SUPPLEMENTAL DATA
OIL DROPLET INGESTION AND OIL FOULING IN THE COPEPOD CALANUS FINMARCHICUS
EXPOSED TO MECHANICALLY AND CHEMICALLY DISPERSED CRUDE OIL
Content of supplemental data:

Analysis of water samples (method).

Analysis of Biological samples (method).

Figure S1. Oil associated with biomass in copepods.

Figure S2. Fluorescence pictures copepods.
Analysis of water samples
Determination of the total extractable organic compounds (total hydrocarbon
content with carbon numbers from 10 to 36; THC (C10-C36)) was performed on
dichloromethane (DCM) extracts by Gas Chromatography–Flame Ionization Detector (GCFID). The system comprised of an Agilent6890N GC fitted with an Agilent 7683B Series
autosampler. The column was an Agilent J&W HP-5 fused silica capillary column (30 m × 0.25
mm ID × 0.25 μm film thickness). The carrier gas was helium at a constant flow of 1.5 ml x
min-1. 1 µl samples were injected into a 310 °C split/splitless injector. The oven temperature
was heated to 40 °C for 1 min, then heated to 315 °C at 6 °C/min and held at 315 °C for
15 min.
Volatile organic compounds (C5-C10) were quantifed by Purge and Trap Gas
Chromatography/Mass Spectrometry, using a modified US Environmental Protection Agency
EPA-Method 8260, with a 50 m (0.20 mm ID, 0.50 µm film thickness) Supelco Petrocol
capillary column. Target analyses were detected with an Agilent 5973B MSD and the data
were acquired using the Agilent EnviroQuant Chemstation software.
Analysis for semi-volatile organic compounds including decalins, naphthalenes, and 3
to 5-ring polycyclic aromatic hydrocarbons were performed by Gas Chromatography–Mass
Spectrometry (GC-MS) operated in selected ion monitoring mode. The system comprised of
an Agilent 6890N GC with an Agilent 5975B quadrupole Mass Selective Detector. The column
was an Agilent J&W HP-5MS fused silica capillary column (60 m x 0.25 mm ID × 0.25 μm film
thickness). The carrier gas was helium at a constant flow of 1.2 ml x min-1. A 1 µl sample was
injected into a 310 °C split/splitless injector. The oven temperature was heated to 40°C for 1
min, then heated to 315 °C at 6 °C x min-1 and held for 15 min. Data and chromatograms
were monitored and recorded using MSD ChemStation (version D.03.00.611) software. The
MSD ion source temperature was 230 °C.
Analysis of Biological samples
To each 10 ml vial containing 20 copepods 3 ml of potassium hydroxide (6.5%) in
methanol (80%) and internal standards (SIS) were added. The mixture was heated for two
hours in an ultrasonic bath at 80 °C to achieve saponification, followed by filtration and serial
extraction with hexane (3 x 3 ml). The combined extracts were dried with anhydrous sodium
sulphate and concentrated to approximately 0.5 ml using a Zymark Turbovap® 500
Concentrator. Cleanup of the extracts was performed by solid phase extraction using 3 ml
columns containing 0.5 g normal phase silica packing (Superclean LC-Si, Supelco). The
samples were eluted through the column with 3 x 2 ml of DCM:hexane (1:3). The purified
extracts were concentrated to 90 µl in an insert GC vial insert and recovery internal
standards (RIS; fluorene-d10, and acenaphthene-d10) were added in 10 µl of DCM
immediately prior to GC-MS analysis. The following target oil compounds were quantified:
decalins (C0-C3), naphthalenes (C0-C3), biphenyl, acenaphtylene, acenaphthene,
dibenzofuran, fluorenes (C0-C3), phenanthrenes/anthracenes (C0-C4), dibenzothiophenes
(C0-C4), fluoranthenes/pyrenes (C0-C3), benz(a)anthracene, chrysenes (C0-C3),
benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene,
indeno(1,2,3-c,d)pyrene, dibenz(a,h)anthracene and benzo(g,h,i)perylene.
O il in b io m a s s (m g /k g )
20000
15000
10000
5000
C - 3 D e c a lin
C 1 -C h ry s e n e
C 2 -C h ry s e n e
0
0 .1
1
10
O il E x p o s u r e ( µ L /L )
Figure S1. Oil associated with biomass in copepods exposed to mechanically dispersed oil
droplets. Based on three different components with high Log Kow (> ~6). Curves generated
by second order polynomial (quadratic) fit, N= 2 and 3 for the high exposure groups and 4
for the other groups, SD indicated.
Figure S2. Fluorescence pictures of the frontal part (indicated in the picture on top) of
randomly selected individuals exposed to various concentrations of oil dispersions.
Fluorescence from algae is seen as white areas. N = 6 for all exposure groups;
CD = chemically dispersed; MD = mechanically dispersed; L = low exposure concentration; M
= medium exposure concentration; H = high exposure concentration .