2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology
P13 ECOTOxICOLOGICAL TESTING AND TEST
METhODS OF ChEMICALS
HELEnA DOLEŽALOVá WEISSMAnnOVá, HELEnA
ZLáMALOVá GARGOŠOVá and MILADA VáVROVá
Brno University of Technology, Faculty of Chemistry, Purkyňova
118, Brno, Czech Republic
dolezalova@fch.vutbr.cz
Introduction
Ecotoxicity involves the identification of chemical
hazards to the environment. The Globally Harmonized System
of Classification and Labeling of Chemicals (GHS)
describes testing for hazards to the aquatic environment in
Part 3, Chapter 3.10 (UnECE, 2004a). Annex 8, Guidance
on Hazards to the Aquatic Environment (UnECE, 2004).
Ecotoxicity tests can be classified into two following categories:
standard and alternative tests.
The ecological testing according to US EPA and
UnECE includes many tests; however, several methods of
ecotoxicological testing are used in the Czech Republic. For
the typical standard and alternative aquatic ecotoxicological
tests crustacea (Daphnia magna, Thamnocephalus platyurus,
and Artemia salina), aquatic plant (Lemna minor), algae
(Desmodesmus subspicatus, Scenedesmus subcapitatus, Selenastrum
capricornutum) are used. In accordance with OECD,
UnECE and Czech legislation the standard tests include
these organisms in following tests – Daphnia magna (OECD
Test no. 202: Daphnia sp. Acute Immobilizations Test, En
ISO 6341:1996), Desmodesmus subspicatus (OECD – Test
no. 201: Alga, Growth Inhibition Test, ISO 8692:2004). The
aquatic organism Lemna minor (OECD Test no. 221: Lemna
sp. Growth Inhibition Test, ISO 20079:2005) is application
in OECD and ISO standard methods for the testing of chemicals.
Experimental
The general principle of ecotoxicological tests is the
determination of effective concentration (EC50), eventually
lethal concentration (LC50) or inhibition concentration
(IC50). The limit test corresponds to one dose level of
100 g dm –3 . A stepwise procedure involves four steps: the
limit test, confirmatory test, basic test and definitive test.
These types of studies produce end points such as LC50,
EC50 and nOEC. EC50, LC50 and IC50 are the effective
concentrations (i.e., the concentration of material in water
that is estimated to produce a specifically quantified effect
to 50 % of the test organisms). The EC50 and its 95 % confidence
interval are usually derived by statistical analysis of
a quantal, “all or nothing”, response (such as death, fertilisation,
germination, or development) in several test concentrations,
after a fixed period of exposure. End point means the
variables (i.e., time, reaction of the organisms) that indicate
the termination of a test, and also means the measurement(s)
or value(s) derived, that characterise the results of the test
(EC50). LOEC (lowest observed effect concentration) is the
s359
lowest concentration tested causing a statistically measurable
effect to the test system. nOEC (no observed effect concentration)
is the highest concentration tested causing no statistically
measurable effect to the test system. The parameters
LOEC and nOEC may be statistically determined.
The aim of statistical analysis of ecotoxicological data is
to obtain a quantitative concentration-response relationship
by regression analysis. For this purpose many models could
be used such as: linear interpolation, polynomial regression,
log–logistic regression, probit model, Weibull model, Dunnett
test, William test, Jokheere-Terpstra test.
E c o t o x i c o l o g i c a l T e s t s f o r
D e t e r m i n a t i o n o f E c o t o x i c i t y o f
S t a n d a r d K 2 C r O 4
The standard chemical K 2 CrO 4 was used for the determination
of ecotoxicity, for th estimation of sensitivity of
testing organisms Dapnia magna Strauss, (Daphtoxkit F
magna – standard test) Thamnocephalus platyurus (Thamnotoxkit
F – alternative test) and Lemna minor (standard
test OECD) and for validation and comparison of obtained
results with the declared values. Table I performs the basic
parameters of tests in coordinance with the directive.
Table I
The parameters of tests
Parameters Daphtoxkit Thamnotoxkit Lemna
F F minor
c [mg dm −3 ] 0.32–3.20 0.032–0.320 10–160
T [°C] 22 ± 2 20 ± 2 24 ± 2
I [lx] 6,000 4,000 –
Results
On the base of analysis of obtained ecotoxicological
results for studied tests the values of EC50, LC50 and IC50
were determined for standard chemical K 2 CrO 4 . The experimental
data with the basic statistic characteristics are summarized
in Table II. The EU-Directive 93/67/EEC classifies
substances according to their ecotoxicological value (EC50).
On the base of obtained data from ecotoxicological tests
(crustacea tests Daphtoxkit F and Thamnotoxkit F) the
study chemical belongs to very toxic for aquatic organisms.
The studied chemical K 2 CrO 4 is also toxic to aquatic organisms
for macrophyta Lemna minor.
The Thamnocephalus platyurus was found
as the test organism of highest sensitivity with
LC50 = 0.092 ± 0.002 mg dm –3 . The effective concentration
(EC50/24 h) from Daphtoxkit F for exposure 24
hours was 0.99 ± 0.022 mg dm –3 and the effective concentration
(EC50/48 h) was confirmed 0.79 ± 0.031 mg dm –3
The inhibition concentration for Lemna minor (IC50/168 h)
was calculated from average specific growth rate and confirmed
via percent reduction in yield, IC50/168 h was equal
to 20.52 ± 0.399 mg dm –3 . For validation and confirmation of
experimental data the probit model was used for determina-