Marine Pollution Bulletin 114 (2017) 860–866

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Marine Pollution Bulletin

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Toxicity of treated bilge water: The need for revised regulatory control
Peter Tiselius a,⁎, Kerstin Magnusson b
a
University of Gothenburg, Department of Biological and Environmental Sciences – Kristineberg, SE-45178 Fiskebäckskil, Sweden
b
IVL Swedish Environmental Research Institute, Kristineberg, SE-45178 Fiskebäckskil, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: Water accumulating in the bottom of ships (bilge water), contains a mixture of oil, detergents and other com-
Received 18 September 2016 pounds from on board activities. To evaluate ecological effects of released bilge water the chemical composition
Received in revised form 8 November 2016 and toxicity of treated bilge water from seven passenger ships was analysed. The oil content was below
Accepted 9 November 2016
15 mg L−1, the threshold for legal discharge, in all but one ship. Still, significant reductions in feeding and repro-
Available online 14 November 2016
duction of Acartia tonsa were found after 48 h exposure in dilutions with 2.5–5% of bilge water. Mortality was sig-
Keywords:
nificant at dilutions of 5–10% in 4 of the 5 bilge water samples. Surfactants were the most significant contributor
Toxicity to the toxicity on copepod vital rates and survival. Toxicity was also tested with Microtox where an EC50 was
Bilge water found at dilutions between 4.3% and 52%. The results show that ecological effects might occur also in diluted sus-
Oil pollution pensions of bilge water.
Surfactants © 2016 Elsevier Ltd. All rights reserved.
Copepods
Microtox

1. Introduction The International Maritime Organization (IMO) regulates handling

Release of oil from various shipping activities has over the past half
century caused enormous problems to aquatic ecosystems. Most atten-
tion has been given to oil spills where marine biota initially are exposed
to high oil concentrations which gradually are reduced through e.g.
evaporation, photo-oxidation and bacterial break-down of the oil com-
ponents. Less attention is given to the effects of the legally accepted re-
lease of smaller volumes of oil to the sea through activities like
discharge of bilge water, ballast water and cleaning of tanks. Recent es-
timates indicate that the total chronic release of oil worldwide to the
ocean averaged 270,000 tons per year over the period 1990–1999,
equal to the largest single oil spills from an oil tanker accident, the At-
lantic Empress in 1979, or about one third of the total release of oil
from the Deepwater Horizon Macondo well in 2010 (Farrington,
2013). The input of oil from ship operations is continuous, so even
though most oil fractions have a fairly rapid half-life they may cause
permanently increased oil concentrations in areas with intense ship-
ping. Marine biota in these areas hence run the risk of being chronically
exposed to low but still elevated concentrations of oil.
Bilge water is the water that accumulates in the bottom of the ship
and it is generated from machinery leakage and wash-down of fresh
water. It may contain fuel, hydraulic oils, lubricant oils, volatile organic
compounds, metals, detergents, degreasers and other chemicals derived
from activities on board a ship (US EPA, 2008).
⁎ Corresponding author.
E-mail addresses: peter.tiselius@bioenv.gu.se (P. Tiselius), kerstin.magnusson@ivl.se
(K. Magnusson).
of bilge water. The focus for regulation is set on the oil content of the
discharged bilge water since this is generally considered to be the
most important toxic component. According to the International Con-
vention for the Prevention of Pollution from Ships, (MARPOL 73/78)
no water may be discharged into the sea if it contains ≥15 mg L−1 of
oil. To meet the IMO regulations the bilge water is either treated en
route, in an oil separation system before being discharged to the sea
or deposited at reception facilities on land. The treatment is complicated
due to its mixed content of chemicals in the water. The most problem-
atic is the mixture of oil and surfactants derived from cleaning, which
prohibits the water from separating into two distinct phases. Despite
this, a recent study indicates that oily water separators mounted on
three container and bulk carriers significantly reduced most substances
for which there are regulated concentration limits (McLaughlin et al.,
2014).
The chemical composition of bilge water varies both between ves-
sels and also from day to day within a vessel. Cruise ships and passenger
ferries produce significantly more bilge water than ships of other cate-
gories due to their complicated constructions and support for many pas-
sengers (US EPA, 2008). In a survey by Det Norske Veritas (DNV) the
median production of bilge water was estimated to be 7500 L day−1
from passenger ships, 360 L day−1 from offshore ships, and
50 L day− 1 from tankers and cargo vessels (Sjøfartsdirektoratet,
2009). The corresponding amount of oil being released from a passen-
ger ship, assuming a maximum allowed oil content of 15 mg L−1,
would be 112 g oil day− 1, from an offshore ship 6 g oil day− 1 and
from tankers and cargo vessels 1 g day− 1 ship− 1. Large cruise ships
with gross tonnage from 20,000 to 78,000 operating off Alaska produced

http://dx.doi.org/10.1016/j.marpolbul.2016.11.010
0025-326X/© 2016 Elsevier Ltd. All rights reserved.
 P. Tiselius, K. Magnusson / Marine Pollution Bulletin 114 (2017) 860–866 861

5–20 m3 of bilge water per day equal to 75–300 g oil day−1 in legal dis-
charges (US EPA, 2008). There is to our knowledge no data on the vol-
umes of other components discharged with the bilge water.
An important group of chemicals present in bilge water are the
surfactants. Many surfactants are known to be toxic in themselves and
mixtures of oil and surfactants may be more toxic than each of the indi-
vidual components, either caused by synergistic effects of the actual
toxicity the two components or as a result of an increased dissolution
of the crude oil by the dispersant making it more bioavailable for the ex-
posed organisms (Greer et al., 2012; Wu et al., 2012; Almeda et al.,
2014b).
In general, toxicity tests of crude oil on planktonic species have been
carried out with just the water-soluble fraction of the oil since this often
has been considered to be the only bioavailable fraction (e.g. Berdugo et
al., 1977; Berrojalbiz et al., 2009). However, oil discharged to sea water
also occurs as droplets either formed by natural factors (wind, waves) or
by dispersants applied after oil spills (Mukherjee and Wrenn, 2009). It
has been shown that suspended oil droplets in the size range of food
particles can be ingested by several species of zooplankton and hence
add to the uptake of oil (Lee et al., 2012; Almeda et al., 2014a, 2014b,
2014c). Surfactants in bilge water are also likely to contribute to the for-
mation of oil droplets, which might hence affect the toxicity of the oil
fraction. Since the bilge water is released in the water column, plankton-
ic species in areas with intense shipping are prime targets for environ-
mental effects. Zooplankton, larvae and phytoplankton are particularly
at risk since they have limited capabilities to avoid areas of oil-contam-
inated water.
The aim of the present study was to investigate the toxicity on the
marine environment of treated bilge water from large passenger ferries
sailing in Swedish waters. No effort has been made to rank different
treatment technologies. The toxicity of the bilge water from seven
ferries collected before and after treatment was tested with Microtox,
a screening test with marine bacteria. Since zooplankton is the prime
group affected by a continuous release of contaminated water to the
sea, further experiments were conducted using treated bilge water
from four of the ferries recording the survival and sub-lethal effects on
the marine copepod Acartia tonsa.
2. Material and methods
2.1. Bilge water sampling
Treated bilge water, after passage through an oily water separator,
was collected between May 2015 and February 2016 from seven
passenger ships (ships A–G) ranging in size from 19,700–
52,000 gross ton. Sampling was done between one and three times
from each ship (Table 1). The oil separating equipment differed be-
tween the ships. Water from ships A and B was pumped from a storage
tank to the treatment system without any mixing. The chemical compo-
sition of the bilge water may therefore depend on the level of the stor-
age tank and on what time in the treatment process the sampling was
carried out. Samples were therefore taken both at early operation (EO,
Table 1) and late operation (LO). The bilge water in ships C to G was
mixed during treatment and samples were taken only once at each sam-
pling occasion. All sampling was done when the ships were in the port
and not during operation at sea. The ship-mounted measuring instru-
ments always reported the oil content to be b15 mg L−1, which is the
upper limit for bilge water to be released into the sea. The collected
bilge water was stored in dark 1 L glass bottles at 4 °C in the dark until
chemical analysis and toxicity tests.
2.2. Chemical analyses
All sampled bilge water was analysed for the total oil index and car-
bon fractions (NC10–C12, NC12–C16, N C16–C35 and NC35 –bC40),
∑PAH16, anionic surfactants and metals including a metalloid (V, Cr,
Mn, Co, Ni, Cu, Zn, Fe, As, S, Cd, Pb and in some samples also Hg). Five
ships were also analysed for cationic and non-ionic surfactants. The
total oil index includes all hydrocarbons, alkanes and aromatic com-
pounds. Accredited laboratories performed all analyses. Total oil index
and oil fractions were analysed using GC-FID (methods EN ISO 9377-2,
Z1 and TNRCC method 1006), PAH using GC–MS (method US EPA
8270 and EN ISO 6468), BTX using GC–MS Headspace and GC-FID
(methods US EPA 624, US EPA 8260, EN ISO 10301, MADEP 2004, rev.
1.1), surfactants using UV–VIS spectrophotometry (methods EN903
and ISO7875-2), BOD7 according to EN1899-1 and CODCr according to
TNV 757520.
2.3. Toxicity tests
2.3.1. Sub-lethal effects on the marine bacteria - Microtox
A standard Microtox screening (SS-EN-ISO 11348-3:2008) was con-
ducted to test the toxicity of the treated bilge water. In this assay, toxic-
ity is measured as the inhibition of bioluminescence of the marine
bacteria Vibrio fischeri. The bacteria are exposed to the bilge water in a
range of dilutions for 5 or 15 min. The dilution at which 20% and 50%
inhibition of the luminescence occurs is recorded and termed EC20
5 min/15 min and EC50 5 min/15 min, respectively. In this study only
EC20 and EC50 after 15 min exposure are reported.

Table 1
Treated bilge water samples collected from ships A–G. Samples from ships A1, A2, B1 and
2.3.2. Lethal and sub-lethal effects on the marine copepod Acartia tonsa
B2 were taken both at beginning of the bilge water treatment, EO (=Early Operation) and Detailed methods and statistical procedures of the copepod experi-
at the end of the treatment, LO (=Late Operation). ments are given in the SI. Briefly, bilge water from ship A (two sampling
Sampling date Ship ID Chemical Test with Test with
occasions A1 and A2), and ships B, C and D (one sampling occasion from
analyses Microtox Acartia tonsa each ship) were tested in three experiments where a dilution series was
created ranging from 0.01 to 10% of bilge water. The animals were fed
7 May 2015 A1EO X X X
8 May 2015 A1LO X X microalgae ad libitum during the experiments (Rhodomonas baltica,
2 June 2015 A2EO X X X 30,000 cells mL−1), which lasted for 48 h. We used egg production as
3 June 2015 A2LO X X a measure of reproduction, pellet production as a measure of feeding
26 May 2015 B1EO X X X and dead females as measure of mortality.
27 May 2015 B1LO X X
15 June 2015 B2EO X X
15 June 2015 B2LO X X 2.4. Statistical treatment
17 Nov 2015 B3 X X
1 June 2015 C1 X X X For the copepods we used ANOVA to determine significant differ-
11 Nov 2015 C2 X X
ences between controls and treatments and the dilutions of the bilge
11 June 2015 D1 X X X
15 June 2015 D2 X X water were used as fixed factors. Vital rates of A. tonsa in the controls
2 Dec 2015 D3 X X varied between experiments and all rates were therefore normalised
26 Nov 2015 E X X to the controls before analysis. In case of significant treatment effects,
27 Nov 2015 F X X Dunnett's post-hoc test was used to compare the controls with each
10 July 2015 G X X
treatment. A significance level of 0.05 was used. Since the mixture of
862 P. Tiselius, K. Magnusson / Marine Pollution Bulletin 114 (2017) 860–866

chemicals was not the same between the ships, a correlation analysis
and a multiple regression was performed to evaluate the importance
of single compounds/group of compounds for toxicity.
The correlation analysis tested whether the Microtox results were
correlated to any of the metals or groups of organic compounds. The
multiple regressions tested which of three chemical components of
the bilge water had any significant effect on the copepods. The normal-
ised egg production (NEPR) and normalised pellets production (NPPR)
were used as dependents and the factors oil concentration (μg L− 1),
total PAH concentration (μg L−1) and concentration of anionic surfac-
tants (mg L−1) were used in a multiple regression:
NEPRi ðor NFPRi ¼ β0 þ β1 oili þ β2 PAHi þ β3 surfactanti þ εi
Data for the multiple regressions were selected based on the results
from the Dunnett's post-hoc test, and limited to data that were signifi-
cantly lower than the controls. This selection assured that data from
the relevant range of toxicity were used, spanning from no effect to a
significant effect.
(EC50 of ≥ 90% and EC20 between 29 and 39%). Samples from ships A
and B were collected during both the early and late part of the bilge
water treatment process, but only at sampling occasion A1 was the
treated water more toxic during early operation (EO) compared to the
late operation (LO). No such difference was found in the other sam-
plings, indicating that the treated water is fairly similar throughout
the treatment operation.
Correlation analyses were done between toxicity to the marine bac-
teria in the Microtox test and the chemical components analysed in the
treated bilge water samples (Table 3). Since EC50 values could not be de-
termined for ship B (values N 90%), the EC20 values after 15 min expo-
sure were used for the correlation analyses. The negative correlations
were strongest for the oil fractions and PAHs with low and medium
number of carbon atoms and for the anionic surfactants. An exception
was the water from ship D at the third sampling occasion, which had
a low toxicity to the Microtox bacteria in spite of relatively high oil con-
centrations (ship D3, Table 2). No correlations were found between
metal concentrations and toxicity.

3. Results 3.3. Copepod responses

3.1. Bilge water content 3.3.1. Mortality

The total oil content in the treated bilge water varied between 0.091
and 69.3 mg L−1 and was below the accepted 15 mg L−1 for all ships but
ship E (Table S2). The oil index differed considerably between sampling
occasions for those ships where bilge water was collected at more than
one occasion (ships A, B, C and D), while samples taken on a single ship
in early or late part of the treatment process (EO and LO, respectively)
showed similar concentrations. For ships B to G the oil content of the
treated bilge water was at most occasions dominated by the fraction
NC16–C35, whereas the water from ship A was dominated by the
smallest defined fraction, N C12–C16. The concentrations of ∑ 16
PAHs, which are a part of the reported total oil content, showed
large differences between the ships. The highest concentrations,
100–240 μg L−1, were found in the treated bilge water from ships A, E
and F and the lowest from ship B, 0.067–0.53 μg L−1. The relative contri-
bution of PAHs to the total oil index was highest for ship A, where the
∑ 16 PAHs made up between 9 and 18%. Naphthalene, which has a
low number of carbon atoms (part of PAH-L), was the dominating
PAH in bilge water of all ships except ship B, where pyrene (part of
PAH-M) was found at the highest concentration (Table S2, Table S4).
Concentrations of anionic surfactants varied between 450 and
14,000 μg L−1, often exceeding the oil index (Table S2). Cationic and
nonionic surfactants were only analysed in samples from ships B3 and
ships C\\F and occurred in concentrations similar to or lower than
those of the anionic surfactants. The ratio between total oil and anionic
Mortality was low (b 8%) in the controls and all treatments, except
the test waters with 5 and 10% bilge water, where most animals died
(Fig. 1). From ship A bilge water was sampled at two occasions (A1
and A2), which gave slightly different results. All animals died when
the exposure water contained 10% of the A1EO bilge water, but only 2
out of 12 animals died in 10% of the A2EO water. Bilge water from ship
B1EO did not affect copepod survival, but all animals died in the 5% dilu-
tion from ship C1 and 11 out of 12 animals in ship D2.
3.3.2. Feeding
The highest test concentration of bilge water was 10% dilution and
this was only from ship A. At the first sampling occasion (A1) feeding
was significantly reduced to 28% of the controls whereas no significant
effect on feeding was found at the second sampling occasion (Fig. 2).
For ship B1EO, significantly reduced feeding was observed at 5% dilution,
while bilge water from ships C1 and D1 reduced feeding significantly al-
ready at a dilution with 2.5% bilge water. The strongest negative effect
was found in ships C1 and D1 where almost no feeding was recorded
at 5% dilutions.
Table 2
Toxicity of the treated bilge water from ships A–G measured with Microtox. Total oil and
surfactant concentrations corresponding to the EC50 values are shown. Samples marked
with * were also used in the tests with Acartia tonsa.

surfactants varied considerably with values ranging between 0.01 and Microtox
29.7 (median 0.28). Ship ID EC20 15 min% EC50 15 min% Total oil, Anionic surfactants,
Metal concentrations varied between ships and also between sam- mg L−1 mg L−1
pling occasions from the same ship (Table S3). Bilge water from ship B
A1EO* 2 6.4 0.10 0.52
generally had the highest concentrations. The concentrations of Zn A1LO 5.6 26 0.32 4.21
(filtered b 0.45 μm) in bilge water from ship B and sample D2 were 10 A2EO* 11 38 0.22 1.73
to 20 times higher than the threshold level of 8 μg Zn·L−1 in the aquatic A2LO 16 51 0.37 2.44
environment which has been suggested by the Swedish EPA (Swedish B1EO* 34 N90 0.13 0.64
B1LO 29 N90 0.14 0.71
EPA, 2008). Also Cu (filtered b 0.45 μm) was elevated in bilge water B2EO 36 N90 0.63 0.41
from ship B at the first two sampling occasions (11–65 μg L−1) com- B2LO 39 N90 0.44 0.53
pared to the suggested threshold value, 4 μg L−1 (Swedish EPA, 2008). B3 38 90 2.30 1.61
C1* 2.3 9.8 0.93 0.35
C2 3.6 18 0.29 0.41
3.2. Microtox
D1* 2.6 14 0.01 1.32
D2 3.4 9.8 0.05 1.13
The most toxic water derived from ship E where EC50 was achieved D3 16 52 3.89 4.02
when the exposure water contained 4.3% of bilge water (Table 2). The E 1.5 4.3 2.98 0.10
least toxic were the samples from ship B where 50% reduction in biolu- F 3.3 14 0.70 1.96
G 6.3 27 0.13
minescence was not reached even in the highest test concentrations
 P. Tiselius, K. Magnusson / Marine Pollution Bulletin 114 (2017) 860–866 863

Table 3 negative effects were typically found at dilutions of 1–5% treated bilge
Correlation analysis of Microtox EC20 15 min and chemical components measured in the water.
bilge water (log-transformed). Pearson product moment correlation coefficient (r), signif-
icance (p) and number of measurements (n) are shown.
The oil content is the only chemical concentration in bilge water reg-
ulated by law. In the present study toxicity in the Microtox assay was
Component r p n also found to best correlate to the oil concentrations, including the
Oil index −0.42 0.117 15 low and medium weight PAHs (Table 3). In the copepod assay, however,
Oil fraction the surfactant concentration was the only analysed component that cor-
C10–C12 −0.83 b0.001 14
related to all three rates (feeding, reproduction and mortality, Table 5).
C12–C16 −0.70 0.004 15
C16–C35 −0.39 0.17 14 Total oil content was significantly correlated with reduced feeding, but
bC35 −0.45 0.13 12 not with reproduction or mortality. Earlier studies have shown that
PAH crude oil has a fairly low acute toxicity. Mortality of mixed copepod
∑16 −0.75 0.001 15 communities were moderately affected by crude oil at 4 mg L−1, show-
∑ low −0.63 0.029 12
∑ medium −0.57 0.027 15
ing 21% mortality after 48 h compared to 11% in the controls (Almeda et
∑ high −0.30 0.41 10
Surfactants
Anionic −0.79 b0.001 15 Mortality
Cationic −0.57 0.24 6 1.2
Nonionic −0.70 0.30 4 Ship A1EO *
1
0.8
3.3.3. Reproduction 0.6
Reproduction was slightly less sensitive than feeding to bilge water
0.4
exposure. Significant reductions were found at the 10% dilution in
ships A1EO and A2EO whereas ship B1EO gave no effects at the highest di- 0.2
lution 5%. Copepods exposed to water from ships C1 and D1 showed sig- 0
nificantly reduced reproduction at 2.5% dilution. 0.01 0.1 1 10
1.2
Ship A2EO
1
3.4. Threshold concentrations for toxic effects 0.8
0.6
The effect of the bilge water on the copepods was evaluated by de-
fining the dilutions at which a significant reduction in feeding, repro- 0.4
duction or survival occurred. The threshold dilutions ranged from 1 to 0.2
5% dilutions with slightly higher values for mortality and lower for feed- 0
ing (Table 4A). 1.25 2.5 5 10
The dilutions can be directly translated to concentrations of any 1.2
chemical analysed. However, it is not possible to determine which of Ship B1EO
1
the chemicals in the bilge water caused the observed responses. To en-
hance the precision, we therefore conducted a multiple regression 0.8
where the three most important components were used as predictors 0.6
for the mortality and vital rates of the copepods. Feeding was negatively 0.4
affected by oil index (p = 0.008) and anionic surfactants (p = 0.031),
0.2
while reproduction was only affected by anionic surfactants (p =
0
0.009, Table 5). Mortality increased with surfactant concentration
0.625 1.25 2.5 5
(p = 0.043), but the oil index did not significantly affect mortality.
1.2
PAHs did not have a significant effect on either vital rates or mortality. Ship C1 *
We conclude that surfactant concentration was the main predictor for 1
copepod responses. 0.8
Since anionic surfactants were the chemical group that appeared to 0.6
have the most important toxic effect, the threshold dilutions for cope-
0.4
pods were multiplied by the concentration of anionic surfactants to
give units of mg L−1 (Table 4B). The threshold concentrations for surfac- 0.2

tants were 0.08–0.14 (≥ 0.23), 0.08–0.23 (≥ 0.036) and 0.045–0.29 0

(≥ 0.45) mg L−1 for feeding, reproduction and mortality, respectively 0.625 1.25 2.5 5
(numbers within parenthesis indicate concentration in samples where 1.2
Ship D1 *
no significant effect could be detected). 1
0.8
0.6
4. Discussion
0.4
In the present study the chemical composition and toxicity of treated 0.2
bilge water from seven passenger ferries in the Baltic Sea was investi- 0
gated. Only one of the ships, ship E, had treated water with a total oil 0.625 1.25 2.5 5
content exceeding the legal limit, 69.3 mg L−1 compared to regulations Concentration, % bilge water
of a maximum of 15 mg L−1. There was a clear correlation between di-
lution of the treated bilge water and biological response and although Fig. 1. Mortality (mean ± SE) of Acartia tonsa after 48 h exposure to different dilutions of
there were some differences in toxicity between ships, significant bilge water collected from ships A–D. Data normalised to mortality in the controls.
864 P. Tiselius, K. Magnusson / Marine Pollution Bulletin 114 (2017) 860–866

Feeding Reproduction Our experimental approach is therefore most suited for sub-lethal ef-
1.2 1.2 fects. The egg production in pelagic copepods was found to be two
Ship A1EO
1 1 times more sensitive than mortality in a study of selected PAHs (Bellas
*
0.8 0.8 and Thor, 2007), and Avila et al. (2010) found a LC50 = 48 μg L−1 for
0.6 0.6 copepods exposed to the water soluble fraction of crude oil, but a 30%
0.4 * 0.4 * decline in both feeding and reproduction at 15 μg L−1 (the LC10 concen-
tration). Thus, there seems to be graded physiological response to the
0.2 0.2
oil, which first affects the feeding then the reproduction. Interestingly,
0 0
0.01 0.1 1 10 0.01 0.1 1 10 there also seems to be a rapid recovery of reproduction in copepods
1.2 1.2 exposed to high oil concentrations when returned to clean water
Ship A2EO (Olsen et al., 2013).
1 1
The PAH fraction of the treated bilge water was not found to corre-
0.8 0.8
late to the observed toxicity of the copepods. In the multiple regression,
0.6 0.6
the PAH fraction correlated strongly to surfactants which caused
0.4 0.4 *
severe collinearity and was removed from the analysis because it had
0.2 0.2 a non-significant contribution if retained together with the oil index
0 0 (Table 5). The total PAH concentration in ships A, E and F varied
1.25 2.5 5 10 1.25 2.5 5 10
between 100 and 240 μg L−1, which is considerably higher than concen-
1.2 1.2
Ship B1EO trations in other studies where toxic effects on zooplankton have been
1 1
observed. Reduced reproduction was found in two Calanus species
0.8 0.8
after exposure to concentrations of 16 PAHs of around 10 μg L−1
0.6 * 0.6 (Jensen and Carroll, 2010). In contrast, reported NOECs from experi-
0.4 0.4 mental studies with individual PAHs are far above our measured con-
0.2 0.2 centrations (Berdugo et al., 1977; Barata et al., 2005; Saiz et al., 2009).
0 0 It is therefore likely that the toxic effect of the surfactants in the treated
0.625 1.25 2.5 5 0.625 1.25 2.5 5 bilge water dominated over the effects of PAHs for the response of the
1.2 1.2 animals. We suggest that surfactants should be given more attention
Ship C1
1 1 in future evaluations of marine pollution.
0.8 0.8 Although no toxic effects of PAHs could be observed for the cope-
0.6 * 0.6 pods in the present study many PAHs were found in concentrations
0.4 0.4 * well above the Maximum Allowed Concentration for the Environmental
0.2 0.2
Quality Standards (MAC-EQS) given in the EU Water Frame Directive
* (2000/60/EC, Table S4). Although the bilge water will be diluted when
0 0
0.625 1.25 2.5 5 0.625 1.25 2.5 5 discharged the continuous release into the shipping lanes might lead
1.2 1.2 to elevated concentrations in the sea water that could have a negative
Ship D1
1 1 impact on the marine ecosystem.
0.8 0.8
The discharge of bilge water to the sea, with its content of oil and
0.6 * 0.6 * surfactants, may be compared to the situation when natural seawater
is treated with dispersant after an oil spill. There is growing evidence
0.4 0.4
that the mixture of oil and dispersing agents is more toxic than the oil
0.2 0.2 *
* alone. The dispersant Corexit 9500A used to treat the Deepwater Hori-
0 0
zon oil spill in the Gulf of Mexico consistently exacerbated the negative
0.625 1.25 2.5 5 0.625 1.25 2.5 5
Concentration, % bilge water

Table 4
Fig. 2. Feeding estimated from faecal pellet production and reproduction estimated from
Threshold dilutions of bilge water on feeding, reproduction and mortality of Acartia tonsa.
egg production (mean ± SE) of Acartia tonsa after 48 h exposure to different dilutions of
A. The threshold dilution for copepod assays is defined as the per cent dilution next lower
bilge water collected from ships A–D. Data normalised to the controls. * indicates
to that which caused a statistically significant difference from the control (Figs. 1 and 2).
significant differences from the controls according to ANOVA with Dunnett's post-hoc-
Values within parenthesis indicate where no significant effect was observed even at the
test (p b 0.05).
highest per cent dilution (10 or 5%). B. Threshold concentrations of anionic surfactants
expressed as concentrations (mg L−1) in the bilge water. Values within parenthesis are
cases where no effect was observed even at the highest surfactant concentration in the ex-
al., 2013). In laboratory experiments only one out of three copepod
posure water.
species showed significantly higher mortality when exposed to
0.85 mg L−1 crude oil for 48 h (Almeda et al., 2014a). These concentra- A. Dilutions
Ship Feeding Reproduction Mortality
tions are in the same range as oil concentrations in the undiluted bilge
water from most ships in the present study. For three of the four ships A1EO 1% 1% 1%
A2EO (≥10%) 5% (≥10%)
(ships B, C and D), copepod feeding activity was more sensitive than re-
B1EO 2.5% (≥5%) (≥5%)
production to the treated bilge water (Fig. 2). Variable responses de- C1 1.25% 2.5% 2.5%
pending on what vital rate is monitored were also reported by Cowles D1 1.25% 1.25% 2.5%
and Remillard (1983) who found reduced feeding in a copepod species
(Centropages hamatus), but no effect on reproduction at 20–80 μg L−1 of B. Anionic surfactants, mg L−1
crude oil water-soluble phase. Ship Feeding Reproduction Mortality
There were indications of a gradual response in both feeding and re- A1EO 0.08 0.08 0.08
production of the copepods. The fact that both eggs and faecal pellets A2EO (≥0.23) 0.23 (≥0.45)
were produced in the bottles where all females died after 48 h (ships B1EO 0.018 (≥0.036) (≥0.036)
C and D) implies that females might have survived for a significant C1 0.045 0.091 0.045
D1 0.14 0.14 0.29
time even though we could not observe this in our 48 h incubations.
 P. Tiselius, K. Magnusson / Marine Pollution Bulletin 114 (2017) 860–866
Table 5
Output from multiple regressions of feeding, reproduction and mortality of the
copepod Acartia tonsa on factors “oil index” and “surfactants”. df = degrees of freedom,
p = significance level, R2 = fraction of the variability explained by the model.
df Slope p R2
Feeding
Oil index 2,24 −0.001 0.008**
Surfactants −0.423 0.031*
Reproduction
Oil index 2,18 −0.0004 0.282
Surfactants −0.538 0.009**
Mortality
Oil index 2,16 0.001 0.106
Surfactants 0.79 0.043*
* = p b 0.05, ** = p b 0.01
effect of crude oil both in field populations of mixed copepods (Almeda
et al., 2013) and in laboratory studies with three species of common co-
pepods (Almeda et al., 2014a). Almeda et al. (2014a) also found that
toxicity of the dispersant on feeding activity and reproduction often
was in the same range as for the oil. The major surfactant component
in Corexit is the anionic surfactant dioctyl sodium sulfosuccinate (Gray
et al., 2014). Anionic surfactants of the type linear alkyl benzene sulfo-
nate (LAS) were found to be toxic to Acartia tonsa affecting both mortal-
ity (LC50 = 1.2 mg L−1) and reproduction (EC50 = 0.74 mg L−1) with
significantly reduced rates at 1 mg L−1 after 48 h (Christoffersen et al.,
2003). In most cases the bilge water in our study contained more anion-
ic surfactants than oil (Table S2) and they were discharged in higher rel-
ative proportions than those recommended to fight oil spills (ratio
dispersant to oil: 0.1–0.02, Almeda et al., 2014a). This may explain
why the most consistent pattern of toxicity was related to the surfac-
tant. During ship operation, the bilge water is treated in oily water sep-
arators where oil is shunted to a separate tank and the treated water is
discharged to the sea (US EPA, 2008). In the treatment process the sur-
factants in the water create an emulsion that prevent separation of oil
and water and compromise the function of separators and oil content
monitors (US EPA, 2008). The treated bilge water therefore often con-
tains a much higher fraction of surfactants than oil. This was also the
case for several of the collected bilge water samples in the present
study.
The effect of legally discharged bilge water (oil concentrations
b 15 mg L− 1) in the sea will depend on the volumes being released
and the toxicity and degradation rate of the compounds in the released
water. In an enclosed area like the Baltic Sea with intense shipping activ-
ity there is a risk that total amount of bilge water released from all ships
could reach concentrations that might be harmful to the plankton and
other organisms. Information on the volumes and composition of the
discharged bilge water in the Baltic Sea is however still too scarce to
draw any conclusions about the toxic effects in situ.
5. Conclusions
Toxic effects of bilge water on Microtox bacteria and copepods were
typically found at dilutions of 1–5%. Anionic surfactants and oil content
correlated best with observed toxic effects, with some correlation be-
tween the low and medium molecular sized PAHs and Microtox toxicity.
Although the variables responsible for the negative effects cannot be
fully determined, the ecological implication is clear: discharges of treat-
ed bilge water from the majority of the passenger ferries would be toxic
to the zooplankton and the microbial community in the ambient water.
The high surfactant content of bilge water compared to untreated or
treated oil spills deserves further attention. Because the function of
the oily water separators used in ships is compromised by surfactants
and the toxicity of the surfactants themselves is so high, we recommend
that the type and use of surfactants should be regulated to a higher
865
degree than at present. A reduced discharge of surfactants through the
bilge water and the development of less toxic surfactants are high prior-
ity to reduce any negative effects on the plankton ecosystem.
Funding
0.41 This work was supported by Wåhlströms Foundation, Wilhelm and
Martina Lundgren Foundation and the Adlerbertska Research Founda-
tion to P.T. and from the BONUS funded project ZEB (Zero emissions
0.36 in the Baltic Sea) to Kerstin Magnusson.
Acknowledgements
0.31
We are grateful for support from Ann-Sofie Allard for performing the
Microtox tests and IVL analytical laboratory for oil analyses. Rodrigo
Almeda provided valuable suggestions and comments from the editor
improved the manuscript significantly.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.marpolbul.2016.11.010.
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