Exposure to Plasticiser DEHP Affects Eggs Spawned by Blue Mussels: A Possible Risk to Fertilisation?

Abstract

The endocrine disruptive chemical DEHP is a plasticiser often found in marine waters. Here, we assessed the effect of this additive on the number and size of eggs spawned by female mussels during a synchronised spawning event. After achieving the ripeness of the gonads, mussels of both sexes were exposed to two environmentally relevant concentrations of DEHP (nominal concentrations 0.5 and 50 µg/L) for one week. A spawning event was then induced and eggs were collected, counted, and their size measured (area and diameter). A slight but not significant effect was observed in lowering the number of eggs spawned when increasing the DEHP concentration. This effect was greater when adding spent gonads (possibly fully spawned females) to the total number of females. A significant effect of the lower dose on the average egg sizes was noticed, with a smaller area and diameter measured with respect to the control and the higher concentrated treatments. These results once again underline the importance for ecotoxicological studies to address the nonlinear dose-response effects of endocrine disruptive chemicals environmentally present at concentrations in the order of just a few µg/L that could not elicit a strong defence mechanism at low levels and be absorbed by filter feeder animals such as mussels.

Keywords: DEHP; blue mussels; egg count; egg size; fertility; reprotoxicity.

Figure 1

Gametogenesis stages of 10 μm gonadal tissue sections after DEHP exposure and the spawning induction in males and females stained with haematoxylin and eosin. Mature gonads in females (A) and males (B), spawning stage 3 in females (C) and males (D), spawning stage 1 in females (E) and males (F), empty spent follicles (G), and resting/spent stage (H). Scale bars represent 200 μm. Images were modified for brightness and contrast.

Figure 2

Effects of DEHP treatments on the gametogenesis stages. Percentage of each stage and sexual maturity index (SMI) of males (A) and females (B) in CTRL (n = 13 (males), 15 (females)) LOW DEHP (n = 17 (males), 10 (females)), HIGH DEHP (n = 9 (males), 14 (females)), and the associated SMIs.

Figure 3

Total eggs for females counted in 10 microlitres (8 replicate aliquots counted in 5 tanks for each treatment). Data are expressed as the mean ± standard error of the mean (SEM). Abbreviations are CTRL (0 µg/L), LOW DEHP (0.5 µg/L), and HIGH DEHP (50 µg/L). Different shapes represent different replicate tanks.

Figure 4

Counted eggs adjusted by adding 50% of the observed spent gonads to the total females in 10 microlitres (8 replicate aliquots counted in 5 tanks for each treatment). Data are expressed as the mean ± standard error of the mean (SEM). Abbreviations are CTRL (0 µg/L), LOW DEHP (0.5 µg/L), and HIGH DEHP (50 µg/L). Different shapes represent different replicate tanks. Kruskal–Wallis p value is annotated in the top right corner.

Figure 5

Egg area (squared micrometre) for each treatment (8 replicate aliquots measured in 5 tanks for each treatment). Data are expressed as the mean ± standard error of the mean (SEM). Abbreviations are CTRL (0 µg/L), LOW DEHP (0.5 µg/L), and HIGH DEHP (50 µg/L). Different shapes represent different replicate tanks. ANOVA p values are annotated and differences between groups are indicated by bars over the histograms.

Figure 6

Egg diameter (micrometre) for each treatment (8 replicate aliquots measured in 5 tanks for each treatment). Data are expressed as the mean ± standard error of the mean (SEM). Abbreviations are CTRL (0 µg/L), LOW DEHP (0.5 µg/L), and HIGH DEHP (50 µg/L). Different shapes represent different replicate tanks. The grey line represents the minimum threshold of fertilised eggs reported by [58]. ANOVA p values are annotated and differences between groups are indicated by bars over the histograms.

Figure 7

DEHP effect on the mg/L water oxygen concentration (consumed oxygen estimation) throughout the 30−s time points on day 2 after an injection of (A) 0.5 µg DEHP/L or (B) 50 µg DEHP/L.Abbreviations are CTRL (0 µg/L, in green), LOW DEHP (0.5 µg/L, in red), and HIGH DEHP (50 µg/L, in blue), n = 17−18. Datapoints are expressed as the mean ± standard error of the mean (SEM). ANOVA significant p values are annotated on the graphs.

Figure 8

Percentages of individuals with (A) open (green) or closed (purple) valves (modal values). “Split” (blue) values indicate mussels that have spent an equal time in open and closed states exposed to each of the two DEHP concentrations. (B) Percentages of individuals with more than one change from open to closed valves or vice versa (green), one change (blue) or no changes from the initial status (purple). (C) Percentages of individuals with more than opening events (from closed to open), measured as none (purple), one (blue), more than one (green). Abbreviations are CTRL (0 µg/L), LOW DEHP (0.5 µg/L), and HIGH DEHP (50 µg/L), n = 17–18.