Title: Maternal transfer of organic pollutants in sea turtles undergoing
extensive oceanic migrations
Members` names:
PhD researcher Cynthia C. Muñoz1,
Prof. Ad Ragas1, Prof. Jan Hendriks1, Dr. Peter Vermeiren1, Prof. Tatsuya Kunisue2
Affiliations:
1. Radboud University, Department of Environmental Science, Heyendaalseweg 135, 6525 AJ
Nijmegen, The Netherlands.
2. Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama,
Ehime prefecture, Japan. 790-8577.
Note: Due to the COVID-19 Pandemic and travel restrictions between Japan and The Netherlands, I(Cynthia C. Muñoz) could not travel to Ehime University to conduct laboratory work. As a solution, theoriginally submitted plan for Joint Usage/Research Center, LaMer in Fiscal Year 2021 was adapted inconsultation with Prof. Kunisue. This report reflects the activities conducted under the revised plan.

1. Aim:The project aimed to quantify the exposure of early life stages of loggerhead sea turtles nestingin Kochi prefecture, Japan, to persistent organic pollutants, (POPs). Specifically, we intended toanswer the following research questions:
1. Do individual adult females differ in their diet and habitat preferences, thereby
potentially affecting POP levels observed in eggs from different mothers?
2. Do POP levels vary among different internal tissues?
3. Which pollutants are maternally transferred from mother towards egg albumen and yolk,
and how does the chemical structure of individual compounds relate to the transfer rate?
2. Procedure:
2.1. Research question 1, I conducted an analysis of ontogenetic shifts in diet and habitat
resource use of loggerhead sea turtles occurring in coastal waters near Kochi Prefecture.
Therefore, I applied stable isotope analysis to time series consisting of consecutive layers ofturtle carapace scute, an inert tissue in which chemical composition is preserved over time.
2.2. Research question 2, I conducted a systematic literature search for publications reporting
POPs in multiple sea turtle tissues. A subsequent meta-analysis of data extracted from thesepublications, then, quantified the tissue distribution of POPs across all sea turtle species.
2.3 Research question 3, in collaboration with the laboratory team of Prof. Kunisue, I analyzed
samples of loggerhead sea turtle eggs (4 albumen and 4 yolk samples), collected at Iburi, Kochi
prefecture. Samples were stored at -20°C until transfer into the es-Bank, where they were storeduntil analysis. These samples, complement analyses of the motherˊs blood (1 sample) and eggs
(4 albumen and 4 yolk samples) were conducted during the 2020 ‘Joint usage of equipment’
collaboration between Radboud University and Ehime University1. All samples were collected1

http://lamer-cmes.jp/performance-en/2766

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under the appropriate permit and ethics approvals in collaboration with Prof. Saito (Usa Marine
Biological Institute, Kochi University).
2.4. Chemical analysis: Chemical analysis targeted PCBs and OCPs in the blood plasma of 1
female loggerhead turtle, and PCBs, OCPs, and PBDEs in 8 of her eggs (yolk and albumen,Table 1). The analysis of eggs followed the protocol reported by Coelho et al. (2016). Yolk
(approximately 20 g) and albumen (6–10 g) samples were freeze-dried and then extracted withhexane/acetone (1:1 v/v) on a SE-100 High-Speed Solvent Extractor (Mitsubishi ChemicalAnalytech, Japan). The extract was cleaned in gel permeation chromatography for lipid removal(GPC) and activated silica-gel, and then concentrated. PCBs and PBDEs were analysed using agas chromatograph (GC: Agilent 7980 A) coupled with a quadrupole mass spectrometer (qMS:Agilent 5975C), and OCPs were analysed using a GC (Agilent 7890 A) coupled with a triple
quadrupole mass spectrometer (MS/MS: Agilent 7000). The blood plasma sample (2.8 g) was
liquid-liquid extracted using hexane/methyl tert-butyl ether (1:1 v/v) according to the protocolby Eguchi et al. (2015), subjected to the same clean-up method described above, and then PCBs
and OCPs were determined using a GC (Agilent 6890A) coupled with a magnetic sector
high-resolution mass spectrometer (HRMS: JEOL JMS-800D) using instrumental settings
reported by Goto et al. (2020).
2.5. Lipid content determination: To allow comparison with other studies, and to assess uptake
relative to lipid-based partitioning capacities of different tissues, we analyzed the lipid content(i.e. total extractable solids) of the blood, yolk, and albumen samples gravimetrically using
aliquots (10%–20%) of the respective sample extracts for chemical analysis.
3. Results and discussion
3.1 . Research question 1: ontogenetic shifts in diet and habitat
Our research identified that loggerhead sea turtles undergo ontogenetic shifts from oceanicsubadults with a pelagic diet toward neritic adults with a benthic diet. Within the population
occurring near Kochi Prefecture, those ontogenetic shifts have occurred at different times, hence
suggesting a mixed-age population structure. Moreover, during the adult stage, individuals havehigh individual specialization in their resource use. Combined, these results suggest that adults
might be exposed to higher pollution burdens than subadults, due to their higher trophic leveland proximity to the coast. Additionally, different individuals likely are exposed to different
levels, depending on their individual-based resource use patterns. (see publication 1 for details)
3.2 . Research question 2: internal pollution distribution
Our meta-analysis identified that the concentrations of POPs in several tissues can be predictedby the partitioning capacities determined by the lipid content of the tissues. However, egg
tissues deviate from this expected equilibrium. This has implications for the toxicity ofpollutants in eggs, and subsequently, to embryos. To explain deviations from equilibrium
partitioning, accurate measurements of lipid contents of different tissues are needed.
Nonetheless, our literature review identified that lipid contents for many sea turtle tissues arepoorly known. Therefore, we developed a lipid content database to support research in this area,

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which includes a limited number of records for lipid contents in egg compartments (Table 1),
relevant for research question 3. (see publication 2 for details)
3.3 . Research question 3: Maternal transfer of POPs in loggerhead turtles
3.3.1. Lipid contents
Lipid contents decreased in the order of yolk > blood plasma > albumen (Table 1). These resultscomplement the limited data available on lipid contents in sea turtles with information forloggerhead turtles and details on yolk and albumen as individual compartments. Our results fallwithin the range of previously reported values. Lipid contents in egg albumen were low (Table1). Measurement of lipid contents at such low quantities is challenging, which is reflected in thevariability across the 8 samples. Nonetheless, the median value corresponds with the known
lipid content of 0.020 % in the albumen of snakes. Consequently, we lipid-normalized allconcentrations in egg albumen samples against the median lipid content of 0.017 %. Lipid
contents in egg yolk were much higher than in albumen (Table 1), as was expected given therole of egg yolk in providing energy reserves to developing embryos. Lipid contents varied little
between yolks, therefore, we lipid normalized measured POP concentrations against thesample-specific yolk lipid content.
Table 1 Known data on lipid percentages (%) reported as median; mean; and range (min. - max.) incompartments of female blood and her eggs for 3 species of sea turtle (Sp.: C.c.: loggerhead, C.m.; green,
D.c.: leatherback turtle).
Study

Sp.

Whole blood

Plasma*Whole egg

Egg yolk

Egg albumen

This report

C.c-

0.62

-8.75; 8.34;

0.017; 0.151;

6.40 - 9.70

0.008 - 0.560------van de Merwe

C.mNa; 1.5 (0.1SE);

et al., 2010
Guirlet et al.,-Na; 8.9;

1.1-2.1
D.cNa

6.8 - 10.9-Na; 12.9;

2010
Stewart et al.,

3.8 - 25.5
D.c0.48; 0.56;

2010*1-5.02; 5.13;

0.22-1.00

4.67 - 5.69

Blood plasma from 1 turtle

3.3.2. Concentrations of OCPs, PCBs and PBDEs
Individual POP compounds reached the highest concentration in wet mass in yolk followed by
the plasma and the albumen. By contrast, median lipid normalized concentrations of POPcompounds recorded in individual albumen samples were one to two orders of magnitude greaterthan in yolk and plasma samples (Fig. 1). The results for OCPs only show measurements for eggs
T1-T4, with the measurement for eggs T5-T8 currently under analysis (OCPs will therefore not
be further discussed in this report). PCBs and PBDEs were analysed across the 8 eggs. PBDEs
were only reported in albumen and yolk, with only PBDE-28, -100, -153, -154, and -155
measured at detectable levels. Meanwhile, nearly all major tri- to deca-chlorinated PCB
congeners were detected in multiple egg yolk samples.

3 / 5Fig. 1 Distribution of lipid normalized concentrations of PCB, OCP and PBDE compounds recorded inindividual samples of blood plasma, albumen and yolk tissue. Measurements <LOD substituted by ½×LOD.
Boxplots: median, 25−75th percentiles, 1.5×interquartile range, outliers as individual points.

3.3.3. Tissue distribution of POPs
Lipid normalized concentrations PCB congeners were higher in plasma than yolk (Fig. 2),
suggesting the presence of a barrier or process that restricts the maternal transfer of PCB
compounds into egg yolk, as I previously pointed out in a systematic review of maternal transfer
in sea turtles (Muñoz and Vermeiren, 20202). Individual measurements of PCBs varied between
yolk samples (notice the wide spread in yolk concentrations for the same plasma concentration).
Nevertheless, the trend in partitioning across the PCB congeners detected in each yolk sample
showed a comparable pattern (coloured lines in Fig. 2 are roughly parallel).

Fig. 2 Lipid-normalized concentrations of individual, detected PCBs in plasma vs. yolk (left) or albumen(right) in 1 female loggerhead turtle and 8 of her eggs. Grey line: equilibrium partitioning, Red, blue
dashed lines: models for green and leatherback turtles derived from a scientific literature meta-analysis2.

A potential hypothesis for the increased variability at lower concentrations of PCBs within yolk is
the difficulty in precisely quantifying such low concentrations. Alternatively, maternal transfer
rates could vary among eggs from the same female. However, turtles produce all eggs within one
clutch at the same time, which is known to reduce variability in POP levels among eggs. Thepattern in maternal transfer observed here for loggerhead turtles overlaps with previous data from2

Muñoz and Vermeiren, 2020, Environmental Toxicology & Chemistry 39:9-29

https://doi.org/10.1002/etc.4585

4 / 5green and leatherback turtles that I extracted during a systematic literature review (Muñoz andVermeiren, 20202, Fig. 2). These results suggest a comparable pattern (and possibly process) of
maternal transfer across sea turtles, which is different from the expected equilibrium partitioning.
3.3.4. Predicting maternal transfer rates of POPs
PCBs with low lipophilicity (approximated as log Kow) have lower maternal transfer rates thanthose with higher lipophilicity (Fig. 3). Additionally, the maternal transfer ratio levels off anddecreases again for compounds with log Kow > 7.5. Previous studies on maternal transfer in sea
turtles only contained data on PCBs with log Kow of close to 7 and higher. Potentially, thosestudies missed important patterns and processes operating for compounds with log Kow of 6 andlower, which were measured in the current collaboration. Planned analyses of OCPs will provide
a valuable contribution to these data as OCP compounds also have lower log Kow.

Fig. 3 Maternal transfer rates (log10 of lipid normalized concentrations in yolk/plasma) for PCB congeners
in individual samples, expressed against the log Kow of these PCBs. Triangles: data < LOD in plasma. Redline: linear regression, Blue line: quadratic regression with standard error.

4. Publication/conference presentation
1. Muñoz C.C, Saito, T., Vermeiren, P. (2021) Marine Ecology Progress Series Vol 671:175-190
https://doi.org/10.3354/meps13767
2. Muñoz C.C, Hendriks, A.J., Ragas, A.M.J, Vermeiren, P. (2021) Environmental Science &Technology 55:10012-10024. https://doi.org/10.1021/acs.est.1c02845 -> also presented atSETAC Europe 31st Annual Meeting
5. Perspective in future① Our results indicated that maternal transfer rates depend on chemical properties (log Kow).
We will include additional chemical properties into further, advanced statistical analyses.
② We plan to analyze POPs in eggs, embryos and hatchlings of green sea turtles from Okinawa.
Such analysis of POP transfer into sea turtle embryos and hatchlings is currently lacking, buthighly relevant for risk assessment of early life stages. These analyses were planned for the2021 fiscal year LAMER project, but could not be conducted due to the Covid-19 Pandemic.

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