Manufacturing doubt about endocrine disrupter science--A rebuttal of industry-sponsored critical comments on the UNEP/WHO report "State of the Science of Endocrine Disrupting Chemicals 2012".

We present a detailed response to the critique of "State of the Science of Endocrine Disrupting Chemicals 2012" (UNEP/WHO, 2013) by financial stakeholders, authored by Lamb et al. (2014). Lamb et al.'s claim that UNEP/WHO (2013) does not provide a balanced perspective on endocrine disruption is based on incomplete and misleading quoting of the report through omission of qualifying statements and inaccurate description of study objectives, results and conclusions. Lamb et al. define extremely narrow standards for synthesizing evidence which are then used to dismiss the UNEP/WHO 2013 report as flawed. We show that Lamb et al. misuse conceptual frameworks for assessing causality, especially the Bradford-Hill criteria, by ignoring the fundamental problems that exist with inferring causality from empirical observations. We conclude that Lamb et al.'s attempt of deconstructing the UNEP/WHO (2013) report is not particularly erudite and that their critique is not intended to be convincing to the scientific community, but to confuse the scientific data. Consequently, it promotes misinterpretation of the UNEP/WHO (2013) report by non-specialists, bureaucrats, politicians and other decision makers not intimately familiar with the topic of endocrine disruption and therefore susceptible to false generalizations of bias and subjectivity.

Recent progress on our understanding of the biological effects of mercury in fish and wildlife in the Canadian Arctic.

This review summarizes our current state of knowledge regarding the potential biological effects of mercury (Hg) exposure on fish and wildlife in the Canadian Arctic. Although Hg in most freshwater fish from northern Canada was not sufficiently elevated to be of concern, a few lakes in the Northwest Territories and Nunavut contained fish of certain species (e.g. northern pike, Arctic char) whose muscle Hg concentrations exceeded an estimated threshold range (0.5-1.0 µg g(-1) wet weight) within which adverse biological effects begin to occur. Marine fish species generally had substantially lower Hg concentrations than freshwater fish; but the Greenland shark, a long-lived predatory species, had mean muscle Hg concentrations exceeding the threshold range for possible effects on health or reproduction. An examination of recent egg Hg concentrations for marine birds from the Canadian Arctic indicated that mean Hg concentration in ivory gulls from Seymour Island fell within the threshold range associated with adverse effects on reproduction in birds. Mercury concentrations in brain tissue of beluga whales and polar bears were generally lower than levels associated with neurotoxicity in mammals, but were sometimes high enough to cause subtle neurochemical changes that can precede overt neurotoxicity. Harbour seals from western Hudson Bay had elevated mean liver Hg concentrations along with comparatively high muscle Hg concentrations indicating potential health effects from methylmercury (MeHg) exposure on this subpopulation. Because current information is generally insufficient to determine with confidence whether Hg exposure is impacting the health of specific fish or wildlife populations in the Canadian Arctic, biological effects studies should comprise a major focus of future Hg research in the Canadian Arctic. Additionally, studies on cellular interactions between Hg and selenium (Se) are required to better account for potential protective effects of Se on Hg toxicity, especially in large predatory Arctic fish, birds, and mammals.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

The "common sense" intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

Transfer of PBDEs and chlorinated POPs from mother to pup during lactation in harp seals Phoca groenlandica.

Seven mother-pup pairs of harp seal (Phoca groenlandica) were sampled during the lactation season near Magdalen Islands, QC, Canada. The blubber and serum of pups and mothers as well as the milk of mothers were analyzed for polybrominated diphenyl ethers and several chlorinated persistent organic pollutants (POPs) to evaluate their transfer prior to weaning. Principal component analysis demonstrated significant variations in contaminant distribution patterns between seal tissues of mothers and pups. The selectivity in the transfer of POPs between mothers and pups appeared mainly driven by their log K(ow) (octanol/water partition coefficient). The most selective transfer step for the POPs examined occurred between the blubber and the serum of the mother. A novel approach to examine temporal changes of POP concentrations in tissues of seals which take into account the contamination of mothers was developed. A general decrease of POP concentrations in pup tissues throughout lactation may suggest a growth dilution. A concomitant rise of POP concentrations was observed in maternal blubber and serum and in milk as lactation progresses. The intensive offloading of contaminants via lactation constitutes a major but selective excretory route for reproductive female seals and also a significant route of exposure for suckling pup seals.

PBDEs in serum and blubber of harbor, grey and harp seal pups from Eastern Canada.

Serum and blubber of pup harbor (Phoca vitulina), grey (Halichoerus grypus) and harp (Phoca groenlandica) seals from the Estuary and the Gulf of St. Lawrence were analyzed for polybrominated diphenyl ethers (PBDEs). Concentrations of ∑PBDEs (on lipid weight basis) in tissues of harbor seal pups inhabiting the St. Lawrence Estuary were about five times higher than in those from a colony located in the northern Gulf. Harp seal pups have the lowest levels of ∑PBDEs among the seal species born in the Gulf of St. Lawrence. Lipid normalized concentrations of ∑PBDEs in serum and blubber were strongly correlated, suggesting that the blood collection as a less invasive method could be used to monitor ∑PBDEs in pups. This study shows that fluvial inputs of PBDEs are important to the St. Lawrence marine ecosystem and that inhabiting harbor seal pups have a substantial exposure to PBDEs at a critical developmental stage. In addition, the observed difference in PBDE levels between harp and grey or harbor seal pups from the Gulf of St. Lawrence is explained by the difference in diets of their mothers which is linked with their residency time in the Gulf and their seasonal migration pattern.

Toxic effects of various pollutants in 11B7501 lymphoma B cell line from harbour seal (Phoca vitulina).

Although, heavy metals and polycyclic aromatic hydrocarbons (PAHs) have been reported at high levels in marine mammals, little is known about the toxic effects of some of these contaminants. In this study, we assessed the immunotoxic and genotoxic effects of seven heavy metals (arsenic, vanadium, selenium, iron, zinc, silver and chromium) and one PAH (benzo[a]pyrene or B[a]P) on a lymphoma B cell line from harbour seal (Phoca vitulina). A significant reduction in lymphocyte proliferation was registered following an exposure to 0.05 microM of B[a]P, 5 microM of arsenic or selenium, 50 microM of vanadium, 100 microM of silver and 200 microM of iron. On the contrary, zinc increased the lymphoproliferative response at 200 microM. Decreased phagocytosis was observed at 20 microM of arsenic, 50 microM of B[a]P or selenium, 200 microM of zinc and 500 microM of vanadium. Micronuclei induction occurred with 0.2 microM of B[a]P, 100 microM of vanadium and with 200muM of arsenic or selenium. Exposure to 50muM of arsenic decreased G(2)/M phase of the cell cycle. Chromium did not induce any effects at the concentrations tested. Concentrations of heavy metals (except silver and vanadium) and B[a]P inducing an toxic effect are within the environmental ranges reported in the blood tissue of pinnipeds. The reduction of some functional activities of the harbour seal immune system may cause a significant weakness capable of altering host resistance to disease in free-ranging pinnipeds.

Phagocytosis in pup and adult harbour, grey and harp seals.

Knowledge on pinniped immunology is still in its infancy. For instance, age-related and developmental aspects of the immune system in pinnipeds need to be better described. The present study examined the phagocytic activity and efficiency of harbour, grey and harp seal leukocytes. In the first part of the study, peripheral blood was collected from captive female harbour seals of various ages. Data showed an age-related decrease in phagocytosis in female harbour seals from sub-adult to adulthood. In the second part of the study, changes in phagocytosis were quantified during lactation in wild newborn harbour, grey and harp seals and in their mothers (harp and grey seals). In newborns of the same age, leukocytes of harbour and harp seals phagocytosed less than those of grey seal pups. The phagocytic activity and efficiency increased significantly from early to mid-lactation in newborn harbour seals, and from early to late lactation in newborn grey seals, which could suggest that the transfer of phagocytosis-promoting factor(s) in colostrum is an important feature of temporary protection for pups. In contrast, no changes in phagocytic activity and efficiency were observed in lactating females of the two seal species, harp and grey, examined. At late lactation, phagocytic activity in both grey and harp seal pups and phagocytic efficiency in grey seal pups were significantly higher than in their mothers. These results could reflect either the capacity of phagocytes of the newborn harp and grey seals to respond to pathogens. Results from this study suggest that the phagocytosis of the seal species examined is not fully developed at birth as it generally increases in pups during lactation. Thereafter, the phagocytic activity of seals appears to decrease throughout adulthood.

Effects of individual polybrominated diphenyl ether (PBDE) congeners on harbour seal immune cells in vitro.

Effects of polybrominated diphenyl ethers (PBDEs) on the immune system of marine mammals are poorly understood. One important innate immune function of granulocytes is the respiratory burst which generates reactive oxygen species (ROS) used to kill engulfed microorganisms. The present study investigates in vitro the effects of BDE-47, -99 and -153, on the formation of ROS, on intracellular level of thiols, on activity and efficiency of phagocytosis and on apoptosis in granulocytes of harbour seals. Compounds were tested at four different concentrations ranging from 1.5 to 12 microM. Results showed that ROS levels, thiol levels and phagocytosis were all affected when harbour seal cells were exposed to the highest concentration (12 microM) of PBDE congeners. Apoptosis was not affected by PBDEs. The observed effects were similar in adults, pups and in the 11B7501 cell line of harbour seals.

Toxic effects of tributyltin and its metabolites on harbour seal (Phoca vitulina) immune cells in vitro.

The widespread environmental contamination, bioaccumulation and endocrine disruptor effects of butyltins (BTs) to wildlife are well documented. Although suspected, potential effects of BTs exposure on the immune system of marine mammals have been little investigated. In this study, we assessed the effects of tributyltin (TBT) and its dealkylated metabolites dibutyltin (DBT) and monobutyltin (MBT) on the immune responses of harbour seals. Peripheral blood mononuclear cells isolated from pup and adult harbour seals were exposed in vitro to varying concentrations of BTs. DBT resulted in a significant decrease at 100 and 200 nM of phagocytotic activity and reduced significantly phagocytic efficiency at 200 nM in adult seals. There was no effect in phagocytosis with TBT and MBT. In pups, the highest concentration (200 nM) of DBT inhibited phagocytic efficiency. A reduction of tumor-killing capacity of adult natural killer (NK) cells occurred when leukocytes were incubated in vitro with 50 nM DBT and 200 nM TBT for 24h. In adult seals, T-lymphocyte proliferation was significantly suppressed when the cells were exposed to 200 nM TBT and 100 nM DBT. In pups, the proliferative response increased after an exposure to 100 nM TBT and 50 nM DBT, but decreased with 200 nM TBT and 100 nM DBT. The immune functions were more affected by BTs exposure in adults than in pups, suggesting that other unsuspected mechanisms could trigger immune parameters in pups. The toxic potential of BTs followed the order of DBT>TBT>MBT. BT concentrations of harbour seal pups from the St. Lawrence Estuary (Bic National Park) ranged between 0.1-0.4 ng Sn/g wet weight (ww) and 1.2-13.4 ng Sn/g ww in blood and blubber, respectively. For these animals, DBT concentrations were consistently below the quantification limit of 0.04 ng Sn/g ww in blood and 0.2 ng Sn/g ww in blubber. Results suggest that concentrations measured in pups are considered too low to induce toxic effects to their immune system during first days of life. However, based on our in vitro results, we hypothesize that BTs, and DBT in particular, could pose a serious threat to the immune functions in free-ranging harbour seal adults.

Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg).

The Pacific oyster, Crassostrea gigas, is extensively cultivated and represents an important economic activity. Oysters are reared in estuarine areas, subjected to various biotic and abiotic factors. One of the limiting factors in aquaculture is mortality outbreaks, which may limit oyster production, and the causes of these outbreaks are not completely understood. In this context, the effects of temperature and salinity on Pacific oyster, C. gigas, haemocytes, were studied. Haemocytes are the invertebrate blood cells and thus have been shown to be involved in defence mechanisms. Flow cytometry was used for monitoring several haemocyte parameters. An increase of temperature induced an increase of haemocyte mortality, in both in vitro and in vivo experiments. Temperature modulated aminopeptidase activity. An in vitro decrease of salinity was associated with cell mortality. During the course of in vivo experiments, an increase of phagocytic activity was reported at 15 per thousand and 50 per thousand. Environmental physical parameters may modulate haemocyte activities.