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New Zealand Journal of Marine and Freshwater Research
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Acute sublethal toxicity of MV Rena contaminants
(heavy fuel oil, oil dispersant and cryolite) to
finfish and rock lobster
A Webby & N Ling
To cite this article: A Webby & N Ling (2016) Acute sublethal toxicity of MV Rena contaminants
(heavy fuel oil, oil dispersant and cryolite) to finfish and rock lobster, New Zealand Journal of
Marine and Freshwater Research, 50:1, 144-158, DOI: 10.1080/00288330.2015.1104366
To link to this article: http://dx.doi.org/10.1080/00288330.2015.1104366
Published online: 27 Apr 2016.
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2. RESEARCH ARTICLE
Acute sublethal toxicity of MV Rena contaminants (heavy fuel
oil, oil dispersant and cryolite) to finfish and rock lobster
A Webby and N Ling
School of Science, University of Waikato, Hamilton, New Zealand
ABSTRACT
The wreck of the MV Rena in October 2011 discharged large
quantities of potentially toxic materials in the coastal marine
environment of the Bay of Plenty, New Zealand. We evaluated
sublethal effects of the water soluble fractions of physically and
chemically dispersed heavy fuel oil (HFO) and the water soluble
fraction of cryolite on the haematology of subadult or adult
snapper (Chrysophrys auratus), spotted wrasse (Notolabrus
celidotus) and red rock lobster (Jasus edwardsii). Small but
transient effects occurred, the most significant of which were
changes in circulating immune cell populations in fish and lobster
that recovered by the conclusion of a 96-h exposure period. No
post-exposure effects were observed during a 10-day recovery in
toxicant-free seawater. Corexit 9500 oil dispersant increased
polycyclic aromatic hydrocarbon (PAH) uptake from HFO in fish
and lobster by around two-fold and four-fold, respectively, and
caused a larger change in immune cell abundance in snapper
than exposure to HFO alone. While depuration of PAHs was more
rapid in fish than lobster, significantly elevated PAH levels were
still evident after 10 days of depuration.
ARTICLE HISTORY
Received 11 January 2015
Accepted 24 September 2015
KEYWORDS
Chrysophrys auratus; Corexit
9500; cryolite; heavy fuel oil;
Jasus edwardsii; MV Rena;
Notolabrus celidotus
Introduction
The wreck of the MV Rena on Astrolabe Reef (37°32′
42′′
S, 176°25′
73′′
E) in October 2011
discharged large quantities of potentially hazardous material into the marine environment
of the Bay of Plenty, New Zealand (Battershill et al. this edition). Approximately 350
tonnes of heavy fuel oil (HFO) was discharged from the breached hull and early attempts
to disperse the resulting surface oil slick involved the application of around 3000 L of the
dispersant Corexit 9500. Thirty-two of the 1368 containers on the ship were documented
to contain dangerous goods and subsequent salvage operations determined that most, if
not all, of these containers had been breached, releasing their contents. Of particular
concern at the time were 20 containers comprising approximately 515 tonnes of cryolite
(sodium hexafluoroaluminate), a mineral used in the smelting of aluminium and sourced
from the Tiwai Point aluminium smelter (New Zealand’s Aluminium Smelter Limited).
Although generally described in media reports as cryolite, this material was more likely
to be waste cryolite in the form of spent pot lining (SPL) given that it was in transit
from, and not to, the smelter and that the Tiwai Point smelter initiated export of this
© 2016 The Royal Society of New Zealand
CONTACT N Ling nling@waikato.ac.nz
NEW ZEALAND JOURNAL OF MARINE AND FRESHWATER RESEARCH, 2016
VOL. 50, NO. 1, 144–158
http://dx.doi.org/10.1080/00288330.2015.1104366
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3. waste material in 2010 (Jamieson 2010). The composition of SPL is highly variable with
carbon, alumina, sodium fluoride and cryolite typically the most abundant components
(Lisbona & Steel 2008). Dispersal of these contaminants throughout the local marine
environment created a potential for toxic effects in marine fauna, primarily due to
acute exposure given the relatively high energy open coastal nature of the wider area
affected by the oil spill and the dispersal of containerised dangerous goods.
Relatively little information is available on the toxicity of HFO or cryolite to marine
organisms, and virtually no research has been undertaken on the toxicity of oil to New
Zealand marine species. HFO is primarily composed of the residue remaining after
much of the lighter components of crude oil have been removed during the refining
process, but is subsequently mixed with lighter oils to produce a product with a viscosity
that is manageable for handling and transport. Heavy fuel oils are highly variable
because their final composition depends on the composition of the crude oil feedstock,
the refining process and the composition of the diluents added to reduce viscosity.
Heavy fuel oil is a complex mixture of many hydrocarbon compounds, but does not
contain as much of the volatile components of crude oil and experiences far less loss
by weathering during spill events. Of most concern toxicologically are the many
species of polycyclic aromatic hydrocarbons (PAHs), with recent studies indicating
that some types of HFO may be up to 50 times more toxic than some crude oils (Born-
stein et al. 2014). Furthermore, chemical dispersant can increase the toxicity of heavy
fuel oil to fish with lower dispersant concentrations having the greatest impact on oil
toxicity (Koyama & Kakuno 2004).
Literature on the toxic effects of pure cryolite, SPL or cryolite recovery sludge to aquatic
organisms, especially marine, is scarce. The US Environmental Protection Agency (EPA)
explains that acute risk is not expected to birds, mammals or aquatic organisms as, in the
presence of sufficient water, cryolite is quickly converted to near natural background levels
of simple inorganic compounds containing its constituent elements (sodium, aluminium
and fluorine). Furthermore, chemical equilibriums in soil and aquatic environments are
said to buffer concentrations of cryolite residue (United States Environmental Protection
Agency 2011).
Exposure of subtidal organisms to oil or its water soluble fraction at high concen-
trations is likely to be of limited duration due to the relatively rapid dispersal of oil and
oil emulsions in the marine environment. Chronic exposure to oil and its components
is more likely in sheltered environments such as mangrove habitats and estuaries,
where intertidal organisms are impacted by significant oil deposition on rocky shores
or where subtidal benthic organisms remain in close association with oil residue. The inso-
lubility of cryolite or SPL material means that large deposits are likely to be present where
sunk containers spilled their contents and some degree of chronic exposure is expected in
organisms at these locations. Due to the local importance of indigenous, recreational and
commercial fishing interests in the region there was concern that major fishery species
could be adversely affected resulting in ecotoxicological concerns for those species or toxi-
cological concerns for human consumers.
Haematology, particularly any change in the immune system, is a potentially useful
sublethal bioindicator for ecotoxicological effects at higher levels of biological organisation
(individual, population or community), but relatively few studies expose large adult or
subadult fish to oil or other toxicants because of the logistical difficulties of doing so.
NEW ZEALAND JOURNAL OF MARINE AND FRESHWATER RESEARCH 145
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4. The aim of this study was to examine acute sublethal effects on the haematology of three
species (two vertebrate and one invertebrate) of subtidal macrofauna that are of major cul-
tural, commercial or ecological importance, in response to an environmentally realistic
exposure (both in concentration and duration) to the physically or chemically dispersed
water soluble fractions of oil and the physically dispersed water soluble fraction of cryolite.
Additionally, we investigated the rate of recovery following such exposure.
Methods
Test species
Test species were chosen on the basis that they were of major recreational or commercial
interest, of significance to Māori, or ecologically important macrofauna. Snapper (Chry-
sophrys auratus [Forster]) are one of the most abundant demersal continental shelf
species in New Zealand and southern Australia, of cultural significance to Māori and
are considered to be one of New Zealand’s most valuable inshore finfish species
(Parsons et al. 2014). Spotted wrasse (Notolabrus celidotus [Bloch & Schneider]) is a
New Zealand endemic and one of the most common reef fish found on open coastal
reefs as well as in harbours and estuaries (Ayling & Cox 1982; Jones 1988). Snapper
and spotted wrasse are both protogynous hermaphrodites. Red rock lobster (Jasus edward-
sii [Hutton]) are abundant throughout New Zealand and southern Australia and are con-
sidered to be a valuable resource as food, revenue and recreation. They are arguably the
most dominant benthic predator on coastal reefs and play an important role in ecosystem
functioning as well as being an important kai moana (sea food) species for Māori (Mac-
Diarmid et al. 2013).
Study animals were collected from the Waikato and Bay of Plenty coastal regions in the
North Island of New Zealand outside of areas affected by the Rena oil spill. Subadult red
rock lobsters (ocular carapace length 86 ± 7.9 mm, weight 428 ± 7.9 g; mean ± SEM) were
collected by SCUBA diving as well as from a commercial fishing vessel. Adult female
spotted wrasse (length 170 ± 3.7 mm total length, weight 85 ± 6.7 g) and subadult
snapper (length 181 ± 2.7 mm fork length, weight 140 ± 5.8 g) were caught by baited
angling.
Animals were held at the University of Waikato Aquatic Research Centre in 5000 L
fibreglass holding tanks supplied with artificial recirculated seawater (Crystal Sea Marine-
mix, Marine Enterprises International, Baltimore, MD). Salinity varied from 32 ppt–34
ppt and water temperature between 15–18 °C with a 12L:12D photoperiod. Water
quality (salinity, temperature, ammonia, nitrite, nitrate and phosphate) was measured
at least every 3 days and water changes made if necessary. The holding tanks contained
cinder blocks, terracotta pots and plastic pipes for refuge. The protogynus spotted
wrasse were maintained as adult females by the inclusion of at least one large adult
male in each holding tank. Snapper are also protogynus but, unlike spotted wrasse, sex
change in this species is not socially mediated and occurs at a size range greater than
that used here so the inclusion of adult males was unnecessary. Animals were fed twice
weekly on chopped green-lipped mussels (Perna canaliculus Gmelin) with excess food
waste removed up to 2 h after feeding, and animals were monitored daily for signs of
injury, changes in behaviour or disease.
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5. Toxicant exposures
Animals were exposed to toxicants individually in 25 L glass aquaria. The aquaria were
constructed without silicon glue because polysiloxane material can strongly absorb
PAHs (Popp et al. 2004). Aquarium walls were edge glued with Dymax Light-Weld to
present virtually no surface for adsorption and supported externally with an aluminium
frame. Aquaria contained 10 L of toxicant solution with 50% replacement every 24 h
and exposures lasted up to 96 h (24, 48 or 96 h) with temperature maintained at 16 ±
0.5 °C in a constant temperature room. Control animals were either sacrificed directly
from the holding tanks or held for 96 h in the exposure aquaria in artificial seawater to
test for the physiological effects of confinement. Aquaria were aerated using standard
aquarium air stones attached to glass tubing to eliminate adsorption of PAHs to plastic
or silicon airline tubing. Toxicant mixtures tested were a water accommodated fraction
(WAF) of Rena heavy fuel oil (HFO), the chemical dispersant (Corexit 9500) used on
the Rena oil slick, a chemically enhanced water accommodated fraction (CEWAF) com-
prising a combination of Corexit 9500 and Rena HFO, and a water soluble fraction of
cryolite.
Heavy fuel oil was obtained from fuel tanks of the MV Rena during salvage, Corexit
9500 was supplied by Maritime New Zealand and cryolite (butt bath material) was sup-
plied by Tiwai Point aluminium smelter (New Zealand’s Aluminium Smelter Ltd).
Exposures involved a single toxicant concentration that was determined to represent a
maximum likely environmental exposure concentration (10% WAF for HFO and cryolite)
but which was estimated to be lower than the relevant 96 h LC50 value. For the purposes
of this experiment, contaminant concentrations needed to be below the LC50 value in
order to examine acute sublethal effects rather than mortality. Rena HFO WAF and
CEWAF were prepared according to the method of Singer et al. (2000). Oil was added
to filtered (0.45 µm) artificial seawater at a concentration of 1 g/L in a 2 L borosilicate
glass vessel and stirred to produce a vortex reaching 25% of the depth of the solution
for 24 h at room temperature because the petroleum hydrocarbon content of WAF and
CEWAF under these conditions achieves a steady state within this time (Singer et al.
2000). The mixed oil was then allowed to settle for 2 h prior to use and any unused
WAF or CEWAF was discarded because of potential bacterial spoilage. Preparation of
CEWAF included the addition of Corexit 9500 dispersant at a ratio of 1:40 to oil (w/w).
The bottom layer of the WAF or CEWAF was aspirated using a glass tube to avoid includ-
ing any surface oil and diluted 1:10 with artificial seawater to produce the final exposure
solutions (10% WAF or 10% CEWAF). A Corexit 9500 control solution was prepared in
the same manner. Cryolite was mechanically sieved through a 25 µm sieve to remove large
particles and provide maximum surface area of the mineral for dissolution. Cryolite mix-
tures were prepared with 1 g/L cryolite:seawater. Two grams of cryolite was added to 2 L of
seawater and mixed following the standard mixing procedure. This suspension was further
diluted to produce a final exposure concentration of 10% WAF.
To examine post-exposure recovery and depuration, animals exposed to toxicants for
96 h were removed from treatment aquaria and held in 200 L recovery tanks supplied
with recirculating artificial seawater for 4 or 10 days. The recirculation system included
a filter of activated carbon to remove any excreted hydrocarbons. The number of
animals exposed was limited by their availability, the logistics of treating a large
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6. number of animals individually, and the limited availability of Rena HFO, of which only a
few hundred millilitres were available. Treatment groups included up to six animals for
each of both controls as well as 96-h exposure and 10-day recovery. Other treatments
(24- and 48-h exposure, 4-day recovery) used three animals each. Following exposure
or recovery, fish were euthanised with an overdose of anaesthetic (0.1 g/L benzocaine)
and rock lobsters were euthanised by hypothermia followed by brain ablation. Organism
weights (±1 g) and lengths (total length for fish and ocular carapace length for red rock
lobster, ±1 mm) were recorded and a visual inspection of the organism was carried out,
checking for any obvious signs of disease.
Blood (400 µL) was taken from the caudal vein of fish with a 1 mL heparinised syringe
and 23 G needle. Blood samples (500 µL) from rock lobster were collected using a 1 mL
syringe (23 G needle) from the pericardinal sinus under the thoracic carapace (cardiac
puncture) and immediately mixed with an equal volume of 10% neutral buffered formalin
fixative. Blood samples were stored on ice until analysis within 2 h. Fish blood was ana-
lysed for total red blood cell counts (RBCC), haemoglobin concentration (Hb), packed
cell volume (PCV), mean cell haemoglobin concentration (MCHC), mean cell haemo-
globin (MCH), mean cell volume (MCV) and differential leukocyte cell counts according
to standard methods. Lobster haematological analysis included total and differential hae-
mocyte counts (HC).
PAH uptake and depuration
Bile and haepatopancreas samples were analysed for pyrene-like fluorescence by synchro-
nous fluorescence spectrometry (SFS) using a Shimadzu RF5301 scanning spectrofluo-
rometer and a 1 cm path length quartz cuvette. Synchronous fluorescence spectrometry
spectra were scanned from 263 to 413 nm (excitation wavelength), scanning both mono-
chromators simultaneously at a fixed wavelength difference (Δλ) of 37 nm and bandwidth
of 5 nm. Quantification of conjugated pyrene was determined by measuring the net peak
area of the SFS spectrum from 372 to 392 nm (emission wavelength). Earlier studies have
identified that the highest concentrations of PAHs are detected in the haepatobiliary
system (Aas et al. 1998; Hosnedl et al. 2003). The bioavailability of pyrene to aquatic
organisms is relatively high and it exhibits a strong fluorescence which enables sensitive
detection (Hosnedl et al. 2003). Although different biotransformation products of
pyrene may be produced in different aquatic species, primarily pyrene-1 glucuronide
and pyrene-1-sulphate in finfish (Ikenaka et al. 2013) and a novel glucose–sulphate con-
jugate in crustaceans (Ikenaka et al. 2006), pyrene-like fluorescence has been quantitat-
ively correlated with PAH exposure in both these groups. Because the relative
fluorescence intensities of these different conjugates have not been determined they
were assumed to equate to that of pyrene-1-glucuronide which has been determined
(Ariese et al. 1993). Because conjugated pyrene standards are not available, peak areas
were calibrated against a series of unconjugated 1-hydroxypyrene standards (Toronto
Research Chemicals Inc., Toronto, Canada) and corrected for the greater fluorescence
intensity and blue shifted emission spectrum (by 5 nm) of pyrene-1-glucuronide using
a calibration factor of 2.2 according to Ariese et al. (1993).
A sample of bile was extracted from the gall bladders of fish using a 0.5 mL syringe (29
G fixed needle) and samples of lobster haepatopancreas (approximately 10 g) were
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7. removed and stored at −20 °C until analysis. Frozen bile samples were thawed and diluted
between 1:500 (controls) to 1:25,000 (treatments) in ethanol: water (1:1) and analysed by
SFS as above. Samples of frozen haepatopancreas tissue were thawed and 1 g was weighed
and placed into a 15 mL plastic centrifuge tube with 5 mL of ethanol:water (1:1). Samples
were then homogenised using an Ultra Turrax T8 homogeniser for 1 min followed by
ultrasonication using an ultrasonic processor Misonix sonicator for a total of 1 min 20 s
on power setting 3, consisting of 20 s of sonication, followed by 20 s of cooling. Homogen-
ates were centrifuged at 3500 RCF for 10 min and the supernatant aspirated from between
the surface fat layer and the tissue pellet using a 1 mL syringe with 19 G needle. 50 µL of
supernatant was diluted in 4950 µL ethanol:water (1:1) and analysed by SFS.
All statistical analyses were carried out in Statistica v11 (StatSoft Inc., Tulsa, OK). Stat-
istical differences between blood parameters and treatments were tested using one-way
analysis of variance (ANOVA) combined with Tukey’s honest significant difference
post-hoc tests. Results for four snapper were removed as they were found to be
anaemic when sampled. Statistics showed that their blood parameters fell below the 5th
percentile and that any causation was not obvious from treatment.
All tests carried out within this study had been approved by the University of Waikato
Animal Ethics Committee.
Results
No mortalities occurred to any of the toxicants tested. Exposure to the water accommo-
dated fraction (10% WAF) of Rena HFO or a chemically enhanced water accommodated
fraction (10% CEWAF) produced no obvious effects on the red cell haematology of either
finfish species (Tables 1 and 2). Similarly, the oil dispersant Corexit 9500 and the alu-
minium smelter product cryolite apparently did not cause any changes in red cell haema-
tology. Confinement of individual fish in a relatively small water volume during the acute
exposures would be expected to elicit stress induced changes in haematology such as hae-
moconcentration and erythrocytic swelling but this was not evident from any significant
difference in values between the control fish and those confined in clean water for 96 h.
Any significant differences observed between treatments and these controls are most
likely a result of the small sample size tested.
Significant changes in circulating immune cell populations were observed however,
both in the finfish species and rock lobster (Tables 1–3). A significant decrease in lympho-
cytes and a corresponding increase in the relative abundance of granulocytes occurred in
snapper in response to the first 48 h of exposure to 10% HFO CEWAF. A similar, non-
significant response occurred in spotted wrasse to the same contaminant and, in both
species, to cryolite. The stress of confinement would be expected to induce a reduction
in the numbers of circulating lymphocytes via cortisol-induced apoptosis but this was
not apparent from the 96 h confinement controls. The lymphocytopenia observed is there-
fore likely a true response to contaminant exposure. Significant changes in immune cell
abundance were also observed in rock lobster although in this species there appeared to
be a large effect of confinement with confinement controls showing a significant reduction
in hyaline cells and a corresponding relative increase in the abundance of granulocytes. Of
interest is the absence of this effect in animals exposed to Corexit 9500 and the possibility
that Corexit 9500 diminishes this stress response in lobster. In all treatments and all
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8. Table 1. Haematological values of snapper (Chrysophrys auratus) exposed to heavy fuel oil (HFO) water accommodated fraction (WAF), chemically enhanced water
accommodated fraction (CEWAF), Corexit 9500 dispersant and cryolite for up to 96 h and following 4 or 10 days depuration.
Treatment and time n Hb (g L−1
) PCV (%) RBCC (×1012
cells L−1
) MCHC (g L−1
) MCH (pg cell−1
) MCV (fl) Lymphocytes (%) Granulocytes (%) Thrombocytes (%)
Control 6 72.6±3.0 33.1±1.2 3.18±0.17 220±11 23.0±1.2 106±8 81.5±2.7 16.3±2.4 2.8±0.5
Control 96 h 6 83.2±5.3 35.0±1.8 3.27±0.12 238±11 25.8±2.4 108±8 75.8±8.1 23.7±8.1 1.6±0.5
HFO WAF 24 h 3 73.3±4.4 41.1±3.9 3.31±0.22 182±21†
22.6±3.0 125±12 67.0±4.5 33.3±5.0 0.5±0.5
HFO WAF 48 h 3 75.4±2.7 41.1±0.8 3.23±0.03 184±10 23.4±0.6 127±4 70.7±5.6 28.7±5.4 0.7±0.2
HFO WAF 96 h 6 66.3±4.6 30.1±2.4 2.82±0.23 223±11 23.7±0.8 107±5 83.2±6.0 14.5±6.0 2.6±0.9
HFO WAF 4 d 3 58.1±7.4†
24.4±2.1†
2.76±0.35 236±12 21.0±0.4 89±4 67.7±3.3 29.7±2.8 3.8±0.7
HFO WAF 10 d 6 70.2±1.8 30.7±0.6 3.14±0.80 228±4 22.4±1.4 98±7 85.8±3.2 12.7±3.5 1.7±0.4
HFO CEWAF 24 h 3 N.D 37.8±3.7 3.08±0.12 N.D N.D 122±7 22.0±5.7*,†
78.3±5.4*,†
0.0±0.0
HFO CEWAF 48 h 3 80.8±2.7 37.8±1.4 2.68±0.35 214±10 30.9±2.7 146±19 51.0±2.6* 48.3±2.3* 1.0±0.5
HFO CEWAF 96 h 5 69.8±5.0 30.3±2.0 2.91±0.32 231±9 23.9±1.2 104±5 88.6±1.4 9.0±0.8 3.2±0.6
HFO CEWAF 4 d 3 76.5±5.1 32.5±3.9 3.58±0.51 239±19 21.9±1.7 92±3 84.0±2.0 14.3±2.3 3.0±1.0
HFO CEWAF 10 d 6 67.8±4.2 29.8±1.6 3.03±0.72 229±18 22.5±1.6 99±7 80.0±4.1 18.7±4.3 2.3±0.4
Corexit 9500 24 h 3 73.5±5.0 34.6±3.6 3.16±0.11 216±18 23.3±1.1 110±13 79.0±4.0 20.7±4.7 1.3±0.7
Corexit 9500 48 h 3 75.7±2.5 31.3±1.5 3.29±0.36 242±4 23.4±1.7 97±6 79.0±8.7 20.3±8.7 2.7±0.3
Corexit 9500 96 h 5 67.5±3.2 30.5±1.7 3.15±0.34 224±14 21.4±0.7 97±6 85.3±2.4 13.8±2.5 1.8±0.3
Corexit 9500 4 d 3 64.4±6.4 25.6±0.7†
3.20±0.50 251±20 21.2±3.7 84±13 86.0±3.1 13.3±3.2 2.2±0.4
Corexit 9500 10 d 6 66.5±5.7 26.8±2.5†
2.92±0.36 249±9 22.9±0.7 92±4 83.7±5.5 13.8±5.1 3.1±0.6
Cryolite 24 h 3 70.6±8.2 36.2±3.5 2.92±0.27 194±5 24.1±1.4 124±7 57.3±7.4 43.0±7.6 0.2±0.2
Cryolite 48 h 3 72.1±1.4 38.2±2.2 3.24±0.25 189±7 22.5±1.4 119±7 56.7±9.7 43.0±10.1 0.7±0.7
Cryolite 96 h 3 74.2±2.9 38.2±2.2 2.78±0.25 195±3 27.3±3.2 140±17 80.3±8.2 17.7±7.4 2.7±1.2
Cryolite 4 d 3 73.2±1.2 35.8±0.4 3.19±0.02 205±4 23.0±0.5 112±1 82.3±1.7 17.0±1.5 2.3±0.3
Cryolite 10 d 3 75.7±4.7 32.5±0.4 3.30±0.25 233±15 23.3±2.6 100±7 85.3±5.7 13.3±6.4 2.2±0.4
Hb, whole blood haemoglobin concentration; PCV, erythrocyte packed cell volume; RBCC, red blood cell count; MCHC, mean cell haemoglobin concentration; MCH, mean cell haemoglobin
content; MCV, mean cell volume.
Values are means ± SEM. Values in bold accompanied by * or † indicates that the value is significantly different (P < 0.05) from the control or 96 h control, respectively.
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9. Table 2. Haematological values of spotted wrasse (Notolabrus celidotus) exposed to heavy fuel oil (HFO) water accommodated fraction (WAF), chemically enhanced
water accommodated fraction (CEWAF), Corexit 9500 dispersant and cryolite for up to 96 h and following 4 or 10 days depuration.
Treatment and time n Hb (g L−1
) PCV (%) RBCC (×1012
cells L−1
) MCHC (g L−1
) MCH (pg cell−1
) MCV (fl) Lymphocytes (%) Granulocytes (%) Thrombocytes (%)
Control 6 62.4±3.0 30.5±1.6 3.42±0.18 206±13 18.3±0.8 90±8 83.2±8.7 14.6±8.5 3.0±0.4
Control 96 h 6 81.5±5.9 39.1±5.3 3.43±0.23 224±30 23.8±1.0 114±15 70.8±1.9 23.5±2.0 5.5±1.0
HFO WAF 24 h 3 77.8±7.7 46.1±6.5 3.97±0.66 173±22 20.4±2.4 117±4 54.7±5.0 28.3±7.1 17.3±6.7
HFO WAF 48 h 3 90.6±17.2 46.5±6.0 4.28±0.74 195±26 21.4±2.2 111±7 52.7±9.6 34.7±3.2 13.0±6.7
HFO WAF 96 h 3 71.6±5.9 55.8±9.7 4.06±1.07 134±15 19.6±4.1 150±37 55.0±2.0 21.0±3.8 23.7±5.2
HFO WAF 4 d 3 64.2±6.1 40.3±2.1 4.05±0.19 159±10 15.8±1.0†
99±1 87.3±2.7 8.7±2.0 4.7±0.9
HFO WAF 10 d 3 57.6±7.7 33.8±5.9 2.83±0.19 177±26 20.3±1.9 118±14 77.7±14.0 7.7±3.9 15.0±12.1
HFO CEWAF 24 h 3 54.0±2.3†
39.4±3.6 3.30±0.11 139±9 16.3±0.4†
119±7 58.0±12.2 40.0±11.6 2.0±0.6
HFO CEWAF 48 h 3 60.3±2.1 30.8±3.2 2.58±0.26 202±29 23.7±1.8 123±23 52.5±0.5 25.5±6.5 23.0±7.0
HFO CEWAF 96 h 3 72.2±1.6 35.8±0.8 3.35 ±0.03 202±4 21.6±0.7 107±3 52.0±12.5 21.7±10.1 26.3±17.6
HFO CEWAF 4 d 3 72.1±2.5 40.7±2.2 3.52±0.40 178±8 20.9±2.2 119±19 83.3±2.0 10.7±1.2 6.7±1.3
HFO CEWAF 10 d 3 74.5±10.7 37.7±4.4 3.23±0.36 198±21 23.2±3.1 117±4 82.7±1.2 13.0±1.0 5.3±1.3
Corexit 9500 24 h 3 56.5±7.5†
29.3±2.1 3.46±0.55 193±22 16.5±0.5†
89±14 68.7±10.9 27.3±10.8 5.0±0.6
Corexit 9500 48 h 3 63.9±3.1 32.5±0.8 2.83±0.45 196±4 23.4±2.6 120±14 63.7±9.0 33.7±8.5 3.7±0.7
Corexit 9500 96 h 3 54.9±7.0†
34.1±3.4 2.86±0.18 160±7 19.2±1.9 119±7 82.0±1.5 13.7±0.7 5.3±1.9
Corexit 9500 4 d 3 62.3±4.7 37.0±4.0 3.29±0.27 174±28 19.0±0.3 116±21 85.0±0.6 10.3±0.9 5.7±0.3
Corexit 9500 10 d 3 65.0±1.6 33.7±2.6 3.35±0.33 195±15 19.7±1.8 102±8 80.0±4.5 14.0±2.5 6.7±2.2
Cryolite 24 h 3 62.5±5.5 38.6±2.1 3.44±0.58 163±17 18.9±2.3 119±20 59.5±8.5 33.0±10.0 8.0±1.0
Cryolite 48 h 3 74.6±13.0 39.0±1.2 3.36±0.42 192±37 22.3±2.6 120±13 59.7±2.3 37.3±4.3 3.3±2.4
Cryolite 96 h 3 65.0±3.6 37.8±1.2 3.27±0.11 172±12 19.9±0.6 116±7 71.7±4.4 21.7±2.7 7.3±2.0
Cryolite 4 d 3 62.1±7.2 42.8±4.8 4.12±0.23 148±23 15.0±1.5†
104±6 85.7±2.3 9.7±1.2 5.7±1.2
Cryolite 10 d 3 56.0±1.8†
29.7±2.9 2.37±0.20 194±26 24.0±2.3 126±15 77.0±7.2 19.0±7.0 4.0±0.7
See Table 1 for abbreviations.
Values are means ± SEM. Values in bold accompanied by * or † indicates that the value is significantly different (P < 0.05) from the control or 96 h control, respectively.
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10. species, recovery during depuration was rapid with immune cell values returning to
control levels within 4 days. Across all species tested a pattern emerges of a generally
greater immune response to HFO (both WAF and CEWAF), a lesser response to cryolite,
and virtually no response at all to exposure to Corexit 9500 alone.
Exposure to 10% HFO WAF caused a large increase in the concentration of the PAH
metabolite pyrene-1-glucuronide (P1G) in the bile of snapper (Figure 1) and spotted
wrasse (data not shown). Chemical enhancement of the water accommodated fraction
Table 3. Haematological values of red rock lobster (Jasus edwardsii) exposed to heavy fuel oil (HFO)
water accommodated fraction (WAF), chemically enhanced water accommodated fraction (CEWAF),
Corexit 9500 dispersant and cryolite for up to 96 h and following 4 or 10 days depuration.
Treatment and time n Granulocytes (%) Semi-granulocytes (%) Hyaline cells (%) Haemocytes (×1010
cells L−1
)
Control 6 11.0±1.8 12.3±1.4 77.5±2.9 2.04±0.20
Control 96 h 6 34.2±5.8* 22.2±4.2 43.7±9.1* 1.39±0.20
HFO WAF 24 h 3 47.0±1.0* 25.3±8.3 28.0±7.0* 1.42±0.37
HFO WAF 48 h 3 52.3±3.3* 30.0±3.8 17.7±1.5* 2.21±0.28
HFO WAF 96 h 6 31.0±8.9 15.2±4.3 54.0±12.6 2.41±0.35
HFO WAF 4 d 3 6.3±2.2 7.0±1.2 87.3±3.4 1.83±0.25
HFO WAF 10 d 6 17.5±6.1 20.3±5.7 62.3±11.3 2.01±0.22
HFO CEWAF 24 h 3 42.7±1.2* 20.3±0.3 37.7±0.9* 2.31±0.25
HFO CEWAF 48 h 3 32.0±3.5* 20.3±5.0 48.0±8.7 1.89±0.46
HFO CEWAF 96 h 5 14.0±2.8†
12±2.1 74.6±4.3 2.63±0.40
HFO CEWAF 4 d 3 12.0±0.6†
8.3±1.5 80.0±1.5 1.65±0.09
HFO CEWAF 10 d 5 13.2±4.3†
12.4±2.2 74.8±5.3 2.17±0.14
Corexit 9500 24 h 3 10.7±4.9†
7.0±1.5†
82.3±5.8†
2.08±0.22
Corexit 9500 48 h 3 16.0±1.5†
16.3±5.5 68.3±3.9 2.05±0.10
Corexit 9500 96 h 6 8.7±1.1†
12.2±2.2 79.5±2.0†
2.15±0.14
Corexit 9500 4 d 3 13.3±1.8†
12.3±0.9 75.0±2.5†
1.86±0.08
Corexit 9500 10 d 6 11.5±1.8†
9.2±1.1†
79.8±2.7†
2.05±0.30
Cryolite 24 h 3 27.3±5.0 40.0±11.8* 33.3±7.0* 2.07±0.16
Cryolite 48 h 3 42.7±5.8* 29.7±7.6 27.3±10.5* 0.90±0.46
Cryolite 96 h 3 46.0±1.5* 23.3±8.0 31.7±7.3* 1.92±0.40
Cryolite 4 d 3 13.0±1.5†
14.0±0.0 72.7±1.8 0.95±0.18
Cryolite 10 d 3 8.7± 4.70†
10.7±1.9 80.7±5.5 1.47±0.12
Values are means ± SEM. Values in bold accompanied by * or † indicates that the value is significantly different (P < 0.05)
from the control or 96 h control, respectively.
Figure 1 Concentration (µg/L) of pyrene-1-glucuronide in bile of snapper (Pagrus auratus) exposed to
heavy fuel oil (HFO) water accommodated fraction (WAF; ○) or chemically enhanced water accommo-
dated fraction (CEWAF; ●) for up to 96 h (shaded) and following up to 10 days depuration (unshaded).
Half-closed circles represent control values at time 0 h and 96 h. Values are means ± SEM.
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11. (CEWAF) with Corexit 9500 caused a two-fold increase in P1G excretion and, presumably
therefore, also in PAH uptake. Levels of P1G had fallen in both treatment groups to equiv-
alent levels after 10 days indicating a possibly faster rate of depuration in animals exposed
to CEWAF. However, P1G concentration was still significantly elevated above baseline
control values after 10 days. Results for spotted wrasse (not shown) were very similar to
those of snapper with bile concentrations of P1G in animals exposed to 10% HFO
CEWAF approximately twice those of animals exposed to 10% HFO WAF and declining
by around 50% by 10 days of depuration. P1G concentration was measured directly in the
hepatopancreas tissue of rock lobster and also showed a significant increase during oil
exposure (Figure 2). Once again this effect was significantly enhanced (around four-
fold) by the addition of Corexit 9500. Metabolism of the parent PAH pyrene into the
metabolite seems to occur more slowly in lobster than in fish with levels in the CEWAF
treatment group, continuing to rise during the first 4 days of depuration and little or no
decline in concentration after 10 days.
Discussion
Ninety-six hour median lethal concentrations (LC50) for Corexit 9500 range from 3.5 to
36 mg/L for crustaceans and from 25 to 350 mg/L for fish depending on species and life
stage (George-Ares & Clarke 2000). Accordingly, the concentration used in this study (2.5
ppm) was not expected to cause mortality and did not cause any measurable effect on any
parameter measured in the three species tested. The addition of Corexit 9500 to oil did,
however, significantly increase the uptake of PAH as measured by pyrene metabolites
in all three species. It is well established that oil dispersants tend to increase the bioavail-
ability of PAHs in oil, potentially increasing toxicity. Ramachandran et al. (2004) found
that Corexit 9500 increased the sublethal effective toxicity of crude oil by between six
and 1000-fold in rainbow trout (Oncorhynchus mykiss [Walbaum]), as measured by ethox-
yresorufin-o-deethylase (EROD) induction, depending on the oil stock, and the relative
increase in EROD induction was greatest for the least soluble and highest viscosity oil
Figure 2 Concentration (µg/L) of pyrene-1-glucuronide in the hepatopancreas of red rock lobster (Jasus
edwardsii) exposed to heavy fuel oil (HFO) water accommodated fraction (WAF; ○) or chemically
enhanced water accommodated fraction (CEWAF; ●) for up to 96 h (shaded) and following up to
10 days depuration (unshaded). Half-closed circles represent control values at time 0 h and 96 h.
Values are means ± SEM.
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12. tested. The implications of this study were that chemical dispersal increases the toxic
potential of less soluble, high viscosity oils to a much greater degree than for lower vis-
cosity oils. Much less studied is the impact that oil dispersants may have on the metab-
olism and clearance of PAH metabolites in fish and our results clearly demonstrate
significant differences in metabolism between finfish and lobster, with much slower
accumulation of P1G in lobster and no discernible loss after 10 days of depuration.
Although no significant effects were observed in the red cell haematology of the
finfish species, exposure to either HFO WAF or HFO CEWAF did appear to have
some effect on erythrocyte physiology because an increase in abundance of melanoma-
crophage centres was observed in the spleen of snapper at 10 days post-exposure in both
treatments (Webby 2014). One of the key functions of melanomacrophage centres
appears to be involvement in the breakdown of erythrocytes and the sequestration
and storage of iron in haemolytic disorders (Agius & Roberts 2003). An increase in mel-
anomacrophage centres at 10 days post-exposure can be explained by the fact that his-
tological changes are slower and more prolonged than haematological changes.
Melanomacrophage centres have been suggested as sensitive and reliable biomarkers
for environmental stress or degraded water quality, and Atlantic cod (Gadus morhua
L.) exposed to water-soluble fractions of Venezuelan and Hibernia crude oils at concen-
trations of 50–300 µg/L for 12–13 weeks resulted in an increase of melanomacrophage
centres in the spleen and kidney (Khan & Kiceniuk 1984). Zbanyszek & Smith (1984)
observed significant increases in haematocrit, haemoglobin and red cell abundance in
rainbow trout exposed to 7.2 mg/L water soluble aromatic hydrocarbons, but this con-
centration is greater than that tested in our study and close to the lethal concentration
for this species.
Stress-induced lymphocytopaenia due to cortisol-mediated apoptosis occurs in fish
(Pickering & Pottinger 1995); however, this did not occur in response to confinement
in either species tested. The observed decline in the relative abundance of lymphocytes
in the treatment groups during the early stages of toxicant exposure and which had, in
most instances, returned to control values by 96 h is therefore presumably toxicant
induced. Crude oil (20% WAF) was found to induce a five-fold rise in cortisol in Mugil
cephalus L. within an hour of exposure and a consequent hyperglycaemia although cortisol
had returned to baseline levels within 12 h and glucose to baseline within 72 h (Thomas
et al. 1980). However, these authors attributed the observed recovery to a measured bioac-
cumulative depletion of PAHs in the exposure water and subsequent replenishment of the
WAF stimulated a further increase in cortisol and glucose. In our study, WAF was replen-
ished regularly throughout the exposure period yet all disturbances in differential immune
cell values returned to normal by the end of the 96-h exposure period indicating physio-
logical acclimation to the toxicant conditions after an initial stress-induced disruption to
homeostasis. Crude oil and its water soluble fraction are known to induce both pathologi-
cal and metabolic changes in the gills of fish and many observed alterations in haemato-
logic indices in fish exposed to oil could be attributed to disruptions of the respiratory and
osmoregulatory functions of the gills. Solangi & Overstreet (1982) observed gross altera-
tions in gill structure to a WAF of crude oil by 7 days post-exposure although their study
used a concentration five times higher than our HFO WAF and exposures lasted up to 30
days resulting in greater than 50% mortality. Despite this, partial to complete recovery of
histopathology occurred during recovery in oil-free water within 17 days.
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13. Although a large number of studies have examined changes in crustacean total haemo-
cyte abundance in response to a range of environmental and physiological stressors, few
studies to date have quantified changes in differential haemocyte abundance. Capture
stress caused a relative decline in abundance of granulocytes compared with hyaline
cells in Panulirus cygnus George (Jussila et al. 1997) whereas the stress of acute severe
hypoxia caused the opposite effect of a proportional decline in hyaline cells in Penaeus sty-
lirostris (Stimpson) (Le Moullac et al. 1998). The observed relative decline in hyaline cells
in our study is not due to hypoxia given that the treatment aquaria were aerated and is
therefore presumably due to confinement stress alone, although the absence of this
response in the Corexit 9500 exposure is interesting and warrants further study.
Exposure to a water soluble fraction of cryolite appeared to have little effect on the hae-
matological variables measured in this study other than possible minor transient changes
in immune cell relative abundance. The Department of Agriculture and Fisheries for Scot-
land (1982) and Johnstone et al. (1982) investigated the impact of cryolite recovery sludge
(CRS) to a range of marine animals and these studies were undertaken because active
dumping of CRS had been occurring at a site in the Moray Firth since 1972. Cryolite
recovery sludge is a waste product resulting from reprocessing of SPL to recover cryolite
but has a similar ultimate mineral composition to the SPL material. Johnstone et al. (1982)
found that CRS had no adverse measurable effects on behaviour of salmon (Salmo salar L.)
although fish exposed to CRS (concentrations above 1%) and CRS filtrate (concentrations
above 0.6%) for up to 1 h showed clear but transient changes in heart rate, gill ventilation
rate and increased oxygen consumption. Transient chemosensory impairment was also
observed. Results of this study indicated that although the fish were able to detect the
CRS they would not actively avoid the solution. The Department of Agriculture and Fish-
eries for Scotland (1982) investigation found no effect on survival of Nephrops sp. caged
inside versus outside the dump area. After evaluation of catch rates of a variety of
benthic organisms inside and outside of the CRS disposal zone they suggested that
catch rates did not vary between areas affected by CRS and those not affected (Department
of Agriculture and Fisheries for Scotland 1982). The USEPA lists fluoride and cyanide as
the two primary ecotoxicological concerns arising from SPL material (Chanania & Eby
2000) with the cyanide content of SPL varying from 18.5 to more than 9000 mg/kg in
material sourced from different aluminium smelters in the USA (Chanania & Eby
2000). The composition of the material on the Rena is unknown; however, cyanide com-
pounds typically have relatively high solubilities and are readily leached from SPL material
(Chanania & Eby 2000) and therefore likely to quickly disperse in the turbulent waters sur-
rounding Astrolabe Reef. The slower leaching of fluoride from the highly insoluble
mineral substances in the pot liner material is therefore the primary ecotoxicological
concern; however, cryolite solubility in seawater is low and the fluoride content of seawater
saturated with the butt bath material supplied for this study was around 50 mg/L (L Trem-
blay, Cawthron Institute, pers. comm., 2014). Hickey et al. (2004), using Australian and
New Zealand Guidelines for Fresh and Marine Water Quality (ANZECC) procedures,
proposed a long-term exposure marine water quality guideline of 5 mg/L which would
be unlikely to be exceeded in the Rena receiving environment given the high level of
dilution. Although SPL has been found to be genotoxic in plants (Andrade-Vieira et al.
2011) this effect occurs at concentrations exceeding those likely to be found anywhere
other than in direct contact with the Rena material. The small effect on immune cell
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14. populations seen in the present study is unlikely to occur in highly mobile reef macrofauna
such as fish and crayfish, unless crayfish were occupying refuges in direct proximity to
large aggregations of the Rena cryolite material. Of much greater concern is acute toxicity
arising from deliberate or accidental ingestion of the material, given that cryolite is acutely
toxic by ingestion with an LC50 value of approximately 2 g/kg in both vertebrates and
invertebrates (USEPA 2014), and it is this route by which cryolite has been used as an
insecticide to protect crops against chewing insect pests since the 1930s.
The physiological effects of soluble hydrocarbons from physically or chemically dis-
persed HFO from the Rena, or of cryolite contributing to the potential fluoride toxicity
from the SPL material, appear to be minor and transient in the three species tested. In
most cases, physiological indices returned to control levels by the end of the 96-h exposure
period after a brief disruption and there were no further signs of haematological disruption
during a further 10 days of depuration. However, animals continued to carry a body
burden of accumulation PAHs following oil exposure that, although declining, had not
reached near to baseline values by 10 days following exposure, and loss of PAHs appeared
to be particularly slow in lobster. Whether accumulated PAHs would contribute to chronic
pathologies in these species is unknown and requires further study, and fishers should be
wary of consuming these species for some time following potential exposure to physically
or chemically dispersed oil.
Acknowledgements
Thanks go to Caleb Valler, Dudley Bell, Warrick Powrie and Wayne Short for assistance with fish
collection, Alice Sharp, Barry O’Brien, Brittany Jaine and Judith Burrows for laboratory assistance,
and to Maritime New Zealand and the Bay of Plenty Regional Council for financial and logistic
support.
Guest Editor: Professor Chris Battershill.
Disclosure statement
No potential conflict of interest was reported by the authors.
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