Eggsposed : impact of maternally transferred POPs on fish early life development

Persistent organic pollutants (POP), with well-known representatives as polychlorinated biphenyls (PCBs), dioxins, and brominated flame retardants as polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecanes (HBCD), are still globally present in the marine environment, This despite the substantial reduction of application and emission that was achieved during the last decades. Apart from their persistency these compounds share low water solubility and a high lipophilicity which make that the highest concentrations in the aquatic environment are found in the organic matrix of sediments and in biota. Dissolved water concentrations are low. Hence, intake of contaminated food items forms the major source for POPs exposure of aquatic organisms, and through biomagnification the highest concentrations can be found in the tissue of top predators. POPs have the potency to cause a variety of toxic effects, among which endocrine disruption and teratogenic effects that especially apply to early life stages. As the early life stages of most fish species develop suspended in the water column, exposure to POPs may be considered relatively low, at least until the larvae start feeding after yolk absorption. However, POPs accumulated in the tissue of the mother are transferred to the eggs. The research presented in this thesis aims at the determination of the impact of such maternally transferred POPs on development and survival of fish early life stages, in order to assess if this exposure route can significantly impact the development of a fish population at current environmental concentrations, especially in combination with high fishing pressure. For this purpose a bioassay was developed with the common sole (<em>Solea solea</em>). The advantages for this research of this new bioassay above standard fish early life stage (ELS) tests are that sole is a native West European species that as all flatfishes undergoes an obvious metamorphosis. The test set-up includes this metamorphosis that is thyroid hormone mediated and therefore expected to be easily disrupted by POPs, based on research with amphibians. The prolonged Early Life Stage test (p-ELS) with sole is presented in chapter 2. Early life stages were exposed to a concentration series of the dioxin-like PCB 126 (3,3',4,4',5-pentachlorobiphenyl) in seawater until 4, 8,10 and 15 days post fertilisation (dpf). Subsequently the development of the larvae was registered under further unexposed conditions. The LC50s at the start of the free-feeding stage (12 dpf) ranged between 39 and 83 ng PCB 126/l depending on exposure duration. After the fish had completed the metamorphosis, the LC50 values ranged between 1.7 and 3.7 ng PCB 126/l for the groups exposed for 4, 8 and 10 dpf respectively. Thus exposure for only 4 days, covering only the egg stage, was sufficient to cause adverse effects during a critical developmental phase two weeks later. This study indicates that ELS fish tests that are terminated shortly after the fish becomes free-feeding underestimate the toxic potential of compounds with low acute toxicity such as PCBs. The internal dosages of these larvae at the end of the exposure, determined by means of an in-vitro gene reporter assays as dioxin-equivalent values (TEQ), revealed an internal lethal concentration, ILC50 of 1 ng TEQ/g lipid, which is within the same order of magnitude as TEQ levels found in fish from highly polluted areas. This suggests that larval survival of fish populations at contaminated sites can be affected by persistent compounds that are accumulated by the female fish and passed on to the eggs. Based on these first results the p-ELS test procedure was improved to reach a better control performance. The exposure period was terminated when all larvae had hatched (6 dpf), this in order to mimic exposure through maternal transfer as good as possible without exposing parent fish or manipulation the eggs. In a second test (Chapter 3) the eggs were exposed to a concentration series of methyltriclosan (MTCS), a metabolite of triclosan (TCS) that is commonly used as bactericide in a wide variety of human care products. MTCS and TCS are discharged with waste water, bioaccumulate in fish tissue, and are known to have the potency to disrupt the thyroid hormone system. Mortality occurred in the higher treatment levels until 20 dpf. Indications for thyroid hormone disruption were not observed; all surviving larvae completed metamorphoses without problems. Internal effect concentrations, reached in larvae at the end of the exposure (6 dpf), were 5.8 mg/g lipid weight (lw) and 2.1 mg/g lw for ILC50 and ILC10 respectively. These internal effect concentrations are at least 200 times higher than concentrations that due to maternally transfer can be expected in the eggs of highly exposed fish in a field situation. Our results thus do not indicate a high risk from maternally transferred MTCS for fish at the current field concentrations. In order to get more insight in the fate of the POPs in the larvae, in Chapter 4 the existing bioaccumulation model OMEGA was adjusted for sole early life stages and validated with experimental data with PCBs. This study revealed, that tissue concentrations of compounds with log K_ow>6, peak in the tissues of developing sole at the end of the yolk-sac stage, when lipid reserves are depleted. As a result, just before the larvae become free feeding, the peak tissue concentrations of the pollutants in the larvae exceed that of the adult fish. This also explains at least partly, the delayed effects that were observed in Chapter 2 (and 5). Chapter 5 assesses the likelihood that early life development of fish from contaminated areas is affected by maternally transferred POPs. Following the p-ELS test protocol, effects on sole larvae were determined for the dioxin-like PCB 126, the technical PCB-mixture Arochlor 1254, PBDEs and HBCDs, for an artificial mixture of PCBs and PBDEs, and for ‘field mixtures’ extracted from sole collected from the North Sea and in the contaminated Western Scheldt estuary. As was earlier observed with PCB126 and MTCS, exposure to PCBs, PBDEs and the artificial and field mixtures caused mortality that started to occur shortly after the larvae became free-feeding and continued to increase until the onset of metamorphoses. The effects induced by the field mixtures correlated well with the ∑PCB concentrations in the tissue of the exposed larvae. No indications were found for synergistic effects or for substantial contribution of other (unknown) substances in the field mixtures. HBCD did not induce toxic effects. POP levels in sole from Western Scheldt estuary are about 20 times lower than the ILC50, the larval tissue concentration that produced 50% early life stage mortality. Levels in North Sea sole are an order of a magnitude lower. Chapter 6 describes a risk assessment for toxicant induced larval survival for European eel (<em>Anguilla anguilla</em>). Eels are considered sensitive for the effect of POPs that can accumulate to high levels in their lipid rich tissue. During spawning migration without feeding high lipophilic dioxin-like POPs in the eel’s tissue were estimated to increase 1.33 or 2 fold, due to weight loss. As no toxicity data are available for eel larvae, the critical egg concentrations for larval survival was estimated from a sensitivity distribution based on literature data of other species. It was assumed that eel larvae belong to the 5% or 1% most sensitive teleost fish species. Given concentrations of dioxin-like pollutants as reported for European eel, and following the worst case scenarios with respect to sensitivity of the larvae and bio amplification during migration, it can be expected that larvae of eel from highly contaminated locations in The Netherlands and Belgium will experience more than 50% mortality due to maternally transferred dioxin-like toxicants. Chapter 7 explores the potential impact of (toxicant induced) early life stage mortality on the population development of sole by application of a simple age structured matrix model. The model is used to explore the population response to a combination of (toxicant induced) larval mortality and fishing-related mortality of mature fish. The results indicate that the impact of larval mortality that occurs before metamorphosis is very low, even in population subject to high fishing pressure. This is the result of the combination of a high fecundity and the fact that the larval mortality occurs before the moment when the number of recruits is limited by the carrying capacity of the nursery areas. When colonising the nursery areas the, until than pelagic sole larvae metamorphose into flatfishes with a benthic life style. The individuals hence concentrate from the three dimensional pelagic environment to a two dimensional benthic environment, which caused density dependent mortality. This concentration of early life stages is typical for flatfish. Mortality that occurs after the nursery areas are populated will have a more pronounced impact on population development. The results further imply that population development of pelagic fish species that do not concentrate in nursery areas, and species with low fecundity is more vulnerable for disturbance through mortality of early life stages. Chapter 8 synthesises and discusses the outcome of the research. It is stressed that short term fish tests, often covering only the embryonic development, will underestimate the real risk of lipophilic substances. Toxicity of these substances will peak after yolk sac absorption when these tests have already been ended. When the characteristics of the test substance are known this risk is predictable with for instance the ELS-OMEGA model. However, especially when mixtures of unknown composition (effluents, sediment extracts) are being tested one must realise that the contribution of lipophilic substances may be underestimated in test that are terminated before, or too soon after the fish larvae are free feeding. The absence of effects on metamorphosis in our P-ELS test is explained by the prediction of the ELS-OMEGA model that the POPs concentrations in the larvae, had reached too low concentrations at the moment of metamorphoses to disrupt the thyroid hormone system. This was due to passive excretion (for substances with log K_ow<6) and growth dilution. It must be realised that the experimental set-up that was followed to mimic the effects of maternally transferred POPs does not include potential effects of maternally transferred metabolites of these POPs that can be formed by the parent fish and that are often more toxic than the mother compounds. Also effects of the mother’s condition on the quality of the eggs and epigenetic effects were not included. This implies that the results of the tests as performed in some cases might underestimate the actual effects of these substances. The species most vulnerable to the effects of maternally transferred POPs share a high exposure, low fecundity and the absence of density dependent mortality of early life stages. According to these criteria sharks and especially the Greenland shark (<em>Somniosus microcephalus</em>) that is highly exposed to POPs can be considered as highly vulnerable. It is therefore recommended to investigate the actual sensitivity of this species, in order to get more insight in the potential vulnerability of the populations.