FAQ

Frequently asked questions

You will find usefull informations regarding to the different products.
See the answers by clicking on the appropriate question.

you may also have a look at the latest LemnaTec presentation of Plant Phenomics

Please find the latest documentation for LemnaLauncher and LemnaMiner here

the LemnaTec scanalyzer3D traing is scheduled for 3 Days and will cover the following issues:

 

  • Introduction to Lemna Launcher,
  • Introduction to LemnaTec Image Processing,
  • Introduction to the LemnaTec DataBase Concept,
  • Advanced Image Processing,
  • Approach for unique Image Processing,
  • Introduction to LemnaMiner Software,
  • Import/Export of Data

Please download the full document here

 Standards FAQ

 

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 (chemicals, plant protection) the final proposal for guideline 221 “Lemna sp. Growth Inhibition Test” is available (OECD 2000a). 37 participants successfully finished an international ring test in December 1998. The final version will be available soon.

The process of ISO standardization (waste water, soil, waste) started in summer 2000 with a complete draft developed and practically checked in Germany. The ISO standard ISO/CD 20079 is now available as “Water quality - Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) - Duckweed growth inhibition test” (ISO 2001). Next decisive steps within the fixed schedule will be made as the beginning of 2002 in Philadelphia. Decision on a ring test will be made there. Comments on this document are invited and should be addressed together with detailed alternative wordings in English to the ISO´s member body in your country. View list here!

The separations of standards for testing chemicals and environmental samples partially vanished. According to the title of the ISO standard single chemicals may be tested as “water constituents”. Other water constituents may be elutriates of soil and waste. For soil elutriates the duckweed test was validated successfully. In the appendix of the ISO standard CD 15799 “Soil quality - Guidance on the ecotoxicological characterizations of soil and soil Materials” (ISO 1999) the listed AFNOR standard will be substituted by the ISO standard to give the formal basis to use duckweed with soil elutriates.

The use of the OECD Duckweed guideline for waste water has some practical concerns using the test-medium. It was developed for the test of substances free of additional nutrients. Additional nutrients of the environmental sample cause additional growth relative to the controls in some cases and reduce sensitivity of the test.
The Duckweed test is mentioned directly in the revised draft guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD 2000b) to test suspensions or turbid and dyed test solutions.
All standards are in full compliance with measurements made by image-analysis as image-analysis include the observation parameter frond number. OECD and the ISO standard include total frond area (alternatively to dry weight (OECD, ISO) or chlorophyll (ISO)) as a second observation parameter too. Most of the time consuming additional observations to be made in all standards (reduction of frond size, chlorosis, necrosis etc.) are quantified reproducibly without any additional workload by image analysis. Even special requirements can be achieved by image-analysis. For example the ASTM standard points out that only fronds with less than 50 percent chlorosis should be counted as living.

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 planttoxicity test using lemna ssp., Tiers I and II , EPA 712-C-96-156}

ISO 1999

ISO/CD 15799 1999 Soil quality - Guidance on the ecotoxicological characterizations of soil and soil materials

ISO 2001

Water quality - Determination of the toxic effect of water constituents and waste water to duckweed (Lemna minor) - Duckweed growth inhibition test” ISO/CD 20079 August 2001

OECD 2000a

Proposal for Guideline 221 Lemna sp. Growth Inhibition Test OECD, October 2000

OECD 2000b

Revised draft guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD, January 2000

SIS 1995

Swedish Institute of Standards, Water quality - determination of growth inhibition (7-d) Lemna minor , duckweed SS 02 82 13

 

Media FAQ

 

Steinberg 


Stoff

Nährmedium nach
Steinberg (modifiziert)

 

 

Makroelemente

    e Molgew.

mg/l

mmol/l

KNO3

    101,12

350,00

3,46

Ca(NO3)2*4H2O

    236,15

295,00

1,25

KH2PO4

    136,09

90,00

0,66

K2HPO4

    174,18

12,60

0,07

MgSO4*7H2O

    246,37

100,00

0,41

Mikroelemente

    Molgew.

µg/l

µmol/l

H3BO3

    61,83

120,00

1,94

ZnSO4*7H2O

    287,43

180,00

0,63

Na2MoO4*2H2O

    241,92

44,00

0,18

MnCl2*4H2O

    197,84

180,00

0,91

FeCl3*6H2O

    270,21

760,00

2,81

Titriplex III (EDTA)

    372,24

1500,00

4,03

  

Solution 1:  

1. Makroelemente (50fach konzentriert)

g/I

Solution 1:

 

KNO3

17,50

KH2PO4

4,5

K2HPO4

0,63

Solution 2:

 

MgSO4*7H2O

5,00

Solution 3:

 

Ca(NO3)2*4H2O

14,75

2. Mikroelemente (1000fach konzentriert)

mg/I

Solution 4:

 

H3BO3

120,0

ZnSO4*7H2O

180,0

Na2MoO4*2H2O

44,0

MnCl2*4H2O

180,0

Solution 5:

 

FeCl3*6H2O

760,00

Titriplex III (EDTA)

1500,00

Start Kultivating with:

  • 20 ml Solution 1 bis 3

  • 1,0 ml Solution 4 und 5

  • aqua bidest ad 1000 ml

autoclave at 121°C, for 20 Min. or use 0.2µm sterilfilter

OECD, 1998 – Culture and Test media for Lemna minor (SIS growth medium)


Substance

Stock Solution
[g/L]

Mediuma
[mg/L]

Element

Stock Solution

MgSO4 – 7 H2O

15

75

NIb

II

NaNO3

8,5

85

NI

I

CaCl2 – 2 H2O

7,2

36

NI

III

Na2CO3

4,0

20

NI

IV

KH2PO4

1,34

13,4

NI

I

H3BO3

1,0

1

NI

V

MnCl2 – 4 H2O

0,2

0,2

NI

V

Na2MoO4 – 2H2O

0,010

0,010

NI

V

ZnSO4 – 7 H2O

0,050

0,050

NI

V

CuSO4 – 5 H2O

0,005

0,005

NI

V

Co(No3)2 – 6 H2O

0,010

0,010

NI

V

Na2 EDTA

1,4

1,4

NI

VIc

FeCl3 – 6 H2O

0,84

0,84

NI

VIc

MOPS (buffer)d

488

488

NI

VIc

PH Adjustment

PH adjustment to 6,5 ± 0,2 by addition of NaOH or HCl

Sterilization

Stock solutions I to V are sterilized by autoclaving (120°C, 15 min.) or by membrane filtration (pore diameter 0,2 μm); stock solutions IV (and optional VII) are sterilized by membrane filtration only (i.e. , these should not be autoclaved).

a

Concentration of substance in Medium

b

Not indicated

c

Added after autoclaving

d

MOPS buffer is only required when pH control of the test medium is particularly important (e.g. when testing metals or substances which are hydrolytically unstable)

Zitiert aus: Report EPS 1/RM/37; Dec. 1998,

Biological Test Method: Test for Measuring the Inhibition of Growth using the Freshwater Macrophyte, Lemna minor

Method Development and Application Section  
Environmental Technology Centre
Environment Canada          
Ottawa, Ontario

ASTM, 1991 – 20X-AAP Mediuma for Culturing and Testing Lemna gibba


Substance

Stock Solution
[g/L]

Medium
[mg/L]

Element

Stock Solution

MgSO4 – 7 H2O

14,70

38,22

S

D

NaNO3

25,50

84,00

N

A

CaCl2 – 2 H2O

4,410

24,04

Ca

F

Na2CO3

15,00 / ---

220,02 / 42,86

Na / C

B / B

KH2PO4

1,044 / ---

9,38 / 372

K / P

C / C

H3BO3

0,18552

0,64920

B

G

MnCl2 – 4 H2O

0,41561

2,30748

Mn

G

Na2MoO4 – 2H2O

0,00726

0,05756

Mo

G

ZnCl2

0,00327

0,0314

Zn

G

CuCl2 – 2 H2O

1,2 x 10^-5

8 x 10^-5

Cu

G

CoCl6– 6 H2O

0,00143

0,00708

Co

G

Na2 EDTA 2 H2O

0,300

---

---

G

FeCl3 – 6 H2O

0,160

0,66102

Fe

G

MgCl2 – 6 H2O

12,164

58,08

Mg

E

PH Adjustment

PH adjustment to 7,4-7,6 by addition 0,1N NaOH or HCl

Sterilization

sterilized by membrane filtration only (pore diameter 0,2 μm)

Zitiert aus: Report EPS 1/RM/37; Dec. 1998,
Biological Test Method: Test for Measuring the Inhibition of Growth using the Freshwater Macrophyte,
Lemna minor

Method Development and Application Section  
Environmental Technology Centre
Environment Canada          
Ottawa, Ontario

ASTM, 1991 – Modified Hoagland's Mediuma (no Sucrose or EDTA) for Culturing and Testing Lemna gibba


Substance

Stock Solution
[g/L]

Mediumb
[mg/L]

Element

Stock Solution

MgSO4 – 7 H2O

NIc

75

NI

Ad

KNO3

NI

85

NI

A

Ca(NO3)2 – 4 H2O

NI

36

NI

A

KH2PO4

NI

13,4

NI

A

H3BO3

NI

1,0

NI

Bd

MnCl2 – 4 H2O

NI

0,2

NI

B

Na2MoO4 – 2H2O

NI

0,010

NI

B

ZnSO4 – 7 H2O

NI

0,050

NI

B

CuSO4 – 5 H2O

NI

0,005

NI

B

FeCl3 – 6 H2O

NI

0,010

NI

A

Tartaric Acid

NI

1,4

NI

A

PH Adjustment

PH adjustment to 5,01 ± 0,1 by addition of 1N KOH or HCl

Sterilization

Autoclave 20 min. at 121°C and 1,1 Kg/cm²

a

This medium is the same as Hoagland´s E+ medium exept the sucrose, bacto-tryptone, yeast and EDTA have been excluded

b

Concentration of substance in Medium

c

Not indicated

d

Add each chemical (A) to distilled or deionized water
Add 1 ml of micronutrient stock solution

Zitiert aus: Report EPS 1/RM/37; Dec. 1998,
Biological Test Method: Test for Measuring the Inhibition of Growth using the Freshwater Macrophyte,
Lemna minor

Method Development and Application Section  
Environmental Technology Centre
Environment Canada          
Ottawa, Ontario

 

LT Colony Counter FAQ

 

Choice of brightness/aperture and acquisition time

Getting the optimum brightness is a result of choosing good aperture and acquisition time pairs. To maximise sharpness of the image independent of the number of sharp objects the auto focus could use, aperture should have values higher than 4 best would be 8. As colonies generally do not move acquisition times up to 0.5 seconds could be accepted before there is a need to open the aperture.

But what is the right brightness to choose? There are two general rules to optimise the images:

First the LT counter software has no problems to analyse images, which are a little bit darker than the value the human eye would like to be optimum for manual count.
As a consequence underexposure is no problem for image processing while overexposure is one. If the background of a petridish is clear white it does not contain any more information. It cannot be assured that e.g. small colonies with bad contrast are not more to be seen on the image and therefore do not exist for the machine.
To assure visibility of all colonies the background even of the whitest or clearest plate in a measurement series should have a light colour.This can be easily achieved for backlight if the area around the plate has a slight grey colour.
If colonies tend to be very bright in top light avoiding overexposed i.e. maximum white colonies if needed if colonies should be separated properly.
Avoid any overexposured bright white area within the whole area of interest is best to assure best image processing possible.
Secondly further optimisation of image processing will be reached by maximising contrasts between colonies and background and borders between colonies according to visual impression. For this reason try if backlight ot toplight gives better contrasts for all colonies.

Focussing with LT Counter standard and digital

Optimising the focus is needed to get sharp images and good counts. The best basis for sharp images is to use a small aperture I. E. using high numbers for aperture in the camera configuration. Inly if time is getting linger than 0.5 s to reach optimum brightness aperture should be opened.

LT Counter standard

The Camera of the LT counter standard has the possibility to use the auto focus once when creating a new measurement series (Click on “set focus recommended”). This may be done to maximise sharpness if enough sharp objects are to be seen. If colonies are very small or do not have sharp rims manual setting of the focus is preferred. Using of permanent auto focus must be avoided as mechanics may be destroyed if the focus is not able to find a sharp object e.g. because no plate is in the counter. Additionally refocusing needs time and may make image acquisition more slowly.

Click on “set focus recommended” for use manual set of focus in each camera configuration to acquire sharp images.

LT Counter digital

The camera of the digital counter does not allow to fix a certain focus once defined. As aconsequence each image is focused individually. Using the fast advanced focusing technology this is no problem as long as the a maximum aperture possible is used (see above) and there are at least some larger sharp objects in the image. This is generally true as long as at least the rim of the petridish is visible on the image. This is of high importance esp. if the agar is very homogenous and colonies are extremely small or have no sharp rims and may appear at very low numbers.

Please ensure that one or more medium sized objects with sharp rims are on each image to be used by the auto focus.

How to handle lables with the LT Colony Counter

Labels on petridishes are generally a sign for automatation already started. If the label contains a barcode this could be used for naming the plate within the LT counter system too. Thus typing of the name of the plate, a time consuming part of sample handling and source of faults can be avoided. The LT counter is easily able to integrate barcode reading as additional option. To provide an optimised barcode reader some samples of the label with barcode, technical specification of the barcode (if available) and information on the nomenclature used should be available from LemnaTec..

But in detail labels can even provide some problems.

Optimally for image acquisition barcode is to be found at the side of the petridish as this opens all options for choosing the best illumination. Placement of the barcodes on the lid of the petridish is good as long as lid may be removed for image acquisition. If the barcode is on the lid not to be removed or on the bottom two different options are available. If the colonies have good contrast and have darker colour than the background they may be counted in Backlight after special adaptation of the image processing. For dark colonies, which may be counted on white background with top light, a white insert for the top light masks is available. This makes white labels almost invisible. In this case plates with label on the lid may be counted up side down while plates with label on the bottom will be counted in normal position. For pale colonies on almost clear agar the only option is to use preferably backlight and to extrapolate the number of colonies in the area of the label, as they could not be counted (except for labels based on black paper would be available).

Installation of the LT Colony Counter Demo software

  1. Start Computer with Administrator privileges. Do not plug in Hardlock.

  2. Insert CD and start setup.exe

  3. The LTCounterSetup-window appears. Select between Deutsch (German) and English. Click on the install-button.

  4. A message box appears with the headline "Update Microsoft Data Components". Click the Ja (Yes) button.

  5. Press the ok button to start the MS-MDAC setup.

  6. After the extraction of the necessary files you have to agree to the Microsoft End User License Agreement. Check the box before "I accept all of the proceeding license agreement" and click afterwards the “Next” button. Click the finish button to begin the installation.

  7. After the setup has completed click the “Close” button and the installation of the Microsoft Jet Engine starts.

  8. Click on the “Next (weiter)” button to start the installation of the Hardlock device driver.

  9. Click on the “Next (weiter)” button to end the installation of the Hardlock device driver.

  10. If you want you can change the installation folder of the LTCounter-Programm and/or the Database folder. Please keep in mind that the database folder might need a lot of free disk-space on your harddisk (~150 MB). We suggest to create an desktop icon by checking the checkbox before "Create desktop icon". Press the OK button to proceed with the installation.

  11. First the LTCounter-Programm will be copied and afterwards the Demo-Database. This will take a few minutes because the size of the Demo-Database is about 125 MB.

  12. After this a message box appears with the headline "Installation completed". Press the OK button.

  13. Plug in the Hardlock. If the hardware wizard tells you that it has detected new hardware. Please click on its next button.

  14. Have a look for the LTCounter desktop icon. By double-clicking the desktop icon you can launch the LTCounter application.

  15. At the first lunch of the system you will see no folders with images in the database browser. Click on Edit/New query and type “%”. “%” is the general wildcard in database queries. Click “Ok” and you will see all folders in the database. From now on all folders are shown in when opening the database which were displayed at the last shut down.

Arabidopsis FAQ

Arabidopsis thaliana is a small flowering plant that is widely used as a model organism in plant biology. Arabidopsis is a member of the mustard (Brassicaceae) family, which includes cultivated species such as cabbage and radish. Arabidopsis is not of major agronomic significance, but it offers important advantages for basic research in genetics and molecular biology.

  • Small genome (114.5 Mb/125 Mb total) has been sequenced in the year 2000 (AGI)

  • Extensive genetic and physical maps of all 5 chromosomes

  • A rapid life cycle (about 6 weeks from germination to mature seed)

  • Prolific seed production and easy cultivation in restricted space

  • Efficient transformation methods utilizing Agrobacterium tumefaciens

  • A large number of mutant lines and genomic resources

  • Multinational research community of academic, government and industry laboratories

Such advantages have made Arabidopsis a model organism for studies of the cellular and molecular biology of flowering plants. TAIR collects and makes available the information arising from these efforts.

 

Arabidopsis

 

History of Arabidopsis thaliana

"Arabidopsis thaliana was discovered by Johannes Thal (hence, thaliana) in the Harz mountains in the sixteenth century, though he called it Pilosella siliquosa (and it has gone through a number of name changes since). The earliest report of a mutant (that I know of) was in 1873 (by A. Braun). F. Laibach first summarized the potential of Arabidopsis thaliana as a model organism for genetics in 1943 - he did some work on it much earlier though, publishing its correct chromosome number in 1907. The first collection of induced mutants was made by Laibach's student E. Reinholz. Her thesis was submitted in 1945, the work published in 1947. Langridge played an important role in establishing the properties and utility of the organism for laboratory studies in the 1950s, as did Rédei and others (such as J.H. van der Veen in the Netherlands, J. Veleminsky in Czechoslovakia and G. Röbbelen in Germany) in the 1960s. One of Rédei's many important contributions was to write scholarly reviews on Arabidopsis, a particularly thorough one is in Bibliographica Genetica vol 20, No. 2, 1970, pp. 1- 151. He wrote a more easily found one in Ann. Rev. Genet. (1975) vol. 9,111-127. Both go through some of the early history of the use of Arabidopsis in the laboratory, though the longer 1970 one has all the details." --from Elliot Meyerowitz, 1998

Assessment FAQ

 

Comparing Size

The human eye can easily err by comparing sizes. The figures you see on the picture are all differnt in size (10 %)

 

Assessment

Fluorescence

Fluorescence original Fluorescence

 

 

 

 

 

Scanalyzer FAQ


Technical Details:

Camera(RGB):
Camera (Fluoreszenz):
Camera high res. (IEEE)

 


Image System

 

Image device

Interline-transfer, 1/3 inch CCD

Effective picture elements

3 x [768 (H) x 494 (V)]= 1.2 Mio

Sensing Area

6.00 x 4,96

Optical system and functions

 

Lens mount

C mount

Signal system

NTSC colour system

Scanning lines

3 x 525 = 1575

Synchronization

Internal / external

Jitter

Within ±50 nsec.

Scanning frequenzy

Horiz.15.734 Hz Vertical 59.94 Hz

Horizontal resulution

3 x 570 TV lines

Vertical lines

3 x 485 lines

Minimum illumination

31 lx (F2.2 GAIN + 18 dB at 100%)

Sensitivity

2000 lx (F5.6)

Video output

real RGB

Video S/N

59 dB

Gain

0 dB to 18 dB (1 dB increments)

Shutter

1/125, 1/250, 1/500, 1/1000, 1/2000,
1/ 4000, 1/10000, 1/12000

Accumulation

FLD / FRM

Withe balance

Auto / manual

Camera Fluoreszenz

 

Image device

1/2" IT CCD

Effective picture elements

752 (H) x 582 (V)

Signal System

CCIR

Scanning frequenzy

Horiz.15.734 Hz Vertical 50 Hz

Minimum illumination

0.1 lx

Frame rate

3.75 frames/s, 7.5 frames/s

Sensivity

20 lx at: F1.4, Gain + 18 dB

SNR

tbd

>Interface (optical):

 

Imaging device

ICX205AK

Size

1/2"

Type

CCD interline transfer, progressive scan

Resolution

H:1280 V:960

Pixel size

H:4.65 µm V:4.65 µm

Lens mount

C-mount

>Interface (electrical):

 

Supply voltage

8 to 30 VDC

Current consumption

approx 280 mA (at 12 VDC)

Sync in

IEEE 1394-1995

Trigger in

separate trigger in

Remote in

IEEE 1394-1995

Video out

IEEE 1394-1995

Sync out

IEEE 1394-1995

>Interface (mechanical):

 

Dimensions

H:50 mm W:55 mm L:110 mm

Mass

approx 250g

>Adjustments (man):

 

Gain (rmt)

0 to 18 dB

>Environmental:

 

Max. temperature (operation)

-5°C to 45°C

Max. temperature (storage)

-20°C to 60°C


Magnification of Objects

The Scanalyzer is able to scan objects from 20 x 15 cm to 2 x 2 mm.


Scanarea

Scantime

Resolution (Pixel)

µm / Pixel

100 x 150 mm

<0,2 sec.

752 x 582 per color

      130

50 x 75 mm

<0,2 sec.

752 x 582 per color

        75


The system can detect effects which are larger than 0,068 mm² or 0,017 mm² (depending on which lens you use)

LemnaTec offers also a high resolution Linecamera (color) This System is able to scan the following areas / Resolutions

Scanarea

Scantime

Resolution (Pixel)

  µm / Pixel

50 mm x 1600 mm

< 2 sec.

2048 x 64000

       25

100 mm x 1600 mm

< 2 sec.

2048 x 64000

       50

200 mm x 1600 mm

< 2 sec.

2048 x 64000

      100


 Illumination

 The Scanalyzer has deferent types of illumination

  • Back light

  • Cloudy Day

  • LED blue

  • LED red

  • LED UV

  • Darkfield

 

 

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

Adjustments (man):

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.


 

Collembola FAQ

 

Collemboles represent the largest Order in the group of insects. They are small, primarily wingless animals, characterised by a typical jumpfork (FURCA) on the underside of their belly. Using the furca they are able to jump, covering comparatively large distances. The Collemboles are grouped together with the Protura and Diplura as Entognatha.

Findings

Worldwide more than 6000 species are known,about 2000 of these are found in Middle-Europe.

Collemboles live in almost every habitat.They are found beyond the ice-border in mountain ranges, as much as in arctic and Antarctic Regions where they feed upon algae and pollen. The majority of species live in the top- layers of soil, on waste-fauna, sometimes they live on the water. Their populations are sometimes enormously big.

Characteristics

  • Body: Small animals, 0.2 to 10 m, mostly 1 to 2 mm, elongated cylinder-shape or stout spherical, often grey to brown, sometimes colourless or tainted. Often very hairy.

  • Mouth: Mouthtypes are of the chewing-biting or stinging-sucking type. The parts are set into the head capsule (endognath). Eyes: The eyes consist of max. 8 ocelles, there are no complex eyes. In soil-dwelling species they are partly reduced (although even these animals are able to react light-sensitive).

  • Abdomen: The abdomen consists of 6 segments. On the first segment a ventratubulus inserts.

Biology

Collemboles develop without metamorphosis. The undergo 6 to 8 (some species up to 40) changes of skin, but reach their maturity before the last stage. Mostly they live in several generations every year. All stages are able to hibernate. Food: They feed upon rotting vegetation, bacteria, fungi, pollen and other organic substances. Some species live raptorial . Under favourable circumstances in one liter of light, forest humus-soil, one finds up to 2,000, on one square meter up to 100,000 individuals. The number decreases fast in deeper layers of oil. It is possible to find them 2 meters deep, but in 15 cm deep soil only a fraction of the population from the top is found. Sometimes mass-populating happens, the surface of the floor becomes a living body. Therefore, in 1918 it was earnestly considered to use the animal’s body-fat in oil-production.

Mating

The collemboles transfer the semen indirectly.

Enemies

Colemboles have many enemies: Spiders, bugs, beetles, and other insects, more than anything else they are hunted by rduberische mites.

Significance

Unobtrusive as they might appear, they play are major role in the rotting of soil and in the food-chain of many ecosystems. Some species may cause problems in green-houses, mushroom plants etc. Sminthurus viridis is e.g. an important varmint in Australia.

Systematics

The collemboles are grouped together in 4 Subclasses and 20 families. Here only the most important ones: Subclasses:

  • Arthropleona lengthy body .

  • Entomobryidae

  • Isotomidae

  • Onychiuridae

  • Poduridae

  • Symphypleona stout body,

  • Sminthuridae

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