Xenobiotics in the environment include a wide variety of compounds, e.g. pesticides, drugs, textile dyes, personal care products, stabilisers, and many others. Among xenobiotics, pharmaceuticals have recently acquired increasing attention [1, 2]. Pharmacological products enter natural waters mainly via wastewater either from manufacturing facilities or from municipal wastewater (excretion of unmetabolised drugs, disposal of unused drugs). The contamination of natural aquatic systems results in adverse negative effects on aquatic organisms.
In surface waters, physical, chemical, and
biological processes contribute to the transformations of polluting substances.
Photoinitiated processes may represent important degradation pathways in
surface waters for compounds resistant to both biological degradation and
chemical reactions such
as hydrolysis [3].
Photochemical degradation may lead to a decrease in contaminant concentration,
and, in some cases, generate photoproducts with even higher harmful effects
than that of the parent compound [4].
Ecotoxicology represents a framework
enabling to test a given compound and to reveal or at least estimate its
potential harmful effect. This study is focused on aquatic organisms. In
autotrophs, algae Chlorella sp. and Desmodesmus sp. are
often used due to their simple laboratory maintenance [5, 6].
The flagship of heterotrophs toxicity testing in surface water is the planktonic
microcrustacean Daphnia magna [7]. It has several characteristics that in toxicological tests,
especially those targeted at acute toxicity estimation – it can be
relatively easily maintained in the laboratory and, when under suitable
conditions, D. magna reproduces parthenogenetically. A common model
of vertebrates in ecotoxicology is the zebrafish Danio rerio. Although
the extrapolation of the obtained results to higher vertebrates is not
straightforward and should be done with care, the response of fish to xenobiotics
is a significant indicator of how a particular compound (or products of its
photodegradation) affect fish assemblages in surface waters.
In this study, toxicity of atorvastatin, a widely prescribed
hypolipidemic drug, and the mixture of its photoproducts were investigated. The
photoproduct mixture was produced by irradiation of the solution of
atorvastatin (c = 50 mg/l) by the radiation in the range between
300 – 350 nm (to imitate the
short-wavelength solar radiation that reaches the Earth´s surface) for 15 minutes.
Then, two toxicity assays based on OECD 202 [8] and 236 [9] guidelines
were performed.
In the case of acute toxicity test on the model organism Daphnia
magna (OECD 202), 2 juveniles not older than 24 h were introduced
into 5 ml
of pure media,
other juveniles in pairs in the media with atorvastatin in the concentration
range from 1 to 10 000 µg/l; photoproducts solutions were tested
at concentration range
of remaining atorvastatin in the
irradiated solution up to 1000 µg/l. During
this test (48 hrs) constant temperature was held at 18.7 ± 0.2 °C; photoperiod
was 16 hrs light and 6 hrs dark and the
juvenils were not fed. Atorvastatin
did not cause any mortality, photoproducts caused
20 % mortality at the highest concentration used. The LC50 value could not
be evaluated from this experiment. The data show
that lethal concentration for 50 % of daphnids is higher than the highest
used concentration. Photoproducts seem to be more toxic than atorvastatin
itself since in addition to
the observed mortality each daphind showed odd swimming at the highest
concentration of photoproducts.
Toxicity assay based on OECD 236 guideline was done on embryos
of Danio rerio. One fertilized egg was
introduced into 2 ml of
ISO water (control) or into 2 ml of atorvastatin or 2 ml of photoproducts (concentration ranges as in the tests with D.magna).
Tested embryos were kept in the incubator Climacell EVO line, for 96 hours at the temperature 25 – 26 °C
and the photoperiod 14 hrs light/ 10 hrs darkness. The
evolution of the embryos was monitored visually every 24 hours. Four key
parameters indicating the lethality were sought for: coagulated embryos, lack
of somite formation, non – detachment of the tail and lack of heartbeat. The value
of LC50 for atorvastatin was determined by
software Prism 6, its value is 1976 µg/l. Regarding
photoproducts, 40 % mortality was observed at the photoproduct mixture
with remaining 500 µg/l of atorvastatin. In the higher concentration there was also retardation in
the development and the drug had adverse effect on blood formation – lack of
cell flow, transparent cells without pigmentation.