Combination of in silico and molecular techniques for discrimination and virulence characterization of marine Brucella ceti and Brucella pinnipedialis.

Introduction: Mammals are the main hosts for Brucella sp., agents of worldwide zoonosis. Marine cetaceans and pinnipeds can be infected by Brucella ceti and B. pinnipedialis, respectively. Besides classical bacteriological typing, molecular approaches such as MLVA, MLSA, and whole-genome sequencing (WGS) can differentiate these species but are cumbersome to perform. Methods: We compared the DNA and genome sequences of 12 strains isolated from nine marine mammals, with highly zoonotic B. melitensis, B. abortus, and B. suis, and the publicly available genomes of B. ceti and B. pinnipedialis. In silico pipelines were used to detect the antimicrobial resistance (AMR), plasmid, and virulence genes (VGs) by screening six open-source and one home-made library. Results and discussion: Our results show that easier-to-use HRM-PCR, Bruce-ladder, and Suis-ladder can separate marine Brucella sp., and the results are fully concordant with other molecular methods, such as WGS. However, the restriction fragment length polymorphism (RFLP) method cannot discriminate between B. pinnipedialis and B. ceti B1-94-like isolates. MLVA-16 results divided the investigated strains into three clades according to their preferred host, which was confirmed in WGS. In silico analysis did not find any AMR and plasmid genes, suggesting antimicrobial susceptibility of marine Brucella, while the presence of the VGs btpA gene was variable dependent on the clade. Conclusion: The HRM-PCR and Suis-ladder are quick, easy, and cost-effective methods to identify marine Brucella sp. Moreover, in silico genome analyses can give useful insights into the genetic virulence and pathogenicity potential of marine Brucella strains.

Legacy persistent organic pollutants among multiple cetacean species in the Northwest Atlantic.

The historical contamination of eastern Canadian shelf waters remains an ongoing concern, predominantly stemming from industrial and anthropogenic discharges in the Great Lakes region. Although legacy persistent organic pollutants (POPs) were banned decades ago, it remains unclear whether their concentrations have sufficiently decreased to safer levels in cetaceans that feed in the continental shelf waters of the northwestern Atlantic. This study compares polychlorinated biphenyl (PCB) and organochlorine pesticide (OC) accumulation in six cetacean species sampled in the Northwest Atlantic from 2015 to 2022. It assesses the influence of relative trophic level and foraging habitat preferences on POP concentrations using stable isotopes and fatty acids as dietary tracers to identify the drivers of differences in POP concentrations among species. It further identifies the species most susceptible to the effects of these contaminants. Killer whales (Orcinus orca) exhibited the highest PCB (~100 mg/kg lw) and OC concentrations, followed by other odontocetes, with lowest concentrations in mysticetes. Stable isotope analysis revealed an unexpected lack of correlation between δ15N values and contaminant levels. However, there was a positive correlation between δ13C values and POP concentrations. Cetaceans foraging on pelagic prey species, as indicated by elevated proportions of the FA markers 22:1n11 and 20:1n9, had lower contaminant loads compared to cetaceans with benthic/coastal FA signatures. PCB and DDT (dichlorodiphenyltrichloroethane) concentrations are lower now in most cetacean species than in the 1980s and 1990s, likely due to regulatory measures restricting their production and use. Although current PCB concentrations for most species are under the thresholds for high risks of immune and reproductive failure, concentrations in killer whales exceed all established toxicity thresholds, underscoring the need for further action to reduce sources of these contaminants to the continental shelf waters of the northwestern Atlantic.

Convergent evolution in high-altitude and marine mammals: Molecular adaptations to pulmonary fibrosis and hypoxia.

High-altitude and marine mammals inhabit distinct ecosystems but share a common challenge: hypoxia. To survive in low-oxygen environments, these species have evolved similar phenotypic pulmonary adaptations, characterized by a high density of elastic fibers. In this study, we explored the molecular mechanisms underlying these adaptations, focusing on pulmonary fibrosis and hypoxia tolerance through comparative genomics and convergent evolution analyses. We observed significant expansions and contractions in certain gene families across both high-altitude and marine mammals, closely associated with processes involved in pulmonary fibrosis. Notably, members of the keratin gene family, such as KRT17 and KRT14, appear to be associated with the development of the dense elastic fiber phenotype observed in the lungs of hypoxia-tolerant mammals. Through selection pressure and amino acid substitution analyses, we identified multiple genes exhibiting convergent accelerated evolution, positive selection, and amino acid substitution in these species, associated with adaptation to hypoxic environments. Specifically, the convergent evolution of ZFP36L1, FN1, and NEDD9 was found to contribute to the high density of elastic fibers in the lungs of both high-altitude and marine mammals, facilitating their hypoxia tolerance. Additionally, we identified convergent amino acid substitutions and gene loss events associated with sperm development, differentiation, and spermatogenesis, such as amino acid substitutions in SLC26A3 and pseudogenization of CFAP47, as confirmed by PCR. These genetic alterations may be linked to changes in the reproductive capabilities of these animals. Overall, this study offers novel perspectives on the genetic and molecular adaptations of high-altitude and marine mammals to hypoxic environments, with a particular emphasis on pulmonary fibrosis.

Viral metagenomic survey of Caspian seals.

Introduction: Viral diseases of pinnipeds cause substantial mortality and morbidity and can influence population demography. Viral metagenomic studies can therefore play an important role in pinniped health assessments and disease surveillance relevant to both individual species and in a "One Health" context. Methods: This study used a metagenomic approach with high throughput sequencing to make the first assessment of viral diversity in Caspian seals (Pusa caspica), the only marine mammal species endemic to the Caspian Sea. Results: Sequencing libraries from 35 seals sampled 2009-2020 were analysed, finding sequences from the viral families Circoviridae, Parvoviridae, Herpesviridae, Papillomaviridae, Picornaviridae, Caliciviridae, Cruciviridae, Anelloviridae, Smacoviridae, and Orthomyxoviridae, with additional detection of Adenoviridae via PCR. The similarity of viral contigs from Caspian seal to sequences recovered from other pinnipeds ranged from 63.74% (San Miguel sea lion calicivirus) to 78.79% (Seal anellovirus 4). Discussion: Some findings represent novel viral species, but overall, the viral repertoire of Caspian seals is similar to available viromes from other pinnipeds. Among the sequences recovered were partial contigs for influenza B, representing only the second such molecular identification in marine mammals. This work provides a foundation for further studies of viral communities in Caspian seals, the diversity of viromes in pinnipeds more generally, and contributes data relevant for disease risk assessments in marine mammals.

Biomagnification potential and health risks of organophosphate esters in prey to humpback dolphins based on dietary correction.

Information on the biomagnification of organophosphate esters (OPEs) is limited, and the results are inconclusive, mainly because precise predatorprey relationships have not been determined. Herein, we first evaluated the biomagnification potential and dietary exposure risk of 15 OPEs in 14 prey species (n = 234) to Indo-Pacific humpback dolphins from the northern South China Sea using quantitative fatty acid signature analysis (QFASA). QFASA identified Chinese gizzard shad as the primary prey of dolphins. Among the 15 OPEs, 86.7 % (13/15) had a diet-adjusted biomagnification factor (BMFQFASA) greater than 1, indicating the biomagnification potential between dolphins and their diet. Moreover, BMFQFASA exhibited a considerable positive correlation with the log octanolwater partitioning coefficient of OPEs, indicating that lipophilicity may affect the bioamplification of OPEs. Risk assessments showed that although current OPE levels may not pose substantial health risks to dolphins via diet intake, the nondiet-adjusted hazard quotient/hazard index underestimated the exposure risk of OPEs to this vulnerable dolphin species. This study provides novel evidence regarding the biomagnification and dietary exposure risks of OPEs in cetaceans, emphasizing the importance of estimating the dietary composition of predators in such analyses.

Anatomical description of a pygmy sperm whale, Kogia breviceps (Cetacea: Kogiidae), pre-term calf using CT scan and 3D reconstructions.

Little is known about the biology of pygmy sperm whales, Kogia breviceps (De Blainville, 1838), being that most anatomical descriptions for the species derive from necropsy after stranding or from osteological material preserved in museums. This species is rarely seen despite its wide distribution, and its reproductive behaviour is still being investigated. The eventual occurrence of pregnant female strandings and the collection and description of foetuses can give clues about the organisms' mostly unknown early development. However, this type of biological material is extremely rare, limiting anatomical analysis due to the risk of damage or loss. Here, we describe the external and internal anatomy of an 84 cm long K. breviceps foetus. The methods utilised were non-intrusive, meaning that no incisions were made on the specimen. The foetus was analysed using computed tomography images and a three-dimensional reconstruction of the skeleton. A great number of features were observed, such as axial and appendicular skeletal structures, internal organs, echolocation apparatus and umbilical cord, as well as diagnostic characters of the species, such as the asymmetrical skull, spermaceti chamber and false gill pigmentation. We suggest that more specimens on different stages of development should be analysed by the same technique, as well as further comparison with specimens from other taxa, in order to facilitate more comparative studies on embryonic and foetal development of cetaceans.

Central Nervous System Disorders of Marine Mammals: Models for Human Disease?

This article deals with Central Nervous System (CNS) disorders of marine mammals as putative neuropathology and neuropathogenesis models for their human and, to some extent, their animal "counterparts" in a dual "One Health" and "Translational Medicine" perspective. Within this challenging context, special emphasis is placed upon Alzheimer's disease (AD), provided that AD-like pathological changes have been reported in the brain tissue of stranded cetacean specimens belonging to different Odontocete species. Further examples of potential comparative pathology interest are represented by viral infections and, in particular, by "Subacute Sclerosing Panencephalitis" (SSPE), a rare neurologic sequela in patients infected with Measles virus (MeV). Indeed, Cetacean morbillivirus (CeMV)-infected striped dolphins (Stenella coeruleoalba) may also develop a "brain-only" form of CeMV infection, sharing neuropathological similarities with SSPE. Within this framework, the global threat of the A(H5N1) avian influenza virus is another major concern issue, with a severe meningoencephalitis occurring in affected pinnipeds and cetaceans, similarly to what is seen in human beings. Finally, the role of Brucella ceti-infected, neurobrucellosis-affected cetaceans as putative neuropathology and neuropathogenesis models for their human disease counterparts is also analyzed and discussed. Notwithstanding the above, much more work is needed before drawing the conclusion marine mammal CNS disorders mirror their human "analogues".

Characterizing offshore polar ocean soundscapes using ecoacoustic intensity and diversity metrics.

Polar offshore environments are considered the last pristine soundscapes, but accelerating climate change and increasing human activity threaten their integrity. In order to assess the acoustic state of polar oceans, there is the need to investigate their soundscape characteristics more holistically. We apply a set of 14 ecoacoustic metrics (EAMs) to identify which metrics are best suited to reflect the characteristics of disturbed and naturally intact polar offshore soundscapes. We used two soundscape datasets: (i) the Arctic eastern Fram Strait (FS), which is already impacted by anthropogenic noise, and (ii) the quasi-pristine Antarctic Weddell Sea (WS). Our results show that EAMs when applied in concert can be used to quantitatively assess soundscape variability, enabling the appraisal of marine soundscapes over broad spatiotemporal scales. The tested set of EAMs was able to show that the eastern FS, which is virtually free from sea ice, lacks seasonal soundscape dynamics and exhibits low acoustic complexity owing to year-round wind-mediated sounds and anthropogenic noise. By contrast, the WS exhibits pronounced seasonal soundscape dynamics with greater soundscape heterogeneity driven in large part by the vocal activity of marine mammal communities, whose composition in turn varies with the prevailing seasonal sea ice conditions.

Persistent plastic: Insights from seawater weathering and simulated whale gut.

Single-use plastics make up 60-95 % of marine plastic pollution, including common commodity films used for packaging and bags. Plastic film breaks down as a function of environmental variables like wave action, wind, temperature, and UV radiation. Here, we focus on how films degrade in cold waters across depths, time, and simulated mammal digestion. Five types of single-use film plastics (HDPE thin & thick, LDPE, PP, PE) were weathered for eight months in temperate waters at surface and depth in the Salish Sea, WA, USA, and subsequently exposed to a laboratory-simulated gray whale stomach. None of the types of plastics examined here fully degraded during the course of this 8 months study. Weathering time and depth significantly impacted many of the physical attributes of plastics, while exposure to a simulated whale gut did not. If unable to degrade plastics through digestion, whales risk long-term exposure to physical and chemical attributes of plastics.

Traversing the land-sea interface: A climate change risk assessment of terrestrially breeding marine predators.

Terrestrially breeding marine predators have experienced shifts in species distribution, prey availability, breeding phenology, and population dynamics due to climate change worldwide. These central-place foragers are restricted within proximity of their breeding colonies during the breeding season, making them highly susceptible to any changes in both marine and terrestrial environments. While ecologists have developed risk assessments to evaluate climate risk in various contexts, these often overlook critical breeding biology data. To address this knowledge gap, we developed a trait-based risk assessment framework, focusing on the breeding season and applying it to marine predators breeding in parts of Australian territory and Antarctica. Our objectives were to quantify climate change risk, identify specific threats, and establish an adaptable assessment framework. The assessment considered 25 criteria related to three risk components: vulnerability, exposure, and hazard, while accounting for uncertainty. We employed a scoring system that integrated a systematic literature review and expert elicitation for the hazard criteria. Monte Carlo sensitivity analysis was conducted to identify key factors contributing to overall risk. We identified shy albatross (Thalassarche cauta), southern rockhopper penguins (Eudyptes chrysocome), Australian fur seals (Arctocephalus pusillus doriferus), and Australian sea lions (Neophoca cinerea) with high climate urgency. Species breeding in lower latitudes, as well as certain eared seal, albatross, and penguin species, were particularly at risk. Hazard and exposure explained the most variation in relative risk, outweighing vulnerability. Key climate hazards affecting most species include extreme weather events, changes in habitat suitability, and prey availability. We emphasise the need for further research, focusing on at-risk species, and filling knowledge gaps (less-studied hazards, and/or species) to provide a more accurate and robust climate change risk assessment. Our findings offer valuable insights for conservation efforts, given that monitoring and implementing climate adaptation strategies for land-dependent marine predators is more feasible during their breeding season.

First record of Orthohalarachneattenuata in Arctocephalusaustralis in mainland Argentina (Parasitiformes, Mesostigmata, Dermanyssoidea, Halarachnidae) with observations on its ambulacral morphology.

Pinniped respiratory mites of the species Orthohalarachneattenuata have been recorded from various locations around the world but not from continental Argentina. In the present work, we document for the first time the presence of O.attenuata on Arctocephalusaustralis on the Argentine mainland. A total of 23 adult and 381 immature mites were collected from the nose and nasopharyngeal cavity during a necropsy. The mite ambulacrum is described in adults and larvae. This structure consists of a pretarsus, an extensible pulvillum, a pair of claws and paradactyli (pretarsus opercula). The ambulacral structures also have some peculiarities, such as the presence of longitudinal furrows in the claws, straight claws in legs II and III (as opposed to curved in legs I and IV), and the fin-shaped paradactyli. The morphology of the ambulacrum of this mite is interpreted as an adaptation for anchoring to different surfaces of the host, and the protective structures present in the larvae as an adaptation for the dispersal phase in the external environment.

Immunolocalization of Two Neurotrophins, NGF and BDNF, in the Pancreas of the South American Sea Lion Otaria flavescens and Bottlenose Dolphin Tursiops truncatus.

In this study, we have investigated the immunolocalization of NGF (Nerve Growth Factor) and BDNF (Brain-Derived Neurotrophic Factor) in the pancreas of two species of marine mammals: Tursiops truncatus (common bottlenose dolphin), belonging to the order of the Artiodactyla, and Otaria flavescens (South American sea lion), belonging to the order of the Carnivora. Our results demonstrated a significant presence of NGF and BDNF in the pancreas of both species with a wide distribution pattern observed in the exocrine and endocrine components. We identified some differences that can be attributed to the different feeding habits of the two species, which possess a different morphological organization of the digestive system. Altogether, these preliminary observations open new perspectives on the function of neurotrophins and the adaptive mechanisms of marine mammals in the aquatic environment, suggesting potential parallels between the physiology of marine and terrestrial mammals.

eDNA-based survey of the marine vertebrate biodiversity off the west coast of Guadeloupe (French West Indies).

Background: In the marine environment, knowledge of biodiversity remains incomplete for many taxa, requiring assessments to understand and monitor biodiversity loss. Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring marine biodiversity, as it enables several taxa to be characterised simultaneously in a single sample. However, the data generated by environmental DNA metabarcoding are often not easily reusable. Implementing FAIR principles and standards for eDNA-derived data can facilitate data-sharing within the scientific community. New information: This study focuses on the detection of marine vertebrate biodiversity using eDNA metabarcoding on the leeward coast of Guadeloupe, a known hotspot for marine biodiversity in the French West Indies. Occurrences and DNA-derived data are shared here using DarwinCore standards combined with MIMARKS standards.

Pinnipeds and avian influenza: a global timeline and review of research on the impact of highly pathogenic avian influenza on pinniped populations with particular reference to the endangered Caspian seal (Pusa caspica).

This study reviews chronologically the international scientific and health management literature and resources relating to impacts of highly pathogenic avian influenza (HPAI) viruses on pinnipeds in order to reinforce strategies for the conservation of the endangered Caspian seal (Pusa caspica), currently under threat from the HPAI H5N1 subtype transmitted from infected avifauna which share its haul-out habitats. Many cases of mass pinniped deaths globally have occurred from HPAI spill-overs, and are attributed to infected sympatric aquatic avifauna. As the seasonal migrations of Caspian seals provide occasions for contact with viruses from infected migratory aquatic birds in many locations around the Caspian Sea, this poses a great challenge to seal conservation. These are thus critical locations for the surveillance of highly pathogenic influenza A viruses, whose future reassortments may present a pandemic threat to humans.

Suspect and non-target screening of chemicals of emerging Arctic concern in biota, air and human serum.

Contaminants of emerging concern receive increasing attention in the Arctic environment. The aim of this study was to screen for chemicals of emerging Arctic concern (CEACs) in different types of Arctic samples including biota, air and human serum. We used a combination of gas chromatography (GC) and liquid chromatography (LC) with high resolution mass spectrometry (HRMS) for suspect and non-target screening (NTS). Suspect screening of 25 CEACs was based on published in-silico approaches for the identification of CEACs and revealed tetrabromophthalic anhydride (TBPA) in pilot whale and air, albeit with low detection frequencies (17 and 33%, respectively). An NTS workflow detected 49, 42, 31 and 30 compounds in pilot whale, ringed seal, air, and human serum, respectively, at confidence level 2 and 3. Although legacy POPs still dominated the samples, 64 CEACs were tentatively identified and further assessed for persistence (P), bioaccumulation (B), mobility (M), toxicity (T), and long-range transport potential (LRTP). While four PBT compounds were identified, 37 PMT substances dominated among these 64 compounds. Our study indicated that many chemicals of potential risk might be present in Arctic samples and would benefit from confirmation and further studies of their transport to and accumulation in the Arctic environment.

Sex-specific diet differences in harbor seals (Phoca vitulina) via spatial assortment.

The lack of recovery of Chinook salmon (Oncorhynchus tshawytscha) in the Pacific Northwest has been blamed in part on predation by pinnipeds, particularly the harbor seal (Phoca vitulina). Previous work at a limited number of locations has shown that male seal diet contains more salmon than that of female seals and that sex ratios at haul-out sites differ spatiotemporally. This intrapopulation variation in predation may result in greater effects on salmon than suggested by models assuming equal spatial distribution and diet proportion. To address the generality of these patterns, we examined the sex ratios and diet of male and female harbor seals from 13 haul-out sites in the inland waters of Washington State and the province of British Columbia during 2012-2018. DNA metabarcoding was conducted to determine prey species proportions of individual scat samples. The sex of harbor seals was then determined from each scat matrix sample with the use of quantitative polymerase chain reaction (qPCR). We analyzed 2405 harbor seal scat samples using generalized linear mixed models (GLMMs) to examine the factors influencing harbor seal sex ratio at haul-out sites and permutational multivariate analysis of variance (PERMANOVA) to examine the influence of sex and haul-out site on harbor seal diet composition. We found that the overall sex ratio was 1:1.02 (female:male) with notable spatiotemporal variation. Salmoniformes were about 2.6 times more abundant in the diet of males than in the diet of females, and Chinook salmon comprised ca. three times more of the average male harbor seal's diet than the average female's diet. Based on site-specific sex ratios and diet data, we identified three haul-out sites where Chinook salmon appear to be under high predation pressure by male harbor seals: Cowichan Bay, Cutts Area, and Fraser River. Our study indicates that combining sex-specific pinniped diet data with the sex ratio of haul-out sites can help identify priority sites of conservation concern.

The buoyancy of cryptococcal cells and its implications for transport and persistence of Cryptococcus in aqueous environments.

Cryptococcus is a genus of saprophytic fungi with global distribution. Two species complexes, C. neoformans and C. gattii, pose health risks to humans and animals. Cryptococcal infections result from inhalation of aerosolized spores and/or desiccated yeasts from terrestrial reservoirs such as soil, trees, and avian guano. More recently, C. gattii has been implicated in infections in marine mammals, suggesting that inhalation of liquid droplets or aerosols from the air-water interface is also an important, yet understudied, mode of respiratory exposure. Water transport has also been suggested to play a role in the spread of C. gattii from tropical to temperate environments. However, the dynamics of fungal survival, persistence, and transport via water have not been fully studied. The size of the cryptococcal capsule was previously shown to reduce cell density and increase buoyancy. Here, we demonstrate that cell buoyancy is also impacted by the salinity of the media in which cells are suspended, with formation of a halocline interface significantly slowing the rate of settling of cryptococcal cells through water, resulting in persistence of C. neoformans within 1 cm of the air-water interface for over 60 min and C. gattii for 4-6 h. Our data also showed that during culture in yeast peptone dextrose media (YPD), polysaccharide accumulating in the supernatant formed a raft that augmented buoyancy and further slowed settling of cryptococcal cells. These findings illustrate new mechanisms by which cryptococcal cells may persist in aquatic environments, with important implications for aqueous transport and pathogen exposure.

[Zoonotic potential of brucellosis in marine mammals]. / Potentiel zoonotique de la brucellose des mammifères marins.

Introduction: Brucellosis in marine mammals (cetacean and pinnipeds) has emerged in a very significant way during the last two decades. Currently Brucella ceti and Brucella pinnipedialis are the two recognized species in marine mammals, but available information is still limited. Several genotypes have been identified, and studies on the relationship between sequence type (ST) and organ pathogenicity or tropism have indicated differences in pathogenesis between B. ceti sequences in cetaceans. The zoonotic potential of this disease is based on the identification of the main sources of introduction and spread of Brucella spp. in the marine environment as well as on the factors of exposure of marine mammals and humans to the bacteria. Bibliographic review: This article is a bibliographical review on marine mammal brucellosis, including the features, sources and transmission modes of each Brucella species, as well as their potential pathogenicity in animals and humans. Conclusion: Different genotypes of marine Brucella spp have been isolated from marine mammal species but without any evidence of pathology induced by these bacteria. Associated lesions are variable and include subcutaneous abscesses, meningo-encephalomyelitis, pneumonia, myocarditis, osteoarthritis, orchitis, endometritis, placentitis and abortion. The isolation of marine B. spp from marine mammal respiratory parasites associated to lung injury has raised the intriguing possibility that they may serve as a vector for the transmission of this bacterium.The severity of marine B. spp remains unknown due to the lack of an estimate of the prevalence of this disease in marine mammals. The number of suspected human cases is still very limited. However, by analogy with other germs of the genus Brucella responsible for abortion in ruminants and for a febrile and painful state in human beings, prevention measures are essential. The significant increase in the number of strandings coupled with a high seroprevalence in certain species of marine mammals must be considered for people in direct or indirect contact with these animals. Ongoing epidemiological monitoring combined with extensive post-mortem examinations (necropsy, bacteriology and sequencing) of all species of stranded marine mammals would deepen knowledge on the zoonotic potential of marine Brucella species.

Total mercury in fur, whiskers and whole blood of Caspian seals (Pusa capsica) from north-east of Caspian Sea (Kazakhstan).

Total Mercury (THg) content was determined in the fur of 64 Caspian seals, in the whiskers of 59 individuals and whole blood of 29 individuals. The THg content in Caspian seal fur varied from 258 to 8511 µg/kg, in whiskers from 954 to 12,957 µg/kg, and in whole blood from 88 to 350 µg/l. There were no statistically significant differences in mercury concentration in biomaterial between males and females (Kruskal-Wallis test, p < 0.05). The 1-2-year-old seals contained less mercury compared to older seals. The THg content in Caspian seal samples was comparable to seals from different regions of North Eurasia. Four individuals had mercury concentrations in their fur above the threshold values that can lead to nervous system disorders (>5400 µg/kg).