Accumulated Metal Concentrations Research Articles

Intensive marine finfish aquaculture impacts community structure and metal bioaccumulation in meso-zooplankton

Commercial aquaculture has a profound impact on coastal marine environments. Here, we investigate the spatial impact of intensive commercial finfish aquaculture on local meso-zooplankton communities and the bioaccumulation of aquaculturally-derived metals (and other elements) within zooplankton samples in the Vourlias Bay, Greece. The results indicate alterations to zooplankton community composition correlate with increased eutrophic compound concentrations in the water column in closer proximity to aquaculture stations (100–300 m from fish cages). During the summer sampling, higher concentrations of accumulated metals within zooplankton samples were found at reference stations furthest from fish cages (>1000 m). During the winter sampling, however, spatial differences in accumulated metal concentrations were limited. We suggest metals are rapidly accumulated at lower trophic levels near aquaculture stations and are then dispersed to greater distances while ascending the trophic chain. This research provides good evidence for future investigations into zooplankton as an environmental impact bioindicator for aquaculture.

Health Risk Assessment and Levels of Heavy Metals in Farmed Nile Tilapia (Oreochromis niloticus) from the Volta Basin of Ghana

Heavy metals (Pb, Cd, As, Mn, Fe, Zn, Cu, Ni, and Cr) are some of the most toxic elements that can bioaccumulate from sources linked to human activities, such as industry and agriculture. This study quantifies the concentrations of several heavy metals in caged tilapia found in Ghana’s Volta Basin and assesses the associated health risks. The levels of heavy metals in the tissues of Oreochromis niloticus from three cage farms (N = 52) were determined using Atomic Absorption Spectrometry (AAS). The implication for human health was assessed using several risk assessment techniques. Fe (50.11 ± 10.22 mg/kg) and Cr (0.31 ± 0.07 mg/kg) had the highest and lowest accumulated metal concentrations, respectively. Heavy metal concentrations in tilapia tissue from fish farms were ordered as follows: Fe > Mn > Zn > Ni > Cr (farm A), Fe > Zn > Ni > Mn (farm B), and Fe > Mn > Zn > Ni > Cr (farm C). All metals had an estimated daily intake (EDI) below the threshold, and mean differences between sample farms were not statistically significant. Similarly, the values of target hazard quotients (HQs) and hazard indices (HIs) were less than one. According to the risk assessment results, eating tilapia from farms posed no risk to human health. The presence of Mn, Fe, and Ni concentrations above the maximum level in the fish, on the other hand, suggests that they may affect fish health.

Brain region- and metal-specific effects of embedded metals in a shrapnel wound model in the rat

The health effects of prolonged exposure to embedded metal fragments, such as those found in shrapnel wounds sustained by an increasing number of military personnel, are not well known. As part of a large collaborative effort to expand this knowledge, we use an animal model of shrapnel wounds originally developed to investigate effects of embedded depleted uranium to investigate effects of military-relevant metals tungsten, nickel, cobalt, iron, copper, aluminum, lead, and depleted uranium compared to an inert control, tantalum. Rats are surgically implanted with pellets of one of the metals of interest in the gastrocnemius (leg) muscle and tracked until 1 month, 3 months, 6 months, or 12 months from the time of implant, at which point they are euthanized and multiple organs and tissue samples are collected for inspection. Here we focus on four regions of the brain: frontal cortex, hippocampus, amygdala, and cerebellum. We examined changes in accumulated metal concentration in each region as well as changes in expression of proteins related to blood brain barrier tight junction formation, occludin and ZO-1, and synapse function, PSD95, spinophilin, and synaptotagmin. We report few changes in metal accumulation or blood brain barrier protein expression, but a large number of synapse proteins have reduced expression levels, particularly within the first 6 months of exposure, but there are regional and metal-specific differences in effects.

Shedding the load: moulting as a cause of variability in whole-body metal concentrations

Abstract Moulting is a biological process shared by aquatic macroinvertebrates, but while the exoskeleton is believed to be a major sink of metal pollutants, the contribution of the moulting of the crustacean exoskeleton to total accumulated metal concentrations is insufficiently considered. We present a conceptual, qualitative model that illustrates the impact of moulting on the whole-body burden of an unspecified metal analyte in a hypothetical moulting invertebrate. The model demonstrates that moult stage is a contributor to the whole-body pollutant concentration, and that this introduces a temporal component even in steady-state exposure conditions. The applicability of this model is illustrated by comparison to published results of pre- and post-moult accumulations. A solution for reducing this variability in the measurement of whole-body metal concentrations is presented, and its potential application to both ex-situ and in-situ studies of biomonitor species is discussed.

Heavy metals concentration and target hazard quotients assessment through the consumption of fish muscle Ctenopharyngodon Idella (Cyprinidae) from markets in Ahvaz province, Iran

Purpose Heavy metals constitute one of the most hazardous substances that could be accumulated in biota coming from human activities such as agricultural and industrial wastes. This paper aims to study the levels of some of the most common heavy metals in muscles of the fish caught in the Karun River (Ahvaz, Iran) and to assess the health risks associated with their consumption. Design/methodology/approach In present study, the concentrations of three heavy metals (Pb, Cd and Cu) in the muscle of highly consumed fish species Ctenopharyngodon Idella (Cyprinidae) collected from three selected markets (Khoram Kooshk, Ahvaz-e Qadim and Padad Shahr; total number of samples was 45) in Ahvaz city and human health risk associated with their consumption were detected by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Findings The results indicated that the highest and lowest accumulated metal concentrations were related to Cu (20.36 ± 0.84) and Cd (0.53 ± 0.02), respectively. In addition, the mean difference for Pb and Cu across two pairwise of sampling sites were significant (Ahvaz-e Qadim and Khoram Kooshk, P for Pb = 0.025, P for Cu = 0.033; Ahvaz-e Qadim and Padad Shahr, p for Pb = 0.018, p for Cu = 0.039). The estimated daily intake of all metals in muscles of fish in this study were below the legal limit, and mean differences were not statistically significant among the sampling sites, except for Cu (Ahvaz-e Qadim and Khoram Kooshk, p = 0.048; Ahvaz-e Qadim and Padad Shahr, p = 0.041). Target hazard quotient values were also all below 1. Originality/value It can be concluded that the fishes from Ahvaz city markets are comparatively clean and do not constitute a risk for human health.

Biomonitoring of trace metals using transplanted mussels, Mytilus galloprovincialis, in coastal areas around Ulsan and Onsan Bays, Korea

Mediterranean (blue) mussels (Mytilus galloprovincialis) collected from a reference site were transplanted to 15 stations in coastal areas around Ulsan and Onsan Bays, an extensively metal polluted area in Korean coastal waters, to assess metal contamination in the coastal oceans of Korea. During the biomonitoring periods (June 30 to July 20, 2003; 21 days), transplanted mussels, seawater, and particulate materials were collected for analysis of 15 metals (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, Se, Sn, and Zn). Transplanted mussels showed metal enrichment compared to initial concentrations and spatial gradients consistent with dissolved and/or particulate metal concentrations in seawaters. Based on Q mode factor analysis, stations were clustered into three groups. The first group, located on Onsan Bay, showed high Ag, Cd, Cu, Hg, Pb, Sb and Zn enrichment, presumably arising from non-ferrous metal refineries and chemical industries in this area. The second group was located near the mouth of the Oehwang River and was enriched in Co from petrochemical industries. The third group comprised a site intermediate between Group 1 and Group 2, an isolated station with independent metal sources located in Jangsaengpo harbor, where a number of ship repairing and building companies operate, and a less contaminated station near a small fishing village. Metal accumulation rates (%·day-1) in mussels were estimated to be between 8% (Cr) and 281% (Pb), based on accumulated metal concentrations over 21 days. The active biomonitoring technique using M. galloprovincialis demonstrated here is a useful monitoring method because it reflects the present status of seawaters; furthermore, physiological factors can be standardized, and bioavailable and time-integrated metal concentrations can be obtained. Furthermore, this method can be applied even in coastal seawaters so heavily contaminated that living organisms would not normally survive.

Physiological and cellular responses of oysters (Crassostrea hongkongensis) in a multimetal-contaminated estuary.

The Pearl River estuary, southern China, suffers from multiple sources of metal contamination as a result of the rapid industrial development in the region; but the biological impacts of contamination remain unknown. In the present study, a euryhaline oyster, Crassostrea hongkongensis, was collected from different sites of the Pearl River estuary; and various physiological (heart rate, alkaline phosphatase as homeostatic regulation, and glycogen as energy reserve) and cytological (lysosomal membrane stability) biomarkers were quantified to assess this species as a potential bioindicator of metal pollution in contaminated areas. Large variations of metal accumulation levels in the oysters were documented, especially for copper (Cu), zinc (Zn), cadmium (Cd), chromium, and nickel (Ni). Among these metals, the authors demonstrated significant correlations between the digestive gland metal accumulation of Cu, Zn, and Ni and the cellular homeostasis (alkaline phosphatase) and glycogen reserves. Heart rate was positively correlated with Cd but negatively correlated with Cu and Zn concentrations in the gills. Lysosomal membrane stability was significantly inhibited at the most contaminated sites but had no relationship with the accumulated metal concentrations. These measurements indicate that multimetal contamination in the Pearl River estuary impacts the physiological and cytological performance of oysters. Environ Toxicol Chem 2016;35:2577-2586. © 2016 SETAC.

Estimation of target hazard quotients for heavy metals intake through the consumption of fish from Sirvan River in Kermanshah Province, Iran

The aim of this research was to investigate concentrations of cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), and zinc (Zn) in the muscle, gill, and liver of common carp ( Cyprinus carpio ), tuwini ( Capoeta trutta ), and Grass carp ( Ctenopharyngodon idella ) from Sirvan River, Kermanshah Province, Iran, during November to December 2014. This investigation was conducted in order to determine the potential health risk of the intake of these metals through the consumption of the edible parts of fish and also to assess the safe dietary intake levels of these metals. The results of the present study indicated that the highest and lowest accumulated metal concentrations were related to Zn and Cd, respectively. Moreover, the metal concentrations in the gill and liver were higher than in the muscles of the three fish species. The target hazard quotients (THQs) for an adult with mean body weight of 71.5 kg were below 1 based on Cd, Pb, Cr, Cu, and Zn levels. In conclusion, the obtained results indicated that the levels of metals in the edible muscle of fish species in this study were below the level of concern for human consumption.

Accumulation and Translocation of Metals in Soil and Different Parts of French Bean (Phaseolus vulgaris L.) Amended with Sewage Sludge

A pot experiment was conducted to study the accumulation and translocation of metals in French bean (Phaseolus vulgaris L.). Plants were grown in soil amended with up to 100 % sewage sludge. Significant (p < 0.01) changes to soil characteristics were observed. The maximum growth of P. vulgaris was noted in the treatment with 40 % of sewage sludge. Metal concentrations were significantly (p < 0.05) higher in P. vulgaris after sewage sludge amendment where Fe > Zn > Cd > Cu > Cr > Pb. The translocation for Fe and Zn was in the order of leaves > shoot > root > fruits, for Cd, shoot > root > leaves > fruits, for Cu and Pb shoot > leaves > root > fruits and for Cr root > shoot > leaves > fruits of P. vulgaris. All accumulated metal concentrations except Cd in the fruit were below the FAO/WHO standard limits. Thus, the amendment of agricultural soil by sewage sludge might be feasible. However, a regular monitoring of metal levels in agricultural products is recommended to prevent their accumulation in the food chain.

Metal accumulation in wild nine-banded armadillos

Nine-banded armadillos (Dasypus novemcinctus) are widespread and abundant New World mammals with a lifestyle that entails prolonged, intimate contact with soils. Thus, armadillos would seem a promising candidate as a sentinel species to monitor chemical contamination in terrestrial ecosystems. Surprisingly, there have been virtually no toxicology studies on armadillos. Here, we provide the first analysis of metal contaminants for wild armadillos. Liver tissues were obtained from 302 armadillos collected at 6 sites in Georgia and Florida, USA that varied in their extent of human disturbance, from rural pine plantations to highly modified military/space installations. Data were stratified by age (juvenile and adult), sex, and site. Temporal (yearly) variation was examined at two of the sites that were sampled over three consecutive years. Concentrations of aluminum, cadmium, copper, nickel, lead, and zinc were measured in liver samples from each site. Although reference levels are not available for armadillos, accumulated metal concentrations were comparable to those reported for other mammals. We found no evidence of sex or age differences in the concentrations of any metal, except for Cd (age) and Pb (sex and age). However, concentrations of most metals varied substantially across sites and over time. Finally, concentrations of many metals were positively correlated with one another, suggesting that they likely co-occurred in some areas. Collectively, this study indicates the utility of armadillos as a sentinel species for studies of metal contamination in terrestrial systems, and highlights the need for further studies of other toxicants in these animals.

Metal toxicity, uptake and bioaccumulation in aquatic invertebrates—Modelling zinc in crustaceans

We use published data on the different patterns of the bioaccumulation of zinc by three crustaceans, the caridean decapod Palaemon elegans, the amphipod Orchestia gammarellus and the barnacle Amphibalanus amphitrite, to construct comparative biodynamic models of the bioaccumulation of zinc into metabolically available and detoxified components of accumulated zinc in each crustacean under both field and laboratory toxicity test conditions. We then link these bioaccumulation models to the onset of toxic effects on exposure of the crustaceans to high dissolved zinc bioavailabilities, using the tenets that toxicity effects are related to the total uptake rate of the toxic metal, and that toxicity is not usually dependent on the total accumulated metal concentration but always on the concentration of accumulated metal that is metabolically available. We dismiss the general concept that there is a critical accumulated body concentration of a metal in an invertebrate at which toxicity ensues, except under specific circumstances involving a rare lack of storage detoxification of accumulated metal. We thus propose a theoretical framework that can be extended to other metals and other aquatic invertebrates (indeed other animals) to explain the variation in the relationship between bioaccumulated body concentrations and toxicity, and subsequently to predict this relationship in many other species for which we have bioaccumulation modelling data.

Biomonitoring of Trace Metals (Fe, Cu, and Ni) in the Mangrove Area of Peninsular Malaysia Using Different Soft Tissues of Flat Tree Oyster Isognomon alatus

It is well documented that marine oysters are net bioaccumulators of trace metals and they can be employed as biomonitors of time-integrated measurements of bioavailable metal fractions over their lifetime. In this study, the Malaysian mangrove flat tree oyster Isognomon alatus collected from the estuaries of Lukut River (five sites), Sepang Besar River (one site), and one metal-polluted site at Kg. Pasir Puteh were dissected into muscle, mantle plus gills, byssus, and remaining soft tissues. All the four different soft tissues were analyzed for Fe, Cu, and Ni. Significant spatial differences in the accumulated metal concentrations of the oysters were found between sampling sites, and these could be attributed to anthropogenic inputs including discharges of shrimp ponds (Lukut), animal husbandry (Sepang Besar), sewage, shipping, and industrial effluents (Kg. Pasir Puteh). For Fe, the tissue distribution followed: byssus > mantle plus gill >muscle > remaining soft tissues, while for Cu and Ni, both followed byssus > remaining soft tissues > mantle plus gill > muscle. This indicated that byssus could act as an excretion route for the metals. Based on a correlation analysis between oyster tissues and sediments, selected tissues of I. alatus were shown to be good biomonitors of Ni and Cu contamination, while high regulative capacity of Fe in the oyster tissues could hardly reflect the actual Fe levels in the surroundings. However, the clustering patterns based on metal levels in the four different soft tissues were not in agreement with those based on the geochemical fractions of sediment data. This phenomenon which was due to the metal contamination might not necessarily create high bioavailabilities of metals to the biomonitor I. alatus, which might involve differences in uptake, excretion, and sequestration of metals. Similarly to the Mussel Watch approach, this study points to the very potential use of the different soft tissues of I. alatus as biomonitors for regular biomonitoring in the mangrove area of Peninsular Malaysia.

Metal toxicity in a sediment-dwelling polychaete: Threshold body concentrations or overwhelming accumulation rates?

We followed the net accumulation of As, Cu and Zn in the deposit-feeding polychaete Arenicola marina exposed in the laboratory to natural metal-contaminated sediments, one exposure leading to mass mortality between day 10 and 20, and the other not causing lethality over a period of 60 days of exposure. The worms showed lower total accumulated metal concentrations just before mortality occurred (<20 days) at the lethal exposure, than after 30 days of exposure to sediments not causing mortality. Moreover rates of accumulation of As, Cu and Zn were significantly higher in the lethal exposure than in the sublethal exposure. Our results show that it is not possible to link mortality to a critical total body concentration, and we add to a growing body of literature indicating that metal toxicity occurs when organisms cannot cope with overwhelming influx and subsequent accumulation rates.

Heavy metal concentrations and toxicity in water and sediment from stormwater ponds and sedimentation tanks

Sedimentation is a widely used technique in structural best management practices to remove pollutants from stormwater. However, concerns have been expressed about the environmental impacts that may be exerted by the trapped pollutants. This study has concentrated on stormwater ponds and sedimentation tanks and reports on the accumulated metal concentrations (Cd, Cr, Ni, Pb, and Zn) and the associated toxicity to the bacteria Vibrio fischeri. The metal concentrations are compared with guidelines and the toxicity results are assessed in relation to samples for which metal concentrations either exceed or conform to these values. The water phase metal concentrations were highest in the ponds whereas the sedimentation tanks exhibited a distinct decrease towards the outlet. However, none of the water samples demonstrated toxicity even though the concentrations of Cu, Pb, and Zn exceeded the threshold values for the compared guidelines. The facilities with higher traffic intensities had elevated sediment concentrations of Cr, Cu, Ni, and Zn which increased towards the outlet for the sedimentation tanks in agreement with the highest percentage of fine particles. The sediments in both treatment facilities exhibited the expected toxic responses in line with their affinity for heavy metals but the role of organic carbon content is highlighted.

The effect of application time of mobilising agents on growth and phytoextraction of lead by Brassica napus from a calcareous mine soil

Phytoremediation of heavy metal contaminated sites is often limited by the low-bioavailability of the contaminants. Complexing agents can help to improve this technique by enhancing heavy metal solubility. Pot experiments were conducted to determine the best time for the application of ethylene diamine tetraacetic acid (EDTA) and sheep manure extract (SME) for phytoremediation of a contaminated soil by Brassica napus. The plant was grown on a mine calcareous soil treated with increasing concentrations of EDTA or SME 30 and 10 days before sowing (T1 and T2) and 10 and 30 days after sowing (T3 and T4). Soil available Pb and lead concentrations in plant organs of Brassica napus increased with EDTA concentration. The EDTA application before seed germination significantly reduced rapeseed seedling emergence and dry weight. However, the actual amount of phytoextracted Pb by rapeseed did not decrease significantly, due to severe growth depression, at high EDTA concentrations when it had applied at a suitable time (T4). SME application after sowing increased plant dry weight and Pb concentration in the soil solution and enhanced the accumulated metal concentrations in shoots and roots. Comparing the effect of EDTA and SME on Pb phytoextraction, the study showed that SME is not more effective than EDTA. The efficiency of EDTA on Pb phytoextraction by Brassica napus depends on the time of application. The most efficient treatment for Pb phytoextraction by rapeseed was application of 2 g EDTA/kg soil at T4.

Metal and metallothionein content in bullfrogs: Study of a whole watershed impacted by agricultural activities

Among the various anthropogenic activities potentially affecting amphibian populations, agriculture has often been evoked. Fertilizers used in agriculture are documented sources of metals that may contaminate nearby rivers. Bullfrogs ( Rana catesbeiana) were collected within the Yamaska River basin (Quebec, Canada) along a gradient of agricultural land use intensity. Hepatic, renal and intestinal metal content, along with hepatic metallothionein content, were determined. In general, dissolved metal concentrations and accumulated metal concentrations in bullfrogs varied little among sites. Hepatic Cu and Zn concentrations were significantly different among subwatersheds and were in accordance with the gradient in agricultural activities in at least one of the three years studied. Liver metallothionein content did not vary significantly among the subwatersheds, but they were correlated with Cu content within two sites and Cd content at one site. We conclude that R. catesbeiana could be used as a biomonitor for Cu contamination and potentially for other metals.

Comparison of trace metal bioavailabilities in European coastal waters using mussels from Mytilus edulis complex as biomonitors

Mussels from Mytilus edulis complex were used as biomonitors of the trace metals Fe, Mn, Pb, Zn, and Cu at 17 sampling sites to assess the relative bioavailability of metals in coastal waters around the European continent. Because accumulated metal concentrations in a given area can differ temporally, data were corrected for the effect of season before large-scale spatial comparisons were made. The highest concentration of Fe was noted in the North Sea and of Mn in the Baltic. Increased tissue concentrations of Pb were recorded in the mussels from the Bay of Biscay and the Baltic Sea. Low concentrations of metals were determined in the mussels from the Mediterranean Sea and the Northern Baltic. Relatively low geographic variations of Cu and Zn indicate that mussels are able to partially regulate accumulated body concentrations, which means Cu and Zn are, to some extent, independent of environmental concentrations.

Differences in metal sequestration between zebra mussels from clean and polluted field locations

Organisms are able to detoxify accumulated metals by, e.g. binding them to metallothionein (MT) and/or sequestering them in metal-rich granules (MRG). The different factors involved in determining the capacity or efficiency with which metals are detoxified are not yet known. In this work we studied how the sub-cellular distribution pattern of cadmium, copper and zinc in whole tissue of zebra mussels from clean and polluted surface waters is influenced by the total accumulated metal concentration and by its physiological condition. Additionally we measured the metallothionein concentration in the mussel tissue. Metal concentration increased gradually in the metal-sensitive and detoxified sub-cellular fractions with increasing whole tissue concentrations. However, metal concentrations in the sensitive fractions did not increase to the same extent as metal concentrations in whole tissues. In more polluted mussels the contribution of MRG and MT became more important. Nevertheless, metal detoxification was not sufficient to prevent metal binding to heat-sensitive low molecular weight proteins (HDP fraction). Finally we found an indication that metal detoxification was influenced by the condition of the zebra mussels. MT content could be explained for up to 83% by variations in Zn concentration and physiological condition of the mussels.

Phytoextraction of lead by Helianthus annuus: effect of mobilising agent application time

Pot experiments were conducted to determine the best time for application of (ethylenediaminetetraacetic acid) EDTA and sheep manure extract (SME) in phytoremediation of a contaminated soil by &lt;I&gt;Helianthus annuus&lt;/I&gt;. The plant was grown in a mine calcareous soil treated with increasing concentrations of EDTA or SME in 30 and 10 days before sowing (T1 and T2) and 10 and 30 days after sowing (T3 and T4). The best time for EDTA application was T4. The EDTA application before seed germination significantly reduced sunflower seedling emergence and dry weight. Soil available Pb and lead concentrations in plant organs increased with EDTA concentration but the actual amount of phytoextracted Pb decreased at high EDTA concentrations significantly, due to severe growth depression. SME application after sowing can increase plant dry weight and Pb concentration in the soil solution, enhancing the accumulated metal concentrations in shoots and roots. However the results showed that the most efficient treatments for Pb phytoextraction by sunflower are applications of 0.5 and 2 g EDTA/kg soil at T3 and T4, respectively.

Metallothionein gene and protein expression as a biomarker for metal pollution in natural gudgeon populations

Gudgeons ( Gobio gobio) from historically Cd and Zn contaminated sites in Flanders (Belgium) were found to be resistant to elevated Cd levels. In previous work, this increased resistance was largely explained by increased metallothionein (MT) expression. Recently, environmental cleanup efforts resulted in a significant decrease in Cd concentrations in the surface water. In this study, we evaluated the use of hepatic metal and metallothionein (MT) concentrations as biomarkers of metal exposure before and after the cleanup. Hepatic MT mRNA levels were determined after the environmental metal levels decreased in order to assess the applicability of MT gene expression as an environmental biomarker in natural fish populations. Our data show that both metallothionein protein and gene expression have the potential to be sensitive biomarkers for metal exposure. Significant correlations were found (a) among accumulated metal concentrations and both MT protein and mRNA levels, and (b) between MT protein and mRNA levels. However, our data illustrated that while MT protein and gene expression give a quantitative picture of metal load at a single time point, quantitative information in natural populations cannot always be obtained when different time points (including different years) are compared, since MT gene and protein expression are affected by many other factors in addition to the metal load. Furthermore, the result of the environmental cleanup was reflected in a decrease of hepatic Cd concentrations. Zn remained the most important factor determining MT concentrations. Finally, two differently sized MT mRNAs were amplified to test the hypothesis that 3′-UTR length can offer a protective advantage in conditions of environmental stress. Our data provided no evidence to support this hypothesis. In contrast, the ratio of the long mRNA variant relative to total MT mRNA was surprisingly constant, and independent of exposure history.