Exotoxicological Report

Ecotoxicological assessment of water and sediment samples
from Dambovic Lake, Romania

Sponsor:
AECOM, s.r.o.
Research institution: Research Centre for Toxic Compounds

in the Environment (RECETOX)
Masaryk University, Faculty of Science
Kamenice 3, CZ-62500 Brno, Czech Republic
Invoice address:
Masaryk University, Faculty of Science

Kotlsk 2, CZ-61137 Brno, Czech Republic
Report No:
RECETOX-TOCOEN Report No. 378 (April 2010)
Period of the study:
March - April, 2010
Responsible person: Doc. RNDr. Ludk Blha, Ph.D.

blaha@recetox.muni.cz, tel. +420-549493194; +420-605510953
Research staff:
Mgr. Ji Novk, Ph.D.; RNDr. Kateina Brtov; Mgr. Petra Mackov,

Mgr. Barbora Jedlikov
Blha, L., Novk, J., Brtov, K., Mackov, P., Jedlikov, B.: Ecotoxicological assessment of water and
sediment samples from Dambovic Lake, Romania (sponsor: AECOM, s.r.o.). RECETOX-TOCOEN REPORT No.
378, April 2010, 83 pp.
Content
INTRODUCTION AND OVERALL EXPERIMENTAL DESIGN.........................................................................3
SAMPLES DESCRIPTION...............................................................................................................................................4
LIST OF SAMPLES TESTED FOR ECOTOXICITY AND GENOTOXICITY............................................................................5
METHODS SUMMARY..............................................................................................................................................6
WATER PROCESSING FOR TOXICITY TESTS FILTRATION...........................................................................................6
CONCENTRATION OF WATER SAMPLES - LYOPHILIZATION...........................................................................................6
SEDIMENT MANIPULATION - FRESH SEDIMENTS WITH CHIRONOMUS.........................................................................6
SEDIMENT LYOPHILILIZATION - CONTACT TEST WITH LUMINISCENT BACTERIA.........................................................7
GENOTOXICITY TESTING - SOS CHROMOTEST...........................................................................................................7
ALGAL GROWTH INHIBITION TEST..............................................................................................................................8
DAPHNIA MAGNA IMMOBILIZATION TEST...................................................................................................................9
VIBRIO FISCHERI BIOLUMINISCENCE INHIBITION TEST...............................................................................................9
KINETIC BACTERIAL BIOLUMINISCENCE TEST FOR CONTACT SEDIMENT TOXICITY....................................................9
CHIRONOMUS CONTACT SEDIMENT TOXICITY TEST..................................................................................................10
STATISTICS................................................................................................................................................................10
RESULTS 1 - RAW WATER ECOTOXICITY........................................................................................................11
RESULTS SOS CHROMOTEST - GENOTOXICITY.........................................................................................................11
RESULTS OF ALGAL GROWTH INHIBITION TEST........................................................................................................12
DAPHNIA MAGNA IMMOBILIZATION TEST.................................................................................................................13
VIBRIO FISCHERI BIOLUMINISCENCE INHIBITION TEST.............................................................................................14
RAW WATER SAMPLES - SUMMARY...........................................................................................................................15
RESULTS 2 - CONCENTRATED WATER ECOTOXICITY...............................................................................16
ALGAL GROWTH INHIBITION TEST - CONCENTRATED WATER SAMPLES....................................................................16
BIOLUMINISCENCE WITH VIBRIO FISCHERI - CONCENTRATED WATER SAMPLES.......................................................18
GENOTOXICITY TESTING - CONCENTRATED WATER SAMPLES...................................................................................26
CONCENTRATED WATER ECOTOXICITY - RESULTS SUMMARY...................................................................................31
RESULTS 3 - CONTACT SEDIMENT TOXICITY...............................................................................................32
IN VIVO SEDIMENT TOXICITY WITH CHIRONOMUS LARVAE......................................................................................32
TOXICITY TESTING WITH THE KINETIC BACTERIAL BIOLUMINISCENCE TEST...........................................................34
SUMMARY AND CONCLUSIONS..........................................................................................................................38
REFERENCES...........................................................................................................................................................39
ANNEXES...................................................................................................................................................................40
ANNEXES 1 - STANDARD OPERATION PROCEDURES..................................................................................................41
ANNEX 2 - RECETOX LABORATORY CERTIFICATE........................................................................................73
ANNEX 3 - PHOTOGRAPHS........................................................................................................................................75
Introduction and overall experimental design

Water and sediment samples were provided by the Sponsor of the study, i.e. AECOM, s.r.o.,
which originated from Romanian contaminated sites (Dambovnic and Suseni lake).
Objective of the present study was to characterize ecotoxicity and genotoxicity of water
samples and ecotoxicity of sediments using a battery of bioassays.
With the aim to provide thorough overview of ecotoxic effects, following biotests were
employed for different samples. Batteries of bioassays are conventionally used for ecotoxicity
testing, and they should represent biota from major trophic ecosystem levels, i.e.
- producers (i.e. algae, plants)
- consumers (i.e. invertebrates, vertebrates - e.g. fish)
- destruents (i.e. bacteria, microorganisms)
Samples of water (N=10) and sediments (N=3) were provided and tested as follows:
1) Raw water samples were tested with series of bioassays using the endpoint single
concentration experimental setup (no dilutions prepared)
Algal growth inhibition test
Daphnia magna immobilization test
Vibrio fischeri bioluminiscence inhibition test
SOS chromotest (genotoxicity test)
2) Concentrated water samples were prepared by lyophilization and freeze-dried materials
were dissolved in milliQ water, diluted (several serial dilutions prepared) and tested with the
same bioassays to derive EC50 values
SOS chromotest (genotoxicity test)
(Daphnia magna immobilization test with concentrated samples could not be finalized within
short experimental period available; complete results will be provided additionally).
3) Fresh sediment samples were tested in contact toxicity test with larvae of midges
Chironomus riparius (10 day sediment test for mortality)
4) Lyophilized sediment samples were tested for toxicity with a contact biotest using kinetic
bioluminiscence bacterial test (also known as "Flash" assay)
Samples description
Samples were collected at study sites in Dambovnic lake and Suseni lake and delivered to
RECETOX laboratories cooled at 4C
List of samples tested for ecotoxicity and genotoxicity

WATER SAMPLES - (10x 1L in dark glass bottles)
Sample I.D.
D1-IN
D2-IN
D3-IN
D3-B
D4-IN
D4-OUT
S-IN
S-A
S-D
S-OUT
SEDIMENT SAMPLES - (3x fresh sediments in plastic boxes; cooled at 4C)

Sample I.D.
D1-IN
D4-C
S-G
Methods summary
Water processing for toxicity tests filtration
The water samples contained varying amounts of organic debris that could negatively
affect accuracy of some of the employed bioassays. Thus, within a scope of standardization, the
samples were filtered using 2 m glass fiber filters. For algal growth inhibition test, it was
necessary to sterile filter the samples using 0.22 m according to ISO 8692 guideline [1]. The 2
m-filtered samples was used either directly for toxicity assessments or they were further
concentrated before the exposure.
Concentration of water samples - lyophilization

Often, acute toxicity of water samples is not high enough to assess dose-response
relationship, which is necessary for EC50 calculation. Thus, it is necessary to concentrate the
tested water samples to mimic the environmentally relevant situation.
Lyophilization, which was employed in this case, is one of the least destructive ways of
concentration of water samples.
800 ml of each filtered water samples was frozen in thick wall glass bottles at -80 oC and
than freeze-dried at temperature -55 C and low atmospheric pressure (210-3 mbar) using Christ
Gamma 1-16 LSC (Martin Christ Gefriertrocknunganlagen, Osterode am Harz, Germany). The
freeze-dried samples were reconstituted in 8 ml of milli-Q water to obtain hundred-times
concentrated samples. Before testing all samples were stored in refrigerator.
Sediment manipulation - fresh sediments with Chironomus

The sediment samples were stored at refrigerator before testing. The overlaying water
was decanted and the remaining water content in the sediment determined. Two 10 g samples of
well homogenized sediment were placed into ceramic pot and dried for 24 hours at 60-70 C.
The dry weight and percentage of water content was then determined. The sediment was sieved
with a 2 mm sieve in order to remove bigger particles or pieces of organic matter. The equivalent
of 40 g of dw is then weighted into each replicate, overlain by test water and allowed to
condition for 24 hours in the exposition room.
Sediment lyophililization - contact test with luminiscent bacteria

A part of the wet sediment samples was spread in thin layer on lyophilization plates and
frozen at -80 oC and than freeze-dried at temperature -55 C and low atmospheric pressure
(210-3 mbar) using Christ Gamma 1-16 LSC (Martin Christ Gefriertrocknunganlagen, Osterode
am Harz, Germany). The drying was stopped after complete water removal and samples were
homogenized in grinding mortar and than sieved using 2 mm-mesh sieve. The sieved samples
were stored in dry and cold place before toxicity testing.
Genotoxicity testing - SOS chromotest

It is a screening genotoxicity biotest widely used for evaluation of genotoxic potential of
pure chemicals as well as environmental samples [2, 3]. Genetically modified bacteria cells (a
strain Escherichia coli PQ 65 harboring a sulA::lacZ fusion) were employed in the study [2].
DNA is a molecular target and the reporter responds directly to the DNA damage. Cytotoxicity as
a result of more general macro-molecular damage can be detected in this test as well. The 96well microplate format was used for slightly modified SOS chromotest [4]. The tester strain was
grown overnight in LB medium containing ampicillin (20 mg ml-1) at 37 oC. After the incubation
period, the culture was diluted 50-fold into a fresh LB medium with ampicillin and it was
incubated for another 2 h. The optical density (600 nm) of the incubated culture was adjusted to
0.15 before test.
The water samples were diluted with concentrated assay buffer and inoculum so they
made 80% of the final volume in the assay. Distilled water was taken as a negative control; a
solution of 4-nitroquinoline-N-oxide was used as a positive control. The mixtures were incubated
for 2 h at 37 oC. Two microplates were prepared for measurements of enzymatic activities. Galactosidase activity (genotoxicity assay) was determined after the addition 25 l of the contain
of the incubated tubes into 100 l of buffer solution (pH 7.0) with o-nitrophenyl--Dgalactopyranoside (2 mg ml-1). Alkaline phosphatase activity (toxicity assay) was determined
after the addition of 25 l of the contain of the incubated tubes into 100 l of a buffer solution
(pH 8.8) with p-nitrophenylphosphate (2 mg ml-1). The microplates were incubated for 45 min at
37 oC and enzymatic activity was determined spectrophotometrically at 420 nm at the beginning
and the end of incubation. Toxic effects were quantified as a percentage of the alkaline
phosphatase activity in comparison with the negative control. The concentrations showing more
than 50% inhibition were excluded. The SOS induction factor (IF) was calculated for every
tested concentration. The samples with the induction factor higher than 1.5 for any tested
concentration were marked as a significant genotoxins.

This bioassay provides basic information on acute toxicity of tested samples to primary
producers in the fresh water aquatic environment. The used freshwater alga growth inhibition test
protocol is a slightly modified version of ISO 8692 procedure [1]. The purpose of this test is to
determine the effects of a substance on the growth of fresh water microalgae Raphidocelis
subcapitata. Exponentially growing test algae are exposed to the test substance in microplates
over a period of 72 hours.
This test assay was modified and miniaturized for 96-well microplates [5]. The water
samples were diluted with concentrated algal growth medium and they made 75% of the final
volume in the bioassay. Cells in the exponential growth phase were used, and light absorbance
(680 nm; corresponding to the amount of chlorophyll) was determined in the beginning of the
exposure and then at the end of the exposure. Growth inhibition was determined using following
formulas:
Average growth rate:
ln ODi-j ln ODi
i-j = ------------------tj ti
i-j
ti
tj
ODi
ODj
is the average specific growth rate from moment time i to j (day-1);

is the moment time for the start of the period (day);
is the moment time for the end of the period (day);
is the optical density (blank substracted) at time i;
is the optical density (blank substracted) at time j.
Percent inhibition of growth rate:

C T
%I = --------- x 100
C
%I
C
T
is percent inhibition in average specific growth rate;

mean value for in the control;
value for growth rate in the treatment.

This bioassay describes acute toxicity of the samples for Dafnia magna, a model
organism representing aquatic consumer trophy level. The protocol of the test is based on ISO
6341 [6] and it is performed with D. magna neonates that are exposed to tested samples for 48 h.
The endpoint of this test is an inhibition rate, a ratio of the dead and inhibited individuals to the
active ones. The percentage of immobilization was calculated for each period when observations
were recorded (24 and 48h).

The bioassay is a screening tool used for a variety of toxicity testing applications. The
advantages of this toxicity bioassay are its speed, simplicity, and relatively low cost, when
compared to the cost of chemical analysis. The used protocol is based on ISO 11348-3 [7]
guideline.
The mechanism of the biotest is based on contact among the bacteria and the sample.
Toxicity of the sample is then determined by measuring the decrease in bioluminescence of
bacteria. The response of V. fischeri to the sample is compared to the response of the bacteria to a
control, reference or blank sample. Freeze-dried luminescent bacteria Vibrio fischeri NRRL B11177 were obtained from Institute of Microbiology, Czech Academy of Sciences (Praha, Czech
Republic). Bacteria were reconstituted according to ISO 11348-3 [7] in ice-cold 2% NaCl. Prior
to testing, aliquots of bacterial suspension were diluted in 2% NaCl and temperated in 15C
water bath. Assay was performed in white 96 well plates and luminescence was determined at
exposure times 0, 15 and 30 min by microplate Luminoscan Ascent luminometer (Thermo,
Finland). Luminescence inhibition was calculated for times 15 and 30 min against negative
control.
Kinetic bacterial bioluminiscence test for contact sediment toxicity

Sediment suspensions (100 mg sediment dry wt per 1 ml of 2% NaCl, pH 7.2) were
prepared by extensive shaking (vortex) for 5 minutes. Serial dilutions (1:1) for each sample were
then prepared in 2% NaCl (tops of the pipette tips were cut by razor blade). Freeze-dried
luminescent bacteria Vibrio fischeri NRRL B-11177 were obtained from Institute of
Microbiology, Czech Academy of Sciences (Praha, Czech Republic). Bacteria were reconstituted
according to ISO 11348-3 [7] in ice-cold 2% NaCl. Prior to testing, aliquots of bacterial
suspension were diluted in 2% NaCl and temperated in 15C water bath. Assays were performed
using a modified method [8, 9] in white 96 well plates and luminescence was determined by
microplate Luminoscan Ascent luminometer (Thermo, Finland) equipped with computercontrolled injectors. Each well in the microplate contained 80 microliters of studied samples
(serial dilutions of sediment suspensions) or negative controls (2% NaCl). Bacteria (20
9
microliter) were injected into each well and immediate luminescence (initial "Peak" value) was
monitored for 2 seconds. Microplate was than shaken (built-in shaker inside the luminometer)
and the signal was recorded again after 30 seconds ("S30"). Inhibition of luminescence (% of
control) was calculated as follows:
where CF is the correction factor (the S30/Peak ratio in Negative controls reflecting natural
attenuation of bacterial luminescence during 30 second exposures). Concentrations of sediment
suspensions that caused 50% inhibition of luminescence (IC50 in mg dry wt/ml) were derived
from four-parametric logistic curve calculated in GraphPadTM Prism (GraphPad Software, San
Diego, CA, USA).
Detailed Standard Operation Procedure for Kinetic bacterial bioluminescence test is in the
Annexes.
Chironomus contact sediment toxicity test

The purpose of this test is to assess sediment toxicity for the larvae of non-biting midge
Chironomus riparius. The endpoint of the 10-day test is survival (%); growth measured as a
body length (mm) may also be evaluated. This method may be used for comparative assessment
of toxicity of natural sediments. The newly hatched larvae of Chironomus riparius are exposed
to the fresh sediment; sediments may be diluted with a clean (reference / artificial) sediment. The
larvae are fed daily and after 10 days of exposure they are recovered from the test sediment,
counted and measured to determine survival and growth. The test system needs to be checked
daily for proper aeration, and physical-chemical parameters of the overlaying water need to be
measured at the beginning and end of the test.
Statistics
To determine the significance of the response to treatments relative to controls, statistical
analyses were performed using a one-way ANOVA with Dunnett post hoc test f (p < 0.05) using
GraphPadTM Prism (GraphPad Software, San Diego, CA, USA). For general calculations MSEXCEL was used.
.
10
Results 1 - raw water ecotoxicity

Results SOS chromotest - genotoxicity
The genotoxicity was assessed using bacterial model E. coli. The exposure was
performed in three replicates for 2 hours at 37oC. From data on genotoxicity and cytotoxicity, an
induction factor has been calculated. The induction factor higher than 1.5 indicates significantly
genotoxic sample.
The results show that most of the samples did not reach the threshold level of induction
factor and thus they were not significantly genotoxic.
The threshold 1.5 was exceeded only by sample S-A. Nevertheless, even in this case, the
value was not significantly higher than 1.5 (ANOVA, Dunnet post test > 0.05) and so it cannot
be considered genotoxic.
Figure 1 Genotoxic potency of water samples expressed as induction factors (mean from three replicates +
standard deviation)
11
Results of algal growth inhibition test

The testing was done according to ISO 8692 guideline [1] with microplate modification
that was shown to provide same results as the original procedure [5]. The tested water samples
were diluted with concentrated algal growth medium and they made 75% of the final volume in
the bioassay. The algal culture was exposed to tested samples in three replicates for 72 hours.
Besides the samples D2-in and D3-B, which showed non-significant stimulation of
growth (in comparison with control), majority of the samples inhibited growth of the algae in the
test (Figure 2).
This inhibition was statistically significant in case of samples D1-IN, D4-IN S-IN, S-A
and S-OUT. Results indicate significant toxic effects of studied samples to primary producers.
Figure 2 Percentage of algal growth inhibition (mean+ standard deviation); * - statistically significant against
control
12

The testing was performed following ISO 6341[6] guidelines. The testing was carried out
in four replicates. Toxic effects were very low.
Slight immolization on D. magna was observed at samples D1-in and D2-in (maximum
10% effect in comparison to control) but these effects do not indicate statistically significant
toxicity.
Figure 3 Percentage of inhibition in Daphnia magna immobilization test.
13

The water samples were tested with well established and widely used test known also as
"Microtox" [7]. Before testing, water samples were filtered with 2 m glass fiber filter to remove
undisolved organic matter such as plant leaf debris. Vibrio fischeri is a marine organism so it was
necessary to supplement the tested samples to contain 2% NaCl. This was done with 10% NaCl
solution, and resulting concentration of the original raw water in the assay was 80%. All samples
were tested in three replicates.
All tested water samples showed statistically significant toxic effect to Vibrio fischeri at
both exposure durations (15 and 30 minutes). The highest toxicity (almost 50% inhibition) was
observed at the samples D2-in.
*
*
*
*
*
Figure 4 Toxicity of water samples to Vibrio fischeri expressed as inhibition of luminescence a times 15 and 30
min (mean+SD). * - statistically significant against control
14
Raw water samples - Summary

Summary results of the ecotoxicity testing presented in previuos paragraphs are presented in the Table.
Toxicity of raw water samples were screened with a biotest testing genotoxic potential (bacterial SOS-chromotest) and three ecotoxicological
assays covering all three trophic levels (producers, consumers and destruents).
While genotoxicity and Daphnia immobilization test did not show any significant results, algal growth was significantly inhibited by half of the
samples and all samples were significantly toxic to V. fischeri bacteria.
Table 1 Raw water toxicity summary of results; asterisks (*) indicate statisticaly significant effects
water sample
Genotoxicity (IF)
Algal growth inhibition (%)
Daphnia immobilization (%)1
V. fischeri inhibition (%)2
1
data from 48 h exposure
2
data from 30 min exposure
D1-in
0.97
28.6 *
10
28.7 *
D2-in
0.83
-38.9
10
44.5 *
D3-in
1.00
12.5
5
32.1 *
D3-B
1.04
-6.2
0
24.4 *
D4-in
1.02
42.4 *
5
22.1 *
D4-out
1.17
25.7
5
20.6 *
S-in
1.44
28.1 *
5
21.0 *
S-A
1.87
47.0 *
0
19.0 *
S-D
1.03
26.6
0
22.0 *
S-out
0.85
59.7 *
5
22.0*
15
Results 2 - Concentrated water ecotoxicity

Algal growth inhibition test - concentrated water samples
Four concentrations of concentrated water (prepared by lyophilization) were tested for
toxicity to algae (30x, 10x, 3x and 1x concentration factors) in a modified test using microwell
plates. Higher concentrations could not be tested due to the presence of particulate matter in
concentrated samples, which interfered with analysis.
After 72h of exposure, growth was evaluated by determination of absorbance and
inhibitions (%) in comparison with controls were calculated (Table 2)
Table 2 Results of the algal growth inhibition test with concentrated water samples. (Negative values indicate
stimulations in growth)
Growth inhibitions / stimulations (% of control)
Concentration
factor
30x
10x
3x
1x
D1-in
-21.3
-42.8
-49.1
-20.7
D2-in
-34.3
-17.8
-14.8
-44.0
D3-in
-89.4
-31.5
-30.9
-52.5
D3-B
-159.0
-116.6
-103.3
-37.7
D4-in
-133.8
-130.5
-59.5
-56.9
D4-out
-321.5
-125.9
-80.8
-15.1
S-in
-26.2
-58.0
-37.7
-45.9
S-A
-99.0
-52.7
-27.1
5.2
S-D
-44.5
-60.4
-25.7
21.1
S-out
60.9
21.4
13.4
10.0
Standard deviation
Concentration
factor
30x
10x
D1-in
2.8
3.2
D2-in
1.3
0.2
D3-in
10.4
11.9
D3-B
1.7
6.8
D4-in
7.4
6.6
D4-out
40.2
1.9
S-in
8.7
3.7
S-A
12.8
0.6
S-D
9.5
1.1
S-out
9.6
5.0
3x
4.3
20.7
2.5
4.4
5.8
2.5
2.9
1.2
3.9
0.9
1x
4.5
1.6
14.9
3.6
0.7
5.9
3.5
3.2
1.5
2.7
16
As it is apparent, most of the concentrated samples were not toxic but caused stimulations
in algal growth. This is a common observation in toxicity testing with natural samples, which
contain both toxicants as well as nutrients promoting the growth of algae. Consequently, toxic
effects on algae may be masked by the stimulations in the growth.
These effects did not fully correspond to the toxicity testing with fresh samples, where
non- concentrated samples (corresponding to "1x" dilution factor) caused inhibitions up to 5060% (see previous sections). Differences may be atributed to possible changes in the sample
composition during lyophilization and back-dilution with milliQ water.
"S-out" was the only sample, which showed a dose-dependent effects, i.e. inhibitions of
the growth with increasing concentration factor. At the highest tested concentrations 60% growth
inhibition was observed. This sample also showed highest toxicity when tested without
concentration.
For this sample, IC50 was calculated by nonparametric sigmoidal regression (Table 3).
Final IC50 value should be carefully interpreted as calculation was based on partial doseresponse curve (effects at higher concentrations than 30x concentration factor; see Figure 5)
could not evaluated.
Table 3 Toxicity of S-out sample (IC50) in algal growth inhibition test (72h exposure)
IC50
(concentration
95% confidence
factor)
interval
S-out
32.8
23.78 to 47.93
A lg a l g r o w th in h ib itio n
(% )
100
S-out
75
50
25
0
0.0
0.5
1.0
1.5
log (concentration factor)

Figure 5 Dose-response curve for S-out sample in the algal growth inhibition test (72h exposure)
17
Bioluminiscence with Vibrio fischeri - concentrated water samples

Water samples concentrated by lyophilization (dry material) were diluted in milliQ water,
and toxicity was tested with five dilutions (final tested concentrations - corresponding to
concentration factors 1x, 3x, 10x, 30x and 75x concentrated water). Similar approach was
previously used by RIVM (National Institute for Public Health and the Environment) in
Netherlands (A.M. Durant et al. 2009, Toxicity measurements in concentrated water samples.
RIVM Report 607013010).
Because the concentrated samples (30x and 75x concentrated in all cases) were turbid due
to concentrated humic substances and other components present in water, toxicity was tested
with V. fischeri kinetic bioluminiscence test. Application of traditional V. fischeri luminiscence
test was not possible because inhibition of the emmitted light would interfere with coloured
samples.
Table 4 shows obtained IC50 values. As it is apparent, the most toxic was sample D2-in
with IC50 around concentration factor 11 (eleven-times concentrated water caused 50%
inhibition). Graphical presentations of the results are in Figure 6.
Table 4 Toxicity of concentrated water samples in the bacterial bioluminiscence inhibition kinetic V. fisheri
test (IC50 values with confidence intervals - concentration factor of water samples)
IC50
(concentratio
n factor)
95% conf. Int.
D1-in
32.11
23.04 to 44.75
D2-in
11.61
7.164 to 18.81
D3-in
55.72
25.96 to 119.6
D3-B
56.61
33.76 to 94.93
D4-in
31.15
15.73 to 61.67
D4-out
27.76
16.08 to 47.91
S-in
48.97
29.21 to 106.35
S-A
48.69
24.58 to 96.48
S-D
49.4
29.66 to 82.29
S-out
55.72
40.61 to 76.46
18
Figure 6 Toxicity of concentrated water samples in the bacterial bioluminiscence inhibition kinetic V. fisheri
test (IC50 values +/- S.D.)
Table 5 shows the source data from the toxicity testing of concentrated water samples
with kinetic V. fisheri test. Graphs demonstrating the full dose response curves are presented in
the following figures.
19
Table 5 Bacterial bioluminiscence inhibition of concentrated water samples with V. fisheri kinetic test (source
data)
D1-in
Inhibition
(% of control)
Concentration Replicate Replicate
factor
1
2
1
-0.08
6.13
3
8.68
10.98
10
26.85
27.08
30
51.54
54.63
75
76.62
79.43
D4-out
Inhibition
(% of control)
Concentration Replicate Replicate
factor
1
2
1
-7.80
-3.60
3
-9.02
1.73
10
17.60
24.38
30
55.71
57.63
75
80.48
82.48
D2-in
1
3
10
30
75
-1.78
11.56
42.71
64.93
82.58
7.54
20.18
44.20
68.07
86.14
S-in
1
3
10
30
75
-0.85
-0.48
11.63
24.21
72.94
-0.66
-1.81
13.81
43.25
77.20
D3-in
1
3
10
30
75
0.39
-0.81
12.80
49.86
81.03
4.64
3.82
17.22
56.14
83.52
S-A
1
3
10
30
75
-4.75
4.73
18.33
38.09
76.30
-1.18
0.76
21.17
51.42
77.41
D3-B
1
3
10
30
75
-1.78
-2.31
11.86
39.78
67.83
1.31
3.32
16.99
41.21
67.77
S-D
1
3
10
30
75
-5.52
-1.09
7.53
46.40
73.39
-4.25
-2.16
13.28
46.13
74.19
D4-in
1
3
10
30
75
-9.27
-8.14
16.77
56.45
83.64
1.90
0.86
25.20
59.42
85.11
S-out
1
3
10
30
75
-3.86
0.70
12.64
43.64
74.49
-4.07
2.75
12.76
45.96
74.97
20
Inhibition of bacterial bioluminiscence (V. fischeri kinetic test) by concentrated water samples full concentration-response curves.
100
D1-in
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
In h ib itio n (% )
100
D2-in
75
50
25
0
0.0
0.5
1.0
1.5
2.0
21
100
D3-in
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
100
D3-B
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
22
100
D4-in
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
100
D4-out
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
23
100
S-in
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
100
S-A
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
24
100
S-A
In h ib it io n
(% )
75
50
25
0
0.0
0.5
1.0
1.5
2.0
In h ib it io n ( % )
100
S-out
75
50
25
0
0.0
0.5
1.0
1.5
2.0
25
Genotoxicity testing - concentrated water samples

Genotoxicity testing in SOS-chromotest was conducted with 4 concentrations of water
(concentration factors 1-30x). Although it was expected that genotoxicity will be induced with
increasing concentration, this was not observed in this study (Table 6; Figures 7). This might be
attributed (similar as with algal growth test) to the presence of nutrients and other natural
compounds in concentrated samples. Higher activities of reporter enzyme (reflecting DNA
damage) were observed with increasing concentration but final induction factors (genotoxicity
parameter) were low, because bacteria in the assay were stimulated to grow, which masked the
final genotoxic effect.
Significant genotoxicity (IF > 1.5) was observed in two samples 3-times concentrated
(D2-in and D4-in) but the effects were still rather low in comparison with e.g. positive control
with IF ~ 36.
Table 6 Genotoxicity of concentrated water samples (induction factors > 1.5 indicate genotoxicity).
Induction factors
4-NQO (positive control)

D1-in
D2-in
D3-in
D3-B
D4-in
D4-out
S-in
S-A
S-D
S-out
4-NQO
35.99
30x
0.689
0.786
0.557
0.163
0.719
0.128
0.123
0.789
0.518
0.779
Concentration factors
10x
3x
0.812
0.693
0.880
0.238
0.659
0.185
0.898
0.107
0.544
1.012
1.112
1.741*
0.646
1.461
1.937*
1.019
0.780
0.897
1.273
0.666
1x
1.489
0.978
0.983
1.060
1.061
1.302
1.118
1.099
0.566
1.163
Standard deviations
Concentration factors
4-NQO
30x
10x
3x
1x
D1-in
0.128
0.199
0.152
0.176
D2-in
0.169
0.123
0.229
0.059
D3-in
0.216
0.198
0.273
0.143
D3-B
0.249
0.207
0.221
0.154
D4-in
0.077
0.193
0.235
0.081
D4-out
0.091
0.185
0.131
0.215
S-in
0.128
0.108
0.178
0.103
S-A
0.309
0.331
0.189
0.223
S-D
0.346
0.311
0.291
0.240
S-out
0.294
0.229
0.069
0.080
4-NQO (positive control)
0.547
26
Figures 7 Genotoxicity of concentrated water samples along with the induction factor of positive control
(reference toxicant 4-nitroquinoline-N-oxide; horizontal lines show the value of the critical induction factor
value ~ 1.5).
27
28
29
30
Concentrated water ecotoxicity - results summary

Summary of ecotoxicity testing with concentrated water samples is presented in Table 7.
Out of the 10 tested samples, highest toxicity (lowest IC50 values) was observed at the sample
D2-in (IC50 ~ 12x diluted water for V. fischeri and significant genotoxicity in the SOSchromotest). Another genotoxic sample was no. D2-in. Significant inhibitions of algae were
observed at the sample S-out.
In general, kinetic bacterial bioluminiscence assay was found to be the most sensitive and
feasible for testing of complex samples as other two tests were affected by nutrients and other
natural components present in concentrated samples.
Table 7 Toxicity results of water samples - IC50 values (for algal growth inhibition and bioluminiscence) and
minimum inhibitory concentration (MIC). Values are given as concentration factors.
Bioluminiscence
D1-in
D2-in
D3-in
D3-B
D4-in
D4-out
S-in
S-A
S-D
S-out
Algal growth
inhibition with V.
inhibition
fischeri
Genotoxicity
(IC50)
na
na
na
na
na
na
na
na
na
32.8
(IC50)
32.11
11.61
55.72
56.61
31.15
27.76
48.97
48.69
49.4
55.72
(MIC)
na
3x
na
na
3x
na
na
na
na
na
31
Results 3 - Contact sediment toxicity

In vivo sediment toxicity with Chironomus larvae
Toxicity of 3 fresh whole sediment samples was tested in vivo in the experimental setup
using midge Chironomus sp. larvae. Experiments used both original sediment samples as well as
samples diluted with the artificial sediment, which also served as non-toxic (reference) control.
In general, high toxicity was observed in all tested sediments (0% survival). Only in the
sample D1-in diluted 1:3 with the reference artificial sediment, survival around 17% of
chironomid larvae was observed. Complete experimental data are summarized in Table 8 and
statistics are in Table 9.
Table 8 Toxicity results of fresh sediments with Chironomus sp.
Replicate
Control (artificial
sediment)
No. of larvae
Initial
Survived
% survival
1
2
3
20
20
20
16
12
15
80
60
75
D1-in whole
1
2
3
20
20
20
0
0
0
0
0
0
D1-in (1:3 diluted)
1
2
3
20
20
20
5
0
5
25
0
25
D4-C whole
1
2
3
20
20
20
0
0
0
0
0
0
D4-C (1:3 diluted)
1
2
3
20
20
20
0
0
0
0
0
0
S-G (whole)
1
2
3
20
20
20
0
0
0
0
0
0
S-G (1:3 diluted)
1
2
3
20
20
20
0
0
0
0
0
0
32
Table 9 Statistics - results of the toxicity testing with Chironomus sp.

Mean
survival
(%)
S.D.
Control (artificial
sediment)
71.7
10.4
D1-in whole
D1-in (1:3 diluted)
0.0
16.7
na
14.4
3
3
D4-C whole
D4-C (1:3 diluted)
0.0
0.0
na
na
3
3
S-G (whole)
S-G (1:3 diluted)
0.0
0.0
na
na
3
3
33
Toxicity testing with the kinetic bacterial bioluminiscence test

Three sediment samples were tested for contact toxicity with novel bacterial kinetic
luminescence assay. Before testing, sediments were lyophilized and sieved (2 mm); testing was
performed with freshly prepared suspensions and their dilutions (maximum tested concentration
80 mg dry wt/mL). Experiment was conducted in two replicates.
Table 10 and Figure 8 shows the final results of toxicity - IC50 values (i.e. concentrations
of sediment in mg dry weight per mL of tested suspension that caused 50% inhibition of bacterial
luminiscence in the kinetic contact assay). Values of the 95% confidence interval for IC50 are
indicated
Table 10 Summary results of contact toxicity testing of three sediment samples with bacterial kinetic
luminescence assay
D1-in
D4-C
SG
IC50 (mg/mL)
0.195
0.312
0.361
95% Conf.Int. (min)
0.149
0.264
0.269
95% Conf.Int. (max)
0.256
0.369
0.484
Figure 8 Graphical presentation of the sediment toxicity obtained with bacterial kinetic luminescence assay
(IC50 +/- S.D.)
As apparent, the most toxic was the sample D1-in with IC50 around 0.2 mg/mL, the other
two samples were less toxic but the values of IC50 (0.31 - 0.36 mg/mL) still indicate high
toxicity. This is clear from the comparison with other toxicity values of river sediments from the
area polluted by industrial, domestic and rural activities [10]. The lowest IC50 value (the highest
toxicity) observed in the mentioned study [10] was 0.7 mg/mL, while median IC50 values for
more than 50 sediment samples ranged 5-35 mg/mL.
34
Complete results of the toxicity testing are presented in :

Table 11 Summary of the toxicity testing with kinetic bacterial bioluminescence assay
Sample:
D1-in
inhibition (fraction)
conc.
[mg/mL]
0.039
0.078
0.156
0.313
0.625
1.250
2.500
5.000
10.000
20.000
40.000
80.000
log(conc)
-1.408
-1.107
-0.806
-0.505
-0.204
0.097
0.398
0.699
1.000
1.301
1.602
1.903
Sample:
D4-C
replicate 1
9.6
24.2
41.0
55.7
67.1
75.2
83.4
88.5
89.7
89.7
95.8
94.8
replicate 2
4.8
20.8
42.2
54.6
67.3
74.9
81.0
87.8
87.8
90.2
101.2
98.3
conc.
[mg/mL]
0.039
0.078
0.156
0.313
0.625
1.250
2.500
5.000
10.000
20.000
40.000
80.000
log(conc)
-1.408
-1.107
-0.806
-0.505
-0.204
0.097
0.398
0.699
1.000
1.301
1.602
1.903
Sample:
SG
replicate 1
2.0
10.5
32.8
47.8
66.5
76.8
85.8
92.0
99.9
100.1
99.7
101.6
replicate 2
5.5
8.5
31.3
41.5
63.8
73.8
91.7
90.9
97.6
98.5
98.8
100.2
conc.
[mg/mL]
0.039
0.078
0.156
0.313
0.625
1.250
2.500
5.000
10.000
20.000
40.000
80.000
log(conc)
-1.408
-1.107
-0.806
-0.505
-0.204
0.097
0.398
0.699
1.000
1.301
1.602
1.903
replicate 1
1.9
19.2
34.8
44.8
52.0
69.7
76.1
83.6
88.6
95.3
91.3
99.2
replicate 2
8.9
17.5
34.1
48.9
63.5
68.9
76.8
88.2
92.5
96.0
92.5
95.2
35
Following figures present the full concentration-response curves for contact toxicity of 3
sediment samples in the kinetic bacterial bioluminiscence assay:
Sample ID - SEDIMENT "D1-IN"
Sample ID - SEDIMENT "D4-C"
36
Sample ID - SEDIMENT "SG"
37
Summary and conclusions

This report documents toxicity of 10 water samples and 3 sediment samples from
Dambovic Lake, Romania.
Fresh, non-concentrated water samples interestingly showed significant inhibitions of
growth in the algal assay. This was not expected with regard to previous experiences of the
research laboratory. These effects thus indicate significant toxicity towards producers in the
ecosystem.
Weaker but still significant effects of fresh water samples were observed also in the
bioluminiscence inhibition assays with bacteria V. fischeri. The most toxic sample was D2-in
(app. 45% inhibition of luminiscence); other samples had minor toxicity around 20% inhibition).
Water samples caused only minor toxicity towards crustacean D. magna but toxicity of
D2-in (and D1-in) samples was confirmed (10% immobilization).
Only weak genotoxic effects, which were not significantly different from the critical
induction factor 1.5 were observed in SOS-chromotest.
Concentrated water samples were prepared by lyophilization and back diluted with
milliQ water with the aim to obtain more insight into toxicity and assessment of IC50. However,
due to presence of nutrients and other components, rather incoclusive results were obtained with
algal test and SOS-chromotest.
In the assay with algae, stimulations of growth were observed, and the only sample which
showed toxicity was S-out. Weak genotoxicity was observed in samples D2-in and D4-in.
Kinetic bioluminiscence assay with V. fischeri was found to be the most suitable for
testing of complex samples. The assay allowed determination of IC50 for all samples, and
showed that D2-in was the most toxic (IC50 ~ concentration factor of 10x).
Tested sediments were highly toxic in both assays demonstrating high contamination in
the samples. IC50 values derived in the kinetic contact bacterial bioluminescence test were very
low (highly toxic) in comparison to previous available studies. 100% mortality of Chironomus
larvae was was observed in all three undiluted sediments. After dilution with non-toxic reference
sediment, sediment "D1-in" was less toxic than other two samples (D4-C and S-G) as some
surviving larvae (~ 15%) were found.
38
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Agency, I.S., International Standardization Agency 8692, Water quality freshwater

algal growth inhibition test with unicellular green algae. 2004.
Quillardet, P. and M. Hofnung, The Sos Chromotest, a Colorimetric Bacterial Assay for
Genotoxins - Procedures. Mutation Research, 1985. 147(3): p. 65-78.
Quillardet, P. and M. Hofnung, The Sos Chromotest - a Review. Mutation Research, 1993.
297(3): p. 235-279.
Skarek, M., et al., Evaluation of genotoxic and non-genotoxic effects of organic air
pollution using in vitro bioassays. Environment International, 2007. 33(7): p. 859-866.
Rojickova, R., D. Dvorakova, and B. Marsalek, The use of miniaturized algal bioassays
in comparison to the standard flask assay. Environmental Toxicology and Water Quality,
1998. 13(3): p. 235-241.
Agency, I.S., International Standardization Agency 6341, Water qualitydetermination
of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)acute
toxicity test. 1996.
Agency, I.S., International Standardization Agency ISO 11348-3:1998. Water quality -Determination of the inhibitory effect of water samples on the light emission of Vibrio
fischeri (Luminescent bacteria test) -- Part 3: Method using freeze-dried bacteria. 1998.
Lappalainen, J., et al., Automated color correction method for Vibrio fischeri toxicity test.
Comparison of standard and kinetic assays. Chemosphere, 2001. 45(4-5): p. 635-641.
Lappalainen, J., et al., A new flash method for measuring the toxicity of solid and colored
samples. Chemosphere, 1999. 38(5): p. 1069-1083.
Blaha, L., et al., Kinetic Bacterial Bioluminescence Assay for Contact Sediment Toxicity
Testing: Relationships with the Matrix Composition and Contamination. Environmental
Toxicology and Chemistry. 29(3): p. 507-514.
39
Annexes
40
Annexes 1 - Standard operation procedures
41
RECETOX
Research Centre for
Environmental Chemistry and EcoTOXicology
Masaryk University
EU-DG Research Centre of Excellence for Environmental Chemistry and Ecotoxicology
_________________________________________________________________________________________________________________
Freshwater Alga and Cyanobacteria, Growth Inhibition Test

June 2005
Daniel Janula, Pavel Babica
1.0 DEFINITIONS AND ACRONYMS
Growth is the increase in biomass concentration over the test period.
Growth rate is the increase in biomass concentration per unit of time.
ECx (e.g. EC50) is the concentration of the test substance dissolved in test medium that results in a x %
(e.g. 50%) reduction in growth of the test organism within a stated exposure period.
Lowest Observed Effect Concentration (LOEC) is the lowest tested concentration at which the substance
is observed to have a statistically significant reducing effect on growth (at p < 0.05) when compared with
the control, within a given exposure time. However, all test concentrations above the LOEC must have a
harmful effect equal to or greater than those observed at the LOEC. When these two conditions cannot be
satisfied, a full explanation must be given for how the LOEC (and hence the NOEC) has been selected.
No Observed Effect Concentration (NOEC) is the test concentration immediately below the LOEC which,
when compared with the control, has no statistically significant effect (p < 0.05), within a given exposure
time.
Test medium is the complete synthetic culture medium on which test plants grow when exposed to the test
substance. The test substance will normally be dissolved in the test medium.
2.0
PURPOSE
Freshwater Alga and Cyanobacteria, Growth Inhibition Test was develloped to detect inhibitive properties
of chemicals.
3.0
SCOPE AND APPLICATION
The test is most easily applied to water-soluble substances which, under the conditions of the test, are
likely to remain in the water. For testing of substances that are volatile, strongly adsorbing, coloured,
having a low solubility in water or substances that may affect the availability of nutrient or minerals in the
test medium, certain modifications of the described procedure may be required.
4.0
SUMMARY OF METHOD
This test is a modification of OECD 201 (ISO 8692) Growth inhibition test. The purpose of this test is to
determine the effects of a substance on the growth of fresh water microalgae (algae and cyanobacteria).
42
Exponentially growing test algae are exposed to the test substance in batch cultures normally over a
period of 72 hours.
The system response is the reduction of growth in a series of algal cultures (test units) exposed to various
concentrations of a test substance. The response is evaluated as a function of the exposure concentration
by comparison with the average growth of replicate, unexposed control cultures. For full expression of
the system response to toxic effects (optimal sensitivity), the cultures are allowed unrestricted exponential
growth under nutrient sufficient conditions and continuous light for a sufficient period of time to measure
reduction of the specific growth rate. Growth and growth inhibition are quantified from measurements of
the algal biomass density as a function of time.
The test endpoint is inhibition of growth, expressed as logarithmic algal biomass increase (average
growth rate) during the exposure period. The ECx (e.g. EC50) is determined and expressed from the
average growth rates recorded in a series of test solutions and the concentration bringing about a specified
x % inhibition of growth (e.g. 50%).
Measurement of biomass by manual cell counting by microscope or an electronic particle counter (cell
counts and/or biovolume, the biovolume correlates directly with biomass) are preferred. Alternative
techniques, eg. in vitro chlorophyll fluorescence, or optical density can be used providing a satisfactory
correlation with biomass (dry weight mg/l).
Several species of non-attached microalgae and cyanobacteria may be used. These strains are listed in
OECD 201 Guideline: green algae Pseudokirchneriella subcapitata (formerly known as Raphidocelis
subcapitata or Selenastrum capricornutum), Scenedesmus subspicatus, diatom Navicula pelliculosa,
cyanobacteria Anabaena flos-aquae, Synechococcus leopoliensis (syn. Anacystis nidulans). If other
species are used, the strain should be reported. It has to be confirmed that exponential growth of the
selected test alga can be maintained throughout the test period under the prevailing conditions. Test media
recommended by OECD 201 Guideline are OECD TG 201 medium (ISO 8692 medium) or AAP medium.
5.0
SAFETY CONSIDERATIONS
It is necessary to pay attention especially during testing of hazardous chemicals.
6.0
7.0
EQUIPMENT, MATERIALS, AND REAGENTS

Equipment:
automatic pipette 10-100 L, 100-1000 L, tips, pipette 5 mL, 10 mL, beakers,

1.5 mL test-tubes, 96-well microplate, source of light, spectrophotometer
Materials:
algal culture of Pseudokirchneriella subcapitata
Reagents:
distilled water
test medium: OECD TG 201 medium (see Appendix 1)
solution of tested substance
METHOD, PROCEDURES, AND REQUIRMENTS
Procedure:
1) Preparation of inoculum culture
In order to adapt the test alga to the test conditions, an inoculum culture in the test medium is prepared 23 days before start of the test. The algal biomass should be adjusted in order to allow exponential growth
to prevail in the inoculum culture until test start. The inoculum culture shall be incubated under the same
conditions as the test cultures.
2) Dilution of inoculum culture to initial optical density
Initial optical density of Pseudokirchneriella subcapitata in the test should be about 0.06-0.07 AU
(measured for 250 L of algal culture in 96-well microplate at 680 nm), which is approximately
corresponding to 104-105 cells/mL (!modified in comparison with OECD 201 Guideline, where is
recommended initial cell concentration of Pseudokirchneriella subcapitata 5 000 50 000 cells/mL).
43
a) Add 3x 250 L of inoculum culture to microplate wells (for estimation of actual OD of inoculum
culture)
b) Add 3x 250 L of test medium without algae to other microplate wells (for estimation of OD of
test medium - blank)
c) Measure optical density at 680 nm
d) Calculate dilution factor according to this equation (use mean values of three replicates):
Actual OD of inoculum culture OD of test medium
------------------------------------------------------------------Required OD of test culture OD of test medium
Dilution factor =
e) Dilute inoculum culture with test medium to required initial optical density (11 mL is volume of
diluted inoculum culture needed to test five experimental concentrations of one substance)
3) Preparation of stock solutions of the test substance
Prepare at least five concentrations of test substance in a geometric series. Concentrations of stocks
solutions shall be 30x greater than final concentration in the test. (Solvents, e.g. acetone, t-butyl alcohol
and dimethylsulfoxide, may be used as carriers to add substances of low water solubility to the test
medium. The final concentration of solvent in the test should not exceed 100 l/L, and the same
concentration of solvent should be added to all cultures in the test series including control).
4) Preparation of test solutions

Add 50 L of each stock solution (or solvent in the case of solvent control) to the 6 separated tubes each
containing 1450 L of the diluted inoculum culture. Mix vigorously.
5) Testing
a) Add 250 L of diluted inoculum culture in six replicates to microplate wells
b) Add 250 L of each test solution (including solvent control) in five replicates to microplate wells
c) Add 250 L of distilled water to the wells surrounding experimental ones
Scheme of microplate:
A
B
C
D
E
F
G
H
1
W
W
W
W
W
W
W
W
2
W
SC
SC
SC
C
C5
C5
W
3
W
C1
C1
C1
C
C4
C4
W
4
W
C2
C2
C2
C
C3
C3
W
5
W
C3
C3
C3
C
C2
C2
W
6
W
C4
C4
C4
C
C1
C1
W
7
W
C5
C5
C5
C
SC
SC
W
8 9
W
W
W
W
W
W
W
W
10 11 12
W
C
SC
C1
C2
C3
C4
C5
distilled water
control
solvent control
concentration 1 (lowest)
concentration 2
concentration 3
concentration 4
concentration 5 (highest)
6) Incubation
Microplate covered with a lid should be maintained at a temperature in the range of 21 to 24 C,
controlled at 2C. Illumination in the range of 6 000-10 000 lux is acceptable (60-120 E/m 2/s of
photosynthetically effective wavelenght range of 400-700 nm). Make sure that the illumination is uniform
over the incubation area. Duration of the test is 72 2 hours.
7) Measurements
The algal optical density (at 680 nm) in microplate should be determined at least at the start and the end
of the test (daily in an ideal case). Resuspend content of each well (using pipette or microplate shaker)
before optical density measurement.
44
6.0
RECORDS, DOCUMENTATION, AND QC REQUIRMENTS (modified!)
For the test to be valid, the following performance criteria should be met:
- the optical density in the control cultures (OD of test medium has to be substracted!) should have
increased by a factor of at least 16 within the test period (this criterion applies to the test algae
Pseudokirchneriella subcapitata).
- the coefficient of variation among daily growth rates in the control cultures during the course of the test
(days 0-1, 1-2 and 2-3) must not exceed 35%
- coefficient of variation of average growth in replicate control cultures must not exceed 15%
7.0
RESULTS CALCULATION (modified!)
1) Substract average optical density of test medium (blank) from each measured optical density value
2) Calculation of growth rates
The average specific growth rate for a specific period is calculated as the logarithmic increase in
biomass from the equation:
i-j =
where:
i-j
ti
tj
ODi
ODj
ln ODi-j ln ODi
------------------tj ti
is the average specific growth rate from moment time i to j (day-1);

is the moment time for the start of the period (day);
is the moment time for the end of the period (day);
is the optical density (blank substracted) at time i;
is the optical density (blank substracted) at time j.
a) Calculate average specific growth rate over the test duration (normally days 0-3).
b) Calculate also the average daily growth rates for each day during the course of the test (days 0-1,
1-2 and 2-3) and examine whether the growth rate remains constant (see validity criteria). A
lower growth rate on day one than the total average growth rate reveals a lag phase. While a lag
phase can be minimized and practically eliminated in control cultures by proper propagation of
the pre-culture, a lag phase in exposed cultures may indicate recovery after initial toxic stress or
reduced exposure due to loss of test substance (including sorption onto the algal biomass) after
initial exposure.
c) Calculate the percent inhibition of growth rate for each treatment replicate from the equation:
%I =
C T
--------- x 100
C
where:
%I
is percent inhibition in average specific growth rate;
C
mean value for in the control;
T
value for growth rate in the treatment.
3) Estimation of EC50 (ECx).
ECx (including confidence interval) is calculated using probit/logit analysis or non-linear regression.
For simplified assessment the EC50 can be estimated by findnig experimental concentration which has
caused growth inhibition tightly above 50% and the second one which has caused growth inhibition
tightly below 50% of (solvent) control. Use simple linear regression to calculate EC 50 from these two
concentrations/two growth inhibition values.
45
4) Estimation of experimental NOEC/LOEC.

For estimation of the LOEC, and hence the NOEC, for effects of the test substance on average growth, it
is necessary to calculate the mean growth across replicates for each concentration and the pooled residual
standard deviation, and this can be done using analysis of variance (ANOVA). The mean for each
concentration must then be compared with the control mean using an appropriate multiple comparison
method. Dunnetts or Williams tests may be useful. It is necessary to check whether the ANOVA
assumption of homogenity of variance holds. It is recommended that this is done graphically rather than
via a formal significance test; a suitable alternative is to run a Bartletts test. If this assumption does not
hold, then consideration should be given to transforming the data to homogenise variances prior to
performing the ANOVA, or carrying out a weighted ANOVA.
9.0
REFERENCES
OECD Guidelines For The Testing Of Chemicals Guideline 201: Freshwater Alga and Cyanobacteria,
Growth Inhibition Test
APPENDIX 1 Test media
One of the following two test media may be used:
a) OECD medium : Original medium of OECD TG 201, also according to ISO 8692
b) US EPA medium AAP, also according to ASTM.
When preparing these media, reagent or analytical-grade chemicals should be used and deionised water.
Composition of the AAP-medium (US. EPA) and the OECD TG 201 medium:
Component
NaHCO3
NaNO3
NH4Cl
MgCl2 6(H2O)
CaCl22(H2O)
MgSO47(H2O)
K2HPO4
FeCl36(H2O)
Na2EDTA2(H2O)
H3BO3
MnCl24(H2O)
ZnCl2
CoCl26(H2O)
Na2MoO42(H2O)
CuCl22(H2O)
pH
EPA
mg/L
15.000
25.500
12.160
4.410
14.600
1.044
0.160
0.300
0.185500
0.415400
0.003270
0.001428
0.007260
0.000012
7.5
mM
0.178550
0.300000
0.059804
0.029984
0.059232
0.005994
0.000591
0.000806
0.003000
0.002099
0.000024
0.000006
0.000030
0.00000007
OECD
mg/L
50.00000
15.00000
12.00000
18.00000
15.00000
1.60000
0.08000
0.10000
0.18500
0.41500
0.00300
0.00150
0.00700
0.00001
8.3
mM
0.59516724
0.28037383
0.05901736
0.12238248
0.06085440
0.00918590
0.00029595
0.00026864
0.00299159
0.00209691
0.00002201
0.00000630
0.00002893
0.00000006
In test with the diatom Navicula pelliculosa both media must be supplemented with Na2SiO39H2O to
obtain a concentration of 1.4 mg Si/L.
The pH-values are adjusted to the specified values using solutions of hydrochloric acid or sodium
hydroxide.
46
ALGAL GROWTH INHIBITION TEST

Time i (day):
Time j (day):
Organism:
Test medium:
Test substance:
Measured wavelenght:
Temperature:
Tested concentrations (unit):
Solvent:
Solvent concentration:
Test medium OD (blank):
c1 =
c2 =
c3 =
c4 =
c5 =
OD (blank
control
solvent c.
c1
c2
c3
c4
c5
unit
control
solvent c.
c1
c2
c3
c4
c5
unit
control
solvent c.
c1
c2
c3
c4
c5
unit
control
solvent c.
c1
c2
c3
c4
c5
unit
substracted)
1
5
3
4
5
6
mean
cv%
OD (blank
substracted)
1
5
3
4
5
6
mean
cv%
1
5
3
4
5
6
mean
cv%
%I
1
5
3
4
5
6
mean
cv%
< 50%
%I
concentration
EC50
> 50%
Date:
Person:
47
RECETOX
Research Centre for
Masaryk University
_________________________________________________________________________________________________________________
Determination of the inhibition of the mobility of

Daphnia magna Straus (Cladocera, Crustacea) Acute toxicity test
Date:
June 2005
Author:
Marie Feldmannov
This Standard operating procedure is based on SN EN ISO 6341
1.0
DEFINITIONS AND ACRONYMS
Immobilization: Those animals which are not able to move within 15 seconds after gentle
agitation of the test container are considered to be immobile.
EC 50 This is the concentration, in terms of initial values, which immobilizes 50% of the
Daphnia in a test batch within a continuous period of exposure.
2.0
PURPOSE
The purpose of this test is to determine the median effective concentration (EC50) of a substance
for immobilization of Daphnia in fresh water.
The directive requirement for the LC50 for Daphnia is considered to be fulfilled by the
determination of the EC50 as described in this test method.
Acute toxicity is expressed in this test as the median effective concentration (EC50) for
immobilization.
3.0
48
This SOPs serves for determination of acute toxicity of the following types of samples for
Daphnia magna:
Chemical compounds easily soluble in the test medium
Waste water from industry
Ground and surface water
4.0
SUMMARY OF METHOD
The Daphnias are exposed to the test substance added to water at a range of concentrations for
24/48 hours.
5.0
No special safety considerations.
Safety considerations depend on physical-chemical properties and predicted toxicity of tested
compound.
6.0

Equipment:
Normal laboratory apparatus and equipment should be used. Equipment which will come into
contact with the test solutions should preferably be made entirely of glass:
- Oxygen meter (with microelectrode or other suitable equipment for measuring dissolved
oxygen in low-volume samples),
- adequate apparatus for temperature control (20 2C)
- pH meter,
- equipment for the determination of hardness of water.
- equipment for the light-dark cycle (16h light:8h dark)
Materials:
Test organism
The test animals shall be less than 24 hours old, at the beginning of the test, laboratory bred, free
from overt disease and with a known history (e.g. breeding -any pretreatments, etc.).
Test water
Reconstituted water is used in this test. To avoid the necessity for acclimation prior to the test, it
is recommended that the culture water should be of similar quality (pH, hardness) as the water
used for the test.
All chemicals must be of analytical grade.
Reagents:
Potassium dichromate K2Cr2O7
Calcium chloride dihydrate CaC12.2 H2O
Magnesium sulphate heptahydrate MgSO4.7H2O
Sodium hydrogen carbonate NaHCO3
Potassium chloride KCl
8.0

Instrument preparation and calibration:
49
For a statistically acceptable evaluation of the effects, each test concentration as well as the
control has to be assayed in 4 replicates. Each multiwell plate is provided with 4 test wells and
one "rinsing well". These rinsing wells serve to prevent dilution of the toxicant in the multiwell
cups during the transfer of the test organisms from the hatching cups to the test plate. The test
wells in each column are labelled A, B, C, D and rows are labelled X (control), 1, 2, 3, 4, 5 for
the five toxicant dilutions.
Rinsing wells
Test wells
Test conditions
Stock solutions of the required concentration are prepared by dissolving the substance in
deionized water.
The substances should be normally only tested up to the limit of solubility.
Ultrasonic dispersion, organic solvents, emulsifiers or dispersants may be used as an aid
to prepare stock solutions of substances with low aqueous solubility or to help to disperse
these substances in the test medium. When such solvent carrier is used, all test
concentrations should contain the same amount of solvent, and additional control where
Daphnia should be exposed to the same concentration of the solvent as that used in the
test series needs to be added to the test. The concentration of such auxiliary substances
(solvents) should be minimized, but in no case should exceed 100 mg per litre in the test
medium or 2% of volume.
Number of animals: at least 20 animals at each test concentration preferably divided into
four batches of five animals each.
Loading: at least 2 ml of test solutions should be provided for each animal,
Test concentration: the test solution should be prepared immediately before introduction
of the Daphnia, preferably without using any solvent other than water.
Temperature: the test temperature should be between 18 and 22 C, but for each single
test it should be constant within 1 C.
Aeration: the test solutions must not be bubble-aerated.
Feeding: none. Only in order to prevent death by starvation in the 48h test, a two hour
"pre-feeding" of the test organisms with mixture of algae, prior to the start of the assay,
proved to be most successful.
50
pH and the oxygen concentration of the controls and of all the test concentrations should
be measured at the end of the test; the pH of the test solutions should not be modified.
Volatile compounds should be tested in completely filled closed containers, large enough
to prevent lack of oxygen.
Daphnia are inspected after 24 hours exposure and again after 48 hours.
The dilution series to be prepared spans the range of the lowest concentration producing
100% effect and the highest one producing less than 10% effect in the range finding test.
Procedure:
Preparation of the test media
Stock solutions
CaC1 2.2 H2O (calcium chloride dihydrate):
dissolve in, and make up to 1 litre with water
MgSO4.7H2O (magnesium sulphate heptahydrate):
NaHCO3 (sodium hydrogen carbonate):
KCl (potassium chloride):
11.76 g
4.93 g
2.59 g
0.23 g
Reconstituted dilution water

Mix 25 ml of each of the four stock solutions and make up to 1litre with water.
Aerate until the dissolved oxygen concentration equals the air-saturation value.
The pH should be 7.8 0.2.
If necessary adjust the pH with NaOH (sodium hydroxide) or HCl (hydrochloric acid).
The prepared dilution water is set aside for about 12 hours and need not be further aerated.
The sum of the Ca and Mg ions in this solution is 2.5 mmol per litre. The ratio of Ca:Mg ions is
4:1 and of Na:K ions is 10:1. The total alkalinity of this solution is 0.8 mmol per litre.
Preparation of test samples, dilution series

Transfer 10 ml of dilution water into each well of the control row and 10 ml of the
respective toxicant concentration.
Transfer the Daphnia neonates into the test wells
Transfer at least 20 (actively swimming) neonates from hatching cup into each rinsing cup. Try
to carry over as little as possible liquid from the hatching cup to the wells during this transfer.
Transfer exactly 5 neonates from each rinsing well into 4 wells of each column. This transfer
shall be also performed in the sequence of increasing test concentration. Count the neonates as
they exit the micropipette to be sure to put exactly 5 neonates per well.
Close the plate and move it into cultivation room
Incubate the test plate
After 24h/48h incubation take the multiwell plate out of the incubation room and check
the wells of each row and record the number of dead and immobilized neonates (Those
animals which are not able to swim within 15 seconds after gentle agitation of the test
container are considered to be immobile) versus that of the actively swimming test
organisms in each well
Report the figures on the results sheet
Total the number of dead and immobile neonates for each concentration and calculate the
% effect for each toxicant concentration
51
8.0
RECORDS, DOCUMENTATION, AND QC REQUIRMENTS
Immobilization in the controls must not exceed 10% at the end of the test.
It is desirable that concentration of dissolved oxygen in the test vessels should remain above 3
mg/L throughout the course of the test.
The pH should not vary by more than 1 unit.
Reference test
In order to check the correct performance of the test procedure and the sensitivity of the test
animals, it is advised to perform a reference test from time to time.
Quality control test can be performed with reference toxicant potassium dichromate K2Cr2O7
(24h-EC 50 = 0.6 1.7 mg/l)
9.0
RESULTS CALCULATION
The percentage of immobilization is calculated for each period when observations were recorded
(24 and 48h), and 24h EC50 and 48h EC50 are determined. Probit analysis is usually used or data
are analysed graphically in Gauss logarithmic diagram.
52
Results sheet
Name.................................................................................
Date of test.....................................................
Toxicant tested...................................................................
Dilution series tested concentration 1....................................................................
concentration 2....................................................................
concentration 3....................................................................
concentration 4....................................................................
concentration 5....................................................................
Control
exposure(h) 24
48
A
conc.5
24
48
conc.4
24
48
conc.3
24
48
conc.2
24
48
conc.1
24
48
B
C
D
Total
% effect
24h EC50..................................
48h EC50..................................
53
RECETOX
Research Centre for
Masaryk University
_________________________________________________________________________________________________________________
STANDARD OPERATING PROCEDURE
Title:Vibrio fischeri luminescence inhibition test

Date: 6/23/2005
Authors: Flegrov Zuzana, Barto Tom
1.0
Acclimatization
Bioluminescence
EC50
Lyophilized
Reference
Toxicant
Test Conditions
2.0
To accustom experimental organisms to the biological, chemical and

physical conditions present during holding, culture and testing.
Production of light by organisms.
Toxicant concentration that results in an effect (generally adverse) among
50% of the test population.
Freeze-dried under vacuum
A toxicant used to assess the condition of the test organism. Deviation
outside established normal ranges indicates changes in the condition of the
test organism population.
Defined limits for biological, chemical and physical parameters applied
during a toxicity test. For example, temperature and salinity; if these
parameters are not achieved at any time during the exposure period the
entire test may be invalid.
PURPOSE
The Microtox system is a screening tool used for a variety of toxicity testing applications. The
advantages of this toxicity bioassay are its speed, simplicity, and relatively low cost, when
compared to the cost of chemical analysis.
3.0
The bioassay's strongest attribute is its usefulness as a primary screening test for a broad
spectrum of toxicants and its monitoring capability over time. The Microtox procedure can be
used for testing either water (marine or fresh) or associated sediments.
54
4.0
SUMMARY OF METHOD
The Microtox assay uses freeze dried luminescent bacteria (Vibrio Fischeri) as the test
organisms. The bacteria's light-producing mechanism is tied to the metabolic processes of the
cell. If these processes are changed or damaged by a toxic substance, a reduction in light output
results. The bioassay is based on detecting these changes in light output. Results can be obtained
within 5 to 30 minutes. The light output readings are then used to calculate an EC50 for each
sample. The EC50 is defined as the median effective concentration, and is a calculated toxicity
value representing the sample concentration (%) estimated to cause a 50% response by the
exposed test organisms. The EC50's are then compared against a control sample to evaluate
relative toxicity.
The principle of the Microtox test is based on contact between the bacterium and the sample.
Toxicity of the sample is then determined by measuring the decrease in bioluminescence of
bacteria (Vibrio fischeri). There is a positive correlation between the reduction in the light
emission and the amount of toxicity.
The response of V. fischeri to the sample is compared to the response of the bacterium to a
control, reference or blank sample. Toxicity of the sample is then expressed as the concentration
causing 50% light reduction as compared to the blank or other control (EC50) after an incubation
time of t minutes at a constant temperature (C). Toxicity is expressed as EC50 after which the
value is corrected for the particulate content.
5.0
For conducting the Microtox toxicity test, the following parameters must comply with the
reported limits: Temperature 15 1C. Incubation Period 10 minutes.
It is strongly recommended that gloves, safety glasses and a laboratory jacket be worn during the
performance of the reference toxicant tests as well as the conduct of the sampes toxicity tests.
6.0

Equipment:
circulation termostate (temperation for 15C), luminometer, cold water bath
Materials:
automatic pipetts 5 50 l, 50 200 l, 200 1000 l, 1000 5000 l, tips, tubes,
gloves
Reagents:
Microtox diluent (sodium chloride diluent 2%), Microtox lyophilized reagent (Vibrio
fischeri), reference toxicant (ZnSO4)
55
9.0

The termostat must be tempered to 15 0,1 oC before measurement and make sure there
is enough high water level in the termostat.
Procedure (step by step):
1. 0.5 ml 2% NaCl is added to freeze dried bacteria immediately after opening and put
this mixture into the cold water bath and keep it there throughout assay
2. After 15 min of rehydratation 80 l of bacteria suspension is added into 2.5 ml of
cold 2% NaCl and let 15 min to temperate.
3. Diluted bacterial suspension is pipeted into 12 tubes, 200 l. to each tube.
4. After another 15 min of equilibration initial intensity of luminescences is measured
with synchronization one well per 30 sec. After each measurment 800 l 2% NaCl
(negative control) respective sample is added into each measured tube. Next
measurement is performed after 15 and 30 minutes.
10.0
To determine the quality of the test organisms and evaluate the performance of the Microtox test
a control sample and reference toxicant should be used.
11.0
RESULTS CALCULATION
Firstly, coeficients of natural bioluminescence extinction must be calculated for particular times.
R(t) = IK (t) / IK (0)
R (t) is coeficients of natural bioluminescence extinction in time t
IK (t) is bioluminescence of control in time t
IK (0) is bioluminescence intensity of control in time t = 0
For quantitative evaluation % of decreased light (inhibition of luminescence) is calculated
Inh % = [R(t) . I (0)- I (t)] / [R(t) . I (0)] . 100
R (t) is coeficients of natural bioluminescence extinction
I (0) is bioluminiscence in time t = 0, without measures sample
I (t) is luminescence measured in time t
56
RECETOX TOCOEN & Associates

Kamenice 126/3, 625 00 Brno, Czech Republic
Microtox protocol
Record number.:
Sample indication:
sample
zkum.
1a Contr.
0
0
Date:
0
15
15
15
30
30:00
1b Contr. 0:30
1:00
2a
15:30
30:30
16:00
31:00
16:30
31:30
3a
2b 1:30
2:00
17:00
32:00
17:30
32:30
4a
3b 2:30
3:00
18:00
33:00
18:30
33:30
5a
4b 3:30
4:00
19:00
34:00
19:30
34:30
6a
5b 4:30
5:00
20:00
35:00
6b 5:30
20:30
35:30
30
Locality :
Sample characterisation :
pH :
colour :
turbidity :
Extraction reagent :
Operator :
Basic number (sensitivity) :
Time from bacterial resuscitation :
57
RECETOX
Research Centre for
Masaryk University
_________________________________________________________________________________________________________________
10-day Sediment Toxicity Test with Chironomus

riparius (Diptera)
Date: October 2009
Author: Zuzana Rbov
______________________________________________________________
This Standard operating procedure is based on OECD Test Guideline 218 and
600/R-99/064
EPA

LC50:
concentration which causes 50% mortality of the test animals
EC50:
concentration at which 50% of the test animals show statistically different body length
in comparison with the control
AS:
artificial sediment
dw:
dry weight
DO:
dissolved oxygen
2.0 PURPOSE
The purpose of this test is to assess sediment toxicity for the larvae of non-biting midge
Chironomus riparius. The endpoints of the 10-day test are survival (%) and growth measured as
body length (mm). This method may be used for comparative assessment of toxicity of natural
sediments or to determine basic ecotoxicological parameters like LC50, EC50, LOEC or NOEC
for tested substances spiked into artificial sediment
.
3.0 SCOPE AND APPLICATION
This SOP serves for determination of toxicity of the following types of samples for the larvae of
Chironomus riparius:
58
natural sediments
natural sediments spiked with a test substance
artificial sediments spiked with a test substance
4.0 SUMMARY OF THE METHOD
The newly hatched larvae of Chironomus riparius are exposed to the test sediment; either in a
concentration range of the test substance spiked into sediments or to natural sediments that may
be possibly diluted with a clean sediment. The larvae are fed daily and after 10 days of exposure
they are recovered from the test sediment, counted and measured to determine survival and
growth. The test system needs to be checked daily for proper aeration, and physical-chemical
parameters of the overlaying water need to be measured at the beginning and end of the test.
5.0 SAFETY CONSIDERATIONS
No special safety considerations.
Safety considerations depend on physical-chemical properties and predicted toxicity of tested
compound. It is always recommended to wear gloves and laboratory clothes especially when
working with natural sediments with unknown toxic effects. When working with semi-volatile
chemicals and solvents, all preparations and test operations should be performed in a fume
hood. The harmful waste must be then disposed into designated waste-containers.
6.0 EQUIPMENT, MATERIALS, AND REAGENTS
Equipment:
Normal laboratory apparatus and equipment should be used.
- oxygen meter
- pH meter
- thermometer
- conductivity meter
- aquarium air pumps
- binocular with a measure device
- scales
- adequate apparatus for temperature control (20 2C)
- equipment for the light-dark cycle (16h light:8h dark)
- multiple 400-600 ml glass beakers, Petri dishes
- graduated cylinders
- pipettes, Pasteur pipettes
- plastic hoses for aquarium aeration
- forceps, stainless spoons, perforated plastic lids (beaker covers), circular plastic discs
Materials:
Test organism
The test animals shall be 1-3 days old (first instar larvae) when introduced into the test
sediment. The laboratory bred organisms should be free from overt disease or parasites and with
a known history (e.g. average emergence time, survival and sex ratio in the culture)
Test water
Dutch Standard water is used as the overlaying water in the test and preferably in the culture as
59
well.
Dutch Standard Water Concentrate:
Substance
Formula
Amout (mg) to be diluted in 1 L of distilled water
Sodium hydrogen carbonate NaHCO3
500
Potassium hydrogen carbonate
KHCO3
100
Calcium chloride dihydrate CaC12.2 H2O 750
The test water is a Dutch Standard Water Concentrate diluted with distilled water in the ratio
1:4.
Reagents:
Reference chemicals
cadmium chloride
Other Reagents
Ethanol > 80%
HCl, NaOH (for pH adjustment if needed), acetone, hexane, chloroform (for non-water soluble
compounds)
7.0 METHOD, PROCEDURES, AND REQUIREMENTS
Preparation of the artificial sediment (AS)
Constituent Characteristics
% of sediment dry weight
Peat Sphagnum moss peat, as close to pH 5.5-6.0 as possible, no visible plant remains, finely
ground (particle size 1mm), dried 4-5
Quartz sand Grain size: 0.5 m 75-76
Kaolin Kaolinite content > 30%
20
Organic carbon
Adjusted by addition of peat and sand
2 0,5
Calcium carbonate CaCO3 pulverized, chemically pure 0,05-0,25
Water Conductivity < 10 S/cm
30
Each replicate encompasses 40 g dw of AS corresponding to 52g of wet weight.
The total amount of artificial sediment should be calculated with some leftover reserve since
some of the AS will be used to measure the pH.
All the dry ingredients of AS are thoroughly mixed in a bowl. The peat is dried and ground to
fine powder prior to adding into the mixture. The corresponding amount of water (30 % of dw)
is added and the mixture thoroughly mixed again.
Three samples of the AS are then taken for pH measurement. 25 ml of distilled water are added
to 10 ml of sediment in a glass, this suspension is then shaken for two hours. The pH value
should be in a range of 6.5 7.5; if it is to acidic then the pH value may be adjusted by adding
more CaCO3, if it is to alkaline then the pH value may be adjusted by adding the main
constituents or just peat. Peat is usually the source of this uncertainty of the AS reaction.
The AS with optimized pH value is weighted into individual beakers. The replicates are then
spiked individually by adding 1 ml of the contaminant stock solution. The spiked sediment is
thoroughly mixed.
When the test substance shows low water solubility, the AS needs to be spiked with a carrier
solvent like acetone, hexane or chloroform. 10 g of quartz sand are mixed with the solvent
60
solution. The solvent is then allowed to evaporate and the sand is added to the suitable amount
sediment per test beaker (prior to adding the contaminated sand, the AS in the beakers had been
prepared with adequately less sand).
AS is then covered by a thin plastic disc to prevent resuspendation of the fine material and
floating of peat while pouring the test water on the sediment. The test water should be poured
very gently over the AS and should reach up to 200 ml in the beaker. The plastic discs are then
removed with a forceps.
The replicates with different test substance concentrations should be distributed randomly in the
exposure system.
Aeration is installed into each individual replicate; introducing smooth bubbling by a Pasteur
pipette. The beakers are covered with a plastic lid to prevent extensive test water evaporation
enhanced by bubbling.
The AS is then conditioned for at least 24 hours under in test conditions in the exposition room.
Preparation of natural sediments
The samples of natural tests sediments should be allowed to defreeze under room temperature if
they had been stored frozen. The overlaying water should be decanted and the remaining water
content in the sediment determined. Two 10 g samples of well homogenized sediment are placed
into ceramic pot and dried for 24 hours at 60-70 C. The dry weight and percentage of water
content is then determined. The sediment is sieved with a 2 mm sieve in order to remove bigger
particles or pieces of organic matter remains. The equivalent of 40 g of dw is then weighted into
each replicate, overlain by test water and allowed to condition for 24 hours in the exposition
room.
Test conditions
Tab. 1: General conditions and characteristics of the 10-d Chironomus test
Parametr
Conditions
1.
Test type
Test of sediment toxicity with Chironomus riparius
2.
Test organism
Chironomus riparius - larvae
3.
Temperature
20 2 C
4.
Light
wide spectra fluorescent light
5.
Photoperiod
16L : 8D
6.
Test vessel
400-600 ml glass beakers, approximately 8 cm in
diameter
7.
Amount of sediment
40 g dw
8.
Age of organisms
1-3 days old larvae
9.
Number of individuals
20
per replicate
10.
Number of replicates per 5
concentration
11.
Feeding
Suspension of finely ground TetrafinR, 0,5 mg/larva per
day, fed daily
12.
Aeration
Smooth continuous aeration with a Pasteur pipette
13.
Test water
Dutch Standard Water
14.
Sediment
Natural or artificial
15.
Water quality
pH 6-9, Dissolved oxygen at least 60% ASV (air
sturatian value) measured at beginning and end of a test
61
16.
17.
Endpoints
Validity
% survival, growth (body length)

Survival after 10 days > 80%
Procedure:
Introduction of larvae into the test system
The egg ropes are collected from the laboratory culture and placed into a Petri dish with test
water. It usually takes 2-3 days for the eggs to hatch.
On the day 0, the newly hatched larvae are introduced into the test system with a Pasteur pipette.
The aeration must be stopped before introducing the larvae and it needs to be put back into
operation only after 24 hours (on the day 1 of the test). The larvae are selected from a Petri dish
under a binocular microscope since they can be hardly seen with the naked eye. If the density of
organisms in the Petri dish is too high, it is necessary to split the population in order to gain an
optimal density for reliable transfer of the exact number of individuals into a pipette. This is a
critical step in the operating procedure since introduction of the exact number of larvae is crucial
for a correct test outcome.
All the larvae selected to the test should be equally big and vital; they should show movement
(typical movement along S-shaped trajectory) and no morphological deformities.
Twenty organisms are then transferred to the test vessel. It is important to dip the Pasteur pipette
under the test water surface and push the larvae smoothly out of the pipette. Failing to do so may
result in the loss of larvae on the water surface since they may not be able to overcome the
surface tension and enter the test water column.
Feeding and further activities during exposure

The organisms need to be fed daily with a suspension of finely ground Tetrafin flocks. 1ml of
food suspension (1 g of finely ground flocks in 100 ml of distilled water) should be added into
each replicate so that each replicate receives 0.5 g of food per capita and day. The food
suspension needs to be shaken before each single pipetting since the grounded flocks tend to
suspend very quickly in the bottom. The food suspension may be kept for several days in a
fridge.
The test vessels should be checked every day for proper aeration and test water level. If the test
water level decreases for some reason it should be refilled with distilled water.
On the day 1, the aeration should be switched on.
Water quality parameters like pH, DO, temperature and conductivity should be measured at the
beginning and end of the test and the values should not deviate from the above mentioned
criteria.
Observations of the behavior (e.g. building tubes, reentering the water column, excessive
movement etc.) should be done daily and any remarkable peculiarities should be reported.
Ending the test
On the day 10, the test is ended and endpoints determined. The overlaying water is decanted;
either poured into a sink or in the case of replicates with higher contaminant concentrations into
designated waste containers. The test sediment is then gently transferred into a large Petri dish
(diameter 20 cm) with a spoon and investigated for the larvae. The individuals are extracted with
forceps and placed into a Petri dish with clean test water. The count of living animals in each
replicate is reported. The immobile individuals or those not found in the sediment are considered
dead. The larvae are subsequently sacrificed by transferring them into a Petri dish or Eppendorf
vial with ethanol. The body length of fixed animals is then measured under binocular microscope
with a measuring device and written into a protocol. It should be born in mind that extracting of
62
larvae from the sediment may be very time-consuming, particularly when their growth is
negatively affected by the test substance.
The contaminated sediment should be allowed to dry and disposed in designated waste
containers.
6.0 RECORDS, DOCUMENTATION, AND QC REQUIREMENTS
Survival of the controls must exceed 80%.
In order to check the correct performance of the test procedure and the sensitivity of the test
animals, it is advised to perform a reference test from time to time. Quality control test can be
performed with reference toxicant cadmium chloride (see ASTM E1706 - 05e1 Standard Test
Method for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater
Invertebrates)
The water quality should meet the prescribed requirements (see tab.1)
63
Example of test reports and results sheet:
vapour pressure
partition
coefficient in soil
(sediment)
method of spiking
length of
conditioning
Water solubility
Purity
Formula
volume of test
water
WW of sediments
in test vessels
Notes
DW of sediment in
test vessels
Mr
Number of source
egmasses
pH
CAS number
Age of animals when
inserted into test vessels
Date of
formulation*
Date of the day when the

eggmasses were laid
Sediment type
Common name
Test animal
Test Sediment
Test substance
Date:
* if natural sediments are tested, the data about the sampling, storage, preparation and
conditioning should be included
Date:
Test conditions beginning of test
Test conditions end of test
Treatment
control A
control E
concentration 1 A
concentration X E
Treatment
Temperature pH
[C]
DO
[ASV]
Conductivity Light cycle

S/cm
and intensity
Temperature pH
[C]
DO
[ASV]
Conductivity Light cycle

and intensity
control A
control E
concentration 1 A
concentration X E
64
Date:
Results
Treatment
Number of
living
individuals
% survival
Length
[mm]
Measured test
concentrations
control A
control E
concentration 1 A
concentration X E
7.0 RESULTS CALCULATION

Mean values and standard deviations are calculated for each category (sediment, concentration).
Data are then analyzed for statistically significant differences using the Analysis of variance
(ANOVA) Fisher LSD test, or any non-parametric equivalent.
The homogeneity of variance can be tested with Leven's-test and normality with KolmogoroffSmirnov's. If all presumptions are fulfilled, ANOVA is performed and followed by post-hoc test
of differences of treatments Fischer LSD test. This will determine the first concentration
significantly different from kontrol (LOEC) and the last concentration significantly not different
from kontrol (NOEC).
If the presumptions of normality and homogenity of variance are not fulfilled, non-parametric
methods like Kruskal-Wallis test need to be applied.
Probit analysis should normally be applied to determine the LC50 for survival. However, in
cases where this method of analysis is unsuitable (e.g. if data from less than three concentrations
with partial mortality are available), alternative methods can be used. These methods could
include moving averages, the trimmed Spearman-Karber method or simple interpolation (e.g.
geometrical mean of LC0 and LC100, as computed by the square root of LC0 multiplied by
LC100. To compute any ECx value, the regression analysis is performed to model dose-response
function. A logistic regression is commonly used model.
8.0 REFERENCES
OECD, Sediment-Water Chironomid Toxicity Test Using Spiked Sediment. OECD Test Guideline
218, 2004.
65
US-EPA (2000). "Methods for Measuring the Toxicity and Bioaccumulation of Sedimentassociated Contaminants with Freshwater Invertebrates." US-EPA EPA 600/R-99/064.
66
RECETOX
Research Centre for
Masaryk University
_________________________________________________________________________________________________________________
Contact toxicity test with

kinetic bacterial bioluminescence assay
Date: November 2008
Authors: Tom p, Zbynk plchal, Ludk Blha
1.0
Bioluminescence Production of light by bacteria Vibrio fisheri

Toxicant concentration inhibits the biological activity (here:
IC50
bioluminiscence) by 50% in comparison with control
Freeze-dried under vacuum
Lyophilized
Defined limits for biological, chemical and physical parameters applied
during a toxicity test. For example, temperature and salinity; if these
Test Conditions parameters are not achieved at any time during the exposure period the entire
test may be invalid.
2.0
PURPOSE
The Kinetic bacterial bioluminiscence assay (also called "Flash") is a screening toxicity testing
tool used for variety of applications. The major advantage over the traditional MICROTOX
assay is the adaptation for testing of coloured or suspended materials. It is also rapid (30 second),
simple to perform and cost efficient toxicity assay.
4.0
The bioassay's characteristics is its ability to be used for toxicity assessment of turbid samples
(e.g. sediment suspensions, solid waste etc.).
4.0
SUMMARY OF METHOD
67
The Kinetic bacterial bioluminiscence assay uses freeze dried luminescent bacteria (Vibrio
Fischeri) as the test organisms. The bacteria physiologically produce light (bioluminiscence) as a
result of overall metabolism, and toxic substances may interfere with this mechanism causing an
inhibition in the emission of light. The bioassay is based on determination of luminiscence
output.
Bacteria are automatically injected into the tested sample, and bioluminescence is monitored
immediatelly after injection (1 s) and after prolonged exposure (30 s or more).
The assay is based on the assumption that the immediate (initial) contact of bacteria with tested
matrix (less than 1 second) may cause inhibition of bioluminiscence not due to the sample
toxicity but more likely due to other factors such as colour or turbidity of the sample. On the
other hand, the prolonged exposure (30 seconds in this case) integrates and reflects the toxic
effects.
Therefore, toxicity is related to the relative decline in bioluminiscence in each sample; i.e.
comparison of the initial luminiscence (rapid Peak after injection) with luminiscence values after
prolonged exposure.
Figure: Presentation of the principle of the kinetic bacterial bioluminiscence assay

1 - no luminiscence
2 - injection of bacteria followed by the initial peak
3 - initial peak in bioluminescence
: height of the initial peak is affected by the colour
(darker/turbid samples show lower bioluminiscence)
4,5 - decline of the bioluminescence in time (relative to the initial peak) reflects toxicity
of the sample
68
Figure: Presentation of the kinetic record (bioluminescence vs. time) in the bacterial assay
demonstrating differences for various types of samples
To evaluate toxicity following values are recorded during the assay:

Initial "Peak" value (max. bioluminescence after injection of bacteria; exposure < 1s)
Bioluminiscence after 30 second of exposure ("S30")
Inhibition of luminescence for each sample (% of control) is calculated as follows:
where CF is the correction factor (the S30/Peak ratio in Negative controls - i.e. 2% NaCl reflecting natural attenuation of bacterial luminescence during 30 second exposures).
Values of inhibitions (INH%) are derived for several dilutions of the tested sample (chemical,
suspension etc.), and they are used to calculate IC50 value, i.e. concentration causing 50% effect
in comparison to the blank after an incubation time of t at a constant temperature (C).
5.0
Safety gloves, glasses and laboratory coat must be used to conduct toxicity tests.
Exposure
6.0

Equipment:
69
circulation thermostat (temperate for 15C), luminometer with computer controlled

injectors allowing injection of bacteria to the tested sample and immediate luminescence
record, ice-cold water bath
Materials:
automatic pipettes 5 50 l, 50 200 l, 200 1000 l, 1000 5000 l, tips, tubes,
gloves
Reagents:
Testing diluent (NaCl - sodium chloride 2% w/v solution; pH 7.2), Lyophilized bacteria
(Vibrio fischeri)
10.0

The thermostat must be tempered to 15 0,1 oC before measurement (make sure there is
enough high water level in the thermostat).
Prepare luminometer with injectors at air-conditioned room (15C):
- load appropriate kinetic programme into controlling PC
- wash injectors with water followed by 2% NaCl
Sample dilutions and preparation:
Prepare dilutions of the tested samples in white 96-well microplate using 2% NaCl
- 1:1 dilution series is recommended
- for sediment samples, suspensions must be diluted in microtubes using serial dilutions
with cut-off tips (use thorough shaking/vortexing)
- all samples should be tested in two replicates
Blank (negative control - 2% NaCl) must be tested on each microplate
Toxicity testing procedure
5. 0.5 ml 2% NaCl is added to freeze dried bacteria immediately after opening and
this mixture is left in the ice cold water bath in the fridge
6. After 15 min of the initiaal rehydratation of bacteria, 80 l of bacterial innoculum is
added into 2.5 ml of ice cold 2% NaCl
7. Suspension in plastic tube is transferred into 15C thermostat bath and left for 15
min to temperate
8. Prepare white microplate with samples to the driver in the luminometer
9. Suspension of temperated bacteria is moved to the the beaker with water (15C)
and prepared at the luminometer
70
- wash the injector with 2% NaCl again

- prime the injector with bacterial suspension
10. Start the kinetic measurement of the luminescence:
- bacteria are injected and initial luminescence recorded (1 second)
- microplate is shaken at 1200 rpm inside the luminometer (25 seconds)
- luminescence is recorded (t = 30 seconds)
12.0
Data are recorded in the computer programme (Ascent) and further evaluated in the Microsoft
EXCEL software.
To determine the quality of the test organisms and evaluate the performance of the test a control
sample and reference toxicant is used (ZnSO4 or K2Cr2O7 dilution)
71
13.0
RESULTS CALCULATION
Following values are recorded during the assay (automatic record by the computer):
Initial "Peak" value (max. bioluminescence after injection of bacteria; exposure < 1s)
Bioluminiscence after 30 second of exposure ("S30")
Inhibition of luminescence for each sample (% of control) is calculated as follows:
where CF
is the correction factor (the S30/Peak ratio in Negative controls or blanks)

For calculation of INH% values FLASH TEST Application Software 1.0 can be used (developed
at Masaryk University by Tom p during his Master thesis project; www.recetox.muni.cz)
Figure: Screenshot of the FLASH TEST Application Software 1.0 (Microsoft Excel
application)
72
Data of the inhibition for each dilution are recorded for each of the replicates (example below)
and used for the IC50 calculation:
Inhibition
Sample / conc.
(mg dry wt/mL)
Blank
0.3
0.6
1.3
2.5
5
10
20
40
80
Rep.1
-0.1%
-2.3%
-1.6%
6.8%
13.6%
30.7%
59.5%
79.7%
85.8%
91.4%
Rep.2
-0.5%
-6.1%
6.3%
15.3%
17.0%
42.7%
59.9%
75.0%
89.8%
87.1%
Calculation of IC50 values uses four-parametric logistic curve regression built in GraphPadTM
Prism (GraphPad Software, San Diego, CA, USA).
IC50 values along with 95% confidence intervals are reported as the final result (example):
Sampleno.
S:2006/1160
S:2006/1161
S:2006/1162
S:2006/1163
S:2006/1164
S:2006/1165
9.0
SampleI.D.
Brat?ejovkaBrat?ejov
LutoninkanadVizovicemi
LutoninkapodJelnkem
Lutoninkap?edsoutokems
D?evnic
D?evniceposoutokusTrnvkou
Sluovice
D?evniceposoutokus
LutoninkouLpauSluovic
IC50
2.43
7.91
54.02
95%CI
1.78
3.33
7.05
8.87
46.89
62.24
0.72
0.35
1.45
6.35
4.86
8.28
1.58
1.18
2.11
REFERENCES
Lappalainen J, Juvonen R, Vaajasaari K, Karp M. 1999. A new flash method for measuring the
toxicity of solid and colored samples. Chemosphere 38:1069-1083
Lappalainen J, Juvonen R, Nurmi J, Karp M. 2001. Automated color correction method for
Vibrio fischeri toxicity test. Comparison of standard and kinetic assays. Chemosphere 45:635641
International Standardization Agency. 1998. International Standardization Agency ISO 113483:1998. Water quality -- Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) -- Part 3: Method using freeze-dried
bacteria.
73
Annex 2 - RECETOX LABORATORY CERTIFICATE
74
75
Annex 3 - Photographs
76
Figure S 1 SOS chromotest- pipeting of the samples into microplates
Figure S 2 SOS chromotest- assessment of the results
77
Figure S 3 Cultivation of algae cultures
Figure S 4 Algal growth inhibition test- cultivation of the microplates
78
Figure S 5 Algal growth inhibition assessment
Figure S 6 Vibrio fischeri bioluminiscence inhibition test- preparation of sample dilutions and luminescence
assessment
79
Figure S 7 Daphnia magna
Figure S 8 Daphnia magna immobilization test - exposure preparation
80
Figure S 9 Larva of Chironomus riparius
Figure S 10 Sediment contact toxicity assessment with Chironomid larvae
81
Figure S 11 Water processing for toxicity tests - filtration
82
83
Figure S 12 Lyophilization of the water samples and the resulting lyophilizate
84
Figure S 13 Freeze-drying of the sediment samples and the resulting lyophilizate
85

Exotoxicological Report

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Exotoxicological Report

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Ecotoxicological assessment of water and sediment samples

from Dambovic Lake, Romania

Research institution: Research Centre for Toxic Compounds

Masaryk University, Faculty of Science

RECETOX-TOCOEN Report No. 378 (April 2010)

Period of the study:

March - April, 2010

Responsible person: Doc. RNDr. Ludk Blha, Ph.D.

Mgr. Ji Novk, Ph.D.; RNDr. Kateina Brtov; Mgr. Petra Mackov,

Introduction and overall experimental design

List of samples tested for ecotoxicity and genotoxicity

SEDIMENT SAMPLES - (3x fresh sediments in plastic boxes; cooled at 4C)

Concentration of water samples - lyophilization

Sediment manipulation - fresh sediments with Chironomus

Sediment lyophililization - contact test with luminiscent bacteria

Genotoxicity testing - SOS chromotest

Algal growth inhibition test

is the average specific growth rate from moment time i to j (day-1);

Percent inhibition of growth rate:

is percent inhibition in average specific growth rate;

Daphnia magna immobilization test

Vibrio fischeri bioluminiscence inhibition test

Kinetic bacterial bioluminiscence test for contact sediment toxicity

Chironomus contact sediment toxicity test

Results 1 - raw water ecotoxicity

Results of algal growth inhibition test

Daphnia magna immobilization test

Figure 3 Percentage of inhibition in Daphnia magna immobilization test.

Vibrio fischeri bioluminiscence inhibition test

Raw water samples - Summary

Results 2 - Concentrated water ecotoxicity

log (concentration factor)

Bioluminiscence with Vibrio fischeri - concentrated water samples

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

log (concentration factor)

Genotoxicity testing - concentrated water samples

4-NQO (positive control)

4-NQO (positive control)

Concentrated water ecotoxicity - results summary

Results 3 - Contact sediment toxicity

D1-in (1:3 diluted)

D4-C (1:3 diluted)

S-G (1:3 diluted)

Table 9 Statistics - results of the toxicity testing with Chironomus sp.

Toxicity testing with the kinetic bacterial bioluminiscence test

95% Conf.Int. (min)

95% Conf.Int. (max)

Complete results of the toxicity testing are presented in :

Sample ID - SEDIMENT "D1-IN"

Sample ID - SEDIMENT "D4-C"

Sample ID - SEDIMENT "SG"

Summary and conclusions

Agency, I.S., International Standardization Agency 8692, Water quality freshwater

Annexes 1 - Standard operation procedures

Freshwater Alga and Cyanobacteria, Growth Inhibition Test