests rapid or mutagenicity genicity in prokaryotic were agents screen for each time and direct application screen that microorganisms that uses of cost-effectiveness cost, accuracy, specificity, sensitivity, for also and lower chemical carcino presented.210 Systems A E. deficient crassa Neurospora variation strain preservation test compound. Saccharomyces were reviewed.212 tests in sponse coli, the Ames practices, choice presented Zeiger213 of D3, Problems with re by test dosages of be may assay cerevisiae solvent, caused and that evidence and the apparent high correlation between mutagenicity and carcinogenicity in the Ames assay (up to 92%) is an oversimplification and may be misleading because different classes of chemicals show dif ferent levels of correlation. Because no single test has been developed for detecting all hazardous chemicals, criteria for selecting the best combina tions of tests for a test battery were proposed.214 This decision point approach involves five sequential steps in evaluating car cinogenic potential of a chemical. For estimation of carcino genic potential of chemicals that have not been subjected to carcinogenisis tical ments assays, structure-activity and compound Enslein and equation molecular weights. a statis developed Craig215 on substructural based The frag of accuracy the carcinogen classification is between 87 and 91%, and between 78 and 80% for noncarcinogens. An approach for estimating the proportion of mutagens in a sample was based on discrete contagious distribution theory.216 Schaeffer et al.211 studied correlations between the number of mutagens, and untested (mutagenicity) compounds environmental samples. vironmental mutagens mermann218 indicated Concerning attempts based solely on DNA that there nonmutagens, identified in complex are many to identify en apparatus, and membranes. or carcinogenic mammalian Zea mutation, in water. substances Water supplies microsome Dose-related assay.221 mutagenicity increases were found in 11 of the drinking water supply ex tracts. Using the Salmonella/microsome test for drinking water et that Heartlein concluded al.222 samples, drinking water con taminated with agricultural and/or industrial chemicals may result in a potential health hazard. Because or planned unplanned reuse water potable may expose a population to some health risk, evaluating the po tential toxicity of specific water sources is desirable. Neal223 suggested laboratory tests and monitoring procedures for eval uating effects of organic, inorganic, and radiological chemicals and particulate matter present in finished drinking water. Kal maz and Kalmaz224 reviewed the health effects and ecological significance of chlorine residuals in water. Aldrich and Peo a correlation found between oc melanoma malignant currence and trihalomethane contaminated drinking water supplies in Brevard County, Florida. The Ames assay was used to assess mutagenic activity in fish {Rhinichthys cataractae) and sediment from the Sheep River, Alberta, Canada, which receives a discharge of chlori nated wastewater-works effluents.226 icant increase in the mutagenic the effluent was there plume, there was While a signif activity in fish collected from no evidence of mu increased tagenicity in the sediments. Oncogenic effects of benzo(a)pyrene on three species of larval flatfish were investigated using con centrations to levels comparable found in polluted harbors.227 effects were found with sand sole and flathead sole eggs; however, English sole eggs did not show morphological anomalies. The DNA attacking ability using Bacillis subtilis, and mutagenic activity using the Salmonella typhimurium as say of aqueous sediment extracts of the Tama and Ayase Riv Adverse ers, were Japan, tacking investigated, of properties samples and and mutagenic at DNA Suzuki confirmed.228 et al.229 determined total mutagenicity of the lower Tama River sed iment to be 10 times higher than the upper river sediment. Thin-layer chromatograms of the sediment fractions indicated that mutagenicity was attributable to polar, rather than poly cyclic aromatic vanced treatment compounds. mutagenic on the mutagenicity The effects of ad of wastewater-works effluents were investigated with the Ames assay.230Water chlo rination correlated and the occurrence with mutagenicity. of brominated Ten wastewater compounds work were and in dustrial waste treatment plant effluents demonstrated muta genicity and were found to contain 243 organic compounds, 20 of which are listed as priority pollutants.231 A mammalian cell culture technique was used to study the effect of water treatment targets fluents.232 primary forward of 12Great Lakes municipalities were extracted using XAD 2 resin and tested formutagenic potential with the Salmonella/ alteration, Zim where transmissable genetic damage can be induced, including components of spindle-fiber enzymes of DNA metabolism, crassa Neurospora mays reverse mutation) and suggested that various genetic test endpoints should be used when testing drinking water. Pfeif fer220reviewed the use of Salmonella and microsomes to detect ples225 of potential application for testing chemicals in water with emphasis on systems used or proposed for use by the Severn Trent Water Authority Mutagenicity Unit (STMU) were dis cussed by Tye et al.2n Microbial mutagenicity tests, including the Ames, the poly merase cerevisiae, romyces mutagenic mental samples, were discussed at the 10thAnnual Symposium on the Analytical Chemistry of Pollutants.206 Organization for Economic Cooperation and Development (OECD) guidelines for chemical testing were promulgated and procedures for lab oratory testing of a property or effect deemed important for health and environmental hazard evaluation of chemicals were formulated.207 Projects under the auspices of the National Cancer Institute and the U. S. Environmental Protection Agency included effects of carcinogens, mutagens, and terato gens DeMarini et al.2x9 studied raw and treated waters from Lake tests Illinois, using four different mutation Bloomington, (Ames assay, mitotic gene conversion in strain D4 of Saccha fective on processes Conventional in removing of secondary reduction toxicity was to be water treatment shown ef ef toxicity. As part of a continuing program to evaluate the toxicity of environmental samples with biological endpoints, Babish et 808 Journal WPCF, Volume 55, Number 6 Pollution _Water the lethality of municipal al.233 examined extracts organic to mice. were Extracts wastewater sludge ex to be observed tremely toxic, but toxicity did not correlate with PCB content or mutagenicity, and the relative hazard of toxic sludge com was ponents gested sludge extracts the Ames, using et al.234 Hopke in acetone responses mutagenic Zea of demonstrated di anaerobically and mays, Tradescantia Bacterial of were bioassays to screen used for the presence and toxins in extracts from groundwaters mutagens in situ coal were Mutagens gasification.235 of located in in present groundwater, persistent for 2 years after gasification had been terminated, and showed an activity change with time. Prelim inary evidence suggested that quinolines and aniline deriva tives were in water, mutagenic were water phenolic while the The compounds. in ground toxicants environmental hazards a laboratory-scale coal gasifier were and the tar reported, stream was more effluent toxic and mutagenic than aqueous Waste effluents condensate.236 produced by an experimental were coal gasifier and aqueous the Ames assessed with assay, from were effluents et Ridlington mutagenic metabolic following al.23% demonstrated hepatic cyto chrome P450 oxidase induction in estuarine sculpin following in vivo exposure to Class B petroleum refinery effluent from two West Coast refineries. Using the Ames assay, frameshift mutagens identified inmicrobially degraded crude oil were not detected in crude oils prior tomicrobial attack.239Mammalian were liver microsomes not as used, microor soil-degrading from a waste oil ing of 36 polynitroaromatic was conducted using storage pond compounds the Ames assay. effluent. Screen Combinations of com pounds to yield a synthetic condensate blend exhibited mu tagenicity in five Salmonella strains (TA98, TA 100, TA 1535, TA 1537, and TA 1538) with and without metabolic activa tion.241 The reduction in mutagenicity plexes has been compared with TNT and implications with respect to in situ treatment of contaminated lagoons and soils discussed.242 The of mutagenicity anthracene, and anthraquinone, four structurally similar compounds was evaluated by the Ames assay.243 The former did not exhibit mutagenic activity, but the other compounds were mutagenic for strain TA 1537. In an investigation of the Ames assay mutagenicity of hydrazine derivatives an and aromatic amin?, between the pK of the compounds Suzuki demonstrated.244 et al.245 a qualitative was occurrence of the reactions of aromatic com mutagenicity by photochemical pounds such as polycyclic aromatics and monosubstituted ben zenes in aqueous nitrate solution. Sayler et al.246 indicated that bacterial degradation of PCBs tends to decrease mutagenic potential of lesser chlorinated PCBs, and p-chlorophenylglyox ylic acid, p-chloromandelic acid, and three monochlorinated benzoic acids were not mutagenic in either the sister chromatid exchange or Ames June 1983 the influence of parameters causing interlabo mutation classical et al.251 microsome S9 increased as in the Ames frequency of analysis an described was variance empirical discussed.250 ana for approach lyzing Salmonella dose-response data, which assumed that the mutation rate at low doses is a linear function of dose. With the assumption of initial linearity, this method provided es of potency timates and icals on based compare slope from potencies that were used different test to rank chem to as systems sess quantitative correlations. The ability of white-spotted char liver homogenate to convert chlorinated nitrobiphenyl ethers their nitroso- and was to mutagens amino-derivatives in vestigated using the Ames assay, and results indicated that liver homogenate had the ability to convert these compounds to The mutagens.252 between relationship polynuclear hydrocar on thin layer chromatography plates and assays indicated that samples demonstrating flu bon fluorescence mutagenicity orescence were For over has five to be mutagenic likely the activation years, a concern been as times than samples.253 several in the to mutagens of mu interpretation the use of radiolabeled of promutagens and analysis et al.254 tests. Gentile suggested promutagens in conjunction with assays of labeled metabolites with microbial genetic indicator organisms, as a technique capable of identifying specific genotoxic metabolites. A new screening in which technique separated by thin-layer and chromatography are components sample complex their mutagenic has assay and appeared, is useful for evaluating large numbers of environmental samples.255 A spiral plating method was used in conjunction with the Ames assay to reduce labor involved in the production of dose-response et al.251 reported oxygen electrode nella a microbial and within completed decrease with mutagen a period for retaining mutagen by of an composed system filter Preliminary 10-hour this method, can be assayed. Karube electrode a membrane revertants. typhimurium With curves.256 nondiffusible and diffusible mutagens correlating Salmo screening current concentration. The usefulness and limitations of bacterial DNA-repair tests inmutagenicity/carcinogenicity screening were investigated in laboratory studies.258 Larsen et reviewed al.259 to literature determine the efficacy of DNA repair assays as screening tech niques for mutagenic carcinogens. Despite the high frequency of positive reports, it was obvious that repair assays fail to detect, or detect with low efficiency, those agents whose main action is either intercalation or induction of strand breaks. Therefore, assay. Evaluation of the Ames Salmonella typhimurium/mam malian microsome mutagenicity assay with regard to signifi cance of information obtained and modification of the assay received high priority in 1982. Friederich et al.247 attempted to determine to compared Bernstein was correlation and their mutagenicity reported studied effect is registered directly on the plates by means of the Ames com of TNT-surfactant Parameters say in the absence of any added mutagen.248 Mitchell249 ad dressed the problem of establishing Ames test significance and discussed approaches and limitations in data manipulation and interpretation with respect to genetic significance. A set of mutagenicity data was analyzed using several different sta tistical procedures, and the appropriateness of the procedures tation in TNT wastewater Liver time. storage plate background nonfluorescent ganisms constituted the enzyme activation system in the study. Brown and Donnelly240 quantified mutagenic potential of wa ter concentrates and per plate, and activation.237 microsomal results. assay included histidine amount, sterilization of histidine and biotin, preparation of minimal medium, amount of minimal medium spontaneous assays. paludosa areas to assess. difficult for Ames variation ratory carcinogens DNA was repair as a basis not considered for useful. for mutagenic screening In another literature review, methodologies and status of the Host-Mediated Assay were evaluated and found to be an important mutagenicity/ carcinogenicity test.260With proper selection of protocols and indicators, valuable information can be obtained that would 809 Water Pollution_ be overlooked by strict in vitro assays. Efficacy of Aspergillus systems for determining chemical effects on mitotic segrega tion and mutation were critically summarized.261 Standard treatment of ization was procedures as was recommended, the use of better-marked diploids. Several assays were for environmental developed mutagens not efficiently detected by the Ames assay. Oberly et al.262 investigated the use of the L5178Y Mouse Lymphoma Assay for detection of metal carcinogens. Evidence that the Bacillus subtilis multigene mutagen screening test can detect mutagens that escape detection by the Ames assay has also been pre is indicated by an increase in the fre sented.263Mutagenicity of quency asporogenic B. The colonies. subtilis "rec assay" was studied in detail using an isogenic set of strains carrying in repair or recombination functions or different mutations rec assay is not presently satisfactory for the both.264 Although promutagens, detecting advantages of the in include assay creased permeability of cells to chemicals, easy maintenance of culture strains, and the possibility of using purified spores than rather vegetative cells. A. F. Maciorowski is with Ecological Analysts, Inc., Sparks, L. L F. Raynor are with L. W. Little W. Little and Md.; Associates, Raleigh, N. C; and R. C. Sims and J. L. Sims are with Utah State University, Logan. Correspondence should be addressed to Anthony F. Maciarowski, Ecological Analysts, Inc., 15 Loveton Circle, Sparks, MD 21152. 1. "Aquatic Assessment." and Hazard Toxicology et al (Eds.), Am. Soc. Test. Mater., Philadelphia, K. L., et al, Environment." The "Modeling Ann Arbor Fate of Chemicals Science, Woburn, inWater "Biological Monitoring N. Y. (1982). Press, Elmsford, Pergamon 4. Mason, C. F., "Biology of Freshwater Pollution." Aqueous Science 29, 248 Toxicol, Method for Testing the Vol W., et al, "A Laboratory Solution: First Results and Comparison atility from Aqueous Environ. with Theory." Ecotoxicol 6, 545 (1982). Safety, 16. Klopfer, 17. Mackay, ron. Sei. of Bioconcentration "Correlation D., Technol, 16, 274 Envi Factors." (1982). 18. Saarikoski, Chemical "Relation Between J., and Viluksela, M., Physico of Phenols and Their Toxicity and Accu Properties in Fish." 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Annual "Second and Human of Health Services, Department Pro on National and Environmental Institute Cancer Report 814 Journal WPCF, Volume 55, Number 6 Pollution _Water tection Cancer Nat. Projects." Agency Inst., Md. Bethesda, Direct 209. 210. of the Cost-Effectiveness of Tier L. B., et al, "Analysis on Tox 1st World for Potential Congr. Testing Carcinogenesis." 1982. icol. and Environ. Health, May 27-30, International Cancer Research Data Bank, "Short-term Test Sys tems for Potential Mutagens and Carcinogens: I.Microbial Tests Overview)." NTIS-PB81-922903, 233. icol, 234. (1981). Tye, R. J., et al, "Mutagens, Carcinogens Water Pollut. Control, 80, 600 (1981). 212. Gericke, "On D., the Problem of Microbial (Ger.\ 5, 30(1982). E., "The Role of Short-term kologie Zeiger, Identification of Hazardous the Water Cycle." TAPPI, 216. Rao, (1982). in a of Genetic Toxicology ACS Symp. Ser, Testing." 237. K., 238. 219. between Schaeffer, D. J., et al, "Is There a Correlation ber of Mutagens and the Number of Nonmutagens vironmental Int., 7, 353 (1982). Sample." Environ. Zimmermann, a Mutagenic F. K., "Can We Potential." Determine Mutat. D. M., et al, "Use of Four DeMarini, uate the Mutagenicity of Municipal Water." 9, 127 (1982). of Mutagenic/Carcinogenic Pfeiffer, E. H., "Detection in Water with the Salmonella/Microsome Test GWF Wassser Abwasser, 123, 39 (1982). 221. Williams, D. T., et al, "Determination and Organic Contaminants of Great 222. 242. Test)." Water." 14, 263 (1982). Chemosphere, Water Ob M. W., et al, "Mutagenicity of Municipal Heartlein, Area." Environ. Mutagen., tained from an Agricultural 3, 519 224. E. V., Aldrich, Drinking Effects and and Kalmaz, G. D., "The Health in Water." of Chlorine Residuals Che Melanoma J., "Malignant Bull. Environ. Contam. and 245. 246. 227. 247. K., et al, "Distribution Hirayama, Rikusui Ayase River Sediments." Series 230. 231. 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Mitchell, Significance to Genetical Ames Tests and Its Relevance Mutat. Significance." Res., (1982). 250. et al, "Occurrence of Brominated M., Alkylphenol in Mutagenic Wastewater Concen Carboxylates Polyethaxy Sei. Technol, trates." Environ. 16, 351 (1982). 104,25(1982). of Testing Against Ordered Alternatives Boyd, M. N., "Examples 147 in the Analysis Data." Mutat. of Mutagenicity Res., 97, 251. Bernstein, Reinhard, Ellis, D. D., ary Effluents Contam. 232. Zasshi, (1981). in an of Organic Matter J., et al, "Mutagenic Activity Environ. River Sediment." Pollut. (Series A), 29, 91 Ca?cergram 229. CL, of Mutagens Gaku Potential 103, 133(1982). (1982). 228. Liberman, Arch. of Fish and Sediments L. L., et al, "Mutagenic Osborne, Activity in the Sheep River, Alberta." Water Res. (G. B.), 16, 899 (1982). on Early Devel J. E., et al, "Effects of Benzo(a)pyrene Hose, Arch. Environ. Contam. of Flatfish." Toxicol, 11, 167 opment from Con In Refinery Wastewater in Pa Oxidase System Environ. Contam. Toxicol, 11, A. M., L., and Kaplan, Surfactant Complexes." Bacterial Tox icol, 28,519(1982). 226. Effluents Environ. (1982). Significance 10, 1163(1981). A. T. E., and Peoples, Water Contamination." Arch. Sculpin." Mutagen, D. Kaplan, nitrotoluene tween Toxic Kalmaz, mosphere, J. Toxicol. Environ. Health, 9, 975 (1982). of Relative S. K., et al, "Evaluation Environmental from a Coal Gasifier." NTIS-PB81-2117648, Spring Contam. Bull. Environ. Toxicol, 28, 424 (1982). in Salmonella R. J., et al, "Mutagenicity Sparggord, typhimu of Wastewater rium and Structure-Activity Com Relationships of Trinitrotoluene." from the Manufacture ponents Emanating nella Health Risks of Consuming Potential Neal, R. A., "Evaluating Reused Water." J. Am. Water Works Assoc, 74, 638 (1982). 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Assay." 257. which estimates of risk can be made for a given population of organisms. Heavy metals have been defined as those with a specific gravity greater than 4 or 5, located from atomic numbers 22 to 34 and 40 to 52 on the periodic table (as well as the lanthanide and actinide series), and having a specific biological response. The most common heavy metals include titanium (Ti), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), silver (Ag), cadmium (Cd), tin (Sn), mercury (Hg), and lead (Pb). Simkiss and Taylor3 reviewed the assumptions and concepts was papers to responses evaluate to use intent with the this and and toxicology aquatic were With icology this definition investigation in mind, their effects the primary transition Pb,13 metals,12 citations and a compilation metal heavy hazards other In addition, concerning As,14 toxicity, concerning pollution these metals. of citations contamination in Seattle evaluated the daily uptake of tox and occurrence."2 data base from tions of determination were concentrations in adult of arsenic microanalysis. zinc and copper, cadmium, home plumbing. the prime objective of any tox is to provide the environmental adverse subsequent of bioaccumulation of were Included assessment.11 contain searches Studies conducted Jr., Stuart J. Spiegel the probability the program," sources.7 anthropogenic hazard for updated Se.15 These lead, icity of heavy metals requires the definition of the subject. In the previous review on this topic,1 toxicology was defined by the Education Committee of the Society of Toxicology as "the science which studies the adverse effects of chemicals on living assesses control conceptual reviews in toxicology, including reports of the ef fects of methyl mercury on killifish, and cadmium and mo lybdenum on aquatic insects. Collected bibliographies with abstracts published by the National Technical Information changes and with environment in a quality from often the in changes The proceedings of the Fifth Conference on Aquatic Tox icology and Hazard Assessment reviewed recent progress in Metal organisms monitoring.7-10 HEAVY METALS IN THE ENVIRONMENT heavy toxicity related and metals trace elements a review require. of shellfish and marine plants was updated.16 of of do of biological aspects information changes containing presentation excreting acts If a metal REVIEW SOURCES and The on published carcinogenicity, B. Murphy, they sol of and For the purpose of extensive review of the field, biological monitoring was defined as "the systematic use of biological and Cornelius of metal methods detoxifying, which in concentrations evolved as a pollutant it may act by disrupting normal homeostatic mechanisms. A series of test guidelines covering health effects,4 environmental effects,5 and chemical fate6 was published. These documents were promulgated under Section 4(a) of the Toxic Substances Control Act (TOSCA), but may prove useful in the preparation of other toxicological protocols. A series of Service Bioaccumulation as bioaccumulators have of associated the and variation1718. the with type of et al}9 evaluated morphological on sunfish green water drinking components closely Sorensen as sodium from arsenate arsenic Cytoplasmic to aqueous exposure using electron were inclusions solu x-ray probe observed that may be indicative of a direct association between arsenic and metals, sulfur-rich was characteristic protein molecules. investigated of St. Louis and Lytle presented Lytle Crassostrea and oyster, 816 in Arsenic, the water, along sediment, with 16 other and biota the Mississippi coast.20 along cuneata for the clam, Rangia an to establish in an effort virginica, Bay data Journal WPCF, Volume 55, Number 6