Presence and biodistribution of perfluorooctanoic acid (PFOA) in Paracentrotus lividus highlight its potential application for environmental biomonitoring

Samples collection

Three sampling campaigns were carried out at the two sample sites (A and B) on the coast of north-western Sicily (Fig. 1a) chosen for this study. The main features of the sites and sampling details are summarized in Table S1 (Supplementary Information). A total of 90 specimens of sea urchins Paracentrotus lividus (45 specimen per each site), 30 l of seawater (15 per site), 40 samples (20 per site) of sea grass Posidonia oceanica (less than 5 cm leaf fragments, according to the institutional and national ethical guidelines) were collected and analyzed together with 30 l of brackish water from site B (15 l from each creek).

The samplings activity was authorized by the Capitaneria di Porto of Palermo with protocol number: 0029430. In the absence of data about PFOA contamination in the most recent report about chemical contamination in the coastal region subjected to this study³¹, the choice of sample sites was based on supposedly different status of pollution based on the site position or proximity to human activities (e.g. restaurants, pipeline, sewages, etc.).

Site A (see Fig. 1d), was chosen assuming a lower state of pollution based on its position in proximity to Capo Zafferano, at the northern extremity of S. Elia’s bay, with an average depth of 11 m and rocky seabed (see Fig. 1b and Supplementary Information: Table S1). Conversely, Site B (see Fig. 1e was chosen in the same coastal area (only 4.7 km away from Site A) assuming a higher state of pollution due to its position located on the southern side of Solanto promontory, nearby a pipeline and the mouths of two small creeks from inland, with a shallow (3 m) sandy seabed and where a bathing prohibition order is in place³² (see Fig. 1b, c and Supplementary Information: Table S1).

The biodistribution of PFOA in the various matrices was evaluated by analyzing sea urchin’s coelomocytes (CC) (90 samples) and coelomic fluid (CF) (90 samples), as well as gonads (G) (63 samples from 32 sea urchins collected in site A and 31 sea urchins collected in site B), or mixed organs (MIX) (27 samples from 13 sea urchins collected in site A and 14 sea urchins collected in site B) consisting of a homogenized mixture of urchin’s inner matrices when gonads were not developed enough for sampling. Due to their mutually exclusive nature the latter two datasets (G and MIX) were merged and labelled as “Gonads or Mixed organs” (GoM) for statistical analysis and graphical representations that needed a uniform dataset of 45 items per site. Further details on the collection of matrices and their labelling are described in the Supplementary Information.

The size of the sea urchins (horizontal diameter without spines) ranged between 30 and 51 mm indicating specimen that have lived in their respective site approximately from 3 to 5 years²⁵.

PFOA extraction and analysis

Materials, equipment and software are described in the Supplementary Information.

PFOA extraction procedures were adapted³³ to the type of matrix to be analyzed. Recovery percentages (R %) were checked per each batch of analyses by spiking blank samples with different amounts of PFOA analytical standard before the extraction procedure³³.

Spiked samples underwent the same extraction procedure of unspiked samples and the percentage of recovery R was calculated according to Eq. 1, where C_spike is the known concentration of spiked PFOA, D_spiked is the instrumental (LC–MS) analytical response of the spiked sample (i.e. the “detected” concentration), D_unspiked is the analytical response of the unspiked sample. R was then used in Eq. 2 to calculate the actual values, [PFOA], of PFOA concentrations in unspiked analyzed samples.

With the exception of [PFOA]_seawater and [PFOA]_creek, which are expressed as nanograms per liter (ppt), all other PFOA concentrations are expressed in nanograms per gram of matrix (ppb).

The PFOA standard was used for calibration before each batch of analyses and a linear response (R² > 0.99) was recorded in the concentration range from 0.1 to 1000 ppb. The RSDs on three replicates were below 10%. LOD (0.1 ppb) and LOQ (1.0 ppb) were quantified by IUPAC method. LC–MS analyses were performed in the negative ion-monitoring mode (see Supplementary Information).

For the analysis of P. lividus specimens, an estimate of the total PFOA concentration, [PFOA]_TOT in ng/g, in each sea urchin has been calculated considering the sampled weight (W) in grams of each matrix (Eq. 3):

Water analysis

During each one of the 3 sampling campaigns, 2 samples of seawater (5 l from Site A and 5 l from site B) and 2 samples of brackish water (5 l from each creek mouths in site B) were collected for a total of 6 seawater samples and 6 brackish water samples.

Samples were checked for the presence of PFOA by solid phase extraction (SPE) (see Supplementary Information) followed by LC–MS analysis³⁴.

The percentage of recovery, calculated according to Eq. 1, was R = 120%. [PFOA]_seawater and [PFOA]_creek concentrations (ng/L) were determined from analytical data according to Eq. 2.

Posidonia oceanica analysis

A total of 40 samples of leaves were collected from different individuals of P. oceanica (20 samples from site A and 20 samples from site B). Each sample was cut in tiny pieces and homogenized using an agate mortar and pestle, weighed (0.5 g) and transferred to a glass tube for extraction (see Supplementary Information).

The percentage of recovery, calculated according to Eq. 1, was R = 70%. [PFOA]_{P. oceanica} concentrations (ng/g) were determined from analytical data according to Eq. 2.

Coelomocytes and coelomic fluid analysis

The coelomic fluid, containing also the coelomocyte population, was taken from all the ninety collected specimens (45 per site) by inserting an ultrathin and sharp needle (32G 0.26 mm × 12 mm) of a 1 mL syringe through the peristomal membrane³⁵. All samples were centrifuged at 4 °C and 1500 rpm for 5 min in a 5804R refrigerated centrifuge (Eppendorf, Germany) thus separating the supernatant coelomic fluid (CF) from the coelomocytes (CC). CF and CC were then weighed and placed in different glass tubes for subsequent PFOA extractions (see Supplementary Information).

The percentage of recovery, calculated according to Eq. 1, was R = 28% for CF and R = 68% for CC. [PFOA]_CF and [PFOA]_CC concentrations (ng/g) were determined from analytical data according to Eq. 2.

Gonads analysis

The extraction of PFOA from 63 samples of gonads (32 from Site A and 31 from Site B) was performed with LC–MS grade methanol following the same procedure used for extraction from CF and CC (5 mL for samples greater than 0.5 g samples; 2.5 mL for samples between 0.1 g and 0.5 g). In case of undetected PFOA (considered as zero-values in graphics and statistical data treatment), analyses were repeated for confirmation on concentrated sample extracts.

Twenty spiked samples were prepared from the most abundant samples of gonads (10 spiked samples per site), by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of gonads samples. The percentage of PFOA recovery from gonads, calculated according to Eq. 1, was R = 73%. [PFOA]_G concentrations (ng/g) were determined from analytical data according to Eq. 2.

Mixed organs analysis

In 27 specimens of sea urchins (13 from Site A and 14 from Site B), the developmental status was not sufficient to collect at least 0.1 g of gonad sample. For these individuals, organs remaining after CF and CC collection, mainly intestine and undeveloped gonads, were mixed together and extracted similarly to the other matrices.

Spiked samples were prepared by adding 25 µL of an aqueous 1 mg/L stock solution of PFOA to 0.25 g of mixed organs (MIX) samples. The percentage of PFOA recovery from MIX, calculated according to Eq. 1, was R = 20%. [PFOA]_MIX concentrations (ng/g) were determined from analytical data according to Eq. 2.

Statistical analyses and graphical data representation

The distribution of PFOA concentrations in all the sampled matrices from collected sea urchins is graphically represented by box and jitter plot (Fig. 2) where the 25–75 percentiles are drawn using a box; minimum and maximum are shown at the end of the thin lines (whiskers), while the median is marked as a horizontal line in the boxfitting. Statistical tests and linear fittings were used to evaluate data significance and correlations (see Supplementary Information).

A permutational multivariate analysis of variance PERMANOVA³⁶ was performed to evaluate the differences in the PFOA concentrations between the two groups of sea urchins collected from site A and site B. The experimental design comprised of one factor (Site) two levels (fixed and orthogonal) and four variables corresponding to the concentrations of PFOA in each type of sample analysed (coelomocytes, coelomic fluid, gonad or mixed organs) including the estimated total PFOA concentration. Each term in the analysis was tested by 999 random permutations.

Finally, Principal Component Analysis (PCA) (see Supplementary Information: PCA tables and graphs) was performed on a dataset, containing five variables. Specifically sea urchin’s size and PFOA concentrations in each type of sample (CF, CC, and GoM) as well as in the entire sea urchin (TOT), to verify the multivariate nature of data in a relatively small number of dimensions, thus limiting the loss of information.

Source: Ecology - nature.com