Acute test with luminescent bacteria

(Vibrio fischeri)

TEST PROTOCOL

Note: Deviations of this SOP when performing the tests with waste eluates are possible as
long as they are well documented and are not in contradiction to the guideline. Details of
the preparation and handling of the test eluates are described in the respective SOP as well
as in ISO Guideline 14735 (2005).

Evaluation of a biotest battery for the ecotoxicological characterisation of waste and waste
eluates by an interlaboratory test

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Title:
Performance of acute tests with Vibrio fischeri (Luminescent bacteria test).

Aim of this Standard Operation Procedure:

Assurance of the consistent performance of tests according to DIN/EN/ISO 11348-1, 11348-2 and
DIN/EN/ISO 11348-3 (1998).

Summary:
This SOP describes the performance of the acute luminescent bacteria test with liquid-dried bacteria (according to DIN/EN/ISO 11348-2), freeze-dried bacteria (according to DIN/EN/ISO 11348-3),
or freshly prepared bacteria (according to DIN/EN/ISO 11348-1) of the strain Vibrio fischeri. This
acute test is performed with eluates in various dilution steps. The inhibitory effect of the eluates on
the light emission of Vibrio fischeri is determined after 30 minutes in order to identify the EC50.

Equipment:
Freezer:

for the storage of the bacteria at a temperature of -18C to 20C

Refrigator:

to maintain the reconstitution solution at a temperature of

3 C 3 C

Refrigerated centirifuge:

appropriate for centrifugation at 4 C 2 C

Incubated shaker:

for incubation of Erlenmeyer flasks

Autoclave:

e.g. Co. Webeco & Co (Bad Schwartau), Type CS Labor

Piston pipettes for plastic sy- 10 l, 500 l and 1000 l

ringes:
Piston pipettes with variable 10 ml to 200 ml and 200 l to 5000 l
volume:
Incubator:

e.g. Binder GmbH (Tuttlingen), Type KBW 240

Spectral- or filter photometer optical path length: 1 cm

and test tubes:

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Thermostatically

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controlled e.g. Dr. Bruno Lange GmbH & Co KG (Dsseldorf), Type

thermo-block:

LUMIS therm LTG 0

Luminometer:

e.g. Dr. Bruno Lange GmbH & Co KG (Dsseldorf),

Type LUMIStox 300

Test tubes appropriate for the e.g. Dr. Bruno Lange GmbH & Co KG (Dsseldorf), LZP 187
luminometer:
Colour-correction tube:

double walled tube, fitting the luminometer

Vessel (15 ml):

to temperate the reconstitution solution in the thermo block

pH-meter:

e.g. Co. WTW (Weilheim), Type 330 T

Conductometer:

e.g. Co. Merck VWR (Darmstadt), Type Qcond 2400

Waterbath:

e.g. Co. GFL (Burgwedel), Type 1032

Magnetic stirrer and magnetic

stirring bar
Inoculating loop (or needle)

Reagents and materials:

Use chemicals of recognized analytical grade quality. Use distilled water or water of equivalent
purity.
Diluent and control:

Sodium chloride solution, (20 g/l)

pH-value adjustment:

Sodium hydroxide solution c(NaOH) = 1 mol/l

Hydrochloric acid, c(HCl) = 1 mol/l

Reconstitution solution for liquid-dried and freshly prepared bacteria

8.0 g/l D(+) Glucose monohydrate (C6H12O6 H2O)
20.0 g/l Sodium chloride (NaCl)
2.035 g/l Magnesium chloride hexahydrate (MgCl2 6H2O)
0.30 g/l Potassium chloride (KCl)
11.9

g/l

N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic

acid)

(HEPES)
Dissolve in water, stir for about 30 min and adjust the pH to 7.0 0.2 with sodium hydroxide solution or hydrochloric acid. This solution may be stored at -20 C.

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Solution for freeze-dried bacteria:

20 g/l Sodium chloride (NaCl)
2,035 g/l Magnesium chloride hexahydrate (MgCl2 6 H2O)
0,30 g Potassium chloride (KCL)
Liquid medium for pre- and main culture for freshly prepared bacteria:
30.0 g/l Sodium chloride (NaCl)
6.1 g/l Sodium dihydrogenphosphate monohydrate (NaH2PO4 H2O)
2.75 g/l Dipotassium hydrogenphosphate trihydrate (K2HPO4 3H2O)
0.204 g/l Magnesium sulfate heptahydrate (MgSO4 7H2O)
0.500 g/l Diammonium hydrogenphosphate [(NH4)2HPO4]
3 ml Glycerol; 5.00 g/l Caso peptone; 0.50 g/l Yeast extract
Dissolve in water and adjust the pH to 7.0 0.2 with sodium hydroxide solution or hydrochloric
acid. Transfer 50 ml each to Erlenmeyer flasks and autoclave at 121 C for 20 min.
Agar for stock culture for freshly prepared bacteria:
Adjust liquid medium for pre- and main culture to pH 7.0 0.2, add
12 g/l of agar, dissolve by gentle warming, sterilize and transfer to
sterile Petri dishes.
Protective medium for freshly prepared bacteria:
66 g/100 ml D(+) Glucose monohydrate (C6H12O6 H2O)
4 g/100 ml Sodium chloride (NaCl)
2 g/100 ml L-Histidine
0.5 g/100 ml Bovine serum albumin, BSA
Dissolve in water at about 37 C and adjust pH to 7.0 0.2 with sodium hydroxide solution or hydrochloric acid at room temperature.
Reference substances for liquid-dried and freshly prepared bacteria
219.8 mg/l Zinc sulfate heptahydrate (ZnSO4 7H2O)
12.0 mg/l 3,5-Dichlorphenol (C6H4OCl2) (purity 99 %)
9.0 mg/l 3,5-Dichlorphenol (C6H4OCl2) (purity 99 %)
4.5 mg/l 3,5-Dichlorphenol (C6H4OCl2) (purity 99 %)
22.6 mg/l Potassium dichromate (K2Cr2O7)

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These solutions are prepared with sodium chloride solution as diluent, without adjustment of pH.
The concentrations are twice the expected EC50-values for the respective reference substances.
The twice concentrated preparation is due to the test set-up.

Reference substances:

19.34 mg/l Zinc sulfate heptahydrate (ZnSO4 7H2O)

(for freeze-dried bacteria)

6.8 mg/l 3,5-Dichlorphenol (C6H4OCl2) (purity 99 %)

105.8 mg/l Potassium dichromate (K2Cr2O7)

These concentrations are approximately twice the expected EC50-value for the respective reference substances. The volume required depends on the test set-up.

Sample preparation:
The preparation of the eluates is described in the SOP Waste Sample Preparation. Water eluates
can be stored for 72 h at the maximum after preparation (also at 4 2 C). Freezing of the samples
is not allowed in order to avoid changes of the sample properties.
Measure the salinity of the sample. NaCl concentrations of less than 15 g/l or more than 35 g/l or
their osmolarity equivalents in the sample, cause osmosis-related light inhibition. Add as much
sodium chloride to adjust the osmolarity to 20 g/l NaCl. If there is no possibility to measure the salinity of the eluate in the lab, refer to the salinity data for the eluate given in the covering sheets of
each sample (results of the range finding tests).

Interferences:
Coloured or turbid samples cause high-bias results due to physical absorption of light. If the luminometer has no integrated colour and turbidity compensation a colour correction may be necessary. Document the clouding and the colouring of the eluates and whether colour compensation
was conducted in the test protocol. Samples with acute high oxygen consumption (e.g. leachate of
landfill sites) can cause inhibition. Measure and document the O2 concentration of the eluates in
the test protocol.

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Cultivation of luminescent bacteria:

The bacteria strain (Vibrio fisheri NRRL B-11177) can be taken from commercially available freezedried or liquid-dried reagents or from culture collections. Document the type of bacteria in the test
protocol.
Stock culturing:
Transfer luminescent bacteria under sterile conditions to Petri dishes containing the agar for stock
culture and incubate for 2 d to 3 d at 20 C 1 C. Mark single colonies by visual observation in
the dark and store dishes in the refrigerator for one to two weeks. Transfer these colonies under
sterile conditions to fresh dishes afterwards.
Preparation of pre-cultures:
Transfer one single colony of a stock culture aged 2 d to 3 d to 50 ml Liquid medium for pre- and
main culture in an Erlenmeyer flask under sterile conditions. Shake for 21 h 1h at 20 C with
180 r min-1 and determine the turbidity of a 1 in 10 dilution (in a sodium chloride solution (20 g/l)) at
578 nm.
Preparation of main cultures:
Inoculate 50 ml Liquid medium for pre- and main culture in 250 ml Erlenmeyerflasks with an appropriate volume of pre-culture to gain an initial turbidity of 10 FAU. Shake for 21 h 1h at 20 C
with 180 r min-1 and determine the turbidity of a 1 in 10 dilution (in a sodium chloride solution
(20 g/l)) at 578 nm.
Preparation of stock suspension:
Pre-cool sodium chloride solution (20 g/l) and protective medium on ice. Centrifuge bacterial suspension from main culture at 4 C 2 C for 15 min to 20 min at 6000 g 2000 g. Decant supernatant and re-suspend pellets of 50 ml main culture in 5 ml to 10 ml ice cold sodium chloride solution. Repeat centrifugation at 4 C 2 C for 15 min to 20 min at 6000 g 2000 g. Decant supernatant and re-suspend pellets in 5 ml to 10 ml ice cold sodium chloride solution. Transfer the bacterial suspension to a pre-cooled 100 ml beaker and place it on ice. Add slowly about 4 ml of protective medium under constant cooling and stirring. Determine the turbidity of a 1 in 100 dilution
(with a sodium chloride solution (20 g/l)) at 578 nm and add an appropriate volume of pre-cooled
protective medium more quickly to gain a turbidity of 2500 FAU 500 FAU. Stir for another 15 min
to obtain a homogeneous mixture and dispense aliquots of 100 l into test tubes. Store stock solution in a freezer at -20 C. The stock suspension may be used for testing purposes as long as the
validity criteria are met.

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Test performance:
Test bacteria:
Strain Vibrio fischeri DSM 7151. The bacterial suspension used for the toxicity tests are prepared
from commercially available liquid-dried or freeze-dried reagents or from freshly prepared stock
suspensions (s.o.). After reconstitution the bacteria start immediately glowing and are ready for
use.

When using a new batch of liquid-dried bacteria or freeze-dried or freshly prepared bacteria the
sensitivity has to be checked with every reference solution. K2Cr2O7 has to be used in any test.
Prepare the required dilution series of the test eluates with a sodium chloride solution and maintain
the samples, the control and the K2Cr2O7-reference solution at 15 C 1 C.

Preparation of stock suspension for freeze-dried bacteria:

Remove the vial of freeze-dried culture from the freezer immediately befor reconstitution in water.
Cool 1ml of distilled water in a glass test tube to 3C 3C. Pour this volume of cooled water at
once into the lyophilized bacteria in the vial. This stock suspension may be used for testing purposes after 10 min or as long as the validity criteria are met.

Preparation of test suspension:

Liquid-dried or freshly prepared bacteria:
Fill 12 ml of the reconstitution solution in a vessel and place it in the thermo-block for about 15
minutes to maintain a temperature of 15 C 1 C.
Thaw the liquid-dried bacteria in a water bath at 20 C 2 C for two minutes and prepare the test
suspension for the test in two steps:
Add 0.5 ml of reconstitution solution, maintained at 15 C 1 C and homogenize by gentle shaking of the test tube.
Wait for about 15 minutes.
a) Preparation of the bacteria suspension with measurement of the undiluted eluates (D1):
Add 4.5 ml of the reconstitution solution to the resuspended bacteria and transfer 200 l of this
test suspension in the test tubes and maintain a temperature of 15 C 1 C. For the D1
measurement an extra control batch is needed. For the diluted eluates, mix the rest of the bac-

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teria suspension with the rest of the reconstitution solution and transfer 0.5 ml to the test tubes
and maintain a temperature of 15 C 1 C.
b) Preparation of the bacteria without measurement of the undiluted eluates:
Mix the resuspended bacteria with the rest of the reconstitution solution and transfer 0.5 ml in
every (depending on number of dilutions) test tube.
Wait for about 15 minutes.
Freezed-dried bacteria:
Prepare the test suspension from the stock suspension into a second corresponding set of test
tubes, maintained at 15 C 1 C. Prepare the test suspension either in conical flasks or directly in
the test tubes. For preparing the test suspension outside the test tubes in conical flasks add 1 volume of stock suspension to 50 volumes of solution for freeze-dried bacteria (maintained at 3 C 3
C). Pipette 500 l of test suspension into the test tubes, maintained at 15 C 1 C. For preparing
the test suspension directly in the test tubes add 10 l aliquots of stock suspension to 500 l solution for freeze-dried bacteria, contained in test tubes at 15 C 1 C and shake the mixture by
hand.
Wait for about 15 minutes.

Test procedure:
Before measurement adjust the luminometer instrument to a convenient, near maximum setting.
Determine and record the luminescence intensity I0 of the samples, the control and the reference
solution. As the contact time for all samples shall be equal measure the luminescence of each
sample at equal time intervals (e.g. 20 s). Immediately after the initial luminescence measurement
of a test suspension, add 0.5 ml (respectively 0.8 ml for D1) of the sample (respectively diluted
sample, reference solution or sodium chloride), mix by hand and place the test tubes back into the
thermo block at 15 C 1 C. After the incubation time of 30 minutes the luminescence intensity I30
is measured in the same way like I0.

Colour correction method:

If there is a visible colour in the dilutions with a concentration at the EC20 concentration and the
luminometer has no integrated colour and turbidity compensation the following procedure is performed. Carry out the complete procedure in a thermostatically controlled incubator at 15 C
0.5 C. Prepare a dilution of the eluate close to the EC20 concentration. Transfer 2.0 ml of 2 % sodium chloride solution to the outer chamber of the colour-correction tube. For the preparation of the

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bacterial suspension use 1.0 ml of bacterial stock suspension/liquid-dried bacteria. Mix the suspension and transfer it with a Pasteur pipette to the inner chamber of the colour correction-tube.
Add as much suspension to obtain the same level as the solution in the outer chamber. Measure
the light level after 15 min (B0) and remove the sodium chloride solution with a pipette from the
outer chamber. Replace it by 2.0 ml of the pre-cooled (15 C 1 C) diluted eluate and measure
the light level (I5) 5 min after the first measurement. Remove the diluted eluate with a pipette from
the outer chamber and replace it with 2.0 ml pre-cooled (15 C 1 C) sodium chloride solution.
Measure the light level (B10) 10 min after the first measurement.

Test Evaluation:
Calculate the correction factor (fk30-value) from the measured luminescence intensity using
Equation (1):

f K 30 =

I k 30
I0

(1)

where

f K 30

is the correction factor for the contact time of 30 min

I k 30

is the luminescence intensity in the control after 30 min

I0

is the luminescence intensity of the control immediately before the addition of the diluent.

Calculate the mean correction factor f K 30 and the deviation from the means in percent:

(( f K 30 f K 30i ) / f K 30 ) 100

(2)

where

f K 30i is one of the two individual values of the correction factor

f K 30

is the mean of f K 30

Calculate I ct using Equation (3).

I c 30 = I 0 f K 30

(3)

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where

f K 30

is the mean of f K 30

I0

is the luminescence intensity of the test sample suspension, immediately before the addition of the sample or the diluted sample

I c 30

is the corrected value of I 0 for test sample tubes immediately before addition of test sample

Calculate the inhibitory effect of a test sample using Equation (4)

H30 =

Ic30-I30
Ic30

x 100

(4)

where

H 30

is the inhibitory effect of a test sample after a contact time of 30 min in percent

I c 30

is the corrected value of I 0 for test sample tubes immediately before addition of test sample

I 30

is the luminescence activity of the test sample after the contact time of 30 min in relative
luminescence units

Calculate the mean of the inhibitory effect H30 for each dilution level in percent.
Calculate the arithmetic difference of the parallel determinations of H30i from their respective mean

H 30 in percent points:
H 30 (%) H 30i (%)
where
H30i

is either of the two individual values of the inhibitory effects of a test sample

H 30

is the mean value

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For evaluation of concentration-effect relationships, evaluate for each dilution level the gamma
value using Equation (5):

30 =

H 30
100 H 30

(5)

where

30

is the gamma value of the test sample after the contact time of 30 min

H 30

is the mean of H30

Calculation for colour correction:

Note: Only necessary if not performed automatically.
Calculate B5 with Equation (6):

B5 = B0

B0 B10
2

(6)

Calculate the absorption of the uncorrected EC20 concentration with equation (7):

At =

B
EC 20
k ln 5
I5
Ck

(7)

where
ck

is the concentration of the sample in the (colour) tested concentration

is an empirically derived system constant

ln

B5
is the absorption of the tested dilution in the colour correction cuvette
I5

Calculate the corresponding transmission (Tt) with equation (8):

Tt =

1 e At
At

(8)

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Calculate the corrected gamma values ( c ) with equation (9):

ct = (5 Tt ) 4
and

(9)

c = ct 0
where

ct

is the correction factor for gamma values at a given exposure time (t)

is the original gamma value

Perform a recalculation of the test results with the corrected values.

At a given exposure time, the absorption At and the transmission Tt for each test concentration can
be calculated, and from this the uncorrected gamma value with equation (10):

c = Tt (1 + 0 ) 1

(10)

The correction factor is the same for each gamma value assuming that the slope of the original line
is correct. It is therefore sufficient to calculate the correction factor for one gamma value only. In
this calculation, the gamma value corresponding to the uncorrected EC20 concentration is used.
Equation (11) for calculation of the correction factor is deduced as follows:

ct =

c Tt (1 + 0 ) 1
=
0
0

where
c

is the sample concentration

is measured bioluminescence value at 30 min

ct

is the correction factor for gamma values at 30 min

is the original gamma value

is the corrected gamma value

(11)

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Validity criteria for liquid-dried or freshly prepared bacteria:

-

the mean of the correction factor (fk30) value for 30 minutes incubation ranges between
0.6 and 1.8;

the parallel determinations do not deviate from their mean by more than 3 % for the
control samples;

For the test samples which determine the EC20/EC50 values, the deviation from their
mean in percent points does not exceed 3 % points.

The reference solution (use all three solutions to check the bacteria batch, for the test
only K2Cr2O7) cause 20 % to 80 % inhibition after 30 minutes contact time at the following concentrations in the final test suspension:

6 mg/l

3,5- Dichlorphenol

25 mg/l

Zn(II) equivalent to 109.9 mg/l zinc sulfate heptahydrate

4 mg/l

Cr(VI) (equivalent to 11.3 mg/l potassium dichromate)

The reference solution 3,5- Dichlorphenol (4,5 mg/l) cause x % inhibition after 30 minutes contact time in the final test suspension

The reference solution 3,5- Dichlorphenol (9 mg/l) cause x % inhibition after 30 minutes
contact time in the final test suspension

The reference solution 3,5- Dichlorphenol (12 mg/l) cause x % inhibition after 30 minutes contact time in the final test suspension

Validity criteria freeze-dried bacteria:

-

the mean of the correction factor (fk30) value for 30 minutes incubation ranges between
0.6 and 1.8;

the parallel determinations do not deviate from their mean by more than 3 % for the
control samples;

For the test samples which determine the EC20/EC50 values, the deviation from their
mean in percent points does not exceed 3 % points.

The reference solution (use all three solutions to check the bacteria batch, for the test
only K2Cr2O7) cause 20 % to 80 % inhibition after 30 minutes contact time at the following concentrations in the final test suspension:

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3.4 mg/l

3,5- Dichlorphenol

2.2 mg/l

Zn(II) equivalent to 9.67 mg/l zinc sulfate heptahydrate

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18.7 mg/l Cr(VI) (equivalent to 52.9 mg/l potassium dichromate)

Additional information for the ringtest:

Characterization, handling and delivery of the test substrate:
Dr. Roland Becker (BAM): Tel. +49 (0)30 8104 1121; e-mail: roland.becker@bam.de
Test performance:
Dr. Jrg Rmbke (ECT): Tel. +49 (0)6145 956450; e-mail: J-Roembke@ect.de
Test organisation:
Dr. Heidrun Moser (UBA): Tel. +49 (0)340 2103 3560; e-mail: heidrun.moser@uba.de

Please note that the colleagues listed above could/should be contacted directly. However, due to the
high numbers of participants answers may be collected before being distributed as round-mails.