Division

The purpose of the test is to determine the effects of the test substance on the vegetative growth of Lemna. This Testing Method describes two response variables, as member countries have different preferences and regulatory needs. In order for the test results to be acceptable in all member countries, the effects should be evaluated using both response variables (a) and (b) described below.

1. (a)

   Average specific growth rate: this response variable is calculated on the basis of changes in the logarithms of frond numbers, and in addition, on the basis of changes in the logarithms of another measurement parameter (total frond area, dry weight or fresh weight) over time (expressed per day) in the controls and each treatment group. It is sometimes referred to as relative growth rate (15).

2. (b)

   Yield: this response variable is calculated on the basis of changes in frond number, and in addition, on the basis of changes in another measurement parameter (total frond area, dry weight or fresh weight) in the controls and in each treatment group until the end of the test.

It should be noted that toxicity values calculated by using these two response variables are not comparable and this difference must be recognised when using the results of the test. EC_x values based upon average specific growth rate (E_rC_x) will generally be higher than results based upon yield (E_yC_x) if the test conditions of this Testing Method are adhered to, due to the mathematical basis of the respective approaches. This should not be interpreted as a difference in sensitivity between the two response variables, simply that the values are different mathematically. The concept of average specific growth rate is based on the general exponential growth pattern of duckweed in non-limited cultures, where toxicity is estimated on the basis of the effects on the growth rate, without being dependent on the absolute level of the specific growth rate of the control, slope of the concentration-response curve or on test duration. In contrast, results based upon the yield response variable are dependent upon all these other variables. E_yC_x is dependent on the specific growth rate of the duckweed species used in each test and on the maximum specific growth rate that can vary between species and even different clones. This response variable should not be used for comparing the sensitivity to toxicants among duckweed species or even different clones. While the use of average specific growth rate for estimating toxicity is scientifically preferred, toxicity estimates based on yield are also included in this Testing Method to satisfy current regulatory requirements in some countries.

Toxicity estimates should be based on frond number and on one additional measurement variable (total frond area, dry weight or fresh weight), because some substances may affect other measurement variables much more than the frond number. This effect would not be detected by calculating frond number only.

The number of fronds as well as any other recorded measurement variable, i.e. total frond area, dry weight or fresh weight, are tabulated together with the concentrations of the test substance for each measurement occasion. Subsequent data analysis e.g. to estimate a LOEC, NOEC or EC_x should be based on the values for the individual replicates and not calculated means for each treatment group.