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REVIEW ON THE ACUTE DAPHNIA MAGNA TOXICITY TEST
EVALUATION OF THE SENSITIVITY AND THE PRECISION OF ASSAYS
PERFORMED WITH ORGANISMS FROM LABORATORY CULTURES
OR HATCHED FROM DORMANT EGGS
R. Baudo (Italy), M.Cotman (Slovenia), C.Blaise (Canada),
K.Cl.Thompson (United Kingdom), M.Moreira Santos (Portugal), B.Vollat (France),
A.Törökne (Hungary), T.Han (South Korea)
36th AQUATIC TOXICITY WORKSHOP
La Malbaie, Charlevoix (Quebec), Canada, September 27-30, 2009
G.Persoone Ghent University, Laboratory for Environmental Toxicology (Belgium)
MicroBioTests Inc. (Belgium)
Also presented on behalf of
Daily maintenance
Daily feeding
with micro-algae
Infrastructure
Space
Labour
Culturing of Daphnias
Dormant egg
Hatched neonates
- No need for culturing/maintenance of live stocks
- Dormant eggs can be stored
- Dormant eggs can be hatched “anytime”
Alternative : use of Daphniashatched from dormant eggs
Development of a Daphnia microbiotestat the Laboratory for Biological Research in Aquatic Pollution
(Laboratory for Environmental Toxicology and Aquatic Ecology)
(Ghent University, Belgium)
Elaboration in 1996 of the
Daphtoxkit F magna
Used to date in about 40 countries worldwide
Acute Daphnia magna toxicity test
Neonates fromlaboratory cultures
Neonates hatchedfrom dormant eggs
SENSITIVITY
PRECISION
Intralaboratory variability of test results of the acute D.magna test
for single chemicals and effluents (data from Parkhurst et al., 1992)
0 - 4924 h or
48h
712 - 37153Effluents
3 - 7248h 532 - 13342Pure
chemicals
Variation
coefficient
(%)
Test
duration
Total number
of tests
performed
Number of tests
performed in each
laboratory
Total number of
chemicals or
effluents analysed
Total number of
participating
laboratories
Number
of studies
Daphnias from laboratory cultures
Interlaboratory variability of test results of the acute D.magna test
for single chemicals and effluents (data from Parkhurst et al., 1992)
1 - 11024h or
48h
7332 - 93Effluents
39 - 14348h3133 - 52Pure chemicals
Variation
coefficient
(%)
Test durationTotal number of
chemicals or effluents
analysed
Total number of
laboratories in all
exercises
Number of laboratories
in each exercise
Number of
exercises
Daphnias from laboratory cultures
Quality control tests on potassium dichromate
1.00-1.6018.031.27222002-2008National Institute
of Envir.Health
Hungary
0.67-1.5620.281.10332002-2008National Institute
of Chemistry
Slovenia
0.62-1.2518.740.97332004-2007CemagrefFrance
0.70-1.8021.511.24271998-2008Grontmij-AquasenseThe Netherlands
0.43-1.6429.960.80312002-2008VitoBelgium(Flanders)
0.61-1.6626.501.01221997-2005LisecBelgium (Flanders)
0.67-2.3120.791.431832001-2008IssepBelgium (Wallonia)
RangeCV%Mean 24h
EC50 (mg/l)
Number of
tests
YearsOrganisationCountry
Daphnias hatched from dormant eggs
0.79-1.7417.821.28552004-2008Institute of Public Health Nova
GoricaSlovenia
0.60-2.0028.581.02701997-2008InterlabSpain
0.75-1.9016.391.152162002-2008MicroBioTests Inc.Belgium (Flanders)
0.88-1.9018.721.26482005-2008Institut Provincial Hyg.-Bact. MonsBelgium (Wallonia)
RangeCV%Mean 24h
EC50 (mg/l)
Number of
tests
YearsOrganisationCountry
Daphnias from laboratory cultures
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
ISO
Vito
Cem
agre
f
Lis
ec
Nat.In
st.Chem
.
Gro
ntm
ij-
Aquasense
Nat.In
st.Env.H
ealth
Issep
Inte
rlab
Mic
roBio
Tests
Inst.Publ.Health
Inst.Pro
v.H
yg.B
act.
Laboratory cultures Dormant eggs
Mean 2
4h E
C50
Mean 24h EC50s (in mg/l, with standard deviation) for quality control tests
with potassium dichromate, with lab cultures or Daphnia microbiotests
Intralaboratory sensitivity comparison studies on the acute D.magna test
performed in parallel with lab cultures and with Daphnia microbiotests
0.8638Industrial effluentsDaniel et al.,
2004
Brixham Environmental
Laboratory
United
Kingdom
2004
0.9730Household productsUlm et al., 2000Zagreb Institute of Public
Health
Croatia2000
0.9712Solid waste leachatesLatif and Zach,
2000
University of Veterinary
Medecine
Austria2000
0.989PesticidesFochtman, 2000Institute of Organic IndustryPoland2000
0.889Effluents textile industryPersoone, 1998Ghent UniversityBelgium1998
0.9819Inorganic and organic
chemicals
Persoone, 1998Ghent UniversityBelgium1998
Correlation
coefficient (r)
Number of
samples
Type of samplesReferenceLaboratoryCountryYear
Major conclusions from the analysis of all the QC data
on the acute D.magna toxicity tests :
1. Virtually all the results of assays performed with Daphnias
taken from lab cultures or with Daphnia microbiotests are
within the acceptability range set by ISO standard 6341
for the reference chemical potassium dichromate
2. The mean 24h EC50s of the Daphnia microbiotests
performed in different laboratories are within the range of
the mean EC50s of the assays based on lab cultures, and
the variation coefficients (20% to 30%) are similar
3. The precision – in terms of the long term in house
variability – of the quality control Daphnia microbiotests is
as good as that of the QC tests based on lab cultures
Recent interlaboratory sensitivity comparison studies on toxicity tests
SeveralSolid waste
leachates
Umwelt Bundesambt
Germany
European Union2006
Acute D.magnaPure chemicalsEnvironmental Agency
APAT
Italy2005
Acute D.magnaPure chemicalEnvironmental Agency
APAT
Italy2003
Acute D.magnaSimulated
wastewaters
National Institute of
Chemistry
Slovenia2002-2008
SeveralIndustrial effluentsFlanders Environmental
Agency
Belgium2001
Type of testsType of samplesOrganiserCountryYear
Waste water T1
0
10
20
30
40
50
60
70
80
90
2002 2003 2004 2005 2006* 2007 2008
Me
an
EC
50
(v
ol%
)
Lab cultures
Daphtoxkits
Waste water T2
0
1
2
3
4
5
6
7
8
9
2002 2003 2004 2005 2006 2007 2008
Mean
24h
EC
50 (
vo
l%)
Lab cultures
Daphtoxkits
Mean 24h EC50s for lab culture tests and Daphnia microbiotests
Proficiency testing on the acute Daphnia magna test in Slovenia
from 2002 to 2008 (with about 20 participating laboratories)
Waste water T1
0
10
20
30
40
50
60
70
80
90
100
2002 2003 2004 2005 2006* 2007 2008
Va
ria
tio
n c
oe
ffic
ien
t %
Lab cultures
Daphtoxkits
Waste water T2
0
10
20
30
40
50
60
70
80
90
100
2002 2003 2004 2005 2006 2007 2008
Va
ria
tio
n c
oe
ffic
ien
t %
Lab cultures
Daphtoxkits
Coefficients of variation of the 24h EC50s for the lab culture tests
and the Daphnia microbiotests, for 7 Slovenian ringtests
Acute D.magna ringtests performed in Italy in 2003 and 2005 with
potassium dichromate and comparison with ISO data of 1994
50.05.01.12361994ISO 6341
23.99.91.20402005
24.517.11.08562003Daphnia
microbiotest
34.28.51.02332005
27.416.21.08162003Lab cultures
Interlaboratory
CV%
Intralaboratory
CV%
Mean 24h
EC50
(mg/l)
Number of
participants
Year
Acute D. magna ringtest performed in Italy in 2005
with potassium chloride (KCl)
20.86.676542Daphnia
microbiotest
43.15.381732Lab cultures
Interlaboratory
CV%
Intralaboratory
CV%
Mean 24h
EC50
(mg/l)
Number of
participants
Major conclusions from the analysis of all the ringtests
on the acute D.magna toxicity test
1. The sensitivity of Daphnia neonates
hatched from dormant eggs is similar to
that of test organisms taken from lab
cultures
2. The precision of the Daphnia microbiotest
is in most cases superior to that of the
assays based on lab cultures
Sensitivity comparison of neonates hatched from ephippia
originating from different D.magna strains.
n.a.1.001Pond in Maaltebrugge park (Flanders,
Belgium)*
17.51.026Pond in Zwijnaarde (Flanders, Belgium)
10.51.014Pond in Assenede (Flanders, Belgium)
111.313Culture from the University of Metz (France)
CV
(%)
24h EC50 (mg/l)Number of
tests
Origin of strain
* = test performed with neonates hatched from the ephippia collected from the pond
n.a. = not applicable
Mean 24h EC50s for potassium dichromate
Sensitivity comparison of lab cultured D.magna from 2 strains
and of neonates hatched from ephippia produced by these 2 strains
311.013360.983NaPCP**Strain F2
260.00372***5270.003695MalathionStrain F2
180.004265190.005515MalathionStrain K5
20274.9***522307.752,4-DStrain F2
16307.6316319.432,4-D*Strain K5
CV
(%)
Mean 24h
EC50 (mg/l)
Number
of tests
CV
(%)
Mean 24h EC50
(mg/l)
Number
of tests
Ephippia neonatesLab cultured Daphnias
* 2,4-dichlorophenoxyacetic acid
** sodium pentachlorophenol
*** tests performed with neonates hatched from the ephippia collected from the pond
Mean 24h EC50s for 3 chemicals (data from Moreira-Santos, 1998)
K5 = strain from France
K2 = strain from the UK
Conclusion from the comparative sensitivityanalysis of Daphnias hatched from dormant eggs
produced by different D.magna strains
The sensitivity of the neonates hatched
from dormant eggs of different strains
is not different from that of Daphnias from
laboratory cultures
CONCLUSIONS FROM ALL THE INTRA- AND INTER-LABORATORY
COMPARISON STUDIES ON THE ACUTE DAPHNIA MAGNA TOXICITY TEST
- The sensitivity of the young Daphnias obtained from dormant eggs
is the same as that of Daphnias from laboratory cultures
- The precision of the Daphnia microbiotest is generally better
than that of lab culture tests
The Daphnia microbiotest is a well-validated, low cost
alternative to the conventional Daphnia magna test
Review on the acute Daphnia magna toxicity test –
Evaluation of the sensitivity and the precision of assays
performed with organisms from laboratory cultures
or hatched from dormant eggs
This extensive review just appeared in the periodical :
Knowledge and Management of Aquatic Ecosystems (KMAE)
(2009, 393, 1-29)
KMAE is a “free access” journal
All articles can be consulted and downloaded from
http://www.kmae-journal.org
All the information and the data given in this presentation
originate from the publication :