Procedures are described for testing freshwater organisms in the laboratory to evaluate the toxicity or bioaccumulation of contaminants associated with whole sediments. Sediments may be collected from the field or spiked with compounds in the laboratory. Toxicity methods are outlined for two organisms, the amphipod Hyalella azteca and the midge Chironomus tentans. The toxicity tests are conducted for 10d in 300-mL chambers containing 100 mL of sediment and 175 mL of overlying water. Overlying water is renewed daily and test organisms are fed during the toxicity tests. The endpoint in the toxicity test with H. azteca is survival and the endpoints in the toxicity test with C. tentans are survival and growth. Procedures are primarily described for testing freshwater sediments; however, estuarine sediments (up to 15% salinity) can also be tested with H. azteca. Guidance for conducting 28-d bioaccumulation tests with the oligochaete Lumbriculus variegatus is provided in this manual. Overlying water is renewed daily and test organisms are not fed during bioaccumulation tests. Methods are also described for determining bioaccumulation kinetics of different classes of compounds during 28-d exposures with L. variegatus.

Methods for measuring the toxicity and bioaccumulation of sediment-associated contaminants with freshwater invertebrates

In the field of aquatic toxicology, quantitative structure-activity relationships (QSARs) have developed as scientifically credible models for predicting the toxicity of chemicals when little or no empirical data are available. In recent years, there has been an evolution of QSAR development and application from that of a chemical-class perspective to one that is more consistent with assumptions regarding modes of toxic action. The objective of this research was to develop procedures that relate modes of acute toxic action in the fathead minnow (Pimephales promelas) to chemical structures and properties. An empirically derived database for diverse chemical structures of acute toxicity and corresponding modes of toxic action was developed through joint toxic action studies, the establishment of toxicodynamic profiles, and behavioral and dose-response interpretation of 96-h LC50 tests. Using the results from these efforts, as well as principles in the toxicological literature, approximately 600 chemicals were classified as narcotics (three distinct groups), oxidative phosphorylation uncouplers, respiratory inhibitors, electrophiles/proelectrophiles, acetylcholinesterase inhibitors, or central nervous system seizure agents. Using this data set, a computer-based expert system has been established whereby chemical structures are associated with likely modes of toxic action and, when available, corresponding QSARs.

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Predicting modes of toxic action from chemical structure: Acute toxicity in the fathead minnow (Pimephales promelas)

The effects of various water chemistry parameters on the toxicity of copper to larval fathead minnows were investigated. Increased pH, hardness, sodium, dissolved organic matter, and suspended solids each caused toxicity to decrease on the basis of total copper concentrations. In contrast, added potassium resulted in increased toxicity. Alkalinity had no observed effect on total copper LC50s, but its effects might have been masked by those of the cations added with it. In most cases, the effects of water chemistry were found to be similar for different endpoints (growth, survival at different durations), but there were differences in the relative magnitude of some effects across these endpoints. Over all experimental treatments, 96-h total copper LC50s varied 60-fold. Every water chemistry parameter also caused toxicity to vary significantly when expressed on the basis of cupric ion selective electrode measurements, sometimes more so than on the basis of total copper. Therefore, this study does not support attributing to cupric ion a singular importance in regulating toxicity to this test organism. A variety of copper species might be contributing to toxicity and it is evident that toxicity is also affected by water chemistry in ways not related to copper speciation.

The effects of water chemistry on the toxicity of copper to fathead minnows

A comparison between the diacetyl monoxime and urease methods for measuring dissolved concentrations of urea in seawater was conducted in artificial seawater, phytoplankton-culture filtrate and both natural and ureaspiked field samples from coastal and oceanic enviroments during 1984. The urease technique underestimated urea concentrations in unbuffered photoplankton-culture filtrate as a result of the inhibition of the urease enzyme, causing the incomplete hydrolysis of urea in these samples. Factors responsible for inhibiting urease included pH, seawater ions, and possibly extracellular metabolites produced in unialgal cultures. Seawater type and time of sample collection were important variables affecting urea measurement by the urease method, and recovery of internal standards ranged from 40 to 100%. Increasing the heating time of the urease assay, or the concentration of urease added to the seawater samples increased the amount of urea determined by the urease method. However, measured values were still less than the concentration of the urea internal standards. The diacetyl monoxime method was suitable for urea determinations in all the seawater samples we examined; it was easily automated, and the results were accurate and reproducible. This modified technique is recommended for measuring disolved concentrations of urea in seawater.

Comparison of methods for the analysis of dissolved urea in seawater

The rate of reaction of trace metal ions is an important consideration when studying the chemistry of trace metals in natural waters. The application of speciation models to natural water systems requires knowledge of kinetics if reactions are slow. Most bioassay and toxicity tests conducted in static and flow-through systems have not taken reaction kinetics into account. Therefore, results from these studies may overestimate the toxicity in the receiving waters. In the present study, the kinetics of the interaction of Cu(II) with humic acid (HA) and its influence on the toxicity of copper to Ceriodaphnia dubia were investigated by both chemical kinetic studies using a copper ion selective electrode and bioassay tests using a continuous flow-through bioassay system. A two-ligand site with first-order rate constants model gave a very good description of experimental kinetic data of the change of free Cu2+ concentration. Average k1 was 1.85/h and average k2 was 0.094/h. Bioassay tests indicated that different reaction times of copper with HA solution produced different toxic effects to organisms. We determined the hydrodynamic characteristics of the bioassay chambers to better describe the exposure of the organisms to free Cu2+. The bioassays supported the free ion activity model that the bioavailability and therefore toxicity of copper was directly correlated to the free Cu2+ concentration rather than to the total copper concentration. It was further shown that conventional chemical kinetics can be used to predict the toxicity of copper in these bioassays. This study supports the importance of considering reaction kinetics when studying the chemistry of trace metals in natural waters.

Diane L. Olson

Papers

A continuous-flow mini-diluter system for toxicity testing

Effect of selected environmental pollutants and other chemicals on the activity of urease (in vitro).