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 :