Toxicity Testing and Evaluation of Toxicity Test Results

Ecological toxicity tests are classified according to duration (short‐term, intermediate, and/or long‐term), method of adding test solutions (static, recirculation, renewal, or flow‐through), and (to satisfy permit requirements for a proposed pollutant discharge elimination system, and to determine mixing zones, etc.). Detailed contemporary testing protocols are summarized in the regulatory literature (USEPA 1985b, c, 1988, 1989).

A number of terms are utilized in expressing toxicity test results. Acute toxicity is toxicity severe enough to produce a response rapidly (typically a response observed in 48 or 96 hours). “Acute” does not necessarily imply mortality. In studies of marine organisms, the LC₅₀ is the concentration of effluent in dilution water that causes mortality to 50% of the test population; the EC₅₀ is the effluent concentration that causes a measurable negative effect on 50% of the test population. The NOAEL (no observed acute effect level) is defined as the highest tested effluent concentration that causes 10% or less mortality.

Chronic toxicity is the toxicity impact that lingers or continues for a relatively long period of time, often 1/10 or more of the target organism’s life span. Chronic effects could include mortality, reduced growth, or reduced reproduction. The NOEC is the highest measured continuous concentration of an effluent or toxicant that causes no observable effect based on the results of chronic testing. The LOEC (lowest observed effect concentration) is defined as the lowest observed concentration having any effect. The LOEC is determined by an analysis of variance techniques.

Toxicity data are analyzed using the procedures developed by Stephan (1977). The LC₅₀ values are determined analytically using the Spearman–Kayber, moving average, binomial, and probit methods. Graphical methods, as illustrated in Example 4.1, can also be used to obtain estimated LC₅₀ values. Typically, LC₅₀ values are computed based on survival at both 48‐ and 96‐hour exposures. Analysis of variance and Duncan’s multiple comparison of means typically are utilized to compare chronic test results. Example 4.2 presents a simple comparison of results from acute toxicity testing.

EXAMPLE 4.2

Analysis of toxicity data

Use the following hypothetical data to determine the 48‐ and 96‐hour LC₅₀ values in percent by volume.

Concentration of waste % by volume	No. of test animals	No. of test animals dead aftera
		48 hours	96 hours
40	20	17 (85)	20 (100)
20	20	12 (60)	20 (100)
10	20	6 (30)	14 (70)
5	20	0 (0)	7 (35)
3	20	0 (0)	4 (20)

^a Percentage values are given in parentheses.

SOLUTION

1. Plot the concentration of wastewater in percent by volume (log scale) against test animals surviving in percent (probability scale), as shown in the accompanying diagram.
2. Fit a line to the data points by eye, giving most consideration to the points lying between 16 and 84% mortality.
3. Find the wastewater concentration causing 50% mortality.

The estimated LC₅₀ values are seen to be 16.5 vol% for 48 hours and 6.5 vol%.

Comment. The estimated values of the LC₅₀ concentrations obtained graphically are usually quite close to the values obtained with formal prohibit analysis (Stephan 1977). It should be noted that confidence limits cannot be obtained in a graphical analysis (APHA 1989).

Toxic Units

The toxic units (TU) approach has become widely accepted for utilizing the toxicity test results. Both federal and state standards and/or criteria have been or are being formulated on the toxic unit basis. In the toxic units approach (USEPA 1985b, c), a TU concentration is established for the protection of aquatic life.

A toxic unit acute (TU_a) is the reciprocal of the effluent dilution that caused the acute effect by the end of the acute exposure period.

(4.1)

A toxic unit chronic (TU_c) is the reciprocal of the effluent dilution that caused no unacceptable effect on the test organisms by the end of the chronic exposure period:

(4.2)

Where, as before, NOEC is the no observable effect concentration.

Formerly, acute to chronic ratios (ACR) were determined by the equation ACR = LC₅₀/NOEC. The chronic data were determined using extrapolation from acute data. Acute to chronic ratios have been found to vary tremendously between species and between different toxicants. Use of the whole effluent approach prevents implementation of overly stringent as well as overly lenient requirements.