Kathryn Brown ORCID iD: 0000-0002-9093-8742

20-00217
Accepted Article
Environmental Toxicology

K. Brown et al.

Fuel impacts on Antarctic sea urchin Sterechinus neumayeri

Impacts of Petroleum Fuels on Fertilisation and Development of

the Antarctic Sea Urchin Sterechinus neumayeri
Kathryn E. Brown*ab, Catherine K. Kinga, and Peter L. Harrisonb
a
Environmental Protection, Australian Antarctic Division, Kingston, Tasmania, Australia.
b
Marine Ecology Research Centre, Southern Cross University, Lismore, New South
Wales, Australia.

(Accepted 13 April 2020; Returned for Revision 06 May 2020; Accepted 16 September
2020)

Abstract: Antarctic marine environments are at risk from petroleum fuel spills as
shipping activities in the Southern Ocean increase. Knowledge of the sensitivity of
Antarctic species to fuels under environmentally realistic exposure conditions is lacking.
In this study, the toxicity of three fuels, Special Antarctic Blend diesel (SAB), marine gas
oil (MGO) and intermediate fuel oil (IFO 180) to a common Antarctic sea urchin
Sterechinus neumayeri was determined. Sensitivity was estimated for early
developmental stages from fertilisation to the early 4-arm pluteus in toxicity tests of up to
24 d duration. The effects of the water accommodated fractions (WAFs) of fuels were
investigated under different exposure scenarios to determine the relative sensitivity of
stages and of different exposure regimes. Sensitivity to fuel WAFs increased through
development. MGO and IFO 180 were more toxic than SAB, with EC50 values for the
most sensitive pluteus stage of 3.5, 6.5 and 252 µg/L THC respectively. Exposure to a
single pulse during fertilisation and early embryonic development shows similar toxicity
patterns to those observed from continuous exposure. This work shows that exposure to
fuel WAFs during critical early life stages affects the subsequent viability of larvae, with
consequent implications for reproductive success. The sensitivity estimates for S.

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/etc.4878.

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 neumayeri generated in this study can be utilised in risk assessments for the management
of Antarctic marine ecosystems.

Keywords: Marine toxicity tests, invertebrate toxicology, oil spills, polycyclic aromatic
hydrocarbons (PAHs), echinoid, larval
Accepted Article
This article includes online-only Supplemental Data.

*Address correspondence to Kathryn.Brown@aad.gov.au.

Published online XXXX 2020 in Wiley Online Library (www.wileyonlinelibrary.com).

DOI: 10.1002/etc.xxxx

Introduction

Increased shipping activities in the Southern Ocean places Antarctic marine environments
at risk of maritime accidents and petroleum fuel spills. Large volumes of fuel are carried
through Antarctic seas each year by research, station supply, tourism and fishing vessels,
both for their own bunkers and for annual refuelling of research stations (Klekociuk and
Wienecke 2017; ATCM 2018). Contamination of Antarctic coastal waters has occurred
as a result of shipping and refuelling accidents, leaks from fuel storage tanks close to
shorelines, and current and past waste management practises (Raymond et al. 2017).

Fuel spills have potentially long residence times in Antarctic waters due to polar
environmental conditions. The presence of sea ice creates a low energy environment that
can reduce spill dispersal by wave action. Spills can be trapped in or underneath sea-ice
and land-fast ice (Fingas and Hollenone 2003). In addition, the cold temperatures greatly
increase the viscosity and density of fuels, resulting in reduced evaporation of volatile
components from the surface of spills, and enhanced dispersion of toxic aromatic
compounds into the water column (Perkins et al. 2005; Brown et al. 2016). These
physical factors, along with slow biodegradation rates, contribute to reduced rates of oil
weathering in Antarctic waters compared with warmer regions, with potential for chronic
exposure of polar marine organisms to PAHs and other oil-related compounds (Camus
and Olsen 2008; Faksness and Brandvik 2008; Brakstad et al. 2018).

As well as potentially long residence time for fuel exposure, the nearshore environment is
susceptible to pulses of contaminant release during the freeze-thaw cycle. Soluble
hydrocarbon compounds from oil encapsulated in ice are slowly released to the ice-water
interface through brine channels formed during melting (Faksness and Brandvik 2008).
Soils contaminated by fuel spilt during past and current activities on stations can release
contaminants from the terrestrial environment into the nearshore environment when
summer temperatures mobilise meltwaters (Tin et al. 2009). To assist in the management
of petroleum in the region, risk assessments and water quality guidelines for fuels are
required. To develop these guidelines, information on the effects of hydrocarbons on

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 Antarctic organisms is needed. This requires toxicity testing with fuels that are used in
the region with local Antarctic species under ambient conditions.

Diesels specifically refined to suit polar conditions, such as Special Antarctic Blend
diesel (SAB) are in widespread use in Antarctica. Marine gas oil (MGO) and intermediate
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residual fuel oils (IFO 180) are commonly used shipping fuels in the region. Marine
organisms are generally more sensitive to diesel and residual fuel oils than to crude oils
due to the high aromatic content in refined fuels (Anderson et al. 1974; Busdosh and
Atlas 1977; Neff et al. 2000; Hatlen et al. 2010).

There are few published studies on the sensitivity of Antarctic marine invertebrates to
fuels. Amphipods, copepods, echinoderms, and the coastal Antarctic zooplankton
community have been demonstrated to be sensitive to SAB diesel and fuel oils at low
concentrations (Lane and Riddle 2004; Payne et al. 2014; Alexander et al. 2017; Brown
et al. 2017). Most of these studies however, examine a single species or life stage, and a
single fuel only. Further ecotoxicological data are required to estimate sensitivities of a
range of species and life history stages to the range of fuels that are carried in large
volumes in Antarctic waters.

The early life stages of sea urchins are frequently used as model test organisms as they
are sensitive indicators of the toxicity of a range of contaminants (Kobayashi 1981),
however most tests to date have been carried out with temperate and tropical species. The
Antarctic endemic sea urchin Sterechinus neumayeri (Meissner 1900) has been
demonstrated to be a suitable test organism for determining effects of contaminants in
Antarctica (King and Riddle 2001; Alexander et al. 2017).

Sterechinus neumayeri has a circumpolar distribution and is one of the most abundant
animals found in shallow benthic communities (Brey and Gutt 1991; Palma et al. 2007).
This ecologically important species is a grazer and scavenger whose feeding activities
may influence the Antarctic shallow benthic community structure (Brey and Gutt 1991).
This urchin displays the low metabolic rate, slow growth, extended reproductive and
developmental periods, and long lifespans typical of Antarctic marine invertebrates
(Bosch et al. 1987; Shilling and Manahan 1994; Brockington et al. 2007; Peck et al.
2007). Sterechinus neumayeri has typical echinoid planktotrophic development.
Reproduction occurs in the austral late spring/early summer and larval stages form a
significant component of the meroplankton in Antarctic coastal waters during December
to February, with metamorphosis and settlement to a mobile benthic juvenile occurring in
March (Bosch et al. 1987; Bowden et al. 2009).

In this study we use S. neumayeri to assess the risk from fuel spills to Antarctic nearshore
marine ecosystems. We investigate the effects of the water accommodated fractions
(WAFs) of three fuels: Special Antarctic Blend diesel (SAB), marine gas oil (MGO) and
intermediate fuel oil (IFO 180), on fertilisation and normal embryonic and larval
development of S. neumayeri. Three exposure scenarios were used to investigate whether

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 effects on early development were dependent on the life stage at which exposure started
(i.e. gametes or embryos), and whether a single pulse of exposure to fuel WAFs during
fertilisation and embryonic development would have the same effect on developing
larvae as continuous exposure. The sensitivity of S. neumayeri to total hydrocarbon
content in WAFs was determined using multiple endpoints: fertilisation, normal
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embryonic development (to 2 cell, 4-8 cell and unhatched blastula), and normal larval
development (to blastula, gastrula and early 4-arm pluteus). The toxicity of different fuel
types and different test stages are compared using critical effect concentrations to provide
information on the risk of these common fuels to this important species within Antarctic
marine communities.

Materials and methods

Test fuels

Special Antarctic Blend diesel (SAB; BP Australia) was obtained from bulk fuel storage

tanks at Davis station in January 2010. This middle distillate is composed of n-alkanes

ranging from n-C9-14 with a peak around n-C12, along with cyclic and branched alkanes

and aromatic hydrocarbons. This diesel is formulated for polar conditions and may

include anti-icing agents and other additives. Marine gas oil (MGO) was obtained in

December 2009 from the bunker of the Aurora Australis, the Australian Antarctic

research and supply vessel. This marine distillate is composed predominantly of n-

alkanes, cycloalkanes and aromatic hydrocarbons from n-C9-25. Intermediate fuel oil (IFO

180) was provided by BP Australia in October, 2009 from Whinstanes Terminal,

Brisbane. Also known as bunker, or residual fuel oil, IFO 180 is composed of the

residuum from refinery processes blended with ~ 6 to 7% of middle distillate gas oil. This

blend creates a complex mixture in the range n-C6 to >C40 of n-alkanes, branched and

cyclic alkanes and aromatic hydrocarbons, including PAHs, and may include cracked

components. Composition is dependent on the source oil, refinery processes and

proportion of gas oil added. All fuels were stored sealed in drums with minimum

headspace until use.

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 Production of water accommodated fractions

Water accommodated fractions (WAF) of SAB, MGO and IFO 180 were prepared using
the methods as outlined in Brown et al. (2016). All WAFs were made with 1:25 fuel to
filtered seawater (FSW) loading in glass aspirator bottles with 20% headspace. Mean
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water quality parameters for FSW (0.45 µm) used for making WAFs (n = 10) were pH
8.1 ± 0.2, salinity 36.1 ± 0.8‰ and dissolved oxygen 11.1 ± 0.5 mg/L. After loading,
aspirator bottles were sealed and set on magnetic stirrers at ~200 rpm (vortex <1 cm
depth) in darkness in a temperature controlled cabinet set at -1±1°C, for 18 h with a 6 h
settling period. Temperature in the cabinet was monitored at 10 min intervals using a data
logger (Maxim ibutton), and the mean temperature recorded was -0.5 ± 0.9°C.

Bottles were removed from the cabinet after the 6 h fuel settlement period and the
aqueous portion was drained into chilled glass bottles. Volumetric dilutions were
prepared from the 100% WAF solution without delay to provide test treatments at the
required WAF dilutions (Barron and Kaaihue 2003; Hodson et al. 2019). Fresh WAFs
were made every 4 d and used for renewal of test treatments as required.

Field collections

Mature S. neumayeri were collected from the outlet of Ellis Fjord, East Antarctica
(68.62°S, 77.99°E) in December and early January 2010/11, within the summer spawning
period for the species (Bosch et al. 1987). Sea urchins were collected from shallow
nearshore waters < 1m deep using hand-held nets. Sea urchins were placed in 20 L
buckets of seawater and transported to Davis station. They were held for 1–2 d in a flow-
through aquarium at -1±1°C, with macroalgae from the collection site as a food source,
before being used for testing. Seawater for experiments was collected ~20 m from the
shoreline north of Davis station (68°34’ S, 77°57’ E), away from any obvious signs or
inputs of contamination. Collected seawater was filtered to 0.45 µm (FSW) and stored in
30 L polyethylene containers at 0°C. Samples of collected seawater were analysed for
hydrocarbons, which were not detected.

Collection of gametes

Spawning of S. neumayeri was carried out within a temperature controlled room at 0°C.
Sea urchins were induced to spawn by injection of 2–3 mL of 0.5M KCl into the coelom.
Individuals were placed semi submerged in separate containers of FSW with genital
pores exposed. Spawning males were kept upright and sperm was collected dry with a
pipette, transferred into tubes and stored on ice until required. Spawning females were
inverted over containers of FSW with their genital pores submerged to allow eggs to be
released directly into FSW.

The condition of eggs and sperm from each individual were examined under a
microscope for quality (uniform profile of eggs and motile sperm) before use in toxicity

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 tests. Gametes from multiple parents (2–3 females and 2–4 males) were pooled to be
more representative of a population of spawners in natural conditions (Byrne 2012;
Environment Canada 2014), with unique sets of parents used for each toxicity test.

Eggs from each individual were collected with a wide-bore pipette and pooled in a 1 L
Accepted Article
beaker of FSW and gently mixed. The egg concentration was determined from counts of
six 1 mL aliquots of the egg suspension. The collected sperm samples were pooled and
sperm density determined using a haemocytometer.

Toxicity tests

The effect of SAB, MGO and IFO 180 WAFs on the fertilisation success, and on normal
embryonic and larval development of S. neumayeri were tested. Test methods were based
on standard protocols e.g. Environment Canada (2014) with adaptations for this Antarctic
species based on King and Riddle (2001). Table 1 shows the test design detailing the test
set up, developmental stages exposed and exposure regime, and the endpoints assessed
for each test.

Fertilisation and early embryo toxicity tests. Effects of WAFs on fertilisation and on

development to the 2 cell stage were determined in static tests in which both eggs and

sperm were pre-exposed to SAB, MGO and IFO 180 WAFs, fertilised within treatments

and developed to the 2 cell stage (G1, G2, G3; Table 1). Gamete exposure and

fertilisation was done in a temperature controlled room at 0°C. The mean water quality

parameters for FSW used in tests were pH 8.1 ± 0.2, salinity 36.1 ± 0.8‰, and dissolved

oxygen 11.4 ± 0.2 mg/L.

Test vessels were 22 mL borosilicate glass vials with foil lined lids holding 20 mL of test
solution. There were 10 vials for each treatment; 5 replicates for fertilisation and 5
replicates for the 2 cell endpoint. To pre-expose eggs, 5 mL of prepared egg solution was
added to vials that contained 5 mL of 2, 20 and 100% WAFs and FSW controls, to give
final treatment concentrations of 1, 10 and 50% WAF dilutions and FSW controls. Vials
were sealed, swirled gently to mix and left standing for 20 min. To pre-expose sperm,
pooled sperm were activated by dilution in FSW to the density required for a sperm to
egg ratio of 800:1 (previously determined as optimal for this species by Ericson et al.
(2010)). One µL of sperm solution was added to vials containing 5 mL of FSW and
gently mixed. Five mL of this solution was then added to vials containing 5 mL of 2%,
20% and 100% WAFs (final treatments of 1, 10 and 50% WAF dilutions) and FSW
controls. The vials were sealed, swirled gently to mix and left for 15 mins. After the

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 gamete exposure period was complete, for each treatment the contents of the sperm vials
were added to the egg vials (Harrison and Ward 2001) with a final target concentration of
~10 eggs per mL.

Vials were sealed and placed into temperature-controlled cabinets set at -1±1°C.
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Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and
averaged -1.3 ± 0.5°C.

Tests were terminated at 4 h for the fertilisation endpoint, and at 11 h for the 2 cell
endpoint by the addition of 1 mL of 2.5% (v/v) buffered glutaraldehyde. Samples were
viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times
magnification. Fertilisation was assessed according to the presence or absence of a
fertilisation membrane in the first 100 eggs counted, to obtain the percentage of eggs
fertilised in each replicate. Test acceptability criteria was ≥70% fertilisation in controls.
The 2 cell endpoint was assessed in the first 100 embryos counted, as the percentage of
embryos in each replicate with normal first cleavage (intact cells, hyaline layer and
fertilisation membranes, regular cell division). Test acceptability criteria was ≥60%
normally developed 2-cell embryos in controls (Environment Canada 2014).

Embryonic and larval toxicity tests. Effects of fuel WAFs on embryonic and larval

development were tested with 1, 10, and 100% WAFs of SAB, MGO and IFO 180 and

FSW control, with 5 replicates per treatment. Eggs and sperm were collected and density

of solutions adjusted as described above to obtain the optimal sperm to egg ratio of 800:1.

Two semi-static tests (EL1, EL2; Table 1) were done to test effects of WAFs on embryos
and larvae when first exposed as zygotes (eggs fertilised in FSW then exposed to
treatments before the first cleavage). To fertilise eggs, sperm were activated by their
addition to 10 mL of FSW, and 1 µL of this sperm solution was added to beakers
containing 700 mL of egg solution and gently mixed. After two hours, the mixture was
stirred gently with a glass rod to maintain a homogeneous suspension while aliquots were
transferred into 100 mL glass vials filled with 80 mL of test treatment, to a final density
of ~10 zygotes per mL.

Three tests (GL1, GL2, GLP; Table 1) were done to test effects of WAFs on larval
development with exposure commencing as gametes. One mL aliquots of egg mixture
were added to vials containing 80 mL of test solution (to a density of ~10 eggs per mL)
and left for 20 min. Sperm were activated in 10 mL of FSW and 0.1 mL aliquots added to
the vials to fertilise eggs within treatments at a sperm to egg ratio of 800:1. Two exposure
regimes were used; continuous semi-static WAF renewal (GL1 and GL2) and a single
static pulse of WAF exposure up to the 4 d unhatched blastula stage, followed by post
exposure recovery in FSW up to the 21 d pluteus stage (GLP).

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 Vials were left uncovered and placed in a temperature controlled cabinet at -1±1°C with
an 18 h light, 6 h dark photoperiod to reflect Antarctic spring/summer conditions. Tests
were under semi-static conditions, with test solutions renewed every 4 d.

Treatment renewals were done by carefully removing and replacing approximately 90%
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of test solution. Disposable syringes with 10 cm of silicon tubing attached to the nozzle,
and with the end of the tubing covered with plankton mesh, were used to slowly
withdraw test solution while preventing embryos/larvae from being removed. The vials
were then refilled to the 80 mL mark with fresh test solutions. Treatment renewals for
tests EL1, EL2 and GL1, GL2 were with freshly made WAFs every 4 d. For the single
pulse WAF exposure test (GLP) on the first treatment renewal at 4 d, treatment solutions
were removed as described above, and replaced with FSW. This process was repeated so
that the remaining small volumes of WAF left in vials on the first renewal were mostly
flushed from replicates. All subsequent 4 d renewals for test GLP were with FSW.

To maintain the volume and salinity of test treatments a small volume of purified and
deionised (Milli-Q) water at -1°C was gently stirred into the vials to the 80 mL mark
every 2 d between water changes to replenish any water lost by evaporation. Water
quality measurements were made at the start of tests and pre and post treatment renewals.
Mean water quality parameters were pH 8.08 ± 0.10, salinity 36.6 ± 0.9‰ and dissolved
oxygen 11.1 ± 0.61 mg/L. Temperature was recorded at 10 min intervals using a data
logger (Maxim ibutton) and averaged -1.0 ± 1.0°C.

In tests where exposure commenced as zygotes, endpoints were the embryonic 4-8 cell
(20 h) and unhatched blastula (48 h) stages, and the larval blastula (6–7 d) and gastrula
(14–15 d) stages (Table 1). In tests with exposure commencing as gametes, endpoints
were the larval blastula, gastrula and early 4-arm pluteus (21–24 d) stages (Table 1). At
each endpoint a sample was taken from each replicate by drawing an aliquot with a glass
pipette and transferring it to a vial, to which 1 mL of 2.5% (v/v) buffered glutaraldehyde
was added. Embryo and larvae were viewed in a Sedgewick Rafter counting cell under a
compound microscope at 10 times magnification. The first 30 individuals in each sample
at the 4-8 cell and unhatched blastula endpoints, and the first 100 individuals at the
blastula, gastrula and pluteus endpoints, were assessed for normality. Test EL1 ended at
the blastula stage and tests EL2 and GL2 at the gastrula stage as there were insufficient
numbers of larvae remaining to continue the test beyond these stages. All remaining
larvae were counted at the final endpoint.

Criteria for normal embryos were; regular cell division, intact cells, hyaline layer and
fertilisation membrane. Criteria for normal larvae were; symmetrical, blastula with a
well-defined blastocoel, gastrula with normal archenteron development and symmetrical
plutei, with normal arm, spicule and gut development. Individuals were scored as
abnormal if they had abnormal morphology, were at an earlier stage compared to controls
(slow development), or were dead (as indicated by degradation). Test acceptability
criteria was ≥60% normally developed larvae in controls (Environment Canada 2014).

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 Chemical analysis of water accommodated fractions

Total hydrocarbon content (THC) in WAFs were derived from replicate tests conducted
under the same conditions but without test organisms. In these tests at 0°C, the
concentrations of freshly made WAFs of each of the three fuels, and the depletion of
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hydrocarbons from 100%, 50%, 10% and 1% WAFs at multiple time points over 7 d
were measured (Brown et al. 2016). Although the uptake of hydrocarbons by test
organisms may reduce toxicant concentrations within WAFs, THC in WAF with and
without organisms was not expected to be significantly different (Temara et al. 1999),
particularly considering the very low biomass in test vials.

Briefly, hydrocarbons from 500 mL samples of 100% WAF, and 50, 10 and 1% dilutions,
were spiked with surrogate standards and extracted with 10 ml dichloromethane. Extracts
were analysed for THC with GC-FID with a split/splitless injector (Agilent 6890N).
Seawater blanks, SAB spiked seawater blanks and sample duplicates were included in
each sample batch. Total hydrocarbon content was reported as the sum of hydrocarbons
(µg/L) in the range <n-C9 to C28 (Brown et al. 2016). The proportion of THC in three
equivalent carbon number fractions F1 (<n-C9), F2 (n-C9-C18) and F3 (n-C19-C28) was
calculated (Tong and Karasek 1984; Gustafson et al. 1997; Brown et al. 2016).

Each fuel WAF contained mostly monoaromatic and diaromatic hydrocarbons with SAB
WAF dominated by 1,2,4 trimethylbenzene, naphthalene and 2-methylnaphthalene, MGO
WAF dominated by benzene, toluene, m-xylene and 1,2,4-trimethylbenzene and IFO 180
WAF dominated by benzene, toluene, m-xylene, 1,2,4-trimethylbenzene, naphthalene and
2-methylnaphthalene. The mean THC (±SD) measured in 100% WAFs (n = 4) were 2912
± 178 µg/L in SAB (F1 = 1%, F2 = 99%); 1515 ± 110 µg/L in MGO (F1 = 26%, F2 =
72%, F3 = 2%); and 567 ± 40 µg/L in IFO 180 (F1 = 14%, F2 = 71%, F3 = 15%) (Brown
et al. 2016).

Toxicity was assessed as responses to total hydrocarbon content, however, it is

acknowledged that there may be additional toxicants in these mixtures that could not be
quantified by the chemical analysis used. These may particularly be present in IFO 180,
including organic and inorganic compounds such as sulphur, metals and phenolic acids
(Uhler et al. 2007; Anku et al. 2017).

Modelling of exposure concentrations

For fertilisation, and 2 cell embryonic development assays that were done in sealed vials,
measured values in freshly decanted 50% and 10% WAF dilutions were used as the
exposure concentrations. It has been demonstrated that depletion of hydrocarbons from
sealed containers is minimal in these WAFs during the first 24 h (Brown et al. 2016)
hence the hydrocarbon concentrations, and the proportions in fractions, in sealed vials at
4 h (fertilisation endpoint) and 11 h (2 cell stage) are not expected to be significantly
different from those at the start of tests.

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 For the embryonic and larval toxicity tests that were done in open vials, the exposure
concentrations of THC in WAFs were modelled from the measured concentrations in the
WAF depletion tests (Brown et al. 2016). Exposure concentrations used to model
sensitivity estimates were derived by calculating the time weighted mean THC between
pairs of successive measurements in the 100% WAFs and dilutions (Brown et al. 2017) to
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give overall exposure concentrations for each time point (Tsvetnenko and Evans 2002;
OECD 2019). These modelled concentrations integrated the loss of hydrocarbons over
time, and renewal of test solutions at 4 d intervals (Supplementary Figure 1). Weathering
behaviours of the three fuel WAFs meant that MGO and IFO 180 had similar modelled
THC exposure concentrations in the 100% WAFs from the 4 d endpoint onwards (~600
and ~400 µg/L respectively), which were considerably lower than for SAB (~1500 µg/L)
(Supplementary Figure 2).

For all sensitivity estimates, the THC concentrations in 1% WAF treatments were
calculated as a percentage of the THC in 100% WAFs, as hydrocarbon concentrations in
the 1% samples were too low to measure accurately with these methods. Relationships
between WAF concentration and dilution at 0°C were strongly correlated through time
for each fuel, and the relative proportions of THC in fractions F1-F3 for all dilutions were
relatively consistent through time (Brown et al. 2016).

Modelling of Critical Effect Concentrations

Statistical analyses were done using the software ToxCalc (v5.0.26, Tidepool Scientific
Software). All sensitivity estimates were derived using the modelled THC concentrations.
The EC10 and EC50 and their 95% confidence intervals were determined for fertilisation,
2 cell, 4-8 cell, unhatched blastula, blastula, gastrula and pluteus endpoints. Calculations
were done according to the maximum likelihood estimation with probit analysis after data
were normalised to the mean response of controls using Abbott’s formula. Trimmed
Spearman Karber or non-linear interpolation was used to calculate the critical effects
concentrations when the assumptions of probit analysis were not met.

Results

Fertilisation

Mean fertilisation success in controls of the three tests was 87, 73 and 76%. Compared to
controls, fertilisation in the highest concentration treatments (50% WAFs) was reduced
by 5 to 15% in SAB (1431 µg/L THC), by 5 to 30% in MGO (712 µg/L THC) and by 9
to 42% in IFO 180 (267 µg/L THC) (Figure 1). The EC50s for fertilisation of S.
neumayeri were greater than the highest concentrations tested for all three fuels (Table 2).

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 Embryonic development

Exposure to WAFs of the three fuels had detrimental effects on cleavage of S. neumayeri
embryos to the unhatched blastula stage. Observed abnormalities included irregular cell
division, disrupted hyaline layer or fertilisation membrane, lysed cells and necrosis
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(Figure 2).

During first cellular cleavage, more abnormalities were observed when exposure
commenced at the gamete stage (tests G1-3) compared to when exposure commenced as
zygotes (EL1-2, Figure 1). In tests with first exposure as gametes, sensitivity could be
estimated for the 2 cell endpoint in test G1 only, as EC50 estimates were greater than the
highest tested concentrations in tests G2 and G3. The 2 cell stage was most sensitive to
IFO 180 WAF (EC50 12 µg/L). Sensitivity to SAB was an order of magnitude lower
(EC50 602 µg/L THC), while EC50s for MGO were above the highest concentration
tested (712 µg/L THC; Table 2). In tests where first exposure was as zygotes (EL1 and
EL2), EC50 estimates for cleavage were greater than the highest tested concentrations
(2346, 1135 and 456 µg/L THC for SAB, MGO and IFO 180 respectively). At the
unhatched blastula stages in tests EL1 and EL2 the EC50s were above tested
concentrations for unhatched blastula for all 3 fuels (Figure 1; Table 2).

Larval development

Exposure to WAFs of the three fuels had detrimental effects on larval development of S.
neumayeri to the early 4-arm pluteus stage. Observed abnormalities included irregular
profiles, asymmetry, disrupted membranes and lysed cells as well as exogastrulation at
the gastrula stage and irregular development of arms and spicules and/or the gut at the
pluteus stage. (Figure 3).

Overall there were less abnormal larvae in IFO 180 than in MGO and SAB treatments at
the blastula and gastrula stages. In contrast, at the pluteus stage, abnormalities were
higher in IFO 180, both with the continuous exposure (GL1), and the single pulse
exposure (GLP) (Figure 4).

EC50s could be estimated for all three fuels at the blastula, gastrula and pluteus
endpoints, although fewer blastula estimates were able to be reported for IFO 180 (EC50
> highest concentrations tested) than for SAB and MGO. However, where estimates for
IFO 180 could be calculated, larvae showed the greatest sensitivity to this fuel. For
example in test EL1 the blastula EC50 for IFO 180 WAFs was 81 µg/L THC compared
to 468 µg/L for MGO and 876 µg/L for SAB (Table 2).

There was a general trend of increased sensitivity as development progressed. This was
most evident in the single pulse exposure test (Test GLP) where blastula, gastrula and
pluteus stages had EC50s of 630, 332, and 252; and EC10s of 202, 96 and 68 µg/L THC
respectively in SAB WAFs (Table 2; Supplementary Table 1). The same trend was

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 observed in MGO and IFO 180 WAFs, with the pluteus stage being by far the most
sensitive (EC50s of 6.5 and 3.5 µg/L THC for MGO and IFO 180), and an order of
magnitude more sensitive than the gastrula stage.

There was some evidence that larvae were more sensitive when exposure commenced as
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gametes compared to when exposure commenced as zygotes. In addition, larvae were
more sensitive in continuous exposure than in single pulse exposure followed by a
recovery period (Figure 4), particularly on development to the pluteus stage. However,
there was variability between the replicate tests with gamete exposure (e.g. GL1 and
GL2). In addition, responses following continuous exposure from the gamete stage in one
test (GL1) were less than from the fertilised egg stage (EL1 and 2), as well as from single
pulse exposures (GLP). This was most notable in MGO and IFO 180 treatments (Figure
4, d, e, g and h). These differences occurred despite all tests having similar percentages of
normal larvae in controls.

Discussion

The management of fuel use in Antarctic waters requires understanding of effects of fuels
used regularly in the region on Antarctic marine species. This study found that WAFs of
the three tested fuels were toxic to the early developmental stages of the Antarctic sea
urchin S. neumayeri, with sensitivity dependant on the fuel type, exposure duration and
the developmental stage when first exposed. Exposure of S. neumayeri to hydrocarbons
from fuels during early development had teratogenic effects, resulting in abnormal
embryos and larvae unlikely to complete development. These abnormalities were evident
in larvae that were exposed to a single static pulse of WAF during fertilisation and
embryonic development, as well as those exposed to continuous semi-static exposure.

Toxicity of WAFs in cold waters

As WAF chemistry is affected by temperature, exposures in toxicity tests with polar biota

at low temperatures differ from those conducted in warmer waters. Initial hydrocarbon

concentrations in cold seawater WAFs are lower than in WAFs made under warmer

conditions (Perkins et al. 2003; Faksness et al. 2008). Although THC may be lower in a

cold seawater WAF, aromatic hydrocarbon compound concentrations are enhanced,

particularly the volatile and semi-volatile 1 and 2-ring compounds (Perkins et al. 2003,

Brown et al. 2016).

This article is protected by copyright. All rights reserved.

 Despite the low THC in WAFs of all three fuels in this study at 0°C (Supplementary

Figure 2), S. neumayeri was sensitive to all fuels at low concentrations, with MGO and

IFO 180 more toxic than SAB (Table 2). However, the low THC concentrations in WAFs
Accepted Article
limited the number of estimates of sensitivity that could be obtained (estimates were >

highest tested concentrations), particularly for IFO 180. The 3 fuel WAFs were made

using the same loading rates and stirring times for comparisons. Fuel loadings used were

at the maximum rates recommended for sub-arctic conditions (Barron and Kaaihue

2003). The low total hydrocarbon content in these cold-water WAFs, and consequently

the number of estimates able to be determined, could possibly be increased by extending

the stirring times during WAF generation, particularly for IFO 180 (Faksness et al. 2008).

The EC50s for fertilisation estimated in this study indicate that it would be unlikely that a

fuel spill would greatly affect fertilisation success for S. neumayeri. However, sensitivity

estimates for larval stages (EC50s ranging from 3.5 to 1397 µg/L THC) suggest that a

major spill of these types of fuels in Antarctic waters during the austral spring/summer

may impact on annual recruitment to S. neumayeri populations in the vicinity of the spill.

Hydrocarbon concentrations similar to those used in the present study have been

measured in the sub-surface water column after oil spills in the Antarctic and in other

regions (Kennicutt II 1991; Seymour and Geyer 1992). The Bahia Paraiso fuel spill in

Arthur Harbour on the Antarctic Peninsula covered an area of 100 km2, with some

shallow near-shore areas subject to repeated pulses of fuel contamination (Kennicutt II

1990; 1991). Concentrations of up to 100 µg/L PAH (which represents a portion of

THC), were measured in the water column under the slick for several weeks and chronic

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 low level fuel contamination emanated from the shipwreck for over 12 years (Kennicutt

II and Sweet 1992; Janiot et al. 2003).

Accepted Article
Differences in WAF composition of the three fuels influenced their relative toxicity.
Fresh MGO and IFO 180 WAFs had substantial proportions of F1 (<n-C9), F2 (n-C9-C18)
and F3 (n-C19-C28) fractions, whereas SAB was almost entirely composed of the F2
fraction (Brown et al. 2016). The toxicity of aromatic hydrocarbons is positively related
to their KOW values, and the contribution to toxicity of different fractions in a WAF
mixture varies over time with changes in concentration and composition with weathering
(Neff et al. 2000; Bellas et al. 2013). The lower molecular weight F1 fraction evaporates
earlier than F2 and F3, and is often considered to contribute less to the effects of fuel
spills on organisms in the water column as it is rapidly lost from fuel spilt in warmer
waters (Camilli et al. 2010). However, weathering proceeds at a slower rate in polar
seawater than in warmer conditions (Perkins et al. 2005; Brakstad et al. 2018). This is
also true for the WAFs used in the present study at 0°C: it was 4 d before the F1 fraction
was depleted from MGO and IFO 180, while only a third of the F3 fraction was depleted
over the same time period (Supplementary Figure 3) (Brown et al. 2016).

The higher sensitivity to MGO and IFO 180 than to SAB is likely driven by the
proportions of F1 and F3 fractions in these WAFs, due to both the acute toxicity of
monoaromatics and the greater toxicity and persistence of heavier PAHs present. The
lower molecular weight aromatics, including BTEX compounds and naphthalenes, can be
the main contributors to toxicity of petroleum WAFs due to their high solubility in
seawater and rapid uptake by organisms (Landrum et al. 1996; Barron et al. 2004;
Gardiner et al. 2013; Hansen et al. 2019). However, the contribution of heavier
hydrocarbons at lower concentrations to overall toxicity can be orders of magnitude
greater than that of lighter aromatic compounds (Di Toro et al. 2007; Engraff et al. 2011).
Neff et al. (2000) found that monoaromatics were the main contributors to toxicity of
fresh crude oils to sea urchin larvae, with the contribution of heavier PAHs increasing
with weathering. The acute toxicity of light fractions to early life stages of the sea urchin
Lytechinus anamesus was reported by Hamdoun et al. (2002) who found, when
normalized for hydrocarbon content, non-weathered Santa Barbara crude oil was more
toxic than weathered oil. In contrast, the toxicity (by WAF dilution) of heavy fuel oil to
sea urchin larvae Strongylocentrotus purpuratus has been shown to increase with oil
weathering, despite a reduction in aromatic hydrocarbon concentrations with weathering,
likely due to the formation of more toxic compounds from the degradation of PAHs
(Bellas et al. 2013). The comparatively high toxicity of bunker fuel oils, such as IFO 180,
to early life stages of sea urchins has also been demonstrated previously: Kobayashi
(1981) and Rial et al. (2013) reported that bunker fuel oil was more toxic than crude oil to
sea urchin larvae. Alkyl-PAHs have been identified as the main determinants of toxicity
of heavy oils, including residual fuel oils (Adams et al. 2014; Bornstein et al. 2014) and
heavy crude (Kang et al. 2014). However, these fuels are highly complex mixtures of

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 hydrocarbons, and additional soluble polar compounds, phenols, and acid extractable
heterocyclic aromatic hydrocarbons may also contribute to toxicity.

Comparisons with other sea urchin sensitivities

Accepted Article
The early life stages of sea urchins are sensitive to hydrocarbon contaminants, although
comparisons between studies are not straightforward due to different hydrocarbon metrics
used in studies, including petroleum products tested, preparation of test solutions,
exposure regimes, and in the reporting of sensitivity estimates. The sensitivities of early
life stages of S. neumayeri to IFO 180 WAF in the present study were similar to those
estimated by Alexander et al. (2017). The EC50 values for IFO 180 in two replicate tests
of 62 µg/L and 71 µg/L THC at the unhatched blastula stage, and 32 µg/L and 340 µg/L
THC at the gastrula stage (Alexander et al. 2017) were close to those estimated for IFO
180 in the present study for hatched blastula (81 µg/L THC) and gastrula (48 and 258
µg/L THC) stages. In contrast, the early 4-arm pluteus was found to be more sensitive in
the present study. Sterechinus neumayeri is of similar sensitivity to Lytechinus anamesus
to WAFs of Santa Barbara crude oil (EC50s of 30 µg/L and 330 µg/L for non-weathered
and weathered respectively) (Hamdoun et al. 2002).

Influence of exposure duration and developmental stage exposed

The sensitivity of early life stages of sea urchins is dependent on the life stage exposed

and the duration and type of exposure, with larval stages often being more sensitive

(Landrum et al. 2011). Although an EC50 could not be estimated in this study for

fertilisation, sensitivity of S. neumayeri increased as development progressed, with the 4-

arm pluteus being the most sensitive stage. Some teratogenic effects of WAFs may not be

obvious at earlier stages when viewed with a light microscope, but manifest at later

stages of larval development. Previous studies have shown exposure of gametes and early

cleavage stages to hydrocarbons may have impacts on subsequent developing embryo

and larvae (Lewis and Santos 2016). Similar results have been reported for S. purpuratus

by Hose et al. (1983), in which environmental concentrations of benzo[a]pyrene did not

inhibit fertilisation, but did induce embryonic cytotoxicity and genotoxicity when

gametes were exposed.

This article is protected by copyright. All rights reserved.

 Hydrocarbons in WAFs affect the integrity of the cellular and embryonic membranes in
developing S. neumayeri embryos (Figures 2 and 3). Disruption of the chorion, and
vitelline membranes leading to irregular cellular arrangement and cell cytolysis has also
been reported for two sea urchin species in response to exposure to petroleum and
individual hydrocarbons (Pagano et al. 1978). Aromatic hydrocarbon compounds bind to
Accepted Article
the ends of membrane structures and disrupt membrane integrity (Sikkema et al. 1994).
In addition, impaired membranes could be inhibited in performing critical functions such
as ionic permeability, with consequent impacts on cellular ion homeostasis in the
developing embryo and in the regulation of intake of calcium that is required for larval
skeletal development (Zhadan and Vaschenko 1993; Tellis et al. 2013).

Severely abnormal larval morphology observed in S. neumayeri, such as exogastrulation,

abnormal shape and alignment of spicules, and lack of arm development in the pluteus
are also described for other sea urchin larvae exposed to hydrocarbons (e.g. Baldwin et
al. 1992; Pillai et al. 2003). Sea urchin larvae commence feeding at the pluteus stage and
those with abnormal gut and arm development would be unable to feed effectively, nor
metamorphose to the juvenile stage. The EC50s and EC10s estimated here for the pluteus
stage indicate high sensitivity of S. neumayeri to fuels used in Antarctica that would
effectively inhibit reproductive success, both through mortality and the development of
non-viable offspring. Furthermore, estimates of the effects of contaminants on
reproductive success can be indicative of population level effects and can therefore be
related to the health of populations and communities (Lenihan and Oliver 1995; Bellas
and Thor 2007).

Sensitivity under different exposure scenarios

The effect of WAFs of the three fuels was in most cases greater on embryos that were
fertilised within WAF treatments, than for embryos that were fertilised in FSW before
being exposed as zygotes. A similar trend was observed for embryos of the oyster
Crassostrea virginica exposed to physically dispersed oil from the Deepwater Horizon
oil spill (Vignier et al. 2015). Greater abnormalities occurred in embryos first exposed as
gametes in comparison to those first exposed as fertilised eggs (Vignier et al. 2015).
There was some between-test variability in the sensitivity of gametes of S. neumayeri in
the present study (GL1 and GL2), and similar trends have been noted previously in other
studies (Byrne 2012; Alexander et al. 2017). Differences in sensitivities between replicate
tests is likely due to variable gamete quality between batches of gametes from different
parents.

The increased sensitivity of larval stages was not simply due to extended exposure
duration. Both continuous exposure to WAF treatments (semi-static renewal), and pulse
exposure at an early stage, generally resulted in similar effects on larval stages. This
study shows that embryogenesis and early embryonic development of S. neumayeri are
vulnerable to the effects of contaminants, and damage from exposure to a single pulse of
fuel WAF severely affects the capacity for ongoing development to viable larvae. These

This article is protected by copyright. All rights reserved.

 findings suggest that the timing of exposure is critical and that transient exposure can
have significant effects that manifest at later stages of development.

For future studies, a larger number of test concentrations is recommended to increase the
ability to derive ECx estimates for larval stages with greater confidence. Because of
Accepted Article
logistical constraints with research in Antarctica, only 3 concentrations of WAFs (100%,
10%, 1%) plus a control were used in each toxicity test, below the minimum 5
concentrations (with partial biological effects) that are generally recommended in
standard tests. Further testing with exposure commencing at different developmental
stages would also give greater insight into the sensitivity of different life history stages of
S. neumayeri.

Conclusion

The critical effect concentrations for S. neumayeri from this present study can be utilised

in risk assessments and in establishing water quality guideline values for Antarctic

marine ecosystems. This work has demonstrated that exposure to fuel WAFs at

environmentally relevant concentrations during early development severely affects the

subsequent viability of larvae, with consequent implications for reproductive success. A

single pulse exposure to fuel WAFs during early development has similar effects to those

from multiple pulses, indicating that an acute pollution event during the early

development period for S. neumayeri could reduce local seasonal recruitment for this

species. The period during which this critical stage of development occurs coincides with

the annual peak in shipping and refueling activity in Antarctica, highlighting risks from

fuel use in the region. The risks to S. neumayeri early life stages may be particularly high

from a spill of IFO 180, both due to the sensitivity of the pluteus stage to this fuel, and

the potential for a residual fuel oil spill to be long lived in an icy environment.

Supplemental data - The Supplemental Data are available on the Wiley Online Library
at DOI: 10.1002/etc.xxxx

Acknowledgment— This research was supported by the Australian Antarctic Division,

and funded through an Australian Antarctic Science grant to P. Harrison (AAS 3054) and

This article is protected by copyright. All rights reserved.

 an Australian Postgraduate Award to K. Brown through Southern Cross University. The
authors would like to acknowledge members of the AAD 2010/11 summer expedition at
Davis for support during field collections, and M. Ho, J. Ericson and S. Payne for
assistance with sea urchin spawning and test preparation. We thank L. Wise and K.
Kotzakoulakis for chemical analysis of samples and we are grateful for the provision of
Accepted Article
fuel samples by BP Australia and the engineering crew of RSV Aurora Australis of the
AAD Voyage 3 of 2009/10. We thank J. Wasley and T. Raymond and the anonymous
reviewers for comments on the draft manuscript.
Disclaimer— K. Brown, C. King and P. Harrison declare they have no conflicts of

interest.

Author contributions statement— C. King and P. Harrison conceived the project design.
C. King, P. Harrison and K. Brown designed experiments. K. Brown performed
experiments and statistical analysis with technical advice from C. King and P. Harrison.
K. Brown wrote the first draft of the manuscript. C. King and P. Harrison provided input
to subsequent drafts and editorial assistance.

Data availability statement— Data pertaining to this manuscript are held at the
Australian Antarctic Data Centre http://data.aad.gov.au/ and at
DOI:10.4225/15/575F873F81A68, DOI:10.26179/5f1684864ca21. Data, associated
metadata, and calculation tools are also available from the corresponding author
(Kathryn.Brown@aad.gov.au).

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Accepted Article
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 Figure 1. Fertilisation and embryonic development: concentration response of the
Antarctic sea urchin Sterechinus neumayeri to THC (µg/L) in water accommodated
fractions of SAB (a, b, c), MGO (d, e, f) and IFO 180 (g, h, i). Plots a, d and g show the
percentage of fertilised eggs in tests G1, G2, and G3. Plots b, e and h show embryos with
normal cleavage at 2 cell stage in tests G1, G2 and G3 (exposure commencing as
Accepted Article
gametes) and at 4-8 cell stage in tests EL1 and EL2 (exposure commencing as fertilised
eggs). Plots c, f and i show the percentage of normal unhatched blastula embryos in tests
EL1 and EL2. Error bars show ±SD. THC = total hydrocarbon content, SAB = Special
Antarctic Blend diesel, MGO = marine gas oil, IFO 180 = intermediate fuel oil 180.

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 Figure 2. Micrographs of Antarctic sea urchin Sterechinus neumayeri embryos exposed
as gametes to the water accommodated fractions of fuels. Upper row shows fertilised egg
and normally developing embryos at the 2, 4 and 8 cell stages and the unhatched blastula
in seawater controls, and lower row shows examples of abnormally developing embryos
at the same stages in WAF treatments. Scale = 100 µm.
Accepted Article

Figure 3. Micrographs of Antarctic sea urchin Sterechinus neumayeri larvae exposed to

water accommodated fractions of fuels. From left to right, upper row shows normally
developing larvae at the blastula, gastrula and pluteus stage in seawater controls, and
lower row shows examples of abnormally developing larvae at the same stages in WAF
treatments. Scale = 100 µm.

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 Figure 4. Larval development: concentration response of the Antarctic sea urchin
Sterechinus neumayeri to THC (µg/L) in water accommodated fractions of SAB (a, b, c),
MGO (d, e, f) and IFO 180 (g, h, i). Plots show percent normal blastula (left), gastrula
(middle) and 4-arm pluteus (right) developed from embryos fertilised in seawater before
exposure in 2 tests (EL1 and EL2), and from gametes fertilised within treatments in 2
Accepted Article
tests (GL1 and GL2). The response from a single test (GLP) is for exposure to WAFs
from gametes to unhatched blastula stage (4d), with development in FSW to pluteus
stage. THC = total hydrocarbon content (µg/L), SAB = Special Antarctic Blend diesel,
MGO = marine gas oil, IFO 180 = intermediate fuel oil 180.

Table 1. Design of fertilisation, embryonic and larval toxicity tests with the Antarctic sea urchin
Sterechinus neumayeri exposed to water accommodated fractions (WAF) of SAB, MGO and IFO
180 fuels.

Endpoint

Test Test set Stage Fertilisation 2 4-8 Unhatched Blastula Gastrula14 Early 4-
up first 4h cell cell blastula 6-7d – 15 d arm
exposed 11 20 48 h pluteus
h h 21 – 24 d

a
G1 Sealed gametes + +
vials
a
G2 gametes + +

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 20 mL
a
G3 treatments gametes + +
of 0, 1, 10,
50%
WAFs
Accepted Article
b
EL1 Open vials zygote + + + NC NC

b 80 mL
EL2 zygote + + + + NC
treatments
a
GL1 of 0, 1, 10, gametes + + +
100%
a
GL2 WAFs gametes + + NC

c
GLP gametes + + +

a
Eggs and sperm pre-exposed to treatments, fertilised within treatments, static exposure.

b
Exposure to treatments commenced after fertilisation but before first cleavage. Semi-

static exposure, treatments renewed every 4 d.

c
Eggs and sperm pre-exposed to treatments, fertilised within treatments Semi-static

exposure; to WAFS for 4 d, then FSW from 4 to 21 d with renewals every 4 d.

NC = not counted as insufficient numbers of larvae remaining in vials.

FSW = filtered seawater.

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 Table 2: 50% effective concentrations (EC50s) of total hydrocarbon (THC) (µg/L) with
95% confidence intervals for the Antarctic sea urchin Sterechinus neumayeri in water
accommodated fractions (WAF) of Special Antarctic Blend diesel (SAB), marine gas oil
(MGO) and intermediate fuel oil (IFO 180). EC50s are for fertilization, embryonic and
larval development at blastula, gastrula and early 4-arm pluteus stages.
Accepted Article
EC50 (µg/L THC)
4-8 4-arm
WAF Test Fertilisation 2 cell Unhatched Blastula Gastrula
cell blastula pluteus

SAB G1 >1431 602 (454

G2 >1431 – 798)
G3 >1431 >1431
>1431

EL1 >2346 >1825 876 (661 –

EL2 >2346 >1825 1160) 1397 (1328
1072 (764 - – 1479)
1504)

GL1 >1449 >1479

GL2 262 (222 - 348 (294 – <14
GLP 309) 410) 252 (215 -
630 (581 – 332 (293 – 292)
683) 375)

MGO G1 >712 >712

G2 >712 >712
G3 >712 >712

EL1 >782 >782 468 (221 –

EL2 >782 >782 1654) >513
194 (134 –
281)

GL1 >490 >513

GL2 62 (44 – 89) 91 (77 – <5
GLP 175 (161 – 107) 6.5 (3.7 –
190) 91 (80 – 11)
104)

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 IFO G1 >267 12 (6.4 –

180 G2 >267 18.4)

G3 >267 >267
>267
Accepted Article
a
EL1 >456 >356 81
EL2 >456 >356 >294 258 (139 –
481)

GL1 >280 >286

GL2 >306 >291 <3
GLP >280 48 (22 – 3.5 (1.03
119) – 6.7)

Where point estimates could be estimated, but were outside the range of tested
concentrations, they are reported as > highest concentration or < lowest concentration.
a
95% confidence intervals not available.

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