Impact of triclosan on behaviour and neural development of Cyprinodon variegatus

The study focussed on the effects of triclosan (TCS) exposure on mobility and hearing capacities of Cyprinodon variegatus larvae. TCS is an omnipresent antimicrobial and contaminant of aquatic ecosystems, which can act as endocrine disruptor, mainly by modifying thyroid functioning. Larval stages are particularly vulnerable to deleterious effects of endocrine disrupters because of potential impairment of fish development and behaviour. Exposure to TCS was conducted at fertilization of eggs at concentrations likely to be found in the environment: 20, 50 and 100 μg.l-1. The analysis of growth parameters of C. variegatus showed no effect of TCS on the fertility of eggs, survival and larval weight. Subsequently, THs concentrations were measured on 15 days post hatching larvae. THs are initially produced as T4 (thyroxine) cells and then converted in the bioactive form of T3 (triiodothyronine) cells. The observed increase of T4 and T3 cells in larvae exposed to 50 and 100 μg.l-1 suggests an increase in THs synthesis as a consequence of TCS exposure. Auditory thresholds of larvae were determined using ABR (Auditory Brainstem Response) technique, and finally larval mobility was measured. For both parameters no significant differences were observed among the three different treatments. Audiograms showed that the auditory system is not yet completely established at 30 days post hatching. However, these results allowed us to consider C. variegatus as an “hearing generalist” because this species have a hearing sensitivity lower than 2000 Hz. Regarding locomotion, our result summarized short time experiences targeting only swimming speed, distance and degree of mobility. It would be interesting to expand the behavioural aspects on other parameters of locomotion and integrate Cyprinodon reaction to different stress (light or touch). In conclusion, our results require an extensive long-term study on the full life cycle of C. variegatus, in order to evaluate the impact of triclosan on neural function and behaviour through several generations

The thyroid gland and thyroid hormones in sheepshead minnow (Cyprinodon variegatus) during early development and metamorphosis

The sheepshead minnow is widely used in ecotoxicological studies that recently, begin to focus on potential disruption of the thyroid axis by xenobiotics and endocrine disrupting compounds. However, reference levels of thyroxin (T4) and 3,5,3’-triiodothyronine (T3) and their developmental patterns are unknown. This study set out to describe the ontogeny and morphology of the thyroid gland in sheepshead minnow, and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Couples of three females and two males were placed in breeding chambers designed for this experiment. More than 1000 eggs were collected and maintained in seawater. Embryos were selected under a dissection microscope and placed in incubation dishes (50 per dish) at 25°C. On day 6, embryos hatched and larvae were transferred to 1L beakers. For one week after hatching, larvae were fed on artemia, and from 8 to 30 days post-hatch they were fed on flaked fish food. Embryos were sampled on day 0, 3, 6 post-fertilization and larvae and juveniles were sampled every three days from day 0 to 28 days post-hatch. The pooled samples were taken from several incubation dishes and divided in three replicate batches of 10-30 individuals. T4 and T3 were extracted from whole fish bodies and an enzyme-linked immunoassay was used to measure whole-body hormone levels. At each sampling point 5 individuals were placed in formalin fixative for histology. Length and body mass were measured. Hatching success, gross morphology, thyroid hormone levels and histology data were recorded. The onset of metamorphosis at 12 days post-hatching coincided with surges in whole body T4 and T3 concentrations. Thyroid follicles were first observed in pre-metamorphic larvae at hatching, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Follicle size and epithelial cell heights varied during the developmental phase, indicating fluctuations in thyroid hormone synthesis activity. The increase in the whole body T3/T4 ratio was indicative of an increase in outer ring deiodination. This study establishes a baseline for thyroid hormones in sheepshead minnows, which will be vital for the understanding of thyroid hormone functions and in future studies of thyroid toxicants in this species

Evaluation of the impact of Triclosan on the functioning of the thyroid system in Cyprinodon variegatus L., 1803.

Triclosan (5-chloro-2-[2, 4-dichlorophenoxy] phenol) is an antimicrobial widely used in various industrial products such as textiles, cosmetics and body care products. It is often detected in aquatic environments. The presence of the main biotransformation product, methyl TCS, indicates that this compound is not only degraded, but also persistent and accumulates in aquatic organisms. In this study, the effects of TCS on the thyroid system during embryonic and larval stages in Cyprinodon variegatus were evaluated. In particular, whole body thyroid hormone levels and the activity of deiodinases, enzymes involved in the activation of the thyroid prohormone T4, were measured. Couples of three females and two males were placed in breeding chambers designed for this experiment from which 3832 eggs were obtained by reproduction in the laboratory. Eggs were collected and maintained in seawater. Embryos were selected under a dissection microscope, randomly assigned to each of five treatment groups: Control, DMSO control (vehicle), 20 μg/L TCS, 50 μg/L TCS and 100 μg/L TCS and placed in incubation dishes (50 per dish) at 25°C. On day 6, embryos hatched and larvae were transferred to 1L dishes. The larvae were fed on artemia and on flaked fish food till day 15 post hatching when the fish were analyzed. The fertilization and survival rate, as well as the larval dry weight did not vary significantly between individuals exposed to 20, 50 and 100 μg/L TCS. T3 and T4 concentrations increased significantly in larvae exposed to 50 and 100 μg/L TCS. The study of the activity of enzymes involved in the deiodination of thyroid hormones (ORD) represents a new aspect in the study of endocrine disruption in C. variegatus. Unfortunately, we were not able to detect a net enzymatic T4 deiodination activity, most likely due to the very small amounts of protein and low specific enzyme activity in brain homogenates, carcass and liver

THE EFFECTS OF TRICLOSAN ON HEARING DEVELOPMENT OF CYPRINODON VARIEGATUS LARVAE

The aquatic environment represents the final sink for many chemicals, including bactericidal agents. Among them Triclosan (TCS) has been shown to affect the thyroid system of teleost. Larval stages are particularly vulnerable to deleterious effects of endocrine disrupters because of potential impairment of fish development and behaviour. Thyroid hormones are critical to the development of the brain and auditory system. Thus, TCS could affect the development of the brain and hearing. The aims of this study were: to investigate hearing development in sheepshead minnows (Cyprinodon variegatus) using the ABR technique (Auditory Brainstem Response) and to investigate the effects of triclosan on hearing development. Exposure to TCS was conducted from fertilization of eggs on at concentrations likely to be found in the environment: 20, 50 and 100 μgl-1. We characterized previously the ontogenic variation of thyroid hormones in embryos and larvae of sheepshead minnows. We observed an increase of thyroid hormones level around the 12th and the 15th day post hatching (dph), that may be associated with the transition from larval to juvenile stage during the development of this species. We concluded, that this period could be defined as a critical exposure window to pollutants. We determined hearing thresholds for sheepshead minnows of different ages. Due to the small size of larvae, first measurements were only possible at 30 dph. Audiograms showed that the hearing ability is not yet completely established at 30 dph compared to adults. So we pursued our experiments in older larvae of 40, 60, 70, and 80 dph when sexual maturity is attained. First results indicate that the hearing ability considerably improves between 50 and 60 dph. The effects of TCS in this development have yet to be determined but will be fully discussed. This study proposes an interesting new endpoint in thyroid disruption research

Do Triclosan affect hearing development of Cyprinodon variegatus larvae?

The aquatic environment represents the final sink for many chemicals, including bactericidal agents. Among them Triclosan (TCS) has been shown to affect the thyroid system of teleost. Larval stages are particularly vulnerable to deleterious effects of endocrine disrupters because of potential impairment of fish development and behaviour. Thyroid hormones are critical to the development of the brain and auditory system. Thus, TCS could affect the development of the brain and hearing. The aims of this study were: to investigate hearing development in sheepshead minnows (Cyprinodon variegatus) using the ABR technique (Auditory Brainstem Response) and to investigate the effects of triclosan on hearing development. Exposure to TCS was conducted from fertilization of eggs on at concentrations likely to be found in the environment: 20, 50 and 100 µgl-1. We characterized previously the ontogenic variation of thyroid hormones in embryos and larvae of sheepshead minnows. We observed an increase of thyroid hormones level around the 12th and the 15th day post hatching (dph), that may be associated with the transition from larval to juvenile stage during the development of this species. We concluded, that this period could be defined as a critical exposure window to pollutants. We determined hearing thresholds for sheepshead minnows of different ages. Our sheepshead minnows show ontogenic variations in the hearing ability during their development. At 30 days post hatching, their hearing ability is quite bad, with a narrow bandwidth of detected frequencies. But their hearing ability considerably enhance during their development to reach the adult hearing ability at around 80 days post hatching when this species reach sexual maturity. So we observe during the developmental phase of this fish species clear ontogenic improvements of the hearing ability and they showed an ontogenetic expansion in the frequency bandwidth they were able to detect. The effects of TCS in this development have yet to be determined but will be fully discussed. This study proposes an interesting new endpoint in thyroid disruption research