Organochlorines in the Vaccarès Lagoon trophic web (Biosphere Reserve of Camargue, France).

During a decade (1996-2006), ecotoxicological studies were carried out in biota of the Vaccarès Lagoon (Biosphere Reserve in Rhone Delta, France). A multicontamination was shown at all levels of the trophic web due to a direct bioconcentration of chemical from the medium combined with a food transfer. Here, the pollutants investigated were organochlorines, among which many compounds banned or in the course of prohibition (or restriction) (PCB, lindane, pp'-DDE, dieldrin, aldrin, heptachlor, endosulfan...) and some substances likely still used in the Rhone River basin (diuron, fipronil). The results confirmed the ubiquity of contamination. It proves to be chronic, variable and tends to regress; however contamination levels depend on the trophic compartment. A biomagnification process was showed. A comparison of investigation methods used in other Mediterranean wetlands provides basis of discussion, and demonstrates the urgent need of modelling to assess the ecotoxicological risk in order to improve the management of such protected areas.

Organochlorine residues in european eel (Anguilla anguilla), crucian carp (Carassius carassius) and catfish (Ictalurus nebulosus) from Vaccarès lagoon (French National Nature Reserve of Camargue) - effects on some physiological parameters.

European eels (Anguilla anguilla), crucian carps (Carassius carassius) and catfish (Ictalurus nebulosus) were collected in three coastal locations of the Vaccarès lagoon (French National Nature Reserve of Camargue). The purpose of this paper is to report results of the first biomonitoring investigation in fish living in this protected coastal wetland. Residues of organochlorine (OC) contaminants (i.e. SigmaPCBs, gammaHCH, HCB, dieldrin, pp'-DDE) were determined in hepatic and muscular tissues, in order to compare geographical and seasonal distribution. Total exposure levels appeared to be more important in fatty fish such as eels than in crucian carps and catfish. The highest OC concentrations in liver (SigmaPCB) and in muscle (gammaHCH) were detected in Spring in some fish coming from a site located near the mouth of a canal draining irrigation waters of rice fields. Morphophysiological parameters (condition factor, organo-somatic indexes and lipid tissue composition) were measured concomitantly. Correlations between the hepatic and muscular burdens of OC and condition factor or organo somatic indexes were infrequent and rather negative. Localization of lipid accumulation (neutral or polar lipids) depended on metabolic rates of different species and appeared related to the fish trophic level.

Assessment of damage to ecosystems: a major issue in ecotoxicological research.

The assessment of the environmental impact of pollutants at the ecosystem level raises theoretical and methodological problems of substantial magnitude. Despite the usefulness of a more reductionist approach related to the measure of the effects of pollutant on populations, such as specific toxicity testing or--regarding the assessment of impact of chronic exposure--the use of various biomarkers, these measures do not provide data relevant to conclusions at the community and ecosystem level. The assessment of effect on entire ecosystems requires two kinds of information: (1) What are the consequences of pollutant exposure on the community structure, and (2) what is its impact on fundamental ecological processes that control the ecosystem functioning? Disturbances of structure may be appraised in terms of the major parameters that currently describe it at the community level, such as species richness, ecological diversity, and dominance. The assessment of pollutant effect on ecosystem biodiversity, though underestimated for a long time, is a major issue in applied ecotoxicological research. Accurate attention must be given to keystone species, which in both terrestrial and aquatic ecosystems control the diversification of the whole trophic web in a community. Therefore, if induced by a given pollutant, the decline of the populations of a keystone species may lead to a major destructing of the community. The second major issue in ecotoxicological research is assessment of damage to ecosystem functioning. Because productivity is of such practical importance, appraising changes in primary and secondary productivity is acutely needed. Indeed, chronic pollutant exposure is a permanent threat to biological natural resources as it impedes their production and renewal. Another major point in the assessment of effects at the ecosystem level is the effect of pollution on natural biogeochemical cycles, as well as the study of the ways through which a number of various and often common contaminants, some of them acting at global scale, interact with such cycles. It is also fundamental to assess the effects of pollutants on decomposer activity in both soil and natural waters and on its interaction with biogeochemical processes such as element recycling. In a larger context, though still almost unexplored, ecotoxicology is related to assessment of effects on complex ecosystem assemblages on a regional scale (i.e., landscape ecology and its relationship to functional ecotoxicology).

Outdoor experimental ponds (mesocosms) designed for long-term ecotoxicological studies in aquatic environment.

Outdoor artificial ponds (mesocosms) of 12 m3 were designed for long-term ecotoxicological studies. Sediment, macrophytes (Typha angustifolia and Elodea canadensis), and free and caged freshwater snails [Lymnaea palustris (Müller)] and wood lice (Asellus aquaticus L.) were collected in nearby natural ecosystems and introduced in the mesocosms. Sixty goldfish (Carassius auratus L.) were caged in each pond. Introduced species developed and reproduced in every mesocosm. Animal species (mainly insects and amphibians) spontaneously colonized the ponds, developed, and reproduced. The resulting communities qualitatively resemble those living in natural lentic systems in the surrounding area. Homogenity in physical and chemical conditions and in abundance of phytoplanktonic, periphytic, and macroinvertebrate communities between the different mesocosms was assessed during the stabilization period (8 months). Except for periphyton biomass, no divergent evolution was observed between the ponds. Mesocosm water was slightly eutrophic, alkaline (mean pH: 8.47 +/- 0.09), and moderately hard and mineralized. The homogenous and realistic environmental conditions and high ecological representativity of the outdoor experimental ponds were suitable for extensive ecotoxicological studies. Considerations on the choice and origin of introduced species and on possible interactive effects of the transfer of organisms from natural environments, maintainance conditions, and pollutant exposure are discussed.

The role of biomarkers in environmental assessment (5). Invertebrate populations and communities.

Although a number of biomarkers of pollutant exposure have been identified in invertebrate species, direct linkage with changes at population and/or community levels are poorly documented and, despite the ecological importance of invertebrates, there is no conclusive evidence that individual measurements of biochemical parameters may allow the effects of pollutants on populations and communities to be predicted. Among the various biochemical parameters used as biomarkers in invertebrates exposed to pollutants in the field, only those for which changes at population or community level can be suggested are discussed in the present review. At population and community levels, the development of resistance to pesticides and changes in behaviour, reproduction and development are analysed as putative consequences of biochemical and physiological alterations. Limits to the use of biochemical parameters as biomarkers of invertebrate exposure to pollutants are discussed. Future research trends and experimental approaches to the validation of invertebrate biomarkers in environmental pollution assessment are suggested.

Effects of early embryonic bursectomy and opotherapic substitution on the functional development of the adrenocorticotropic axis.

Functional development of the adrenocorticotropic axis was inferred from plasma ACTH and corticosterone levels in intact and embryonically bursectomized (BFX) embryos and chicks from 8 days before to 56 days after hatch. Bursectomy was surgically made at 80 h of incubation and resulted in various alterations in developing adrenocorticotropic axis: ether stress-induced hormonal stimulation could be detected more precociously in BFX (day-6) than in intact embryos; the non stress-responsive period of newly hatched controls did not appear in BFX chicks; BFX young adult chicken exhibited quite smaller responses to stress than controls. In ovo injection of bursin (Lys-His-Gly-NH2) to 6- and 9-days old BFX embryos could restore normal adrenocorticotropic development provided convenient doses of tripeptide were used: administration of 100 fg or 100 pg of bursin was effective to restore normal hormonal levels at all stages studied whereas 100 micrograms was effective at embryonic stages only. The tripeptide Lys-His-Gly-NH2 is suggested as a possible signal from the immune B system directed at the hypothalamo-hypophysial-adrenocortical axis.

Adaptation of the adrenocorticotropic response to chronic intermittent stress was altered by intracerebroventricular infusion of hemicholinium-3.

The role of diencephalic cholinergic neurotransmission in regulating hypothalamic-pituitary-adrenocortical (HPA) axis was investigated by means of administration of hemicholinium-3 (HC-3), a blocker of acetylcholine synthesis, in the third ventricle of hemispherectomized pigeons. Except for an early increase in ACTH and corticosterone levels following injection as a bolus of HC-3 that was ascribed to some stress-like situation, all data indicated that hypothalamic acetylcholine depletion resulted in inhibiting effects on the HPA axis. Twenty-four hours after injection of 6 micrograms of HC-3, the response to acute stress was markedly reduced in both magnitude and duration. Permanent instillation of HC-3 in the third ventricle at the rate of 0.25 microgram/h for 9 days led to lowered basal resting HPA activity and severely affected the development of adaptation to chronic intermittent stress. The anticipatory conditioned, endocrine response did not appear whereas attenuation of the poststress component was amplified. It is suggested that cholinergic mechanisms are involved in modulating the HPA function and particularly the conditioning process that takes place in the course of adaptation of the HPA response to chronic intermittent application of the same stressor.

[Effects of systemic administration of muscimol (gamma-aminobutyric acid agonist) on the corticotropic adenohypophysis]. / Effets de l'administration systémique de muscimol (agoniste de l'acide gamma-aminobutyrique) sur l'adénohypophyse corticotrope.

A short and moderate increase in plasma ACTH and corticosterone levels was observed after i.v. injection of the GABA-agonist, muscimol as a bolus of 0.5 mg . kg-1. Doses lower than 0.3 mg . kg-1 were ineffective. 6 hrs. after i.v. injection of 1 ml of an undiluted immune serum raised against oCRF41, resting hormone levels were markedly diminished, stress-induced responses were almost completely suppressed, whereas i.v. injection of muscimol still resulted in an unaltered adrenocorticotropic stimulation. Adenohypophysectomized animals displayed a low basal adrenal function, no stress-induced response and interestingly enough no longer responded to i.v. muscimol injection. Consequently, acute GABA-ergic stimulation appears to act directly onto the pituitary corticotroph cells. It cannot be inferred so far whether this action is of physiological importance but it can at least contribute to elucidate some controversial data prevailing in this field.

[Comparison of plasma ACTH and corticosterone levels after embryo bursectomy]. / Comparaison des taux plasmatiques d'ACTH et de corticostérone après bursectomie embryonnaire.

Plasma levels of both adrenocorticotropic hormone (ACTH) and corticosterone (B) were determined in embryos (day 15 of incubation), chicks (day 3 after hatch) and young chickens (8 weeks). Experimental animals were bursectomized at 80 hr of incubation, i.e., before any anlage of the bursa of Fabricius could develop. Bursectomized (BFX) animals were compared to sham-operated controls (T), in basal, resting condition and 7 (ACTH) or 14 min (B) after ether stress was delivered for 30 sec. Basal B and ACTH levels seemed not to be significantly modified in BFX embryos, chicks and chickens. Hypophysial and adrenocortical response to stress appeared more precociously in BFX embryos (day 15 of incubation) than in intact ones (day 19). The non stress-responsive period that was observed for one week after hatch of T birds did not appear in 3-day-old BFX chicks whose both B and ACTH stress-induced levels were as high as in intact adults. In contrast, adrenocortical and pituitary corticotropic responses to stress were markedly impaired (by 50%) in adult BFX chickens as compared to intact controls.

[Effect of ACTH injection and graft of bursa of Fabricius on the adrenocortical activity of bursectomized chicks at 80 hours of incubation]. / Effets de l'injection d'ACTH et de la greffe de bourse de Fabricius sur l'activité corticosurrénalienne des poussins et poulets bursectomisés à 80 heures d'incubation.

Early embryonic bursectomy (BFX) disturbed the adrenocortical functioning. The stress-unresponsive period that occurred in controls, and lasted for 2-3 weeks after hatching, no longer appeared in BFX chicks. In contrast, the magnitude of the stress-induced hypercorticosteronemia was much lower in BFX than in sham-operated 5 week-old chicken. It was assumed that such adrenocortical dysfunction was due to bursal deprivation, since grafting bursal buds onto the chorio-allantoic membrane of BFX embryos restored all the parameters under study, i.e., the post-hatching stress unresponsive period and the high magnitude of stress-induced responses in adults. Factor(s) involved in such interregulation are not known but do not seem to affect directly adrenocortical cells because intramuscular injection of a moderate dose of ACTH resulted in the same hypercorticosteronemia whether 3 day-old and 5 week-old chicks had been bursectomized or sham-operated.

Effect of GABAergic treatment on resting and stress-induced adrenocortical activities in the thalamic pigeon.

Muscimol was injected (0.5 mg kg-1) intravenously to either intact or thalamic-lesioned animals through a chronic catheter. Plasma samples were obtained at 7-min intervals and exhibited a moderate and transient increase in corticosterone levels in both groups, together with some symptoms of light discomfort (e.g., ptiloerection or panting). All these signs disappeared within 45 to 60 min. Intraventricular injection of bicuculline (3.5 micrograms) provoked a corticosterone profile quite similar to a stress-induced polyphasic pattern. Acute stress (electrical footshocks for 30 sec) was applied 2 hr after drug treatment. In controls, the stress-induced adrenocortical response appeared to be markedly modified after muscimol injection. The usual polyphasic rebounding profile was replaced by a monophasic one. This effect of muscimol did not occur when bicuculline had been injected in the 3rd ventricle. Bicuculline alone did not alter the stress-induced profile. GABAergic treatment had no effect on the monophasic response of thalamic-lesioned birds.

[Development of blood testosterone in the embryo and young chick after precocious bursectomy]. / Evolution de la testostéronémie chez l'embryon et le jeune Poulet après bursectomie précoce.

The effect of bursa of Fabricius on the endocrine function of the chick testes was studied in vivo by comparing plasma testosterone levels from 48 h before hatch to 16 weeks of age in both intact and bursectomized chicken. Early bursectomy was performed at 80 h of incubation. Post surgery survival was low (12% at 1 week). In controls, plasma testosterone levels were found to be low (100-200 pg. ml-1) from 48 h before to 48 h after hatch, then to raise up to a plateau (2,200 pg. ml-1) at 6 weeks. After bursectomy, values were first higher than in intact (210-440 pg. ml-1 from 48 h before/after hatch and 515 vs 300 pg.ml-1 at 3 days) but no difference could be further detected from 1 to 16 weeks of age. It is suggested that, in addition to the effect of androgen on bursa of Fabricius, the later reciprocally influences the gonadotropic axis during the early stage of development.

[Persistence of the acquisition of an endocrine response to conditioned stress in the thalamic pigeon by chronic GABAergic treatment]. / Persistance de l'acquisition d'une réponse endocrine au stress conditionnée chez le pigeon thalamique sous traitement GABAergique chronique.

The thalamic pigeon was taken as a model to investigate mechanisms of adaptation to chronic intermittent stress. Muscimol was infused into the 3rd ventricle at the rate of 0.25 microgram.hr-1 by means of an osmotic minipump. In controls, pumps were filled in with saline. Animals were placed under constant light regimen and electrical foot shocks were delivered, thrice a day, at fixed time interval following a short dark period (3 min). In controls, adaptation of the adrenocortical response to chronic intermittent stress was achieved within 7 days, including both attenuation of the post-stress hypercorticosteronemia and occurrence of a pre-stress conditioned peak of corticosterone. The anticipating conditioned constituent subsisted in muscimol treated animals whereas no attenuation of the post-stress peak could be detected. GABAergic chronic treatment also resulted in decreased basal plasma corticosterone levels. In controls, extinction of the conditioned endocrine response was not obtained after conditioning stimulus (dark period) was presented alone for 2 weeks.

Effects of chronic intracerebroventricular GABAergic treatment on basal and chronic or acute stress-induced adrenocortical activities in the thalamic pigeon.

Muscimol was chronically administrated to the third ventricle of thalamic pigeons by means of osmotic minipumps at the rate of 0.25 microgram X h-1 for 28 days. No abnormal behavioural sign was noted. The animals were subjected daily to chronic intermittent stress for the same 28-day period. Basal and stress-induced adrenocortical activities were evaluated by recording serial plasma corticosterone levels at the end of the experimental session. Untreated controls exhibited both components of the adaptation of the adrenocortical response to chronic stress: attenuation, i.e., a decrease in magnitude and disappearance of the late rebounding phenomenon, and anticipation, i.e., the occurrence of a conditioned component before stress itself. The adaptation to chronic stress was partly impaired by GABAergic treatment. The anticipatory conditioned peak subsisted but the magnitude of the post-stress peak was found not to be reduced whereas rebounding events were suppressed after chronic as well as acute stress. The basal resting levels of corticosterone were significantly lowered in muscimol-treated animals. A lesion placement in the anterior dorsomedial thalamus (ADMT) resulted in the same profile of stress-induced plasma corticosterone levels as seen after muscimol administration. Adaptation did not develop in ADMT animals and GABAergic stimulation, either acute or chronic, had no effect on their response to stress.

[Effect of muscimol (GABA-agonist) on thalamo-hypothalamic regulation by corticotropic efferents (thalamic model)]. / Influence du muscimol (GABA-agoniste) sur une boucle de régulation thalamo-hypothalamique à efférence corticotrope (modèle thalamique).

It can be assumed from previous data that the stress-induced polyphasic adrenocortical response involves two phenomenons. First, direct hypothalamic afferents stimulate the adrenocorticotropic axis resulting in a rapid increase in plasma corticosterone levels up to a maximum of about 40 ng . ml-1 at 15 min. Then, a thalamic-hypothalamic loop generates the late rebounding components. Intravenous injection of muscimol (0.5 ml . kg-1) produces only slight and short duration (45 min) disturbances (piloerection; panting; corticosteronemia). Stress application, 1 hr after muscimol administration, elicits only the first peak of corticosterone with magnitude and delay similar to that in control. However, the late rebounding component is no more visible. Thus, GABA-ergic stimulation leads to a situation similar to that seen after section of neural connections between thalamic nuclei and infundibular complex. The hypothalamic-adrenocorticotropic reactivity itself is not affected whereas the function of the regulating thalamo-hypothalamic loop is markedly impaired.

Effect of short exposure to cold on plasma thyroxine in Coturnix quail: role of the infundibular complex and its neural afferents.

Exposure of control quail to low ambient temperature (4 degrees) for a short duration (15 min) led to a rapid increase in plasma thyroxine (T4) levels. A peak appeared 40 min after the cold began and was followed by a progressive and slow decline. T4 levels were elevated in birds maintained for up to 3 hr at 4 degrees. Restraint stress could be accompanied by a slight and late decrease in thyroxine concentration, indicating that glucocorticoids could partly inhibit thyroid function. Both cold and restraint stresses elicited sustained adrenocortical activation. No thyroid response to cold appeared after complete or partial neural deafferentation of the hypothalamus, indicating that cold signals were conveyed to the thyrotropic centers from peripheral and/or deep thermoreceptors through neural posterior-lateral afferents to the hypothalamus. No thalamic relay appeared to be necessary since normal thyroidal stimulation was observed after thalamic-hypothalamic disconnection. Such a response persisted in hemispherectomized quail.

Adaptation of the adrenocortical response during repeated stress in thalamic pigeons.

Chronic vascular catheterization allowed to obtain serial blood samples before and after stress application to thalamic pigeons. Daily repetition of the same stress, at the same hour, for 5 weeks led to drastic changes in the stress-induced adrenocortical reaction. The rebounding, long-lasting rise in plasma corticosterone occurring after initial presentation of electrical footshocks was replaced by a single peak of corticosterone, lower and shorter than the initial response. Moreover, an anticipatory conditioned rise in corticosterone appeared before stress. Random distribution of stressful stimuli, thrice a day, for 5 weeks resulted in the single peak pattern of post-stress adrenocortical reaction, without any anticipatory component. When electrical footshocks were omitted after 5 weeks of daily regular presentation of stress, only the anticipatory peak in plasma corticosterone occurred. Shifting the daily lighting from the usual 06.00 a.m. to 04.00 a.m. led to a shift in the anticipatory conditioned endocrine response which appeared 2 h sooner than usual. Thalamic involvement in the process of adaptation to chronic stress is discussed.

Adrenocortical adaptation to chronic intermittent stress in hemispherectomized pigeon.

Hemispherectomized pigeons were exposed daily to electrical footshocks delivered for 15 sec, at the same hour, for 8 weeks. Serial blood samples were obtained through a chronic vascular catheter. The adrenocortical response to chronic intermittent stress was measured kinetically at one week intervals. The initial response including several successive peaks of plasma corticosterone progressively adapted: Late peaks disappeared and only the first one subsisted 12-14 min after stressor application; this first peak diminished in magnitude; furthermore, an anticipatory peak occurred, starting 14 min before stress. In pigeons lesioned in the anterior dorsomedial thalamus, the only response to the stressor was of the single peak (12-14 min) type without any development of anticipatory conditioned response. This phenomenon was consistant all over the experimental period. Thalamic-hypothalamic interrelationships may be suggested to provide neuronal loops that underlie the long lasting, pulsatile repetitive components of the adrenocortical response to acute stress and also the adaptative process of such a response to chronic intermittent stress, including a conditioned, anticipatory endocrine activation.

The adrenocortical response to stress from the pre-hatching period to the adult state in the pigeon: Thalamic participation.

The stress-induced adrenocortical response was kinetically evaluated for 100 min after ether vapor exposure in late embryos (30 h before hatching) and in 1, 2, 3 and 6-wk-old squabs. An-adult-pattern of corticosterone profile was seen in intact 6-wk-old pigeons, i.e. a polyphasic increase of corticosterone including successive peaks at 12-15, 35, 60 and 90 min after stress application. In 1-wk-old squabs, a small adrenocortical response to stress included only a single peak of corticosterone at 12-15 min. The early peak was found again, with the same magnitude, 2 wk after hatching but it was followed by two later and smaller peaks 35 and 60 min after stress. The adult pattern was visible in 3-wk-old stressed pigeons, although the magnitude of the response was still smaller than in adult controls. A single, high peak of corticosterone occurred 12-15 min after stress application, without any later peak, in embryos and a similar monophasic response was seen in thalamic lesioned adult birds. Therefore, it can be suggested that the polyphasic reverberating adrenocortical response to stress depends upon relationships between the adrenocorticotrophic hypothalamus and the anterior mediodorsal thalamus. Such thalamic-hypothalamic interrelations appear to mature during the early post-hatching weeks.