2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures
2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures
2. ENVIRONMENTAL ChEMISTRy & TEChNOLOGy 2.1. Lectures
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Chem. Listy, 102, s265–s1311 (2008) Environmental Chemistry & Technology<br />
P13 ECOTOxICOLOGICAL TESTING AND TEST<br />
METhODS OF ChEMICALS<br />
HELEnA DOLEŽALOVá WEISSMAnnOVá, HELEnA<br />
ZLáMALOVá GARGOŠOVá and MILADA VáVROVá<br />
Brno University of Technology, Faculty of Chemistry, Purkyňova<br />
118, Brno, Czech Republic<br />
dolezalova@fch.vutbr.cz<br />
Introduction<br />
Ecotoxicity involves the identification of chemical<br />
hazards to the environment. The Globally Harmonized System<br />
of Classification and Labeling of Chemicals (GHS)<br />
describes testing for hazards to the aquatic environment in<br />
Part 3, Chapter 3.10 (UnECE, 2004a). Annex 8, Guidance<br />
on Hazards to the Aquatic Environment (UnECE, 2004).<br />
Ecotoxicity tests can be classified into two following categories:<br />
standard and alternative tests.<br />
The ecological testing according to US EPA and<br />
UnECE includes many tests; however, several methods of<br />
ecotoxicological testing are used in the Czech Republic. For<br />
the typical standard and alternative aquatic ecotoxicological<br />
tests crustacea (Daphnia magna, Thamnocephalus platyurus,<br />
and Artemia salina), aquatic plant (Lemna minor), algae<br />
(Desmodesmus subspicatus, Scenedesmus subcapitatus, Selenastrum<br />
capricornutum) are used. In accordance with OECD,<br />
UnECE and Czech legislation the standard tests include<br />
these organisms in following tests – Daphnia magna (OECD<br />
Test no. 202: Daphnia sp. Acute Immobilizations Test, En<br />
ISO 6341:1996), Desmodesmus subspicatus (OECD – Test<br />
no. 201: Alga, Growth Inhibition Test, ISO 8692:2004). The<br />
aquatic organism Lemna minor (OECD Test no. 221: Lemna<br />
sp. Growth Inhibition Test, ISO 20079:2005) is application<br />
in OECD and ISO standard methods for the testing of chemicals.<br />
Experimental<br />
The general principle of ecotoxicological tests is the<br />
determination of effective concentration (EC50), eventually<br />
lethal concentration (LC50) or inhibition concentration<br />
(IC50). The limit test corresponds to one dose level of<br />
100 g dm –3 . A stepwise procedure involves four steps: the<br />
limit test, confirmatory test, basic test and definitive test.<br />
These types of studies produce end points such as LC50,<br />
EC50 and nOEC. EC50, LC50 and IC50 are the effective<br />
concentrations (i.e., the concentration of material in water<br />
that is estimated to produce a specifically quantified effect<br />
to 50 % of the test organisms). The EC50 and its 95 % confidence<br />
interval are usually derived by statistical analysis of<br />
a quantal, “all or nothing”, response (such as death, fertilisation,<br />
germination, or development) in several test concentrations,<br />
after a fixed period of exposure. End point means the<br />
variables (i.e., time, reaction of the organisms) that indicate<br />
the termination of a test, and also means the measurement(s)<br />
or value(s) derived, that characterise the results of the test<br />
(EC50). LOEC (lowest observed effect concentration) is the<br />
s359<br />
lowest concentration tested causing a statistically measurable<br />
effect to the test system. nOEC (no observed effect concentration)<br />
is the highest concentration tested causing no statistically<br />
measurable effect to the test system. The parameters<br />
LOEC and nOEC may be statistically determined.<br />
The aim of statistical analysis of ecotoxicological data is<br />
to obtain a quantitative concentration-response relationship<br />
by regression analysis. For this purpose many models could<br />
be used such as: linear interpolation, polynomial regression,<br />
log–logistic regression, probit model, Weibull model, Dunnett<br />
test, William test, Jokheere-Terpstra test.<br />
E c o t o x i c o l o g i c a l T e s t s f o r<br />
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<br />
S t a n d a r d K 2 C r O 4<br />
The standard chemical K 2 CrO 4 was used for the determination<br />
of ecotoxicity, for th estimation of sensitivity of<br />
testing organisms Dapnia magna Strauss, (Daphtoxkit F<br />
magna – standard test) Thamnocephalus platyurus (Thamnotoxkit<br />
F – alternative test) and Lemna minor (standard<br />
test OECD) and for validation and comparison of obtained<br />
results with the declared values. Table I performs the basic<br />
parameters of tests in coordinance with the directive.<br />
Table I<br />
The parameters of tests<br />
Parameters Daphtoxkit Thamnotoxkit Lemna<br />
F F minor<br />
c [mg dm −3 ] 0.32–3.20 0.032–0.320 10–160<br />
T [°C] 22 ± 2 20 ± 2 24 ± 2<br />
I [lx] 6,000 4,000 –<br />
Results<br />
On the base of analysis of obtained ecotoxicological<br />
results for studied tests the values of EC50, LC50 and IC50<br />
were determined for standard chemical K 2 CrO 4 . The experimental<br />
data with the basic statistic characteristics are summarized<br />
in Table II. The EU-Directive 93/67/EEC classifies<br />
substances according to their ecotoxicological value (EC50).<br />
On the base of obtained data from ecotoxicological tests<br />
(crustacea tests Daphtoxkit F and Thamnotoxkit F) the<br />
study chemical belongs to very toxic for aquatic organisms.<br />
The studied chemical K 2 CrO 4 is also toxic to aquatic organisms<br />
for macrophyta Lemna minor.<br />
The Thamnocephalus platyurus was found<br />
as the test organism of highest sensitivity with<br />
LC50 = 0.092 ± 0.002 mg dm –3 . The effective concentration<br />
(EC50/24 h) from Daphtoxkit F for exposure 24<br />
hours was 0.99 ± 0.022 mg dm –3 and the effective concentration<br />
(EC50/48 h) was confirmed 0.79 ± 0.031 mg dm –3<br />
The inhibition concentration for Lemna minor (IC50/168 h)<br />
was calculated from average specific growth rate and confirmed<br />
via percent reduction in yield, IC50/168 h was equal<br />
to 20.52 ± 0.399 mg dm –3 . For validation and confirmation of<br />
experimental data the probit model was used for determina-