David
Boyle
,
Nathaniel J.
Clark
,
Benjamin P.
Eynon
and
Richard D.
Handy†
*
School of Biological and Marine Sciences, University of Plymouth, Plymouth PL4 8AA, UK. E-mail: R.Handy@plymouth.ac.uk; Tel: +44 (0)1752 584630
First published on 30th June 2021
The dietary bioaccumulation potential of engineered nanomaterials (ENMs) remains poorly understood. The aim of the current study was to assess the dietary bioaccumulation of copper from copper oxide (CuO) ENMs or dissolved copper (CuSO4) exposure in fish. Animals were fed a nominal diet of 750 mg kg−1 Cu in the respective forms for 4 weeks, then fed the control diet for a further 2 weeks in a depuration phase. Fish were sampled at weeks 2, 4 and 6 for total Cu analysis. Samples were also taken at week 4 for plasma ions, biochemistry and histology. An in chemico digestibility assay simulating the gut lumen showed the total Cu could be leached (i.e., was bioaccessible) from both diets. Fish from all treatments showed normal growth and survival, and with healthy histology of gills, intestines and liver. At the end of the 4 week exposure, both Cu materials caused an elevated tissue total Cu concentration with the highest in the mid intestine, hind intestine and liver. For example, the week 4 liver total Cu concentrations were 82 ± 7, 259 ± 19 and 281 ± 17 μg g−1 in the control, CuSO4 and CuO ENM treatments, respectively, with no significant differences between the Cu exposures. Compared to the controls, both forms of Cu caused induction of MT in the hind intestine, as well as some depletion of total GSH in the liver. In the post-exposure phase, there was evidence of depuration of total Cu from the mid and hind intestine, and also the carcass, but not the brain or kidney. The liver also maintained similar total Cu concentrations, in keeping with this organ as a central compartment for Cu excretion. There was limited evidence of nanomaterial-specific effects. Overall, the CuO ENMs showed similar patterns of bioaccumulation to CuSO4, with negligible physiology effects.
Environmental significanceUnderstanding the dietary bioaccumulation potential of engineered nanomaterials (ENMs) in fish is an important aspect of environmental risk assessment. Trout fed diets containing excess copper sulphate or copper oxide nanomaterial for 28 days showed no effects on growth, plasma ions or organ histology, despite some total copper accumulation in the internal organs. The animals managed the exposure with some loss of the antioxidant, glutathione, and some metallothionein induction; and showed clearance of copper from the organs in the post-exposure phase. A digestibility assay showed that copper from both forms of exposure was bioaccessible. Crucially, the bioaccumulation hazard from the nano form was similar to the metal salt, implying the existing metal risk assessment would be protective of the nano form. |
Copper (Cu) is an essential trace metal and dietary Cu requirements in freshwater fish such as rainbow trout (Oncorhynchus mykiss) are in the region of ∼3 mg per kg body weight per day, and aquaculture feeds routinely contain ∼10 mg kg−1 of Cu.7 At concentrations considerably higher than this, Cu has been shown to accumulate in internal tissues and sometimes cause toxicity.8,9 The threshold for the effects of dietary Cu on growth and survival is about 750 mg kg−1 of Cu in the food, or slightly more, depending on the ration size and species of fish (reviews,7,10). At high dietary inclusion rates, CuSO4 have been shown to affect ion homeostasis, cause oxidative stress, and pathologies at sites of Cu accumulation in trout.8,9 At dietary concentrations of 750 mg kg−1 of Cu or less as CuSO4, trout show good maintenance of body system homeostasis, and compensate for the cost of exposure by adjusting the bioenergetics of swimming behaviours.11,12 Only a few studies have considered dietary exposure to Cu ENMs, but total Cu concentrations in tissues have been shown to increase in tissues during dietary exposures.13,14 However, the form(s) of the bioavailable fraction of Cu ENMs in fish tissue are unclear.
The uptake of dissolved Cu at the intestine is well characterised in fish. At the apical membrane of the enterocytes, transport of the Cu ion is via high-affinity Cu uptake proteins (likely the CTR family and DMT1 proteins) with the rate limiting step in Cu assimilation being the ATP-dependent vesicular export of Cu into the blood.15–17 As such, dietary exposures to excess dissolved Cu are characterised by retention of Cu in the intestinal tissues in fish.8,18 The uptake mechanisms for Cu ENMs in the gut of fish remains to be established. Recently, we demonstrated negligible tissue accumulation of total Cu from CuO ENMs using an ex vivo gut sac technique in rainbow trout with a simple physiological saline; CuO ENMs were associated with the mucosa, and unlike dissolved Cu, did not transfer to the blood compartment.19 While CuO ENMs were dispersed in the simple physiological saline, the lack of uptake was unexpected, and further experiments in the same study explored the addition of amino acids that would be present in the chime in vivo. By adding the amino acid, cysteine, Cu was shown to accumulate in the blood compartment of the gut sacs. This was likely caused by increased dissolution of the CuO ENMs in the gut lumen in the presence of cysteine, and transport of Cu-cysteine into the tissue via dissolved Cu uptake pathways.19 Together, these data supported the hypothesis that intact nanoscale particulates of Cu are too large for uptake on solute transporters [see discussion Handy et al.;20 as demonstrated for silver (Ag) ENMs21], but also demonstrate that the chemical composition of gut lumen can drive the transformations of metallic ENMs and subsequent total Cu uptake.19
Notably, the bioaccumulation potential of the CuSO4 and CuO ENMs in fish has been shown using the ex vivo gut sac technique.19 In as little as 4 hours, total Cu from both materials accumulated in the mucosa of the gastrointestinal tract of rainbow trout, with the highest concentrations in the mid and hind intestines.19 To investigate this further, the present study explored the bioaccumulation of CuSO4 and CuO ENMs in juvenile rainbow trout exposed via the diet for 28 days. It also remains unclear whether newly accumulated material can be excreted; therefore, following the exposure period, fish were depurated on the control diet for a further 2 weeks (total 6 weeks) to assess alterations in tissue Cu concentrations. It is also important to link any total Cu accumulation to biological effects; CuSO4 exposure has a well understood aetiology, with the liver as the central organ for metabolism/detoxification, and molecular interactions with reduced glutathione (GSH) and metallothionein (MT). However, the handling process remains unknown for the nano form and so measurements of these biomarkers as well as histological structure of the intestine and liver were made at the end of the exposure. To aid data interpretation, an in chemico digestibility assay was also used to help understand the bioaccessible fraction from the feed in conditions analogous to those found in the gastrointestinal tract.
The dietary exposure was conducted following the essence of the Organisation for Economic Development and Cooperation (OECD) test guideline, TG 305,1 but with a triplicated study design and additional measurements. Fish were fed one of three diets for 28 days: a control diet (no added Cu), or 750 μg g−1 Cu as CuSO4 or CuO ENMs. Following this, a 14 day depuration period occurred where all treatments were fed the control diet. The total duration of the experiment was 42 days. Fish were fed a 2% body weight ration per day whereby the amount of food was altered each week based on the tank biomass. The ration was carefully fed at approximately 09:00, 13:00 and 17:00 each day and observed to ensure the food was eaten immediately to minimise contact time of the diet with water and any leaching of Cu from the diet into the water. There were no residual food pellets in the water after each feed. Faecal matter in the tanks was removed by careful siphoning after each feed.
The water chemistry was monitored in tanks throughout the experiment. Tanks were supplied continuously with Plymouth tap water with a water turnover rate of (mean ± SD): 0.52 ± 0.12 L min−1 to minimise the build-up of ammonia in the tanks (there was no significant difference in water turnover between treatments, one-way ANOVA, p = 0.919). The measured concentrations of total ammonia [NH3] in the tanks were 13.9 ± 5.2, 14.7 ± 5.6, and 13.8 ± 4.9 μM in control, CuSO4 and CuO ENM tanks, respectively. The water was pH 6.92–7.30, temperature was 17 ± 1 °C, dissolved oxygen was >100% saturation throughout the experiment and the ionic composition of the water, based on weekly measurements using inductively coupled optical emission spectrometry (ICP-OES, Varian 725-ES, Agilent Technologies Inc.), were (in mM): Ca2+, 0.41 ± 0.10; K+, 0.06 ± 0.04; Mg2+, 0.09 ± 0.05; Na+, 0.58 ± 0.23. Concentrations of Cu were also measured in tank water for the duration of the experiment (see section 2.4). The aquatics facility had a set 12 h light and 12 h dark photoperiod.
Fish from the experimental tanks were sampled at days 14 and 28 during the exposure phase and at the end of the depuration phase on clean food (day 42) for trace metal analysis (see section 2.4). Samples for tissue biochemistry and histology were also taken at day 28 (see sections 2.5 and 2.6, respectively). In order to facilitate dissection and to ensure animal welfare during handling, fish were not fed on the morning of the sampling days. This allowed for evacuation of the gut after the final feed the previous day, ∼15 h earlier. To achieve a daily ration of 2% body weight per day throughout the experiment, the half daily ration not fed on sampling days was given to fish on the subsequent days. All procedures with live fish were conducted in accordance with ethical approvals from the Home Office, UK, under the Animals (Scientific Procedures) Act 1986 and in compliance with the EU directive 2010/63/EU.
The diet used throughout the study was the same as used by Clark et al.6 The diet was a commercial fish food (Aller Futura, EX, Kaliningrad, Russia), with a pellet size of 1.5 mm. The intact food pellets were supplemented with CuSO4 or CuO ENMs that was allowed to soak into the pellets and this was then sealed with a topcoat of 10% porcine gelatine (Sigma-Aldrich, UK). A nominal concentration of 750 μg g−1 Cu (as CuSO4 or CuO ENMs) in the diets was chosen based on previous studies of dissolved Cu in trout that have shown tissue Cu accumulation.12,24 To prepare the diets, a stock suspension of the CuO ENMs of 1.064 g 100 mL−1 was prepared first in ultrapure H2O (18.2 MΩ, ELGA, UK) in an acid-washed volumetric flask and dispersed in an ultrasonic bath for 1 h (50/60 Hz, 35 Watts, FB15048, Fisherbrand) before spiking the diets. Nanoparticle tracking analysis has demonstrated this approach gives good dispersions of CuO ENMs, with typical aggregate sizes of around 110 nm depending on concentration and water pH.19,23 In any event, the dispersion effort was mainly to make a uniform sample that could be added to the food, and would not reflect the aggregation state in the final food matrix. A stock solution of CuSO4 was prepared in the same manner but was not sonicated. The diets were prepared in a single batch by adding a total volume of 100 mL of either CuO ENMs or CuSO4 directly to 1 kg of diet and gently mixing the diet with a commercial food mixer for five minutes (model XKM810, Kenwood, UK). The diet was then coated with a solution of 10 g of gelatine in 100 mL of ultrapure water at 60 °C (per kg diet) and mixed into the diet for several additional minutes. Control diets were supplemented with ultrapure water in place of the Cu material and prepared as above. All diets were dried overnight at 40 °C to remove excess water and then stored at 4 °C until fed to fish. Total Cu concentrations of the diets were measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Electron Corporation X-Series II quadrupole, see section 2.4). Copper was homogeneously distributed in the exposure diets and were close to the nominal 750 μg Cu g−1 with 9.5 ± 0.3, 685.6 ± 41.5 and 713.3 ± 33.6 μg per g dry weight (means ± SD, n = 5) in the control, CuSO4 and CuO ENMs treatments, respectively. There was no significant difference between the Cu concentrations in the two exposure diets (Student's t-test, p = 0.28).
An in chemico digestibility assay was used to aid data interpretation over the form of Cu in the gut lumen of trout. Two compartments of gastrointestinal tract at different pH values were simulated with artificial solutions: the stomach (0.1 M HCl, 0.9% NaCl, pH 2) and the intestine (0.9% NaCl, pH 7.8). 1 g samples of the experimental diets (n = 3 per treatment) were ground in a ceramic mortar and added to 20 mL of the artificial solutions. Samples were placed on a tube roller (Denley Spiramix 5 Tube Roller Mixer) for 1 h. Afterwards, the samples were centrifuged at 6000 × g for 10 min and the Cu concentration measured in the supernatant by ICP-MS (see section 2.4).
Concentrations of Cu in tissues were measured at days 14, 28 and 42 of the experiment. Concentrations of other elements were measured in tissues on day 28, i.e. only at the end of the exposure period. Care was taken to avoid cross-contamination between tissues and also between fish. Tissues were dissected and weighed into tubes in the following order: gill, brain, liver (after gall bladder removal), kidney, with the mid- and hind-intestine being dissected last to avoid cross contamination between feed/faeces in the intestine and other tissues. The mid and hind intestine were rinsed in ultrapure water and blotted dry to remove residual feed/faeces before wet weight determination. Tissues and carcasses were freeze dried (Lablyo freeze dryer) for 24 h and stored at room temperature for metal analysis (see section 2.4). Measurements based on tissue dry weight were preferred to eliminate possible artefacts associated with osmotic stress (altered tissue moisture content) during metal exposure.
Concentrations of MT in liver, kidney, mid- and hind-intestines were analysed according to Scheuhammer and Cherian.25 Tissues were homogenised in five volumes of 0.25 M sucrose solution. Homogenates were centrifuged at 13000 rpm for 2 min and 100 μL of the supernatant (50 μL of liver) was diluted to 800 μL with glycine buffer (0.5 M, pH 8.5). Five hundred μL of AgNO3 (185.4 μM in glycine buffer) was then added and samples were incubated at room temperature for 5 min to allow the Ag+ to displace other metal ions bound to MT. To remove unbound Ag+ from samples, 100 μL horse haemolysate (Sigma-Aldrich, UK) was added, the samples were boiled for 1.5 min, centrifuged at 1200 × g and the supernatant removed to a second tube. This step was repeated once more and then the supernatant was spun at 15000 × g for 15 min. The final supernatant was diluted with ultrapure water and the Ag concentrations in samples were measured with ICP-MS and compared to matrix matched element standards (Fisher, UK). The concentrations of MT in samples were expressed per unit mass of tissue and were calculated by multiplying the measured mass of Ag by 3.55.
During the 28 day exposure, trout fed CuSO4 and CuO ENMs showed significant increases in concentrations of Cu compared to controls in all tissues examined (one-way ANOVAs or Kruskal-Wallis tests, p < 0.05; Table 1). With the exception of the kidney at day 28, there were no significant differences in Cu accumulation between the Cu treatments. Overall, the increases in Cu concentrations were highest in the mid and hind intestines, consistent with exposure via the diet, and also in the liver, which is the central tissue for metal metabolism. Smaller but significant increases were evident in the kidney, gill, brain and the remaining carcass. In all tissues except the liver, which showed significant increases in Cu accumulation between days 14 and 28 days, there were no further increases in Cu accumulation after the first 14 days of exposure.
Tissue | 14 days (exposure) | 28 days (exposure) | 42 days (depuration) |
---|---|---|---|
Data are means ± SEM (n = 8/9 samples). Different uppercase letters indicate significant differences between time-points within treatment; different lowercase letters indicate significant differences between treatments within time-point (one-way ANOVA or Kruskal-Wallis, p < 0.05). | |||
Mid intestine | |||
Control | 5.52 ± 0.77Aa | 6.03 ± 0.60Aa | 6.58 ± 0.99Aa |
CuSO4 | 29.84 ± 2.76Ab | 32.12 ± 7.36Ab | 8.09 ± 0.97Ba |
CuO ENMs | 27.09 ± 6.93Ab | 28.48 ± 6.06Ab | 6.70 ± 0.65Ba |
Hind intestine | |||
Control | 9.10 ± 1.25Aa | 11.00 ± 0.92Aa | 16.67 ± 1.99Aa |
CuSO4 | 210.75 ± 28.03Ab | 226.77 ± 28.32Ab | 12.87 ± 1.17Ba |
CuO ENMs | 140.42 ± 34.06Ab | 200.15 ± 16.18Ab | 9.08 ± 0.73Ba |
Liver | |||
Control | 87.38 ± 10.43Aa | 82.01 ± 6.97Aa | 102.62 ± 13.48Aa |
CuSO4 | 170.98 ± 11.15Ab | 259.30 ± 19.22Bb | 236.25 ± 19.37Bb |
CuO ENMs | 184.65 ± 7.33Ab | 281.14 ± 16.73Bb | 255.71 ± 21.84Bb |
Kidney | |||
Control | 5.55 ± 0.42Aa | 4.51 ± 0.24Aa | 5.35 ± 1.07Aa |
CuSO4 | 10.65 ± 1.39Ab | 7.21 ± 1.04Ab | 8.23 ± 1.86Ab |
CuO ENMs | 9.15 ± 0.98Ab | 12.01 ± 1.99Ac | 7.54 ± 0.71Aab |
Gill | |||
Control | 4.08 ± 0.40Aa | 3.88 ± 0.15Aa | 3.77 ± 0.16Aa |
CuSO4 | 7.83 ± 1.16Ab | 5.39 ± 0.43Aab | 4.61 ± 0.30Aa |
CuO ENMs | 6.27 ± 0.37Aab | 8.36 ± 0.52Ab | 3.64 ± 0.08Ba |
Brain | |||
Control | 4.48 ± 0.23Aa | 4.18 ± 0.20Aa | 4.94 ± 0.19Aa |
CuSO4 | 5.66 ± 0.34Ab | 5.35 ± 0.26Ab | 7.14 ± 0.28Bb |
CuO ENMs | 5.45 ± 0.31Ab | 5.98 ± 0.25ABb | 6.62 ± 0.33Bb |
Carcass | |||
Control | 1.31 ± 0.12Aa | 0.99 ± 0.04Aa | 1.34 ± 0.10Aa |
CuSO4 | 3.38 ± 0.73ABab | 3.39 ± 0.27Ab | 1.37 ± 0.07Ba |
CuO ENMs | 3.83 ± 0.19Ab | 2.78 ± 0.51Ab | 1.53 ± 0.04Aa |
After the 14 day depuration phase (day 42 of the experiment), Cu concentrations in the mid and hind intestine, gill and carcass had decreased to control levels (one-way ANOVAs or Kruskal-Wallis tests, p > 0.05; Table 1). The same trend was not evident in other tissues. In the kidney and especially the liver and brain, Cu concentrations were elevated compared to controls and there was no change compared to day 28 of the exposure.
Concentrations of Na+ and K+ were measured in blood plasma at days 14, 28 and 42 of the experiment (Table 2) and in tissues at day 28 of the exposure. Blood plasma ion concentrations showed some deviations during the experiment but these appeared to be unrelated to treatment. There were also a few significant effects of the Cu exposures on tissue Na+ and K+ concentrations, with the exception of the hind intestine and the kidney. In the kidney, concentrations of Na+ were significantly decreased in fish treated with CuSO4 or CuO ENMs, but there was no nano-effect (Kruskal-Wallis test, p = 0.004). Measured Na+ concentrations were 248 ± 14, 194 ± 10 and 197 ± 6 μmol g−1 in fish fed control, CuSO4 and CuO ENMs. In the hind intestine, concentrations of both Na+ and K+ were decreased by both Cu treatments compared to controls and there was also a small nano-effect with K+ significantly lower in the hind intestine of fish fed CuO ENMs compared to CuSO4 (one-way ANOVAs, p < 0.05). Concentrations of Na+ in the hind intestine were 107 ± 8, 72 ± 4 and 70 ± 5 μmol g−1 in fish fed control, CuSO4 and CuO ENMs. Concentrations of K+ were 265 ± 10, 213 ± 11 and 182 ± 10 μmol g−1 in fish fed control, CuSO4 and CuO ENMs.
Treatment | Day 14 | Day 28 | Day 42 | |
---|---|---|---|---|
Data are means ± SEM of n = 4–9 samples. There were no treatment or time related effects of dietary Cu exposure on plasma Na+ or K+ concentrations (Kruskal-Wallis tests, p > 0.05). | ||||
Na+ | Control | 160.4 ± 1.2 | 145.7 ± 2.6 | 157.6 ± 6.1 |
CuSO4 | 149.8 ± 2.7 | 138.7 ± 2.2 | 142.7 ± 3.3 | |
CuO ENMs | 148.5 ± 5.1 | 139.1 ± 2.8 | 149.2 ± 2.0 | |
K+ | Control | 1.93 ± 0.51 | 1.10 ± 0.17 | 1.46 ± 0.23 |
CuSO4 | 2.74 ± 0.69 | 1.98 ± 0.35 | 1.43 ± 0.33 | |
CuO ENMs | 1.87 ± 0.32 | 1.48 ± 0.26 | 1.38 ± 0.31 |
Copper is an essential nutrient for fish, and as expected, there was a detectable background of total Cu present in the control tissues (Table 1). The control values for Cu ranging from around 3–11 μg g−1 dw in most organs, and around 100 dw μg g−1 dw in the liver (Table 1), being consistent with previous reports (e.g., ref. 26 and 29). The dietary exposure to the CuSO4 resulted in significant elevations of Cu in the mid intestine, hind intestine and liver in keeping with the route of exposure, and some small increases in the kidney and brain (Table 1), which is broadly similar to other reports for dietary copper exposure in trout.11,24,29 For instance, following a five week exposure to around 1 g Cu kg−1 food as CuSO4, the liver total Cu concentration reached 224 μg g−1 (∼3.5 μmol g−1) compared to 52 μg g−1 (∼0.8 μmol g−1) in controls for juvenile trout.24 The liver of fish exposed to dietary CuSO4 showed a similar trend in the present study, with livers from exposed fish reaching concentrations roughly three times those of the controls (Table 1). This is in keeping with the notion of the liver being a central compartment in Cu metabolism.30
There is less data on dietary exposure to CuO ENMs in fish. In the present study, the target organs and accumulation of total Cu from the dietary CuO exposure was the same as that for CuSO4, although the kidney accumulated more total Cu from exposure to the nano form (Table 1). There have been only a few other studies on dietary exposure to Cu ENMs or CuO ENMs in fish (sea bream;13 Russian sturgeon;14 snow trout31); and these studies can be regarded as preliminary because the material characterisation was often not reported and/or metal salt controls were not included in the study designs. Nonetheless, at least one study showed that fish fed diets containing Cu ENMs had elevated total Cu in the carcass and the liver compared to unexposed controls,14 in keeping with the findings here. In terms of accumulation in different regions of the gut (Table 1), the total Cu concentration of the hind intestine was higher than the mid intestine, indicating the former is the site of accumulation from CuO ENM exposures. This is generally consistent with finding that the distal regions of the gut are involved in the uptake of metal (form unknown) from exposures to ENMs in trout (TiO2,;32 Ag,6).
A crucial question for hazard assessment is whether the hazard from nano forms of Cu are different from the metal salt. The present study shows there were no significant differences between the total Cu concentrations in the organs following exposure to the CuSO4 or CuO ENM treatments, except for the kidney which had a higher total Cu concentration in the latter (Table 1). Similarities in organ concentrations of total Cu were found in red sea bream fed equal mass concentrations (4 mg kg−1) of CuSO4 or CuO ENMs. After 60 days of exposure, there was no significant difference in the total Cu concentration of the muscle, liver or gills.13 There appear to be no other reports of Cu accumulation in the brain or kidney following dietary exposure to Cu ENMs. Both Cu treatments caused increases in total Cu in the brain, but there was no additional elevation in total Cu associated with the nano form (Table 1). Nonetheless, with the brain as a target organ for Cu from CuO ENM exposures, the neurological deficits observed with excess dietary Cu salts, such as changes in circadian rhythms,12 could also be a similar concern for the nano form. For the kidney, more total Cu was accumulated in the exposure to the nano form, perhaps suggesting some additional hazard over the metal salt for this organ. The elevated total Cu might arise from melanomacrophage activity trapping particulate Cu in the parenchyma as observed in aqueous exposures to Cu ENMs in trout.28 However, Cu excretion via the urine is usually minimal in trout, with the liver being the main excretory organ,30 and with normal plasma ions (Table 2), it suggests that renal function was not lost.
However, any bioaccumulation hazard might be transient since the intestines and the carcass showed decreases in total Cu concentrations back to control levels in the post-exposure phase (Table 1), in keeping with dissolved Cu being an essential nutrient that is homeostatically controlled. Notably, total Cu from the CuO ENM exposure was also eliminated by the intestines (Table 1). Copper uptake and excretion is regulated by vesicular trafficking systems involving Cu-ATPase(s) that load Cu into the Golgi apparatus with subsequent vesicle formation.15,33 Increased turnover of the Cu trafficking in the gut epithelial cells by this mechanism might contribute to an apparent excretion of total Cu from the intestines. However, more likely, the normal process of sloughing of the intestinal epithelial cells would contribute to the decrease in total Cu in the intestines in the post-exposure phase. In contrast to the intestines and carcass, neither the liver, kidney nor brain showed decreases of total Cu in the post-exposure phase, regardless of the type of Cu exposure (Table 1). While there was no difference between CuSO4 and CuO ENMs in that regard, it highlights that the clearance of Cu from some organs is usually slower than uptake, as expected.27 For the kidney, as outlined above, the melanomacrophage activity might prolong the apparent retention of Cu in the organ. In the case of the brain, Cu concentrations increased in the post-exposure phase following exposure to CuSO4, but not for CuO ENMs (Table 1). Some redistribution of the Cu body burden is expected post-exposure.27 However, fluxes of metals across the blood brain barrier of fishes is poorly understood, although retention of Cu by metal binding ligands such as melatonin is probable.30 Whether or not intact CuO particles are taken up by the brain of fishes and retained requires investigation.
The total Cu from the CuO diet was much less digestible than that of CuSO4 in the simulated intestine region, as measured by the appearance of total Cu in the supernatant at pH 7.8 (Fig. 2). However, this apparent lower bioaccessibility of the nano form in the intestinal lumen conditions could be offset by other factors in vivo. For example, the dissolution of the CuO ENMs at circumneutral pH was accelerated by the presence of essential amino acids in the diet, cysteine and histidine.19 Thus overall, in vivo, the total metal accumulated from either CuO ENMs and CuSO4 are similar (Table 1). Interestingly, the gut sac studies of Boyle et al.19 also showed that the initial rates of total Cu uptake to the serosal compartment was slower for CuO ENMs compared to CuSO4. In the same study on gut sacs, the dissolution of the ENMs was also influenced by amino acids in the gut lumen. Clearly, the particle transformations in the gut lumen in the presence of complex mixtures of electrolytes, amino acids, gut enzymes and other colloids, and how this influences the uptake rate, requires further investigation. Transformations are also possible in the internal organs, since trout fed excess Cu as metal salts, show metal granules in the liver.35
Copper is an oxidising metal that also binds to –SH groups and so there are concerns for oxidative stress during exposures to dissolved Cu.9 In this regard, fish have biochemical defences; including the total GSH pool and MT (Fig. 3). The total GSH pool is a first line of defence against oxidative stress, with the reduced form of GSH as the anti-oxidant, although it is also a Cu carrier in the cytoplasm. The total GSH was unaffected in the gill, kidney and brain (Fig. 3A). The highest tissue total Cu concentrations in the present study were the hind intestine and the liver, in keeping with the route of exposure, and the two tissues showed some decreases in total GSH (∼40% in the liver, Fig. 3A). However, the pool was not depleted and there was no evidence of inflammation or oxidative damage in the organ histology (Fig. 4), indicating that the fish were moderating the effects of the exposure. Crucially, there were no differences in the total GSH response between the CuSO4 and the CuO ENMs exposures (Fig. 3), so no additional hazard from the nano form. El Basuini et al.13 also found that exposure to either 4 mg kg−1 dietary Cu as CuSO4 or Cu ENMs increased reactive oxygen metabolites in the blood plasma compared to unexposed controls, but there was no material-type effect. Wang et al.14 made similar observations for total antioxidant capacity in the livers of Russian sturgeon.
There were no effects on the MT concentrations in the organs, except for some induction of MT in the hind intestine compared to unexposed controls for both the CuSO4 and CuO ENM treatments (Fig. 3B). Again, there was no difference between the latter, implying no additional hazard from the nano form. The increase in MT is consistent with its role as a metal chelator, and in the hind intestine where higher total Cu concentrations were observed (Table 1). Some MT induction is expected in the intestine and liver of trout during dietary exposure to CuSO4.11 In the present study, the latter organ showed no exposure-dependent increases in MT, perhaps because the liver also has the ability to chelate Cu in copper–sulphur rich particles,35 or a sufficient threshold of intracellular dissolved Cu was not reached in the liver cells. Regardless, it is curious that CuO ENMs also induce MT, albeit only in the hind intestine (Fig. 3B). This implies that some dissolved Cu is generated inside the intestinal tissue by dissolution of the particles, or from uptake of dissolved Cu via particle dissolution in the lumen. This aspect that requires further investigation, but at least one study showed increased expression of the genes for the epithelial Cu channel, Ctr1, and Cu-ATPase in Caco-2 cells during exposures to CuO materials.38
During aqueous exposures, dissolved Cu interferes with osmoregulation and Na+ homeostasis via the gill.39 This is not observed in dietary exposures to CuSO4, because the gills are not directly exposed to dissolved Cu and so plasma electrolytes are largely unaffected.11 Similarly, in the present study, there were no effects of either CuSO4 or CuO ENM exposures on plasma ions (Table 2). There were some transient decreases on Na+ and/or K+ in the kidney and hind intestine, but these are likely not of physiological importance because electrolyte homeostasis of the plasma was maintained. Similar observation were made for dietary Ag and Ag ENMs in trout.6 So it seems that, like CuSO4, dietary exposure to the nano form of Cu is not an osmoregulatory hazard to trout.
Finally, a tiered approach to bioaccumulation testing has been proposed,2 with an ‘in vitro’ tier applying the gut sac technique19 in combination with the in chemico digestibility assay used here. The digestibility assay showed that both forms were bioaccessible at stomach pH, less so for the CuO EMNs at the neutral pH of the intestine (i.e., CuSO4 ≥ CuO ENMs). The gut sac study on the same material used here19 showed marginally less total Cu accumulation in the muscularis from the nano form (i.e., CuSO4 > CuO ENMs). Both those data would predict that the dietary hazard of the nano form is not greater than the metal salt. This is indeed the case, with total Cu bioaccumulation in vivo being largely the same in the present study (i.e., CuSO4 = CuO ENMs). This tiered approach has also been applied to Ag ENMs, where the metal salt is also protective of the nano forms (AgNO3 = Ag ENMs > Ag2S ENMs,6,21). Together, with the data here, this adds to the weight of evidence in support of a tiered approach to bioaccumulation testing that also seeks to minimise the use of vertebrate animals in vivo.2
Footnote |
† Visiting Professor, Department of Nutrition, Cihan University-Erbil, Erbil, Kurdistan Region, Iraq. |
This journal is © The Royal Society of Chemistry 2021 |