Food Chain Transport of Nanoparticles Affects Behaviour and Fat Metabolism in Fish methods
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Materials and Methods
We designed a bioassay protocol which as far as possible ensures that the nanoparticles are taken up through food and the intestinal wall. A three-day process was set up for the food chain as follows: day 1) addition of nanoparticles to the algal culture, day 2) feed zooplankton with algae, day 3) feed fishes with zooplankton. This three day process was repeated throughout the 6 week long experiment ( ). Two parallel food chains were constructed, one with nanoparticles added during algal growth (test) and one with no nanoparticles added (control). The individual steps were performed as follows. On the first day of the process 250 mL of a green algal laboratory culture ( Scenedesmus sp., 25 µm in diameter) was distributed to each of eight glass bottles. Four bottles were set aside for the control group. Four bottles were used for the test group and supplemented with 25 mg polysty...
Materials and Methods
Zooplankton ( Daphnia magna; approximate size 3 mm) were taken from culture and placed in each glass jar (30 adult Daphnia per 250 ml jar). On the second day of the process, the filtered algae were added to the respective Daphnia jars (test or control) and the jars were incubated at 20°C for 24 hours ( ). Four control and four test aquaria were filled with 15 liter of water and aerated. Four Crucian carp ( Carassius carassius ) were randomly assigned to each of the aquaria. On the third day of each experimental cycle, Daphnia were collected on a 50 µm net and presented to the fish. Fish feeding times were monitored five times (Day 18, 21, 24, 27, and 30), by recording the time it took the fish to eat 95% of the Daphnia added to the tank. Differences in feeding time between treatments were tested with Repeated Measures ANOVA.
Materials and Methods
Nanoparticle uptake and transfer through the food chain was studied in a parallel experiment using 28 nm diameter polystyrene particles (1% w/v) with encapsulated fluorophore (Duke Scientific Corp., Palo Alto, CA). Green algae ( Scenedesmus sp., 25 µm in diameter) were grown in the absence (control) or presence of the fluorescent nanoparticles (test) at a concentration of 0.01% (w/v) for 24 hours at 20°C followed by bright field and fluorescence microscopy. Imaging of samples was performed with an inverted Nikon Eclipse TE2000-U microscope (Nikon Corporation, Tokyo, Japan) using an Andor Ixon EMCCD camera (Andor Technology, Belfast, Northern Ireland), with bright field or epifluorescence illumination with white light or Deep Blue filter cube set (Ex/Em 455/520 nm), respectively. Image acquisition was performed with IQ software (Andor Technology, Belfast, Northern Ireland)....
Materials and Methods
Fish blood from Crucian carp ( Carassius carassius ), Bleak ( Alburnus alburnus ), Rudd ( Scardinius erythrophthalmus ), Tench ( Tinca tinca ), Pike ( Esox esox ), and Atlantic salmon ( Salmo salar ), was collected and sampled according to the same procedure as for the Crucian carp (see above). From each fish, 100 µl serum was mixed with 1 mg polystyrene particles, 200 nm (to allow pelleting by centrifugation), in PBS and incubated at 23°C for 1 hour. The mixtures were centrifuged, 13000 rpm, for 10 min and the supernatants discarded. The pellets were dispersed in 0.5 ml PBS and the samples centrifuged again. This was repeated once. Proteins bound to the particles in the pellets were desorbed by adding SDS-PAGE loading buffer, and separated on a 12% SDS-PAGE. Proteins migrating at a position corresponding to around 25 kDa were cut out and digested with trypsin.
Materials and Methods
Trypsin digested peptide extracts were resuspended in 10 µl of 0.1% TFA, and 0.5 µl of each extract was dispensed directly on a MALDI-TOF sample support. The samples were allowed to dry prior to addition of 0.5 µl of matrix solution (5-mg/ml α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 0.1% TFA, 25 mM citric acid) to each sample. MALDI-TOF mass spectrometry was performed using a 4700 proteomicsanalyzer (Applied Biosystems, Framingham, MA) mass spectrometer in positive reflector mode. For MS and tandem MS (MS-MS) analyses, approximately 1,000 and 2,000 single laser shot spectra were summed up, respectively.
Materials and Methods
Samples of muscle and liver from Crucian carp were weighed and 1 ml PBS/100 mg tissue was added and the tissue was homogenized. The homogenized tissue was centrifuged, 13000 rpm, 6 min, and the supernatant transferred to new tubes. The absorbance of the supernatant was measured at 280 nm as a control that the procedure resulted in similar levels of homogenization. The triglyceride and cholesterol concentrations were measured in serum, muscle and liver homogenates. Serum Triglyceride Determination Kit, Sigma TRO 100 and Amplex Red Cholesterol Assay Kit, Sigma A12216, were used to determined triglyceride or cholesterol concentrations, respectively, according to the manufacturer's instructions.
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Evidence quotes (6)
We designed a bioassay protocol which as far as possible ensures that the nanoparticles are taken up through food and the intestinal wall. A three-day process was set up for the food chain as follows: day 1) addition of nanoparticles to the algal culture, day 2) feed zooplankton with algae, day 3) feed fishes with zooplankton. This three day process was repeated throughout the 6 week long experiment ( ). Two parallel food chains were constructed, one with nanoparticles added during algal growth (test) and one with no nanoparticles added (control). The individual steps were performed as follows. On the first day of the process 250 mL of a green algal laboratory culture ( Scenedesmus sp., 25 µm in diameter) was distributed to each of eight glass bottles. Four bottles were set aside for the control group. Four bottles were used for the test group and supplemented with 25 mg polystyrene nanoparticles each, yielding a nanoparticle concentration of 0.01% (w/v). The solutions were mixed by shaking for 5 minutes. Algae were then grown together with nanoparticles for 24 hours at 20°C and at a light/dark cycle of 14/10 h ( ). The 0.01% concentration was chosen to allow a signif...
Zooplankton ( Daphnia magna; approximate size 3 mm) were taken from culture and placed in each glass jar (30 adult Daphnia per 250 ml jar). On the second day of the process, the filtered algae were added to the respective Daphnia jars (test or control) and the jars were incubated at 20°C for 24 hours ( ). Four control and four test aquaria were filled with 15 liter of water and aerated. Four Crucian carp ( Carassius carassius ) were randomly assigned to each of the aquaria. On the third day of each experimental cycle, Daphnia were collected on a 50 µm net and presented to the fish. Fish feeding times were monitored five times (Day 18, 21, 24, 27, and 30), by recording the time it took the fish to eat 95% of the Daphnia added to the tank. Differences in feeding time between treatments were tested with Repeated Measures ANOVA.
Nanoparticle uptake and transfer through the food chain was studied in a parallel experiment using 28 nm diameter polystyrene particles (1% w/v) with encapsulated fluorophore (Duke Scientific Corp., Palo Alto, CA). Green algae ( Scenedesmus sp., 25 µm in diameter) were grown in the absence (control) or presence of the fluorescent nanoparticles (test) at a concentration of 0.01% (w/v) for 24 hours at 20°C followed by bright field and fluorescence microscopy. Imaging of samples was performed with an inverted Nikon Eclipse TE2000-U microscope (Nikon Corporation, Tokyo, Japan) using an Andor Ixon EMCCD camera (Andor Technology, Belfast, Northern Ireland), with bright field or epifluorescence illumination with white light or Deep Blue filter cube set (Ex/Em 455/520 nm), respectively. Image acquisition was performed with IQ software (Andor Technology, Belfast, Northern Ireland). The algae were filtered and added to Daphnia (test or control) in the same proportions as above and the jars with Daphnia were incubated at 20°C for 24 hours. Daphnia were collected on a 50 µm net and washed to remove free nanoparticles, followed by bright field and fluorescence microscopy...
Fish blood from Crucian carp ( Carassius carassius ), Bleak ( Alburnus alburnus ), Rudd ( Scardinius erythrophthalmus ), Tench ( Tinca tinca ), Pike ( Esox esox ), and Atlantic salmon ( Salmo salar ), was collected and sampled according to the same procedure as for the Crucian carp (see above). From each fish, 100 µl serum was mixed with 1 mg polystyrene particles, 200 nm (to allow pelleting by centrifugation), in PBS and incubated at 23°C for 1 hour. The mixtures were centrifuged, 13000 rpm, for 10 min and the supernatants discarded. The pellets were dispersed in 0.5 ml PBS and the samples centrifuged again. This was repeated once. Proteins bound to the particles in the pellets were desorbed by adding SDS-PAGE loading buffer, and separated on a 12% SDS-PAGE. Proteins migrating at a position corresponding to around 25 kDa were cut out and digested with trypsin.
Trypsin digested peptide extracts were resuspended in 10 µl of 0.1% TFA, and 0.5 µl of each extract was dispensed directly on a MALDI-TOF sample support. The samples were allowed to dry prior to addition of 0.5 µl of matrix solution (5-mg/ml α-cyano-4-hydroxycinnamic acid, 50% acetonitrile, 0.1% TFA, 25 mM citric acid) to each sample. MALDI-TOF mass spectrometry was performed using a 4700 proteomicsanalyzer (Applied Biosystems, Framingham, MA) mass spectrometer in positive reflector mode. For MS and tandem MS (MS-MS) analyses, approximately 1,000 and 2,000 single laser shot spectra were summed up, respectively.
Samples of muscle and liver from Crucian carp were weighed and 1 ml PBS/100 mg tissue was added and the tissue was homogenized. The homogenized tissue was centrifuged, 13000 rpm, 6 min, and the supernatant transferred to new tubes. The absorbance of the supernatant was measured at 280 nm as a control that the procedure resulted in similar levels of homogenization. The triglyceride and cholesterol concentrations were measured in serum, muscle and liver homogenates. Serum Triglyceride Determination Kit, Sigma TRO 100 and Amplex Red Cholesterol Assay Kit, Sigma A12216, were used to determined triglyceride or cholesterol concentrations, respectively, according to the manufacturer's instructions.
Machine-readable layer
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