Development of practical methods for assessing the chronic toxicity of effluents


  • See Table S1 for the number of citations and rank of all the “Top 100” papers, which in this essay is reference [ Mount et al., 1].

The paper that prompted this essay 1 had its roots in the development of the water quality–based approach to permitting effluent discharges to surface waters in the United States under the National Pollutant Discharge Elimination System (NPDES) program. Prior to that development, limits for toxic chemicals in effluents were derived from technology-based standards, which were established based on the effluent quality that could be achieved for a particular type of discharge using the treatment technology available at the time. While implementation of technology-based standards achieved considerable reduction in the loading of toxic chemicals to receiving waters, aquatic life was not intrinsically protected because the standards were not based on the effects of discharged chemicals on aquatic life, nor was the in-stream dilution considered in determining acceptable quality of the discharge. The implementation of the water quality–based approach to permitting toxic chemicals in effluents added a new focus to permitting decisions by establishing discharge limits so that concentrations of toxic chemicals in the receiving waters would not exceed those concentrations causing unacceptable effects on aquatic life.

A mainstay of the water quality-based approach was the application of numerical standards for individual toxic chemicals, often based on water quality criteria established by the U.S. Environmental Protection Agency (U.S. EPA). Effluent limits for individual chemicals were determined by calculating the amount of chemical discharge that would not exceed state standards in the receiving water, after accounting for the flow available for dilution. If this calculated concentration was below the technology-based standards, additional treatment would be required to reduce chemical concentrations in the effluent to a level sufficient to protect aquatic life in the receiving water.

While reducing concentrations of individual chemicals in effluents sufficiently to meet state standards provided considerable additional protection to aquatic communities, the U.S. EPA recognized that this alone would not be sufficient to protect surface waters from effluent contaminants for three primary reasons. First, insufficient information was available to derive chemical-specific effluent standards for more than a small fraction of the toxic chemicals potentially present in effluents. Second, the influence of site-specific water quality might alter the toxicity of those chemicals from that assumed by state standards. Third, chemical-specific standards could not reliably predict the effects that might occur from simultaneous exposure to the complex mixtures of chemicals typically present in effluents. To address these shortcomings, the concept of “whole effluent toxicity” was introduced, wherein the acceptability of an effluent for discharge would be determined not only from measured concentrations of known contaminants but also by directly assessing the aggregate toxicity of the intact effluent.

As a monitoring tool, testing the whole effluent for toxicity had the advantages of requiring neither knowledge of the chemical(s) or chemical mixture responsible for toxicity (any toxic chemical is detected if present in toxic amounts) nor full understanding of the effects of bioavailability on those chemicals. However, the practicalities of routinely testing the toxicity of effluents and establishing permit limits for toxicity demanded the development of test methods that could be highly standardized and available as a commodity service from contract laboratories, conducted using relatively small volumes of effluent (so that off-site testing was practical), and completed in a relatively short time frame, both for cost-efficiency and to allow real-time monitoring of toxicity.

The 7-d chronic test with Ceriodaphnia reticulata or Ceriodaphnia dubia 1 (the latter being more common today) was developed specifically to meet these demands. At the time, the most common chronic test with a cladoceran was a 21-d test with Daphnia magna, which was too long and labor-intensive to be a good option for monitoring whole effluent toxicity. Daphnia magna was also under some criticism at the time for being not widely distributed, with the implied suggestion that it would not be a good surrogate for the response of organisms when applied over a broad geographic range. After evaluating some alternatives, Ceriodaphnia species emerged as a promising alternative. Because of their rapid development, a life-cycle chronic test, beginning with neonates and continuing through maturation and production of three broods of offspring, could be completed in only 7 d, and a full dilution series with 10 replicates per treatment could be tested using only about 1.5 L of effluent. After experimentation with different diets and culture methods, laboratory culture of large numbers of organisms was also shown to be practical.

An important companion to the Ceriodaphnia 7-d chronic test was the 7-d larval survival and growth test using fathead minnows 2. Because no single organism is highly sensitive to all contaminants, it was important to complement the Ceriodaphnia chronic with a test using a phylogenetically distant organism. While early life stage exposures with fish species were well established as predictors of life-cycle toxicity at that time, these methods were too long (30–60 d) and required too much effluent (generally flow-through exposures) to be appropriate for effluent monitoring. Building from the observation that effects in early life stage exposures often occurred during early larval development, the 7-d fathead test was developed with an emphasis on having a short test duration and requiring only limited quantities of effluent sample. Both the Ceriodaphnia and fathead minnow 7-d tests, along with an algal test, were later issued as standard methods by U.S. EPA 3 and incorporated into routine toxicity monitoring of effluents discharged to fresh waters. To address the needs of estuarine and marine discharges, similar 7-d sub-lethal effluent toxicity methods were developed in other laboratories using mysid shrimp and a couple of species of marine fish 4.

While the Ceriodaphnia chronic test was applied with success in the effluent monitoring program for which it was originally conceived, it also has been used in a broader range of applications. Experience has shown that Ceriodaphnia are fairly sensitive to a number of toxicant types, which increases their appeal in some circumstances. Perhaps more importantly, the ability to evaluate toxicity in a full life-cycle exposure in just 7 d with a minimum of specialized facilities has made the test a very practical means of efficiently generating chronic toxicity data for individual chemicals, which has broadened its application further and is likely to sustain its continued use into the future.


Table S1 (49 KB PDF).


The views expressed in this paper are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency.