FAQ Duckweed

Duckweed FAQ

The Lemna-Test is the most standardized test using higher plants in bio testing. Till present it is applied mainly for testing of chemicals (OECD-REVISED DRAFT DOCUMENT 221) and sewage water (ISO/WD 20079, DIN-Normung) for possible adverse effects on higher plants. The adaptation of the test procedure to enable testing of soil elutriates and extracts is a demand of the German act on soil protection legislation (Bundes-Bodenschutzgesetz). The whole test procedure and especially the test medium to be used has to be validated on the basis of the ISO working draft. An inter-laboratory comparison will be performed testing different standard-, contaminated- and decontaminated soils along with different parameters as effect-endpoints. An automatic image processing system (LemnaTec Scanalyzer) is included in this testing program for further enhancement of the test performance and test results.

Research Grants
Deutsche Bundesstiftung Umwelt (DBU), German Federal Foundation Environment

Comparison of the sensitivity algae vs. Duckweed
A simulation study

The toxicity of substances or mixtures is based on the interaction of the substances and the respective organisms. As organisms can be structured quite differently their different sensitivity is just logic consequence.
The sensitivity different organisms must be compared if substances should be detected with a maximum of sensitivity or biotest batteries should assess the hazards of environmental samples.
For this reason quite a lot of comparative studies between algae and daphnids have been done. In many cases the EC-values were calculated from inhibition values based on area under the growth curve (agc) or final biomass. But EC-values of almost exponentially growing systems, calculated from final biomass or age depend on test duration and the absolute growth rate of the controls (Nyholm 1985, Nusch1982). Figures 1 and 2 show the dependence of the EC20 and EC50 for growth rates typically found with duckweed (µ = 0.275 d-1; 0.325 d-1; 0.375 d-1) and single cell green algae (µ = 0.9 d-1; 1.4 d-1; 1.9 d-1) on test duration. EC-values based on growth rates drastically change with the test duration while the EC-values depending on growth rates remain constant. The longer the test and the higher the growth rate the higher is the EC-value based on agc and final biomass.

Fig 1: Influence of the absolute growth rate of the control and of the test duration on the inhibition of final biomass for 20 % inhibition of growth rate.

Fig 2: Influence of the absolute growth rate of the control and of the test duration on the inhibition of final biomass for 50 % inhibition of growth rate.
To get a quantitative impression of this phenomena and its consequences on the evaluation of comparative studies, the basic scheme of some studies was used for numerical simulations. The aim of the work was to answer of the question in how far comparisons on the basis of the more sensitive calculation method final biomass results in different EC-values than the less sensitive but ecologically more relevant growth rate.
Three scenarios of comparison were chosen from literature. Some US-studies (Fairchild et al. 1997) used a test duration of 4 days for algae and duckweed (scenario D4A4), in the context of a screening test battery for pesticides (Grossmann et al. 1992) a one day test with algae was compared to a 7 days test with duckweed (scenario D7A1) and a third scenario used a 3 days test with duckweed (OECD, ASTM). In all simulations only the values based on final biomass were calculated as agc calculation result in relatively similar values if it is done on a non logarithmic basis (Nyholm 1985).Figure 3 shows the dependence of inhibition values on the absolute value of the growth rate for the D4A4-scenario. Especially inhibition of final biomass of algae can rise between different tests if growth rate rises from the minimum allowed level (0.9 d-1) to the high but common value of (1.8 d-1) from 57 to 77 % for algae (ECµ20). This results in EC-value differences of about factor 1.5 for steep (like 3,5 DCP) and about factor 3 for shallow (like K2Cr2O7) concentration-response-curves (crc).
For duckweed the difference of ECbiomass-values for different growth rates (0.275 d- and 0.375 d-1) is only 3% for ECµ20 and 4 % for ECµ50.
If both organisms are compared the inhibition of final biomass for ECµ20 of algae is 35 % higher (30 % for ECµ50) than the value of duckweed if medium growth rates (0.325 d-1 for duckweed and 1.4 d-1 for algae) are considered.
35 % differences in inhibition result in an ECbiomass-value for duckweed about factor 2 higher than that of algae if a steep concentrations-response-curve is presumed. For shallow curves the difference of EC-value can reach factor 7 for the same EC-value based on growth rate

.
Fig. 3: Dependence of the inhibition of final biomass from growth rate of the controls for duckweed (left) and algae (right) for the scenario D4A4 (test duration duckweed 4 days, algae 4 days)
This little example already shows that only inhibition values calculated on growth rates allow a scientifically solid comparison of different organisms sensitivities. Studies based on areas under the curve or final biomass can only demonstrate that one test design has a larger tendency to produce smaller EC-values than another test design.
In addition the scenarios with algae at different growth rates compliant to standards show high ostensible differences in sensitivity. This could explain some test to test or interlaboratory differences finally based on different but not documented growth rates. Generally the scenario D4A4 structurally tends to evaluate toxicity towards algae much higher than toxicity towards duckweed. But in a comparative study of 16 herbicides with selenastrum capricornutum and lemna minor calculated on final biomass the duckweed system led to smaller EC50 values for 8 substances (Fairchild et al. 1997).
Going beyond that again a rectilinear comparison of sensitivity of a 4 days duckweed test, 4 days tests with different algae and a 14 day test of submersed rooted macrophytes is not appropriate to measure sensitivities of test species. If the test design is a 7 days duckweed test compared to a 1 day test with algae (scenario D7A1), duckweed seems to be more sensitive if final biomass is the basis of calculation. Figure 4 shows that for a given growth rate inhibition and medium absolute growth rates inhibition duckweed has an inhibition between 2 and 10 % higher than algae. The fact that in real tests duckweed leads for most of the tested substances to EC-values equivalent or lower than that for algae just illustrates that the result is more a product of the test design than one of different sensitivities.

Fig. 4: Dependence of the inhibition of final biomass from growth rate of the controls for duckweed (left) and algae (right) for the scenario D7A1 (test duration duckweed 1 days, algae 1 days)
In the third scenario D7A3 a 7 days duckweed test is compared with a 3 days test with algae. In this case the test design for algae is 2 - 18 % more sensitive than that for duckweed (Figure 5).

Fig. 5: Dependence of the inhibition of final biomass from growth rate of the controls for duckweed (left) and algae (right) for the scenario D7A3 (test duration duckweed 7 days, algae 3 days)

Depending on the slope of the concentration-response curve 18 % difference correspond to EC-values differing between factor 1.5 for steep crc and factor 4 for flat crc.
Future research projects, standardisation procedures and comparative studies should take these connections into consideration. As a consequence the allowed range of growth rate for standard tests with algae should be more limited than it is today if final biomass and area under the curve should be used.
The more consequent solution however would be to calculate at least additionally EC-values based on growth rates. Only growth rate based data are a valid basis for comparisons between different organisms. As Fig. 6 shows, data based on final biomass (or area under the curve) do not provide any basis for an appropriate comparison.

Fig. 6: Dependence of inhibition of final biomass for ECµ20 (left) and ECµ50 (right) from the growth rate of the controls for duckweed and algae.
Waiting for a broad consensus for the appropriate method of data calculation test results should be presented with all calculation methods or the raw data should be easily accessible.

Bassi M., Corradi M. G. Favali M. 1990 Effects of chromium in freshwater algae and macrophytes, 204-224, in: Plants for toxicity assessment ASTM STP 1091, Wang W., Gorsuch J. W., Lower W. R. (eds.) ASTM Philadelphia
Cowgill U. M., Milazzo D. P. and Landenberger B. D., 1989. A comparison of the effect of triclopyr triethylamine salt on two species of duckweed (Lemna) examined for a 7- and 14-day test period. Water Research 23: 617 - 623.
Cowgill U. M., Milazzo D. P. and Landenberger B. D., 1991. The sensitivity of Lemna gibba G-3 and four clones of Lemna minor to eight common chemicals using a 7-day test. Research Journal of the Water Pollution Control Federation 63:991 - 998.
Fairchild J.F., Ruessler D.S., Haverland P.S. and A.R. Carlson 1997 Comparative sensitivity of Selenastrum capricornutum and Lemna minor to sixteen herbicides. Archives of Environmental Contamination and Toxicology 32, 353-357.
Fletcher J.S. 1991 Assessment of published literature concerning pesticide influence on nontarget plants. In: "Plant tier testing: a workshop to evaluate nontarget plant testing in Subdivision J pesticide guidelines". U.S. Environmental Protection Agency, EPA/600/9-91/041, Corvallis, OR, pp. pp. 28-36.
Fletcher J.S. 1990 Use of algae versus vascular aquatic plants to test for chemical toxicity. In: Plants for Toxicity Assessment, edited by W. Wang, J.W. Gorsuch and W.R. Lower. ASTM STP 1091, American Society for Testing and Materials, Philadelphia, PA, pp. 33-39.
Fletcher J.S. 1990 Use of algae versus vascular plants to test for chemical toxicity. In: "Plants for Toxicity Assessment", ASTM STP 1091, Wang W., J.W. Gorsuch and W.R. Lower (eds) American Society for Testing and Materials, Philadelphia, pp. 33-39.
Garten C.T. and M.L. Frank 1984 Comparison of toxicity to terrestrial plants with algal growth inhibition by herbicides. Publ. No. 2361. Environmental Sciences Division, Oak Ridge National Laboratory, TN.
Grossmann K. Berghaus R. Retzlaff G. 1992 Heterotrophic plant cell suspension cultures for monitoring biological activity in agrochemicals research. Comparison with screens using algae, germination seeds and whole plants Pesticide Science 35, 283-289
Hartman W.A. Martin D.B. 1985 Effects of four agricultural pesticides on Daphnia pulex, Lemna minor and Potampogeton pectinatus
Nusch EA (1982) Evaluation of growth curves in bioassays. ISO/TC 147/SC 5/WG5 N62. Nederlands Normalisatie-instituut, Delft, The Netherlands.
Nyholm N (1985) Response variable in algal growth inhibition tests - biomass or growth rate? Wat Res 19:273-279.
Nyholm N (1990) Expression of results from growth inhibition toxicity tests with algae. Archives of Environ Contam Toxicol19:518 - 522.
Peterson H.G., C. Boutin, K.E. Freemark, P.A. Martin 1997 Toxicity of hexazinone and diquat to green algae, diatoms, cyanobacteria and duckweed. Aquatic Toxicology 39: 111-134.
Peterson H.G., C. Boutin, P. Martin, K.E. Freemark, N.J. Ruecker, and M. J. Moody 1994 Aquatic phyto-toxicity of 23 pesticides applied at Expected Environmental Concentrations. Aquatic Toxicology 28: 275-292
Roshon R.D., McCann J.H., Thompson D.G., and G.R. Stephenson (in press) Effects of seven forestry management herbicides on Myriophyllum sibericum, as compared with other non-target aquatic organisms. Canadian Journal of Forest Research
Sloof W.J., H.Canton and J.L.M Hermens 1983 Comparison of the susceptibility of 22 freshwater species to 15 chemical compounds. I. (Sub)acute toxicity tests. Aquatic Toxicology 4, 113-128.
Wang W.C. 1990 Literature review on duckweed toxicity testing. Environmental Research 52, 7-22
Wang W.C., Williams J.M. 1988 Screening and biomonitoring of industrial effluents using phytotoxicity tests. Environmental Toxicology and Chemistry 7, 645-652

Duckweed growth inhibition tests and standardisation

The duckweed growth inhibition test is standardised in a number of states like France (AFNOR 1996), Sweden (SIS 1995, USA (ASTM 1991, EPA 1996, APHA 1992) and Canada (Environment Canada 1998).On the level of OECD a guideline was completed as draft in June 1998 (OECD 1998). 37 participants successfully finished an international ring test in December 1998. A revised draft document (10/2000) is available.
The process of ISO standardisation started in summer 2000.The separations of standards for testing chemicals and environmental samples successively vanishes. The OECD-guideline will offer the opportunity to test not only chemicals but even environmental samples but there are some concerns about the test medium used for L. minor. On the other hand DIN and ISO standards classically made for water samples are more and more opened for soil elutriates and all substances dissolved in water. This includes even pure chemicals. All standards are in so far compatible to measurements made by image-analysis as image-analysis includes the observation parameter frondnumber.
OECD and the DIN/ISO standard will contain total frond area as a second observation parameter at least equal or more sensitive than frond number. Even special requirements can be achieved by image-analysis. For example the ASTM standard points out that only fronds with less than 50 % chlorosis should be counted as living. Switching to new fields of growing importance the duckweed growth inhibition test is an integrated part of standards for soil quality (ISO/CD 15799 1999) and difficult substances (OECD 1999). In these fields the duckweed test shows its power in being able to cope with suspensions and turbid or coloured test material.

Standards and guidelines:

· AFNOR 1996 Determination of the inhibitory effect on the growth of Lemna minor XP T 90-337
· APHA 1992 Toxicity Part 8000 8-32-8-39 in: Standard methods for the examination of water and wastewater 18th ed., APHA, AWWA, WEF, Washington
· ASTM, 1991. Conducting static toxicity tests with Lemna gibba. Guide E 1415-91. Annual book of ASTM standards. Section 11 Water and environmental technology. Vol. 11.04. ASTM, Philadelphia, U.S.
· Environment Canada 1998 Biological test method: test for measuring the inhibition of growth using the freshwater macrophyte lemna minor Report EPS 1/RM/37
· EPA 1996 Ecological effects test Guidelines OPPTS 850.4400 Aquatic plant toxicity test using lemna ssp., Tiers I and II , EPA 712-C-96-156
· ISO/CD 15799 1999 Soil quality - Guidance on the ecotoxicological characterisations of soil and soil materials
· OECD 1998 Draft Test Guideline: Lemna growth inhibition test
· OECD 1999 Difficult substances
· SIS 1995 Swedish Institute of Standards, Water quality - determination of growth inhibition (7-d) Lemna minor), duckweed SS 02 82 13

Using the Duckweed Growth Inhibition Test to detect and evaluate soil contamination

Besides the classic scope of biotests chemical notification procedures and the control of sewage water- the ecotoxicological analysis of presumably contaminated soils gains more and more interest. The detection of biological available contamination as well as assessing the hazards of contaminated or remediated soil are the main focuses of biotests. To assess any risk in ground and surface water but also just as sensitive, readily available detectors aquatic organisms like algae, daphnids and duckweed are used for soil elutriates. Duckweed can be used in a wide range of pH-values. Even intensively coloured, turbid solutions or even suspensions of solids do not interfere with the test. This makes testing with duckweed more robust and flexible than tests with algae if soils or sediments are to be tested. To maximise sensitivity even organic extracts can be used. The great advantage of the duckweed test over germination and growth tests with e.g. cress or lettuce, often used in testing elutriates lies in the highly homogenous plant material. While all duckweed plants are clones, in the seeds different charges weight distribution and the general heterogeneity of the genetic make-up leads to large standard deviations of seed. Generally speaking, comparing the sensitivity of different organisms/biotests has to be done with care. Besides, the intrinsic sensitivity of the organism, test duration, growth rate of the control and the growth model underlying the calculation method of inhibition have great influence on the results(see: Comparison of the sensitivity algae vs. Duckweed - A simulation study, LemnaTec 1999).All studies on hand comparing test systems on a rational basis indicate that none of the systems algae, duckweed or cress is more sensitive in all classes of tested toxicants then any other. This is readily explained by the different classification as lower plant, monocotyledon and dicytoledon. Biotest batteries often leave a gap where a fast biotest with higher plants is wanted; duckweed offers efficient possibilities of testing there. A large number of standards (see below) allow the use of duckweed for soil elutriates. But not in all cases the recommended growth media are suitable. As our experience shows these should not contain organic additives to minimise the growth of bacteria and they should be concentrated enough to minimise hormesis caused by nutrients in the elutriate. Good experience were made by us with the Steinberg-medium which will be the standard medium DIN. Further research on duckweed and soil elutriates is under way at moment. As a part of a larger DBU-project (Deutsche Bundesstiftung Umwelt) "Ecotoxicological test-batteries" the Chair of Biology V of the Technical University of Aachen, supported by LemnaTec, is developing a test procedure adapted to elutriates.

· APHA 1992 Toxicity Part 8000 8-32-8-39 in: standard methods for the examination of water and wastewater 18th ed., APHA, AWWA, WEF, Washington
· ASTM, 1991. Conducting static toxicity tests with Lemna gibba. Guide E 1415-91. Annual book of ASTM standards. Section 11 Water and environmental technology. Vol. 11.04. ASTM, Philadelphia, U.S.
· Environment Canada 1998 Biological test method: test for measuring the inhibition of growth using the freshwater macrophyte lemna minor Report EPS 1/RM/37
· ISO/CD 15799 1999 Soil quality - Guidance on the ecotoxicological characterizations of soil and soil materials
· OECD 1998 Draft OECD test guideline: Lemna growth inhibition test

Observation Parameters in the Duckweed Growth Inhibition Test
The Colour of duckweed

The colour of the plant, in this case duckweed, is as important for the assessment of plant vitality as the number and area of fronds and their habitus. Often chlorotic and necrotic areas but also fronds coloured in a lighter green occur in biotesting besides reduction of fronds area and number. This shows that substances have a direct or indirect negative impact on the photosystem. But even the intensification of the green colour which occurs with low concentrations of triazines can indicate that the plant tries to compensate a toxic impact on its photosystem. This gradual classification of vitality by colour in biotests can be done in three ways if no image-analysis system is used.

1) Measurement of extracted chlorophyll

At the end of the test the duckweed has to be crushed in hot ethanol and extracted overnight. The next day chlorophyll will be quantified photometrically. This analysis needs additional work but if all conditions are held exactly, the results lead to a very sensible and reproducible toxic endpoint. As the measurement is destructive and no aliquots can be made no curves of growth are attainable. Only in combination with total frond area, deepened colour can be distinguished from a higher amount of biomass.

2) Manual report

According to test guidelines all changes in the test organisms should be noticed at any measurement of the parameters. This kind of qualitative description has a highly subjective part and significantly rises the time needed. But nevertheless, these data are not adapted in toxicological endpoints. Furthermore, later reanalysis or reconstruction of the real test event remains difficult, because of subjectively classification of fronds.

3) Visual frond classification
To avoid the equal assessment of healthy, damaged and dead fronds, ASTM and OPPTS-guidelines have decided that only healthy fronds with less than 50 % chlorosis must be counted as living. This method enhances the counting method but on the other hand it results in another increase of time and a significant amount of subjective influences.

What the LemnaTec Scanalyser can achieve by image-analysis of colour

Image-analysis systems are by far superior to human eyes if reproducible detection and quantitative classification of colours is necessary. For this reason it is very important to transfer the colour information of every image pixel into ecotoxicological relevant information.

1) Distribution of colours of total frond area

To assess the vitality of the fronds the pixels are divided into colour-classes corresponding for example to healthy (green), lightly damaged (pale green), chlorotic (yellow) and necrotic (grey or brown) frond areas. Without additional work this method leads to an objective integrative assessment of chlorotic and necrotic structures in relation to the influence of the testing material (Fig.1).

Fig. 1: Change in the areas of different colour classes with rising concentration of potassiumdichromate.

2) Colour classes and single fronds

The LemnaTec Scanalyser is able to separate single fronds. The colour classes can be analogously determined for every single frond. This vast amount of information can be handled if the colour classification is combined with the distribution of frond area. The result is an objective and evident information if small or fully grown fronds are primarily damaged. Final reports can therefore be made on a highly and well documented basis. Fig. 2 shows the different steps from the original image to the frond area distribution combined with colour class information. Especially younger fronds and very large fronds are damaged by chlorosis and necrosis.

Fig. 2: Original image, colour class image and frond area distribution combined with colour classes. Brown colour represents chlorosis and necrosis.

3) Frond classification
Following the ASTM guideline image-analysis fully supports the classification of fronds in living and dead. For this purpose only the percentage of colour classes has to be fixed according to the guideline. Without additional work the fronds can be classified in living and dead using data for the calculation of growth inhibition or rates of death.

Fig.3: Classification of fronds in living and dead (more than 50 percent chlorosis or necrosis)

4) Specific green-values
If colour-analysis is made in fine steps and the results are correlated with chlorophyll an "image-analysis green-value" can be assessed. This allows to use a value similar to chlorophyll contents without additional work and -more important- in a non destructive method. This makes it possible to use this sensitive endpoint in kinetic studies. Like chlorophyll, green-values integrate vitality in a single value.

Summary:

Colour is an important parameter to assess vitality of duckweed in ecotoxicological test systems. The LemnaTec Scanalyser provides the unique possibility to use this colour information in compliance with international standards. Instructive colour quantification combined with analysis of frond area and frond number leads to comprehensive, objective and comfortable test documentation and toxicity assessment.