Page 20 - Volume 3, Issue 1, January 2010
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© Benaki Phytopathological Institute
Toxicity of olive mill wastewater
19
logical Institute. All rats were housed in
groups of five animals per cage under con-
trolled environmental conditions (accord-
ing to the national and European legisla-
tion) with
ad libitum
consumption of food
and water. The bacterium
V. fischeri,
com-
mercially available from Azur Environmen-
tal (USA), was kept at -20
o
C until the time of
the experiment and the cysts of the crusta-
cean
A. fransiscana,
obtained from Micro-
BioTests Inc (Belgium), were kept at 4
o
C be-
fore hatching.
A limit test assay was conducted to esti-
mate the acute oral toxicity of OMW on rats
according to the protocol of OECD 420 (15).
The test sample was administrated orally at
a limit dose of 2,000 mg OMW per kg body
weight (b.w.) by gavage to 5 female Wistar
rats (Group A). Another group, served as a
negative control (Group B), was also con-
sisted of 5 female Wistar rats to which only
the vehicle (tap water) was administrated.
All animals were observed individually after
dosing at least once during the first 30 min-
utes, periodically during the first 24 hrs and
thereafter daily for 14 consecutive days. Ob-
servations included the presence of signs of
toxicity such as tremor, convulsion, saliva-
tion, diarrhea, sleep, changes in skin, fur,
eyes, mucous membranes, and also chang-
es in respiratory, autonomic and central ner-
vous system, somatomotor activity and be-
havioral changes. Additionally, the weight of
the animals was recorded weekly during the
experimental period. At the end of the ob-
servation period all animals were sacrificed
by CO
2
and were subjected to necropsy.
The Microtox® Analyzer Model 500 (Stra-
tegic Diagnostics Inc., Delaware, USA) was
used for the toxicity assay of OMW. The as-
say is based on the inhibition of the natural
luminescence emitted by the bacterium
V.
fischeri,
when exposed to toxic compounds.
The bacterial suspension was exposed to
the test samples and the acute toxicity was
expressed as inhibition of bioluminescence.
The protocols used for the conduction of
the assays were the “81.9% Basic Test” for
the untreated and treated OMW and the
“90% Basic Test for Pure Compounds” for
the polyphenolic mixtures. The end-point
for the assessment of toxicity was the medi-
an effective concentrations (EC
50
) at 5 and 15
min. The pH of all sample solutions was ad-
justed within the range of 6-8. Each sample
was tested at least at three replicates. Zinc
sulfate (ZnSO
4
.7H
2
O) was used as reference
material (positive control) for the toxicity
tests performed on
V. fischeri
.
The nauplii of the marine crustacean
A.
fransiscana
were hatched from commercial-
ly available cysts. The cysts were incubated
in artificial seawater (Instant Ocean) at sa-
linity of 35 ppt. The first nauplii usually ap-
peared after 24 hrs of incubation at 25
o
C un-
der aeration and illumination of 1,000-4,000
lux. They were transferred into new seawa-
ter and incubated for 24 hrs under similar
conditions and then transferred into a multi-
well test platewith the respective concentra-
tions of the test samples. The toxicity of the
samples to
A. fransiscana
nauplii was tested
after 24 and 48 hrs of exposure at 25
o
C in the
darkness. The end-point for the assessment
of toxicity was the mortality incidence after
24 and 48 hrs of exposure. The toxicity test
performed on
A. fransiscana
was based on
the principles of the OECD 202 (14) test for
Daphnia
spp. adapted for the marine crus-
tacean. Potassium dichromate (K
2
Cr
2
O
7
) was
used as reference material (positive control)
for the toxicity tests on
A. fransiscana
.
The determined EC
50
values were calcu-
lated using linear regression analysis as nat-
ural logarithm of sample concentration ver-
sus percentage of mortality or percentage
of growth inhibition.
Results and Discussion
Acute Oral Toxicity on Wistar rats
Mortality of female Wistar rats was not
observed in the acute limit test (2,000 mg/
kg b.w.) of OMW. Moreover, no signs of tox-
icity or abnormal behavior were observed
on any of the test animals during the 14-day
observation period following the adminis-
tration. The body weight development was
normal for all animals (Table 1). The macro-
