Dioxin-like PCB 126

Exposure to the dioxin-like PCB 126 did not cause any dose related effects on larvae survival, morphological abnormalities (Fig. 3), or developmental penalty points (Table 3) for both experiments.

Larvae hatched normally in all dose groups, with larvae reaching the gastrula stage 1 dpf. For sampling 1 (2-4 dpf) no dose related effects were observed. At the end of the observation period of sampling 2 (9 dpf) approximately 50% of the larvae were at the 6-armed pluteus stage, which was similar to control larvae. Finally, at the end of the observation period of sampling 3 (16 dpf), at least 90% of the larvae were at 8-armed pluteus stage in all dose groups.

Because of the unexpected absence of toxic effects resulting from exposure up to and including 0.3 nM of the dioxin-like PCB 126 (Fig. 3; Table 3), a short term exposure to the actual dioxin 2,3,7,8-tetrachlorodibenzodioxin (TCDD) was performed from 0 to 4 dpf, equivalent to sampling 1 in the 16 day ELS assay, to further investigate the previously observed lack of dioxin-like sensitivity. Results for sampling 1 (2-4 dpf) indicated a slight statistically significant increase (20%) in morphological abnormalities at the highest exposure concentration of 3 nM TEQ only (Fig. 4). The observed effects included the absence or abnormal shape of the arms (Fig. 2), while no increase in mortality was observed.

Figure 3. Morphological abnormalities (%) of P. miliaris larvae exposed to PCB 126 from 0-16 dpf (experiment A). Each bar represents the average percentage from 3 repeated observations of 20 larvae for each sampling plus the standard deviation (n=2).

Figure 4. Morphological abnormalities (%) of P. miliaris larvae exposed to TCDD from 0-4 dpf. Each bar represents the average (%) from 3 repeated observations of 20 larvae plus the standard deviation (n=2). *p <0.05 (One-way ANOVA with Bonferroni's Multiple Comparison Test).

3.2.3. Triclosan

Exposure of fertilized eggs to 500 nM (experiment A) or 1000 nM (experiment B) Triclosan (TCS) resulted in complete hatching failure. Of the larvae surviving 500 nM TCS in experiment B, more than 80% developed morphological abnormalities (p<0.001) (Fig 5). Observed abnormalities were related to the development of the arms (i.e. short or deformed) and a slight edema around the larval body, which appears to result from abnormal skeletogenesis of skeletal rods (Fig. 2). The lowest estimated EC50 values obtained from sampling 1 (2-4 dpf) ranged between 308 (95% C.I. 107-886 nM) and 313 nM TCS (95% C.I. 128-568 nM) for experiment A and B, respectively (Table 2). These larvae were also delayed in their development (Fig. 6), thus resulting in a higher number of developmental penalty points at 16 dpf (Table 3). More than 90% of the larvae exposed to 250 nM TCS or lower developed normally and reached the 8-armed pluteus stage at 16 dpf.

Figure 5. Morphological abnormalities (%) of P. miliaris larvae exposed to TCS from 0 -16 dpf (experiment B).

Each bar represents the average (%) from 3 repeated observations of 20 larvae for each sampling plus the standard deviation (n=2). ***p<0.001 (Two-way ANOVA with Bonferroni's Multiple Comparison Test). (†) No hatching occurred.

Figure 6. Developmental stages of P. miliaris larvae at 16 dpf exposed to TCS (experiment B). Each bar graph represents the average percentage of larvae at a given development stage plus the standard deviation (n=2).

***p<0.001 (One-way ANOVA with Dunnett's Multiple Comparison Test). Values for 1000 nM TCS are not included since no hatching occurred.

3.2.4 HBCD

Exposure of fertilized eggs up to the highest HBCD test concentration of 100 nM did not cause any adverse effects on hatching success in both experiment A and B (data not shown). However, larvae exposed to 100 nM HBCD developed a significantly higher percentage of morphological abnormalities in sampling 1 (2-4 dpf) and 3 (2-4 dpf) in both experiment A (p< 0.01 and p<0.001 respectively) and B (p<0.05 and p<0.001 respectively), while this effect was only statistically significant for sampling 2 (7-9 dpf) in experiment B (p<0.001). Morphological abnormalities in larvae exposed to HBCD included the presence of short or deformed larval arms and a slight edema around the larval body that appears to result from abnormal skeletogenesis of skeletal rods as observed for TCS (Fig. 2). The estimated EC50 values were the lowest for sampling 3 (14-16 dpf) varying between 88 (95% C.I. 72-109 nM) and 54 nM HBCD (95% C.I. 31-95 nM) for experiment A and B, respectively (Table 2). Larvae in the 50 nM HBCD group had a slightly higher number of developmental penalty points, which strongly increased in the 100 nM HBCD group (Table 3) where most of the larvae were arrested in the 4-armed pluteus stage (Fig. 8).

Figure 7. Morphological abnormalities (%) of P. miliaris larvae exposed to HBCD from 0 -16 dpf (experiment B). Each bar represents the average (%) from 3 repeated observations of 20 larvae for each sampling plus the standard deviation (n=2). *p<0.05; ***p<0.001 (Two-way ANOVA with Bonferroni's Multiple Comparison Test).

Figure 8. Developmental stages of P. miliaris larvae at 16 dpf exposed to HBCD (experiment B). Each bar graph represents the average percentage of larvae at a given development stage plus the standard deviation (n=2). ***p

<0.001 (One-way ANOVA with Dunnett's Multiple Comparison Test).

3.2.5 TBBPA

Even the highest TBBPA exposure (1500 nM) did not cause adverse effects on hatching success; larval development was normal during the observation period of sampling 1 (2-4 dpf) and 2 (7-9 dpf). However, sampling 3 (14-16 dpf) revealed a statistically significant increase in the percentage of morphologically abnormal larvae exposed to 1500 nM TBBPA in experiment A (p<0.01) (Fig. 9), while in experiment B the observed increase was not statistically significant.

Abnormal larvae displayed shorter or deformed arms and a mild edema around the larval body, which appears to result from abnormal skeletogenesis of skeletal rods as previously mentioned for TCS and HBCD (Fig. 2). The estimated EC50 value for sampling 3 (14-16 dpf) was around 1500 nM TBBPA in experiment B. However, this value could not be precisely quantified due to the steep dose response curve since the concentration below 1500 nM TBBPA did not induce a clear effect. Also, the development stage of larvae at 16 dpf was significantly delayed (Fig. 10), which resulted in a significant increase in the number of developmental penalty points for the 1500 nM exposure group in both experiments (Table 3).

Figure 9. Morphological abnormalities (%) of P. miliaris larvae exposed to TBBPA from 0 -16 dpf (experiment A). Each bar represents the average (%) from 3 repeated observations of 20 larvae for each sampling plus the standard deviation (n=2). **p<0.01 (Two-way ANOVA with Bonferroni's Multiple Comparison Test).

Figure 10. Developmental stages of P. miliaris larvae at 16 dpf exposed to TBBPA (experiment B). Each bar represents the average percentage of larvae at a given development stage plus the standard deviation (n=2). ***p

<0.001 (One-way ANOVA with Dunnett's Multiple Comparison Test).

3.2.6. PFOS

Exposure of fertilized eggs to PFOS concentrations up to 743 nM had no effects on hatching success, nor did it induce any morphological abnormalities in larvae (Fig. 11). At 9 dpf, a small dose related acceleration in larval development was observed (Fig. 12), which resulted in negative values

for the number of developmental penalty points. However, by the end of experiment (16 dpf), such advancement of larval development was no longer observed even when expressed as developmental penalty points (Table 3).

Figure 11. Morphological abnormalities (%) of P. miliaris larvae exposed to PFOS from 0 -16 dpf. Each bar represents the average (%) from 3 repeated observations of 20 larvae for each sampling plus the standard deviation (n=2).

Figure 12. Developmental stages of P. miliaris larvae at 9 dpf exposed to PFOS. Each bar represents the average percentage of larvae at a given development stage plus the standard deviation (n=2). *p <0.05; **p <0.01 (One-way ANOVA with Dunnett's Multiple Comparison Test).

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50 values estimated for Triclosan (TCS) and HBCD. TCSHBCD ABAB

4. Discussion

This study describes the successful development of a novel 16-day echinoid ELS bioassay for the detection and evaluation of developmental effects resulting from exposure to environmentally relevant marine POPs (i.e. PCB 126, TCDD, TCS, HBCD, TBBPA, PFOS). In this prolonged ELS bioassay, P. miliaris embryos and larvae were continuously exposed to test compounds for 16 days and toxic effects were quantified at different time points using the following endpoints: larval development stages, morphological abnormalities and mortality.