Duckweed (Lemna minor) growth inhibition test (based on frond counts and chlorophyll content) for ecotoxicity assessment of water samples (surface and groundwater, leachate and waste water) according to OECD 221

The duckweed species Lemna minor is used as model organism for higher water plants. Duckweeds are monocotyledonous, free-floating angiosperms and belong to the Arales within the subclass of Aridae. Duckweeds are fast growing higher plants, spreading from the tropic to the arctic zone. As primary producers they are a food source for waterfowl, fish and small animals and serve as physical support for a variety of small invertebrates.

Duckweed can be damaged by water constituents and effluents. The subsequent inhibition of growth is calculated from the observation parameters (frond number, frond area, chlorophyll, dry weight) by a number of defined calculation methods.

EC values are determined to allow for an assessment of toxic effects of water constituents (e.g. chemicals, plant protection products). The evaluation for at least two observation parameters is based on the average specific growth-rates.

The test is designed for measurement of response of substances dissolved in water. This includes the definition of a fixed dilution step, or a concentration of the test sample at which a parameter of observation (endpoint) is inhibited relative to a control for a defined percentage.

Description of the test

Preparation of the Micronutrient Solution and of the Fe-EDTA solution (Missouri Botanical Garden)

Composition of the stock solution                                      Fe-EDTA solution

H3BO3                       2.86 g/l                            FeCl3·6H2O         0.121g/250ml

MnCl2·4H2O               1.82 g/l                               EDTA                0.375g/250ml

ZnSO4·7H2O              0.22 g/l                                  

Na2MoO4·2H2O           0.09 g/l                                  

CuSO4·5H2O              0.09 g/l                                  

Lemna minor Hoagland’s E-medium (Missouri Botanical Garden):

The below indicated autoclaved medium is added to 1 liter sterile distilled water, then after thorough shaking the pH is adjusted to 5.8 with sterile NaOH or HCl. The following amounts are added into the final medium:

 Additions per Liter of Final Medium (Missouri Botanical Garden)

MgSO4·7H2O (24.6 g/100ml)                                                          1.0 ml/l

Ca(NO3)2·4H2O (23.6 g/100ml)                                                     2.3 ml/l

KH2PO4 (13.6 g/100ml)                                                                  0.5 ml/l

KNO3 (10.1 g/100ml)                                                                       2.5 ml/l

Micronutrient Solution                                                                      0.5 ml/l

Fe-EDTA solution                                                                            20.0 ml/l

 Performance of the test:

The test has two parts:

During the first 7 days the growth of duckweeds is monitored based on fond counts.

During the seventh day the total chlorophyll content of the duckweeds is measured. 

The test is run in 150 mL beakers in 3 replications. The nutrient solution is the control. 50-50 ml water sample is introduced into each beaker, 10-10 pcs of 2-frond, green coloured duckweed is put on top into each beaker. In order to prevent evaporation and contamination during the test the beakers are covered by plastic foil.  The experimental system is incubated in the thermostate at 21.5±1°C for 7 days exposed to light (8:16 hours dark-light cycles, Daylite tubes). The duckweed fronds are measured every 24 hours during testing.

Total chlorophyll content of the tested samples is measured on the 7th day. First, we remove from each beaker the duckweeds and dry them on filter paper until reaching a constant weight.  Then the duckweeds belonging to a given sample are placed into test tubes with covers and 5 ml of 96% ethanol is added to it. The samples are then kept for 24 hours at room temperature in the dark.

After one day the optical density of each sample is measured by Sanyo SP55 UV/VIS spectrophotometer at 3 wavelengths (480, 647, 664 nm).

Evaluation of the results

The evaluation is performed based on the frond number and total chlorophyll content.

Average specific growth rate

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 ECx should be based on the values for the individual replicates and not calculated means for each treatment group.

The average specific growth rate for a specific period is calculated as the logarithmic increase in the growth variables -frond numbers and one other measurement variable (total frond area, dry weight or fresh weight) - using the formula below for each replicate of control and treatments:

 μ_i-j = (ln(N_j)-ln(N_i))/(t_j-t_i)

where:

-  μ_i-j  : average specific growth rate from time i to j

- N_i: measurement variable in the test or control vessel at time i

- N_j: measurement variable in the test or control vessel at time j

- t_i -j : time period from i to j

For each treatment group and control group, calculate a mean value for growth rate along with variance estimates.

The average specific growth rate should be calculated for the entire test period (time in the above formula „i” is the beginning of the test and time “j” is the end of the test). For each test concentration and control, calculate a mean value for average specific growth rate along with the variance estimates.

In addition, the section-by-section growth rate should be assessed in order to evaluate effects of the test substance occurring during the exposure period (e.g. by inspecting log-transformed growth curves).

Substantial differences between the section-by-section growth rate and the average growth rate indicate deviation from constant exponential growth and that close examination of the growth curves is warranted.

In this case, a conservative approach would be to compare specific growth rates from treated cultures during the time period of maximum inhibition to those for controls during the same time period.

Percent inhibition of growth rate (Ir) may then be calculated for each test concentration (treatment group) according to the following formula:

 Ir= 100*(μC-μT)/μC

where:

% Ir : percent inhibition in average specific growth rate

μC : mean value for growth rate in the control

μT: mean value for growth rate in the treatment group

Total chlorophyll content of the tested sample (C_a+b) is calculated with the below formula:

 C_a+b = (5.24*A664+22.24*A647)*V

where

C_a+b: total chlorophyll content of the measured sample (μg total chlorophyll/sample)

A664: absorbance value at 664 nm wavelength

A647: absorbance value at 667 nm wavelength

V: volume of ethanol used for extraction (5 ml)

For the validity of the test the following conditions should be complied with:

The average doubling time of the plant in the control solution shall not exceed 2.5 days. The average doubling time will be calculated with the following formula:

T_D=ln2/μ_i-j

T_D – doubling time

μ_i-j – general growth rate between time i and j