Environ Monit Assess (2013) 185:3783–3793 DOI 10.1007/s10661-012-2827-2
Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals G. V. Aguirre-Martínez & S. Buratti & E. Fabbri & T. A. Del Valls & M. L. Martín-Díaz
Received: 1 February 2012 / Accepted: 2 August 2012 / Published online: 7 November 2012 # Springer Science+Business Media B.V. 2012
Abstract The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked G. V. Aguirre-Martínez : T. A. Del Valls : M. L. Martín-Díaz Cátedra UNESCO/UNITWIN/WiCop, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, Polígono Río San Pedro s/n, Puerto Real, Cádiz, Spain G. V. Aguirre-Martínez (*) : M. L. Martín-Díaz Andalusian Center of Marine Science and Technology (CACYTMAR), Campus Universitario de Puerto Real, 11510 Puerto Real, Cádiz, Spain e-mail:
[email protected] S. Buratti : E. Fabbri Interdepartmental Centre for Research in Environmental Sciences (CIRSA), University of Bologna, Via S. Alberto 163, 48123 Ravenna, Italy
every 2 days with various concentrations (from 0.1 to 50 μg·L−1) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p5 mg L−1, 60 % saturation) were routinely monitored, under a 12-h:12-h light/dark regime. The experiment was performed in duplicate. Six specimens were used for each individual exposure treatment, and each group was maintained in a 20-L glass aquarium containing filtered seawater. Physical and chemical parameters were strictly controlled during the 28-day experiment. Crabs were fed with frozen mussels, followed by a change of water every 24 h during the 28-day semistatic assay. Stock solutions of pharmaceuticals were prepared in dimethyl sulfoxide (DMSO; 0.001 % by volume) to ensure solubility. Groups of specimens were exposed separately during this period to filtered seawater diluted with different concentrations of caffeine (0.1, 5, 15, and 50 μg·L−1), ibuprofen (0.1, 5, 10, and 50 μg·L−1), carbamazepine (0.1, 1, 10, and 50 μg·L−1), and novobiocin (0.1, 1, 10, and 50 μg·L−1); in addition, a control treatment with seawater was included in order to compare results. In parallel with the pharmaceutical experiments, DMSO was also tested to ensure that there was no solvent effect, as recommended by Eades and Waring (2010)
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and Quinn et al. (2008a, b). The full experiment, therefore, comprised a total of 18 different exposure treatments, each duplicated. Selected pharmaceuticals were purchased from Sigma-Aldrich (Spain). On a world scale, caffeine, a psychoactive stimulant, is one of the most commonly and frequently consumed alkaloid drugs (Fent et al. 2006; Palo and Choudhury 2006). Ibuprofen, carbamazepine, and novobiocin are common pharmaceuticals prescribed for the prevention and/or treatment of several human diseases and disorders. Doses of these chemicals were selected based on environmental concentrations reported in studies carried out in ME, sewage treatment plant effluents, WWTP effluents, surface water, lakes, and seawater worldwide (Table 1). Caffeine concentrations have been detected in the range from 0.07 to 293 μg·L−1. Ibuprofen concentrations have been recorded ranging from 0.01 to 24.6 μg·L−1. Carbamazepine has been found in the environment at concentrations ranging from 0.03 to 6.3 μg·L−1, and novobiocin concentration has been measured in WWTP and ME waters, resulting in a concentration of 0.33 μg·L−1 (Table 1). Lysosomal membrane stability LMS was studied in crab hemolymph by application of the NRR assay, following the methodology proposed by Lowe et al. (1995) and reported in detail by Martínez-Gómez et al. (2008). Hemolymph analysis of crabs was performed at the beginning (day 0) and at the end (day 28) of the bioassay. The methodology was adapted taking into consideration the use of an anticoagulant buffer with crab hemolymph to prevent clotting (Astley et al. 1999). The crab physiological saline (4.66 mM NaCl, 10.9 mM KCl, 12.6 mM CaCl2, 19.5 mM MgCl2, and 10 mM HEPES sodium salt) and the anticoagulant solution (0.45 M NaCl, 0.1 M glucose, and 30 mM trisodium citrate) were prepared following the protocol proposed by Söderhall and Smith (1983). Crab hemolymph was extracted from articulation joints with a 1-mL syringe, and then mixed with 0.1 mL of anticoagulant and 0.1 mL physiological saline. Two replicates of 40 μL samples were transferred at room temperature onto microscope slides and were placed in a lightproof humidity chamber, where the hemocytes were left to settle and attach to the slide surface, for 30 min. Then, 40 μL of 0.2 mM neutral
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Environ Monit Assess (2013) 185:3783–3793
Table 1 Range of MEC of pharmaceuticals in marine and freshwater Pharmaceutical
Caffeine
Ibuprofen
Carbamazepine
Novobiocin
MEC (μg L−1)
Water type studied
Reference
EC50 (μg L−1) 19.90
PNEC
MEC/PNEC
Assessment
0.01
SW
Weigel et al. (2004a)
0.0199
0.50
Not necessary
0.09
SW
Weigel et al. (2004a)
0.0199
4.54
Necessary
4.42
WWTP
Santos et al. (2005)
0.0199
222.11
Necessary
13.9
WWTP
Kosma et al. (2010)
0.0199
6.98
Necessary
293.0
STP
Weigel et al. (2004a)
0.0199
14,723.62
Necessary
0.01
Lake
Tixier et al. (2003)
0.0007
14.29
Necessary
0.15
Surface water
Gross et al. (2004)
0.0007
214.29
Necessary Necessary
0.7
6.3
WWTP
Santos et al. (2005)
0.0007
9,000.00
10.1
WWTP
Santos et al. (2005)
0.0007
144,428.57
Necessary
24.6
WWTP
Miège et al. (2009)
0.0007
35,142.86
Necessary
0.03
WWTP
Gagné et al. (2006)
0.1
Surface water
Gross et al. (2004)
0.316
1.1
WWTP
Kosma et al.(2010)
0.000316
3,481.01
Necessary
6.3
ME
Ternes et al.(1998)
0.000316
19,936.71
Necessary
0.33
ME
Gagné et al. (2006a)
0.002235
147.65
Necessary
2
0.000316
94.94
Necessary
0.000316
316.46
Necessary
EC50 95 % interval after 28 days of exposure to pharmaceuticals and analysis assessment based on the MEC/PNEC results of this research ME municipal effluent, STP sewage treatment plant effluent, WWTP wastewater treatment plant effluent, SW seawater
red solution was added. Finally, the cells were examined under microscope after 15, 30, 60, and 90 min to determine the NRR time, established as the time when 50 % of the cells display leakage from the lysosomes into the cytosol (Martínez-Gómez et al. 2008). Statistical analyses Data were analyzed using the SPSS/PC+ statistical package. Significant differences between exposed and control samples were determined using a oneway analysis of variance followed by the multiple comparison Dunnett’s test. Pairwise correlations were obtained using the Pearson’s rank order correlation test. The significance level was set at p
