after 21 days exposure to 2.00 Æ 0.30 mg/L and a significant reduction of egg
production at the lowest exposure concentrations of 0.50 mg/L [25]. A study by de
Oliveira and coauthors [32] calculated a diclofenac (EC 50 ¼ 123.3 mg/L) in
D. magna, but no effect on population increase was noted after the exposure to
increasing DCF concentrations (range 29.5–75 mg/L). Toxic effects of 21 days
exposure to DCF (5, 50, 500, and 5,000 μg/L) on survival, growth rate, and
reproduction, as well effects on the expression of the genes related to the detoxification metabolism, growth, development, and reproduction, such as HR96, P-gp,
CYP360A8, CYP314, GST, EcR, and Vtg after 96 h exposure, were investigated in
D. magna specimens [33]. Significant toxic effects of DCF to D. magna were
observed at 50 μg/L, whereby the expression of the selected genes was inhibited
after 24 h of exposure, while they were increased after 48 h. Despite modulation of
gene expression, no significant effects were observed in molting frequency, number
of eggs produced in the first brood, total number of eggs per individual, total number
of broods per individual, body length, and growth rate. In contrast, the exposure to
increasing concentrations of DCF (2, 4, 8, 16, and 32 mg/L) affected the population
growth curves of the rotifer Plationus patulus and the cladoceran Moina macrocopa,
leading to a decrease in organism density with increasing levels of drug, as well as a
negative effect on the daily rate of population increase [27]. Another research
investigated the toxicity of DCF at biochemical level in D. magna by assessing the
modulation of hsp70 level as a biomarker for proteotoxicity [34]. Hsp70 induction
occurred at high levels of DCF, as the LOEC was calculated at 40 mg/L. The
cytogenotoxicity of DCF was investigated through an in vitro approach by exposing
hemocytes from the zebra mussel D. polymorpha for 1 h to 60, 126, and 250 μg/L
[30]. Cytotoxicity was evaluated by the neutral red retention assay (NRRA) while
genotoxicity by SCGE (single cell gel electrophoresis) and DNA diffusion assay. A
significant cytotoxic effect was noted only after the exposure to 250 mg/L of DCF,
while genotoxicity occurred after the exposures to all the tested concentrations. A
further in vitro experiment [35] investigated the toxicity of increasing DCF concentrations (0.001, 0.01, 0.1, 1, and 10 mg/L) on three different cell typologies from the
zebra mussel (Dreissena polymorpha), namely, hemocytes, gill, and digestive gland
cells. At the end of the exposure (i.e., 96 h), viability of DCF treated gill cells was
significantly reduced already at the lowest concentration with respect to baseline
levels. Viability of DCF-treated digestive gland cells was significantly reduced
already after 48 h exposure to 0.01 mg/L, while hemocyte viability was affected
already at the lowest concentration (0.001 mg/L). Zebra mussels specimens were
exposed for 96 h to increasing concentrations (95, 318, and 637 ng/L) of DCF
through an in vivo approach [36]. Cytogenotoxicity was assessed by means of the
single cell gel electrophoresis assay, the apoptotic frequency, the micronucleus test
(MN test), and the lysosomal membrane stability (neutral red retention assay) in
mussel hemocytes. Moreover, the activity of catalase, superoxide dismutase, glutathione peroxidase, and the phase II detoxifying enzyme glutathione S-transferase
was measured as oxidative stress biomarkers. Negligible cyto- and genotoxicity of
DCF was noted towards the zebra mussel hemocytes; in fact only a slight decrease of
lysosomal membrane stability was observed at the end of exposure to the highest
154
M. Parolini
production at the lowest exposure concentrations of 0.50 mg/L [25]. A study by de
Oliveira and coauthors [32] calculated a diclofenac (EC 50 ¼ 123.3 mg/L) in
D. magna, but no effect on population increase was noted after the exposure to
increasing DCF concentrations (range 29.5–75 mg/L). Toxic effects of 21 days
exposure to DCF (5, 50, 500, and 5,000 μg/L) on survival, growth rate, and
reproduction, as well effects on the expression of the genes related to the detoxification metabolism, growth, development, and reproduction, such as HR96, P-gp,
CYP360A8, CYP314, GST, EcR, and Vtg after 96 h exposure, were investigated in
D. magna specimens [33]. Significant toxic effects of DCF to D. magna were
observed at 50 μg/L, whereby the expression of the selected genes was inhibited
after 24 h of exposure, while they were increased after 48 h. Despite modulation of
gene expression, no significant effects were observed in molting frequency, number
of eggs produced in the first brood, total number of eggs per individual, total number
of broods per individual, body length, and growth rate. In contrast, the exposure to
increasing concentrations of DCF (2, 4, 8, 16, and 32 mg/L) affected the population
growth curves of the rotifer Plationus patulus and the cladoceran Moina macrocopa,
leading to a decrease in organism density with increasing levels of drug, as well as a
negative effect on the daily rate of population increase [27]. Another research
investigated the toxicity of DCF at biochemical level in D. magna by assessing the
modulation of hsp70 level as a biomarker for proteotoxicity [34]. Hsp70 induction
occurred at high levels of DCF, as the LOEC was calculated at 40 mg/L. The
cytogenotoxicity of DCF was investigated through an in vitro approach by exposing
hemocytes from the zebra mussel D. polymorpha for 1 h to 60, 126, and 250 μg/L
[30]. Cytotoxicity was evaluated by the neutral red retention assay (NRRA) while
genotoxicity by SCGE (single cell gel electrophoresis) and DNA diffusion assay. A
significant cytotoxic effect was noted only after the exposure to 250 mg/L of DCF,
while genotoxicity occurred after the exposures to all the tested concentrations. A
further in vitro experiment [35] investigated the toxicity of increasing DCF concentrations (0.001, 0.01, 0.1, 1, and 10 mg/L) on three different cell typologies from the
zebra mussel (Dreissena polymorpha), namely, hemocytes, gill, and digestive gland
cells. At the end of the exposure (i.e., 96 h), viability of DCF treated gill cells was
significantly reduced already at the lowest concentration with respect to baseline
levels. Viability of DCF-treated digestive gland cells was significantly reduced
already after 48 h exposure to 0.01 mg/L, while hemocyte viability was affected
already at the lowest concentration (0.001 mg/L). Zebra mussels specimens were
exposed for 96 h to increasing concentrations (95, 318, and 637 ng/L) of DCF
through an in vivo approach [36]. Cytogenotoxicity was assessed by means of the
single cell gel electrophoresis assay, the apoptotic frequency, the micronucleus test
(MN test), and the lysosomal membrane stability (neutral red retention assay) in
mussel hemocytes. Moreover, the activity of catalase, superoxide dismutase, glutathione peroxidase, and the phase II detoxifying enzyme glutathione S-transferase
was measured as oxidative stress biomarkers. Negligible cyto- and genotoxicity of
DCF was noted towards the zebra mussel hemocytes; in fact only a slight decrease of
lysosomal membrane stability was observed at the end of exposure to the highest
154
M. Parolini