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_Water Pollution

Bioassays? procedures and results A. F. Maciorowski, L. W. Little, L. F. Raynor, R. C. Sims, J. L. Sims

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 procedures have appeared, as have 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 icology and hazard assessment, new concepts in aquatic tox

icology, biological availability and sediment toxicity, and haz ard assessment and water quality criteria.1 A state-of-the-art

overview pertinent to modeling the fate of chemicals in the aquatic environment propounded the concept that hazard as

sessment requires understanding the relationships between chemical concentrations that cause adverse affects on 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

processes, water quality, microbial transformation and bio

degradation; and modeling as a tool in assessing chemical haz

ard. Cairns et al? provided methods, advantages, difficulties, and future possibilities of biological monitoring in water pol lution condensed into six categories including: early-warning systems; receiving system functional methods, relationships, and indexes; receiving system methodology based on com

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, including an account of the work of the water in

dustry in pollution control, were presented in a new textbook.4

Specific pollutants toxic to aquatic organisms were reviewed

including petroleum and specific petroleum hydrocarbons,5 copper,6 zinc,7 lead,8 cadmium,9 chlorine residuals,10 haloge nated hydrocarbons,11 oil well drilling fluids,12 and acidic de position on aquatic ecosystems.13

DESIGN, INTERPRETATION, STRATEGY

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 toxicity to seven aquatic species was examined,14 and acute

toxicity differed up to a factor of 1000 for the different organ isms tested. However, different samples of one test compound

may result in marked toxicity differences, and all compounds were biodegradable by more than 80% within 4 weeks. Bluzat

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 exposed to the same suspension, 48 and 96 hours after prep aration. Toxicity decreased 71 and 45%, respectively, in the

48- and 96-hour old suspensions. As demonstrated by such

studies, physico-chemical data are often useful adjuncts for

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 cluded vapor pressure, water solubility, adsorption and de

sorption phenomena, partition coefficient, volatility, hydro lysis, and photochemical reactivity. Physicochemical factors influencing bioconcentration of organic solutes were examined suggesting a direct proportional relationship between biocon centration factors and octanol-water partition coefficients.17

The correlation can be extended 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 the 1 -octanol/water system, and to APKa 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 correlation of 10 chlorophenols as expressed by a molecular

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

way to predict the approximate acute toxicity values of en

vironmental pollutants and toxic chemicals. Based on exper iments with Mytilus edulis and organic chemicals, Geyer et

al.20 suggested that water solubility and n-octanol water 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 statistical estimation used parameter differences

between single metal and mixture exposures and assumed that

uptake and release are described by a reversible reaction as a

two-compartment model. Based on PCB uptake experiments with plankton, a mathematical model was developed to predict PCB accumulation and determine the roles of feeding and sorption on uptake under various conditions.22 An allometric

model for pesticide bioaccumulation was also reported using body size frequency distribution and appropriate allometric relationships to permit a more operational approach to pes ticide bioaccumulation than the traditional trophic level con

cept.23 The qualitative similarity between experimental pat terns and those in the literature from laboratory and field ex

periments suggested that empirical relations describing contaminant flux as functions of body weight form the basis for predicting contaminant body burdens in natural systems.

June 1983 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 vironments are related to exposure differences, size, rate of

growth, and metabolic parameters. Pollutants rarely occur singly in the environment, and 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 a Mixture Toxicity Index.25 The toxicity of the mixtures was near concentration addition, and chemical

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 regression coefficients for mixture components and the mix

ture. A /-test was developed such that insignificant t values support additivity, negatively significant values support antag

onism, and positively significant values support synergism.

Heavy metal antagonism was also reported for 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 was decreased in the presence of Zn.28

Correlations between laboratory toxicity test results and ef fects from actual environmental exposure requires consider

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 computation of expected environmental concentration, fate, and persistence of chemicals.29 The Exposure Commitment

Method for pollutant exposure evaluation was described as a time-independent approach to exposure evaluation measured

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 relationships. Frische et al.31 examined

criteria to assess environmental behavior of chemicals with

respect to selection and preliminary quantification. Factors of prime importance included the quantity entering the environ

ment, mobility, accumulation, persistence, and direct or in

direct toxic effects. These factors were quantified using pro duction volumes, physicochemical data, results of degradation and accumulation experiments, and toxicity values. Quanti fied criteria for "well-known" organic chemicals were evalu ated using appropriate weighing factors demonstrating that persistence and accumulation were most important; mutage

nicity and carcinogenicity had to be strongly weighted; and mobility, although indispensible for estimating chemical dis

tribution and persistence, was not included because it can 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 Profile Analysis, included consideration of bioaccumulation potential with algae and fish tests; mammalian retention and elimination with rat tests; information of biodegradability,

metabolism, accumulation, and bound residues of the acti

vated sludge test; and photomineralization by irradiation with UV light.32 The cumulative data are subsequently ranked and

correlated with respective physicochemical properties, and test results compared to complete the assessment.

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 salts. The system used rainbow trout egg and larval develop

ment studies. An inexpensive device for continuous monitor

ing of dissolved gases and supersaturation termed a gasometer was described and may be used to activate an alarm system

and, therefore, protect fish from hyperbaric or hypobaric gas pressures.34

MICROORGANISMS

A compendium of microbiological aspects of water quality included various laboratory techniques, including bioassay

procedures and screening tests.35 A bioassay method based on

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 reference compound. This procedure was sug

gested as a surrogate parameter analysis for organic priority

pollutants. The application of four bacterial screening proce dures to assess changes in the toxicity of chemicals in mixtures 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

fluorescens exposed to pentachlorophenol, and growth was a

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 degradation or weathering.42 Sayler et al.43 evaluated func

tional responses and recovery of sediment microbial com

munities using 20 estimates of microbial population density, biomass, and activity over a 15-month period. Multivariate

analysis of variance and discriminant analysis were used to determine variation between contaminated sites. It was con

cluded that multiple functional measures of microbial com munity responses are required to evaluate the effect and re

covery from environmental contamination, and certain phys

iological traits may 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

_Water Pollution

cultures or in water from a contaminated nutrient-rich res

ervoir.44 Mahaffey 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 pure cultures.46

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 the sequence of chlorophenol addition. Cell suspensions 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. It was 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 computed from cell density changes, bio-volumes, 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 the methods 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 concentration.50 The method was used

to examine water samples from various water treatment stages and piping material, demonstrating that AOC increased if water was ozonated or placed in contact with polyvinyl chlo ride pipes. Gordon et al.51 assessed calorimetry as a technique for studying marine microbial metabolism and organic nu trient concentrations found in marine waters. Microcalori

metric measurements of heat production from glucose by Vib rio alginolyticus demonstrated that glucose metabolism by this bacterium was measurable at submicromolar concentrations

and that a constant metabolism efficiency was indicated over

the wide range of glucose concentrations studied.

Determination of organic compound biodegradability has 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 for measuring biod?gradation (BOD, shake flask, C02 evolution, activated sludge, and the Gledhill test) using five

organic compounds. Certain compounds were rapidly metab

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.

Several investigators used microorganisms other than bac

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

which was not affected by K, Na, Ca, or Fe additions. A re

liable, accurate technique for identifying and analyzing sol vent-pesticide interactions in bioassays was described that uses the fungi Pythium ultimum, Sclerotina homeocarpa, and Pes

talotia sp., acetone, and the fungicides metalaxyl and captan.55 Interaction responses with any given set of bioassay parameters

were dependent on both the acetone and fungicide concentra tions, but the method compensates for discrepancies and in dicates the most suitable test parameters,to use in subsequent

bioassays. A bioassay technique using the protozoan Tetra

hymena pyriformis was described that uses the rate of change of oxygen uptake, over a 10-minute period, as the 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 cartap was examined using a Paramecium primau relia assay providing a 24-hour LC50 of 2.5 mg/L.57

ASSAYS WITH ALGAE AND OTHER PLANTS

New approaches to algal assays were proposed by several

investigators. Domotor et a/.58 demonstrated that autoradiog

raphy facilitated detection of species-specific thermal stress in phytoplankton communities. An automated biological mon

itoring system based on changes in natural fluorescence of algae was designed and evaluated.59 Comparison of cell counts, in vivo fluorescence, phaeophytin a, and chlorophyll a mea

surement as indicators of toxicity to Selenastrum capricor nutum showed that cell count data alone failed to indicate adequately toxic stress while in vivo fluorescence gave an ac

curate and rapid indication.60 According to Sellner et al.,61

natural fluorescence can be enhanced by adding DCMU, sug gesting that the almost instantaneous response of alga fluo rescence to stress may provide a replacement for time-con

suming algal assay procedures. Testing of volatile substances is difficult in the Algal Assay Procedure Bottle Test and a

modification that maintains a constant concentration of vol

atile test substances in culture containers was devised.62

Algal assays continue to be used extensively in determining the limiting nutrient in waters and assessing eutrophication potential. Results of such studies were reported for Balaton

Lake (Hungary),63 English lakes,64 Hastings Lake (Canada),65 the Rideau River (Canada),66 Lake Michigan,67 deep ocean

waters,68 Memphremagog Lake (Canada),69 and Wolderwijd Lake (Netherlands).70 Young et al11 used Scenedesmus bioas says to evaluate phosphorus availability in municipal waste

waters. The problem of poor growth of many marine algae in

June 1983 803

Water Pollution_

artificial seawaters was examined and it was suggested that selenium deficiency may be a problem.72 Visser and Couture73

demonstrated that Selenastrum capricornutum growth in water from natural sources was influenced by the nature and amount

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 phyton strips.

The difficulties in using a single bioassay method to deter mine nutrient status of a water were stressed by several in

vestigators. Several bioassays for determining nitrogen and phosphorus demand were compared, and a combination of

techniques allowed useful conclusions about nutrient status, but no single technique could be considered conclusive.75,76 Chiaudani and Vighi77 preferred a multistep 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 bioassays could be misleading. One reason for erratic results

from 14C methods was the presence of trace metals in shock

solutions and containers used in the assay, and procedures to

eliminate such contamination were reported.80 Yallop81 ob

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 uptake, it was suggested that oxygen uptake measurements be

made in the presence of dichlorophenyl-dimethylurea. Imaoka et a/.82 used red tide organism algal assays to demonstrate that phosphorus removal from municipal wastewater was an effec

tive control 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 wastewaters,86 coal liquids and oils,87"89 detergents,90

ammonia,91 hexa- and polychlorinated biphenyls,92,93 pen

tachloropheol,94 and humic materials.95 Walsh et al.,96 com

pared the sensitivity of Selenastrum capricornutum and Skel etonema costatum to that of Daphnia and Mysidopsis and to 10 wastewaters, and found algae to be more 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 capricornutum assays were used to 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 its metabolites 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 microorganisms were 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 in mixed culture using 24- to 96-hour and 28-day bioassays. Mixed 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 algal species. Five algal genera, as well as a natural

phytoplankton assemblage were used in determining effects of a mixture of 10 heavy metals using primary productivity re duction and other responses.107 Foster108 used 200 isolates,

representing 87 species of green algae, in sensitivity tests with Cu, Pb, Zn, and Cd, and found that isolates were generally resistant to metals normally present in their habitats. In related research,109 species of algae present in metal-polluted rivers

seem to be determined by the degree of metal pollution. Fisher110 observed that conditions in a polluted estuary selected for heavy metal tolerance in marine diatoms. As part of a long term study to evaluate the algal species composition trends in

the Great Lakes, toxicity studies of heavy metals on various algal size fractions were conducted.111

The influence of Co, Ni, Cu, and Cd on Anacystis nidulans was examined to assist in response interpretation of blue-green

algae at mining sites resulting in mutants tolerant to high metal levels.112 Of 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 algae were investigated,113 as were those of Se, Hg, and

Cu in tests with Dunaliella.114 Petersen115 addressed the ap plicability of metal speciation in studies of the joint action of Cu and Zn on Scenedesmus quadricauda and proposed a

model to account for observed biological effects as a function of metal speciation. Several studies concentrated on mecha

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 algae. For the marine diatom Thalassiosira rotula, chain

length was a useful indicator because the chains tended to break apart at toxic Cd and Ni levels.120 Usefulness 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 Burdon-Jones,122 who obtained unsatisfactory results with the

brown algae Padina as a monitor for 10 metals.

The role of plants is becoming more common in toxicity, biomonitoring, and mutagenicity tests. Onion root tips were 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 Test results with

Lemna demonstrated its similarity to animals in responses to

pentachlorophenol.127 To facilitate use of macrophytes for monitoring stream 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 Pollution

western Europe,130 and U. S. waters.131 For short-term genetic

toxicology assays, the Arabidopsis plant seems to be suitable for in situ tests of soil- and water-borne pollutants.132 Italian

researchers suggested that a root tip assay with the bean Vicia faba is sensitive, reliable, and inexpensive as a screening test

for mutagens.133 To assess effects of pollutants on seagrass. Walsh et al.134 devised a flow-through system containing whole plants or leaves of Thalassia testudinum. Plant genetic and cytogenetic assays with respect to protocols, use in screening

programs, and recommended research have been discussed,135 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 and mutagens/carcinogens in the production mechanism of plastid-free Euglena mutants was used for rapid determination of mutagenic activity.138

INVERTEBRATES AND FISH 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 is placed on procedural and methodological literature. For spe 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").

Freshwater cladocerans, specifically Daphnia magna, and

D. pulex represent the most commonly employed freshwater

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 toxicants, resulting in recommendations for procedure im

provements.140 M?ller141 examined the interference of insol 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 commonly used responses of

survivorship, number of juvenile molts, age at reproductive maturity, and neonate body length.143 The former two re

sponses and neonate body length were reduced at Cu concen trations that did not reduce longevity or reproduction, while juvenile molt number, age at reproductive maturity, and mean

brood size responses were erratic to Cu exposure. To assess

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 population size and the level of radiation exposure, as

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. Use of Paramecium tetraurelea for detecting gen

otoxicity and cytoxicity of oil shale process water was discussed as a rapid detection system for health hazards.145 The use of

sponges as model organisms for the detection of carcinogens in marine environments was evaluated, using benzo(a)pyrene

equivalent activity in the Ames test.146 A host-mediated assay for the detection of mutagenic compounds was also developed using mussels grown in clean and polluted areas and clean

mussels transferred into polluted water.147 The direct muta

genic activity of mussel extract on yeast and bacteria was dose

dependent, however, the induced mutagenic frequency never

exceeded a value corresponding to about five times the spon

taneous frequency. Benoit et al.148 evaluated the 32-day fathead minnow early

life stage toxicity test in determining estimated maximum ac

ceptable toxicant concentrations of four untested organic chemicals. Toxic effects on early developmental stages 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 velop water quality criteria when used to predict 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

ployed to examine young-of-the-year striped bass from the 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

June 1983 805

Water Pollution_

icity test using ventilation frequency of steelhead as the re sponse variable. Ventilation frequencies were enumerated

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 ventilatory test was at least as sensitive as chronic toxicity tests.

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 effects of carcinogens, mutagens, teratogens, and geno toxins on fish species. Laboratory and field studies of aquatic animals were reviewed indicating that fish have metabolic pathways similar to mammals for disposition of certain car

cinogens.155 After screening 20 fish species, Boleopthalmus dussumieri, an edible mud skipper, was studied as a cytoge netic model for in vivo mutagen detection.156 Utility of the killifish Nothobranchius rachowi in a sister chromatid ex change test was described157 as was the use of sister chromatid exchange frequency in flatfish exposed to carcinogens.158 The suitability of a micronucleus test for genotoxic compound de tection using the eastern mudminnow Umbrea pygmea was

also reported, and represents an alternative to chromosome

aberration tests.159 Other 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 time, clearance parameters, and dosing regimes for crude oil;160 induction of hepatic microsomal cytochrome P450 and aldrin epoxidase in the sculpin Leptocottus armatus exposed to petroleum refinery wastewater;161 and the use of

adenylate concentrations and adenylate energy charge as in

dicators of hypoxic stress in estuarine fish.162

COMPARATIVE TESTING, MICROCOSMS, AND IN SITU TESTS

The 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. Brusick and 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 vitro mammalian clonal toxicity assays, and consideration of the role of aquatic ecological and terres trial ecological assays in environmental assessment. The sec

ond manual outlined the rationale and proposed methods for performing Level 1 health effects and ecological effects bioas 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 to assess potential environmental

hazards were described. In addition to tests for mammalian, fish, terrestrial insect, aquatic 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

including bacteria, algae, crust?cea, fishes, and amphibians,

using a variety of lethal and sublethal endpoints.165 The lowest "no-toxic-effect-level" was determined to be 0.37 /?g Cd2+/L.

Dauble et al.166 examined effects of coal-liquor water soluble

fraction (WSF) on growth and survival of two freshwater midges, a daphnid, and a green alga. Suppression of growth, reproduction, and survival was observed at concentrations of 0.02 to 0.42% WSF for the invertebrates, with algal populations being the least sensitive of the species tested. The acute toxic responses of species pairs tested simultaneously were deter

mined for sodium lauryl sulfate, cadmium, and 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. Mysids were most sensitive to cadmium and

methanomyl, with algae being most sensitive to sodium lauryl sulfate. Payne168 examined metabolism of oil spill surfactant compounds by rainbow trout, a crustacean Cancer irroratus, and the mollusk Chlamys islandicus demonstrating that each had the capacity for enzymatic hydrolysis of the complex fatty acid ester mixtures.

Comparative aquatic organism evaluation aids in assess

ments, but microcosm tests often allow more realistic simu

lation of environmental exposure conditions. Three-phase microcosm systems (sediment, water, and gas) for evaluating substances under a variety of ecological conditions were de scribed by Porcella et al.169 Response variables were related to general ecosystem functional processes (photosynthesis, res

piration, anaerobic degradation, carbon cycling, and 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 tabolism,173 crude oil on simulated natural lakes using gas

water-sediment laboratory microcosms;174 the water-soluble

fraction of a crude oil liquefaction product on laboratory pond microcosms;175 and no. 2 fuel oil on natural phytoplankton assemblages.176 Microcosm studies were also conducted to de

termine the significance of interfaces in the distribution and metabolism of di-2-ethylhexyl phthalate in a static laboratory ecosystem.177 Interface adsorption occurred rapidly and ac

cumulations occurred in organisms inhabiting and/or feeding at the interfaces. Dickson et al.178 used three-phase microcosms

to study the aquatic fate and effect of benz(a)anthracene, which had no acute effect on aquatic organisms as measured by com

munity structure and function. After 60 days, most

benz(a)anthracene remained in the sediment with no evidence

of metabolism. Atrazine toxicity to submerged vascular plants was investigated by Correll and Wu179 using simulated estu arine microcosms. Macrobenthic estuarine communities de

veloped in sand-filled aquaria in the laboratory and field were exposed to chlorpyrifos to assess community structure ef

fects.180 Molluscan larvae colonizing laboratory aquaria 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

_Water Pollution

tration in laboratory aquaria and at the latter concentration

in field communities. Strachan et al.m developed a teflon che mostat for studies of trace metals to overcome problems of

trace metal contamination from culture vessels and medium

reservoirs. The only non-teflon components were glass pH and

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 port, and toxicity.182 The effects of Cu on an aquatic micro

cosm containing bacteria, algae, protozoa, rotifers, and oli

gochaetes demonstrated that stress responses were greater in

early stages of heterotrophic succession and high nutrient con

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 culture, and Cd removal from the water phase.184 Capone and

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 microcosms were used to study the effects of temperature and salinity,186 and salinity and or ganic nutrient concentration,187 on the survival and growth of Vibrio cholerae, which suggested that this species is an au

tochthonous member of the estuarine microbial 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 litter. The ATP increase occurred when microcosms were 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 separately with chlorine, Cu, and dextrose.191 Protein, carbo

hydrate, and organic content of aufwuchs 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 invertebrate communities that colonize sands in running seawater were examined using biochemical methodology to assess impact.193

In contrast to microcosm studies designed to simulate ex

perimentally more natural conditions in the laboratory, in situ test methods provide an opportunity 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

high mortality of insects and crust?cea was observed.194 Ap

plication of 5 mg/L trichlorphenol and 1 mg/L penta chlorophenol into duplicate compartments of a natural ex perimental pond demonstrated that Daphnia populations were entirely eliminated after 8 days for the former and 3 days for the latter.195 Both chemicals resulted in decreased autotrophic phytoplankton populations, increased flagellate and microor

ganism populations, and significant decreases in dissolved ox ygen from balance shifts of autotrophic and heterotrophic pop ulations. Tsushimoto et al.196 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 column and aquatic vegetation. Treatment of an outdoor ar

tificial pond with 15 fig/L permethrin indicated rapid loss of the compound from water and while readily absorbed by duck

weed, residues were not persistent.197 The long-term fates 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 was observed at any of the treatment levels.

McLachlan and Hartz200 surface dosed the supralittoral 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 subsequently monitored meiofauna. Meiofauna

was reduced 1 month 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 affecting distribution of the bivalve Macoma balthica on a

mudflat receiving wastewater effluent, and demonstrated that

unchlorinated 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 freshwater macroinvertebrate drift density in a mountain 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

HEALTH EFFECTS ASSAYS Test development, evaluation, and 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;

June 1983 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 ropean Environmental Mutagen Society.205 Mutagenicity test ing, bioassay procedures, and analytical techniques for de tecting trace amounts of carcinogenic substances in environ

mental samples, were discussed at the 10th Annual 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 on aquatic species.208

Several tiered-testing scenarios were developed to screen chemicals that are potentially hazardous to public health.209

A systems approach for analysis of cost-effectiveness that uses

sensitivity, specificity, accuracy, cost, and time for each screen

ing test was presented. The development and direct application of short-term, rapid tests or assays in prokaryotic and lower

eukaryotic microorganisms that screen for chemical carcino

genicity or mutagenicity agents were also presented.210 Systems 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 A deficient E. coli, Saccharomyces cerevisiae D3, and

Neurospora crassa tests were reviewed.212 Problems with re

sponse variation in the Ames assay may be caused by test

strain preservation practices, choice of solvent, and dosages of

test compound. Zeiger213 presented evidence that 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 assays, Enslein and Craig215 developed a statis

tical structure-activity equation based on substructural frag ments and compound molecular weights. The accuracy of 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, nonmutagens, and untested (mutagenicity) compounds identified in complex environmental samples. Concerning attempts to identify en

vironmental mutagens based solely on DNA alteration, Zim mermann218 indicated that there are many primary targets where transmissable genetic damage can be induced, including enzymes of DNA metabolism, components of spindle-fiber apparatus, and membranes.

DeMarini et al.2x9 studied raw and treated waters from Lake Bloomington, Illinois, using four different mutation tests (Ames assay, mitotic gene conversion in strain D4 of Saccha romyces cerevisiae, Neurospora crassa forward mutation, Zea

mays reverse mutation) and suggested that various genetic test endpoints should be used when testing drinking water. Pfeif fer220 reviewed the use of Salmonella and microsomes to detect

mutagenic or carcinogenic substances in water. Water supplies of 12 Great Lakes municipalities were extracted using XAD 2 resin and tested for mutagenic potential with the Salmonella/

mammalian microsome assay.221 Dose-related mutagenicity increases were found in 11 of the drinking water supply ex tracts. Using the Salmonella/microsome test for drinking water samples, Heartlein et al.222 concluded that drinking water con taminated with agricultural and/or industrial chemicals may result in a potential health hazard.

Because planned or unplanned potable water reuse 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 ples225 found a correlation between malignant melanoma oc

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 While there was a signif icant increase in the mutagenic activity in fish collected from the effluent plume, there was no evidence of increased mu

tagenicity in the sediments. Oncogenic effects of benzo(a)pyrene on three species of larval flatfish were investigated using con centrations comparable to levels found in polluted harbors.227

Adverse 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 ers, Japan, were investigated, and mutagenic and DNA at

tacking properties of samples confirmed.228 Suzuki 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 mutagenic compounds. The effects of ad

vanced treatment on the mutagenicity of wastewater-works

effluents were investigated with the Ames assay.230 Water chlo rination and the occurrence of brominated compounds were

correlated with mutagenicity. Ten wastewater work 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 processes on toxicity reduction of secondary ef

fluents.232 Conventional water treatment was shown to be ef

fective in removing 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

_Water Pollution

al.233 examined the lethality of municipal wastewater sludge organic extracts to mice. Extracts were observed to be ex

tremely toxic, but toxicity did not correlate with PCB content or mutagenicity, and the relative hazard of toxic sludge com ponents was difficult to assess. Hopke et al.234 demonstrated

mutagenic responses in acetone extracts of anaerobically di

gested sludge using the Ames, Zea mays, and Tradescantia

paludosa assays. Bacterial bioassays were used to screen for the presence of

mutagens and toxins in extracts from groundwaters located in areas of in situ coal gasification.235 Mutagens were present in

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 mutagenic in water, while the toxicants in ground

water were phenolic compounds. The environmental hazards

from a laboratory-scale coal gasifier were reported, and the tar

effluent stream was more toxic and mutagenic than aqueous condensate.236 Waste effluents produced by an experimental coal gasifier were assessed with the Ames assay, and aqueous effluents were mutagenic following metabolic activation.237

Ridlington et al.23% demonstrated hepatic microsomal 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 in microbially degraded crude oil were not detected in crude oils prior to microbial attack.239 Mammalian liver microsomes were not used, as soil-degrading microor

ganisms constituted the enzyme activation system in the study. Brown and Donnelly240 quantified mutagenic potential of wa ter concentrates from a waste oil storage pond effluent. Screen

ing of 36 polynitroaromatic compounds in TNT wastewater was conducted using the Ames assay. 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 of TNT-surfactant com

plexes has been compared with TNT and implications with respect to in situ treatment of contaminated lagoons and soils discussed.242

The mutagenicity of anthracene, anthraquinone, and 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 and an aromatic amin?, a qualitative correlation

between the pK of the compounds and their mutagenicity was demonstrated.244 Suzuki et al.245 reported the occurrence of

mutagenicity by photochemical reactions of aromatic com 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 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 the influence of parameters causing interlabo

ratory variation for Ames assay results. Parameters studied

included histidine amount, sterilization of histidine and biotin, preparation of minimal medium, amount of minimal medium per plate, and plate storage time. Liver microsome S9 increased

spontaneous background mutation frequency in the Ames as

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 compared to classical analysis of variance was discussed.250

Bernstein et al.251 described an empirical approach for ana

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 timates of potency based on slope that were used to rank chem

icals and compare potencies from different test systems to as

sess quantitative correlations. The ability of white-spotted char liver homogenate to convert chlorinated nitrobiphenyl ethers and their nitroso- and amino-derivatives to mutagens was in

vestigated using the Ames assay, and results indicated that liver homogenate had the ability to convert these compounds to

mutagens.252 The relationship between polynuclear hydrocar bon fluorescence on thin layer chromatography plates and mutagenicity assays indicated that samples demonstrating flu orescence were over five times as likely to be mutagenic than

nonfluorescent samples.253 For several years, the activation of promutagens to mutagens

has been a concern in the analysis and interpretation of mu

tation tests. Gentile et al.254 suggested the use of radiolabeled

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 technique in which complex sample components are

separated by thin-layer chromatography and their mutagenic effect is registered directly on the plates by means of the Ames assay has appeared, and 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 curves.256 With this method, nondiffusible and diffusible mutagens can be assayed. Karube et al.251 reported a microbial electrode system composed of an

oxygen electrode and a membrane filter for retaining Salmo

nella typhimurium revertants. Preliminary mutagen screening was completed within a 10-hour period by correlating current

decrease with mutagen concentration.

The usefulness and limitations of bacterial DNA-repair tests in mutagenicity/carcinogenicity screening were investigated in laboratory studies.258 Larsen et al.259 reviewed literature to

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, DNA repair as a basis for screening for mutagenic

carcinogens was not considered useful. In another literature

review, methodologies and status of the Host-Mediated Assay were evaluated and found to be an important mutagenicity/ carcinogenicity test.260 With proper selection of protocols and indicators, valuable information can be obtained that would

June 1983 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 ization of treatment procedures was recommended, as was the

use of better-marked diploids. Several assays were developed for environmental 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 sented.263 Mutagenicity is indicated by an increase in the fre quency of asporogenic colonies. The B. subtilis "rec assay" was studied in detail using an isogenic set of strains carrying different mutations in repair or recombination functions or both.264 Although the rec assay is not presently satisfactory for detecting promutagens, advantages of the assay include in

creased permeability of cells to chemicals, easy maintenance of culture strains, and the possibility of using purified spores rather than vegetative cells.

A. F. Maciorowski is with Ecological Analysts, Inc., Sparks, Md.; L. W. Little and L F. Raynor are with L. W. Little 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.

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205. "Progress in Environmental Mutagenesis and Carcinogenesis,

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Testing for Potential Carcinogenesis." 1st World Congr. on Tox icol. and Environ. Health, May 27-30, 1982.

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218. Zimmermann, F. K., "Can We Determine Mutagenicity or Only a Mutagenic Potential." Mutat. Res., 92, 3 (1982).

219. DeMarini, D. M., et al, "Use of Four Short-Term Tests to Eval uate the Mutagenicity of Municipal Water." /. Toxicol. Environ.

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Urban River Sediment." Environ. Pollut. (Series A), 29, 91

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June 1983 815

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Bioaccumulation and

toxicity of heavy metals and related trace elements Cornelius B. Murphy, Jr., Stuart J. Spiegel

The presentation of a review of bioaccumulation and tox

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 organisms and assesses the probability of their occurrence."2

With this definition in mind, the prime objective of any tox icology investigation is to provide the primary data base from

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 involved in the use of organisms as bioaccumulators of metal

ions. They conjectured that cells have evolved methods of sol

ubilizing, transporting, regulating, detoxifying, and excreting metals in concentrations which they do require. If a metal acts

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

papers was published on aspects of biological monitoring.7-10 For the purpose of extensive review of the field, biological monitoring was defined as "the systematic use of biological responses to evaluate changes in the environment with the

intent to use this information in a quality control program," with the changes often from anthropogenic sources.7

REVIEW SOURCES The proceedings of the Fifth Conference on Aquatic Tox

icology and Hazard Assessment reviewed recent progress in

aquatic toxicology and hazard assessment.11 Included were

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 Service were updated for the transition metals,12 Pb,13 As,14 and Se.15 These searches contain citations concerning toxicity,

carcinogenicity, environmental pollution and other hazards

and adverse effects of these metals. In addition, a compilation

containing citations concerning heavy metal contamination

of shellfish and marine plants was updated.16

HEAVY METALS IN THE ENVIRONMENT

Studies conducted in Seattle evaluated the daily uptake of lead, cadmium, copper, and zinc from drinking water and the

subsequent determination of components of variation1718.

Metal concentrations were closely associated with the type of

home plumbing. Sorensen et al}9 evaluated morphological changes in adult green sunfish on exposure to aqueous solu

tions of arsenic as sodium arsenate using electron probe x-ray

microanalysis. Cytoplasmic arsenic inclusions were observed

that may be indicative of a direct association between arsenic and sulfur-rich protein molecules. Arsenic, along with 16 other

metals, was investigated in the water, sediment, and biota

characteristic of St. Louis Bay along the Mississippi coast.20

Lytle and Lytle presented data for the clam, Rangia cuneata

and oyster, Crassostrea virginica, in an effort to establish an

816 Journal WPCF, Volume 55, Number 6