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Trihalomethanes in marine mammal aquaria:
Occurrences, sources, and health risks
Jun-Jian Wang a, Alex T. Chow a,b,*, Joelle M. Sweeney b,c,d,
Jonna A.K. Mazet b
a
The Belle W. Baruch Institute of Coastal Ecology & Forest Science, Clemson University, SC, USA
Wildlife Health Center, One Health Institute, School of Veterinary Medicine, University of California, Davis, CA,
USA
c
The Marine Mammal Center, Sausalito, CA, USA
d
Moss Landing Marine Labs, Moss Landing, CA, USA
b
article info
abstract
Article history:
Disinfecting water containing the high levels of dissolved organic carbon (DOC) commonly
Received 4 December 2013
generated during pinniped husbandry may cause the formation of carcinogenic disinfec-
Received in revised form
tion byproducts (DBPs). Little information is available on DBP levels, sources, and health
26 February 2014
risks in marine mammal aquaria. Using the commonly observed trihalomethanes (THMs)
Accepted 4 April 2014
as a DBP indicator, we monitored concentrations for seven months at The Marine Mammal
Available online 16 April 2014
Center in Sausalito, California, one of the largest pinniped rehabilitation facilities in the
world. Concentrations of THMs ranged 1.1e144.2 mg/L in pool waters and generally
Keywords:
increased with number of animals housed (P < 0.05). To identify the sources of THM pre-
Chlorination
cursors in marine mammal aquaria, we intensively monitored the mass flows of potential
Bromide
THM precursors (i.e. food and wastes) in an isolated system with nine individual California
California sea lion
sea lions to evaluate the sources and reactivity of dissolved organic carbon (DOC) for 2e5
Disinfection byproducts
weeks. The common frozen foods used in feeding pinnipeds, including herring, sardine,
Food
and squid, produced an average of 22e34 mg-DOC/g-food in water and 836e1066 mg-THM/
Waste
g-food after chlorination, whereas the fecal materials, including fresh scat, decomposed
scat, and urine, produced 2e16 mg-DOC/g-waste and 116e768 mg-THM/g-waste. Food not
eaten by animals could cause a sharp increase of DOC and DBP production and therefore
should be removed rapidly from pools. Marine mammal husbandry staff and trainers are at
risk (5.16 104 to 1.30 103) through exposure of THMs, exceeding the negligible risk
level (106) defined by the US Environmental Protection Agency.
ª 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
Around the world, there are at least 240 large marine aquaria/
ocean life centers in w40 countries displaying marine
mammals or serving as rehabilitation facilities listed by
MarineBio.org (2012). Chemical disinfectants are widely used
in such facilities to maintain water quality and minimize
transmission of pathogens among animals and to personnel
* Corresponding author. The Belle W. Baruch Institute of Coastal Ecology and Forest Science, Clemson University, P.O. Box 596, Georgetown, SC 29442, USA. Tel.: þ1 843 546 1013x232; fax: þ1 843 546 6296.
E-mail addresses: [email protected], [email protected] (A.T. Chow).
http://dx.doi.org/10.1016/j.watres.2014.04.007
0043-1354/ª 2014 Elsevier Ltd. All rights reserved.
220
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
(Huguenin et al., 2003; Spotte, 1991). However, these disinfectants can react with water constituents, such as dissolved
organic matter, ammonia, and bromide (Br), to form a variety
of potentially hazardous disinfection byproducts (DBPs) (Croue
et al., 2000; Xie, 2004). Among more than 600 DBPs identified in
disinfected water (Krasner et al., 2006), the trihalomethanes
(THMs), typically comprised of chloroform (CHCl3), bromodichloromethane (CHCl2Br), dibromochloromethane (CHClBr2),
and bromoform (CHBr3), make up the largest class of DBPs. The
THMs have been documented to be potentially genotoxic and
mutagenic, and may result in adverse health effects with longterm exposure, such as bladder cancer and spontaneous
abortions (Bull et al., 2001; Plewa et al., 2002; Villanueva et al.,
2004). Health risk assessments of THMs in tap water and recreational swimming pools have been increasingly reported
(Dyck et al., 2011; Glauner et al., 2005; Kim et al., 2002; Lee et al.,
2009; Zwiener et al., 2007), but little information is available for
marine mammal aquaria (Shi and Adams, 2012).
Dissolved organic carbon (DOC) has been well recognized
as a DBP precursor (Xie, 2004), suggesting that DOC from
marine mammal foods and wastes left in pools can produce
DBPs during water disinfection (likely at levels exceeding wellstudied municipal water supplies and recreational swimming
pool waters; Spotte, 1991) may produce DBPs during water
disinfection. In marine mammal aquaria for porpoises and
pinnipeds (Huguenin et al., 2003), a disinfecting chlorine residual level of 0.4e0.7 mg/L (sometimes >1.0 mg/L) is usually
maintained. Also, seawater containing approximately 65 ppm
bromide could contribute to brominated DBP formation if
applied (e.g., oceanaria) (Chow et al., 2005; Hua et al., 2006).
With the required precursors in place, DBP formation will
occur and may pose health risks to marine mammals and
their trainers in captive facilities. However, distinct from
municipal water supplies, in which DOC commonly origins
from humic substances from soil or litter leachates (Chow
et al., 2007; Zhang et al., 2009), or recreational swimming
pools, where DOC are mainly from swimmers’ hair, sun block
lotion, saliva, and urine (Kim et al., 2002; Zwiener et al., 2007),
the main sources of DOC in marine mammal pools are probably the uneaten food (e.g., fish), urine, and scat. As the
reactivity of these readily biodegradable organic matters for
forming THMs is unclear, understanding their THM formation
potential (THMFP) (Chow et al., 2005; Xie, 2004) is important to
quantify and control the contribution of different DOC sources, facilitating better engineering and treatment processes.
Typical marine mammals workers, including animal
trainers, veterinarians, biologists, and volunteers in the marine
mammal rehabilitation centers, as well as field researchers and
workers at aquaria and oceanaria that exhibit marine mammals
to the public (Hunt et al., 2008), are likely to be exposed to DBPs in
water and are at risk of health effects. The public may also be at
risk when attending recreational activities like ‘swim-with-thedolphin’ programs by contact with the water directly in marine
mammal aquaria. For example, more than 8000 people in 1989
participated in these programs in the USA alone (National
Marine Fisheries Service, 1990).
Regulations are currently lacking for THMs in swimming or
working pool environments, even though the concentration of
THMs has been regulated in drinking water to a maximum
contaminant level (MCL) of 80 mg/L by the US Environmental
Protection Agency (US EPA, 2004). Trihalomethanes can be
taken up by the swimmers via the skin and inhalation, along
with the unintentional swallowing of water, due to the high
dermal permeability and volatility of THMs (Erdinger et al.,
2004). The health risks of THM exposure for people in recreational swimming pools have been increasingly reported
(Dyck et al., 2011; Glauner et al., 2005; Kim et al., 2002; Lee
et al., 2009; Richardson et al., 2010; Zwiener et al., 2007);
however, quantified exposure assessment and risk characterization of THMs for marine mammal workers or captive
marine mammals are still lacking.
In the present study, we monitored the DOC and THMs
generated in different compartments of two large tank systems housing California sea lions (Zalophus californianus) for
seven months to evaluate the exposure and potential health
risk for marine mammal workers. To identify the THM precursors, we set up an isolated tank system to house each of 9
sea lions and recorded the material flows (food input and
waste production) in detail and determined the THM formation potential from DOC of different sources. Our objective
was to systematically understand THM formation in marine
mammal aquaria and its potential impact on health risk for
marine mammal workers.
2.
Materials and methods
2.1.
Monitoring water quality in large captive facilities
2.1.1.
Study site and water sample collection
To investigate the occurrence of DOC and THM in facilities
housing marine mammals, we monitored the water quality at
The Marine Mammal Center (TMMC) located in the Golden Gate
National Recreation Area, Sausalito, California, USA. One of the
largest marine mammal rescue and rehabilitation facilities in
the world, TMMC had the capacity to care for 200 sick and
injured pinnipeds, including California sea lion (Z. californianus), elephant seal (Mirounga angustirostris), and habour
seal (Phoca vitulina), and had two independently closed freshwater systems maintaining 45,000 gallons of freshwater with
settling tanks, sand filters, and protein skimmers. Water was
primarily disinfected by ozone with supplemental chlorine and
bromine. A flow chart of the water system is depicted in Fig. A.1.
The pool water samples were collected three to five times
per month from February to August for DOC and THM determinations. Water samples from four other sampling locations through the treatment systems were also collected once
monthly from April to June, when the number of housed animal was greater than 100. These other four sampling locations
were: the drain line (from the animal pools to the water treatment system), the settling tank, water storage tank (after the
sand filter treatment), and the return line (from the treatment
system to the animal pools). Water samples were always
collected without headspace in two clean amber glass bottles
(one for ultra-violet absorbance and DOC and one for THM
quantification), stored at 4 C, and analyzed within 2 weeks.
2.1.2.
DOC and THM determination
Water samples for ultra-violet absorbance and DOC were
filtered through a 0.45 mm polyethersulfone membrane filter
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
(Supor-450, Pall Gelman Science). The ultra-violet absorbance
was measured by a diode array spectrophotometer (Hewlett
Packard P8452A). Using heat-promoted persulfate oxidation,
DOC was determined using a total organic carbon analyzer
(O.I. Analytical 1010) (Eaton et al., 1995). Quantification of THM
was accomplished using a purge and trap gas chromatograph
(Hewlett Packard 5890 II), a 63Ni electron capture detector, and
a RESTEK Rtx-502.2 column (105 m 0.53 mm 3 mm) (Chow
et al., 2007). The GC oven was programmed to hold an initial
temperature of 35 C for 10 min, increased to 140 C at a rate of
3.5 C/min and to 220 C at a rate of 5 C/min, and finally held
at 220 C for 3 min. Precision, accuracy and detection limits of
all chemical analyses (e.g., DOC and THM quantification) were
tested for QA/QC using repeating tests on blank and standard
solutions and spike-and-recovery tests. The values of percent
relative standard deviation (%RSD) were all within 5%, and the
recovery values were within 100% 5% for DOC (method
detection limit 0.1 mg/L) and 100% 10% for four THMs
(method detection limit 0.055 mg/L).
2.2.
Exploring THM sources in an isolated tank
2.2.1.
Animals and husbandry
To quantify the THM precursors and their THM formation
potential in pinniped husbandry, isolated tank with freshwater was used to house each of nine California sea lions, with
feeding and waste production recorded in detail. All sea lions
were in the final rehabilitation stages, being held only for final
observation prior to release after being determined to be
clinically recovered by veterinarians. Observers recorded sex,
age class, and weights of all sea lions.
Each meal being fed to sea lion was detailedly weighed and
recorded. The market squid (Loligo opalescens), Pacific sardine
(Sardinops sagax), and Atlantic herring (Clupea harengus) were
among the common prey items in the present controlled study
(Sweeney and Harvey, 2011). Meals were fed once or twice per
day depending on meal size, and the total amount (kg) per day
for individual sea lion remained constant, varying from 2.8% to
6.5% of body weight depending upon different individual needs
and optimal target weights for release (Table A.1).
The freshwater tank housing individual sea lions was 6.1 m
in diameter and 1.1 m in water depth with a 2.1 m width haulout site (Fig. A.2). The tank floor was slanted to facilitate the
collection of all fecal remains when the pool was drained. Scat
and spew deposited (noted as pool scat) on the haul-out site
and in the water during the monitoring periods were collected
immediately with pertinent data recorded. The tank was
drained every 24 h to collect drainpipe scat in a screened
drain-basket, located externally to the tank (noted as drain
scat). Volume and weight were recorded for each collected
scat. A small portion (<30% in mass) of solid wastes were
collected from the drain screen, but the majority of solid
wastes were collected directly from the pools by husbandry
personnel. Husbandry staff (or researchers) closely monitored
each sea lion for 12 h per day for 2e5 weeks. Because sea lions
commonly micturated in the pool, making it difficult to collect
urine from the pool, comparative urine (n ¼ 18) samples were
collected from recently deceased sea lions at necropsy
(collected within 2 days of death). All scat and urine samples
were immediately frozen at 20 C until analysis.
221
2.2.2. Organic carbon extraction and trihalomethane
formation potential
We examined three commonly frozen foods used in feeding
pinnipeds (Atlantic herring, market squid, and Pacific sardine;
n ¼ 9 each), as well as pool scat (n ¼ 18), urine (n ¼ 18), and
drain scat (n ¼ 10), to estimate their potential water extractable organic carbon (WEOC) yield in the marine mammal pool.
Five grams of each solid sample (on a wet-weight basis) were
mixed vigorously with 45 mL deionized water in a 50 mL
centrifuge tube for 1 min to prepare a 1:10 (w:v) extraction
(Seguin et al., 2004; Wu and Ma, 2002). Then, the mixture was
centrifuged for 20 min at 250 g relative centrifugal force. The
supernatant was withdrawn and filtered through a 0.45 mm
polyethersulfone filter, and stored at 4 C within one week
before analyses.
All water extracts and urine samples were analyzed for
DOC and ultra-violet absorbance at 254 nm (UVA254),
whereas water extracts/samples of each potential source
(herring, sardine, squid, pool scat, drain scat, and urine;
n ¼ 5 each) were chosen for THM formation potential
(THMFP) measurement. We employed the dose-based
THMFP method developed by Brtye Laboratory of the California Department Water Resources (California Department
of Water Resources, 1994). Briefly, samples were chlorinated
with freshly prepared NaOCl/H3BO3 buffered at pH 8.3 0.1,
representing the pH of natural seawater. Each sample was
diluted to a DOC value of less than 10 mg/L and treated with
a constant chlorine dosage (120 mg/L). The high chlorine
dosage was used to ensure that there was enough reactive
chlorine for complete chlorination because high ammonia
levels. Samples were stored in a 40 mL borosilicate amber
vial without headspace. The vials were incubated for 7 days
(20 1 C) and then 0.15 mL of 10% sodium sulfite solution
was added to quench residual chlorine. In parallel to the
direct THMFP measurement, an aliquot of 1 ppm bromide
was added to the 30 chosen samples prior to chlorination in
order to evaluate the effects of bromide in the THM formation. All samples were refrigerated at 4 C and analyzed
for THM concentrations using GC-ECD within two weeks.
The reactivity of DOC in forming THM, as determined by
specific THMFP (STHMFP, in mg-THM/mg-C), was also
calculated as the amount of THM formation divided by DOC
concentration of the tested water.
2.3.
Health risk assessment
Lifetime cancer risk commonly refers to the chance a person
has, over the course of his or her lifetime (from birth to death),
of being diagnosed with or dying from cancer. Based on the US
Environmental Protection Agency (EPA) guidelines on carcinogen risk assessment and the Swimmer Exposure Assessment
Model (SWIMODEL 3.0 (US EPA, 2006)), we estimated the lifetime cancer risk for marine mammal workers exposed to
THMs from marine mammal pool water through dermal absorption, inhalation, and incidental oral ingestion. Using
measured aqueous concentrations of THMs and ambient
vapor concentrations, the lifetime risk of THMs can be estimated according to SWIMODEL using Henry’s law. Briefly, the
cancer risks through dermal absorption, inhalation, and oral
ingestion were calculated as:
222
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
RiskDermal ¼ LADDDermal SFDermal
¼
CW KP SA ETDermal EF ED 0:001 L
cm3
1 mg
1000 mg
BW AT 365 d=yr
determine the differences between means of different groups
(significance level of P < 0.05). Pearson correlations were performed when appropriate (ShapiroeWilk’s test, P > 0.05).
SFDermal
3.
RiskInhalation ¼ LADDInhalation SFInhalation
¼
CVP IRInhalation ETInhalation EF ED 1 mg
1000 mg
BW AT 365 d=yr
SFInhalation
RiskOral ¼ LADDOral SFOral
¼
1 mg
CW IROral ETOral EF ED 1000
mg
BW AT 365 d=yr
SFOral
where LADD is lifetime average daily dose (mg kg1 day1) and
SF is the slope factor (an upper bound of the approximated 95%
confidence limit on the increased cancer risk from a lifetime
exposure to an agent by route, in kg mg1 day). The slope factors for different THM species were obtained from US EPA
(2005). The components of the LADD are: CW for chemical
concentration in water (mg L1) or CVP for chemical concentration in vapor (mg m3); KP for the chemical-specific dermal
permeability constant (m h1); SA for skin surface area available for contact (m2) or IR for intake rate (L h1 for ingestion and
m3 h1 for inhalation); ET for exposure time (h event1); EF for
exposure frequency (event year1); ED for exposure duration
(years); BW for body weight (kg); and AT for average lifetime of
marine mammal worker (yr). Based on the recommendation of
SWIMODEL (US EPA, 2006) and exposure risks for marine
mammal workers who work in the water with the animals,
such as cetacean rehabilitation staff, marine mammal trainers,
or swim-with-the-dolphin program workers (hereafter
“trainers”) (Hunt et al., 2008), we assumed the parameters for
maximal risk exposure as follows: body surface areas (SA:
male ¼ 1.94 m2, female ¼ 1.69 m2), inhalation rate (IRInhalation ¼
1.00 m3/h); oral ingestion rate (IROral ¼ 0.010 L h1), swimming
exposure frequency (EF ¼ 250 events year1), career exposure
duration (ED ¼ 20 years), body weight (BW: male ¼ 78.1 kg,
female ¼ 65.4 kg), swimming exposure time (ET ¼ 2 h event1
for dermal and oral working in pool; 6 h event1 for inhalation
considering the addition of a 4 h event1 working around the
pool), and average life span (AT ¼ 70 years). Note that when
swimming in a marine mammal pool or ocean, people are
much more conservative and careful about letting water get in
their mouth than when playing in a swimming pool, and thus
we adopted a lower IROral of 0.010 L h1 compared to that of
0.025 L h1 recommended in SWIMODEL.
Noncarcinogenic risks of THMs were also evaluated using
the hazard index (HI) for different exposure routes (Lee et al.,
2009). This index is calculated as the ratio of the average daily
doses to the reference doses (RfDs). As US EPA recommended,
the RfD values are 1.00 102 mg kg1 day1 for chloroform,
and 2.00 102 mg kg1 day1 for bromodichloromethane,
dibromochloromethane, and bromoform.
2.4.
Statistical analyses
All statistical analyses were conducted using the software SPSS
13 for windows. One way analysis of variance (ANOVA) and
subsequent multiple comparisons (Tukey test) were used to
Results
3.1.
Occurrences of DOC and THMs in the rehabilitation
facility
As presented in Fig. 1, the monthly THM concentrations of
pool water in the large freshwater systems of TMMC ranged
from 1.9 0.6 mg/L in August to 105.3 23.8 mg/L in May. Across
different months, mean THM concentrations generally
increased with both DOC concentrations and the number of
patients admitted in TMMC (P < 0.05). For speciation, the
bromoform dominated all THM species (>90%). Concentrations decreased along the line in the water treatment systems
(Fig. 2) from pool water, drain line, and settling tank (averages
of 25e31 mg/L) to storage tank and return line (averages of
9e11 mg/L; P < 0.001). The most significant drop (58% on
average) occurred from settling tank to post-sand filter.
Dissimilarly, the THM concentrations in the storage tank and
return line after disinfection (averages of 120e130 mg/L) were
higher than those in the diluted in pool water, drain line, and
settling tank (averages of 40e55 mg/L) (P < 0.001).
3.2.
Information on California sea lions in the isolated
tank study
The sub-adult female weights (n ¼ 2) were 45e50 kg (kg); the
sub-adult male weights (n ¼ 3) were 77e119 kg; the adult female weights (n ¼ 2) were 53e80 kg; and the adult male
weights (n ¼ 3) were 190e215 kg (Table A.1). The amount of
solid wastes (including pool and drain scat) generated by a
California sea lion in captivity generally increased with the
body weight (R2 ¼ 0.81, P ¼ 0.001), as daily meals were fed with
a fixed ratio to body weight. When the ratios of meal weight to
body weight stood between 2.8% and 6.5%, California sea lions
converted a range of 3.2e7.7% (5.4% on average) of their meal
weights to solid wastes (Table A.1).
3.3.
Waste and DOC production per animal housed
The amounts of DOC produced from food items (herring,
sardine, and squid) and wastes (pool scat, urine, and drain
scat) were determined by water extraction methods (Chow
et al., 2005). Foods yielded higher DOC concentrations in
pool water with averages of 22e34 mg-C/g-wet sample,
whereas wastes showed averages of 2e16 mg-C/g-wet sample
(Fig. 3). The levels of water extractable organic carbon among
the 5 groups of solid samples were all significantly different
(P < 0.01) with squid > herring > sardine > pool scat > drain
scat. The concentration of DOC in urine was 12 1 g/L.
3.4.
Reactivity of DOC in THM formation
The reactivity of DOC in forming THMs for different wastes is
summarized in Fig. 3. The WEOC from solid wastes (pool and
drain scat) had higher reactivity in forming THMs compared to
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
223
Fig. 2 e Dissolved organic carbon (DOC) and
trihalomethane (THM) concentrations in different locations
of the water treatment systems at The Marine Mammal
Center, Sausalito, California, USA. Different letters on top
of the bars indicate significant differences (P < 0.05)
among groups (uppercase for DOC and lowercase for THM).
Error bars show the standard errors (n [ 6).
Fig. 1 e The concentrations of trihalomethane (THM) in pool
water of the freshwater systems at The Marine Mammal
Center and its correlations with dissolved organic carbon
(DOC) (a) and the number of sea lions admitted (nsea_lions)
(b). Values are presented as monthly mean ± SD (n [ 10).
waste extracts (Fig. 4a) and between DOC concentrations and
UVA254 in pool water (Fig. 4c) were observed without statistically
different slopes or intercepts (P > 0.05). Significant but weak
correlations (Figs. 4b and d) were found between THMFP and
UVA254 of all extracted samples (R2 ¼ 0.40), as well as between
THM concentration and UVA254 in pool water at TMMC (R2 ¼ 0.44).
3.6.
Health risk assessment of THMs in large
rehabilitation facility
food items (P < 0.05). The drain scat samples showed the
highest formation potential (58 8 mg-THM/mg-C), followed by
pool scat (48 10 mg-THM/mg-C). Among food sources, herring
and sardine had comparable STHMFP (P > 0.05). Although squid
had the highest WEOC potential, its STHMFP (31 4 mg-THM/
mg-C) was significantly lower than that of herring (41 5 mgTHM/mg-C) or sardine (38 6 mg-THM/mg-C; P < 0.05). The
urine samples had the lowest STHMFP (14 6 mg-THM/mg-C)
compared to solid wastes and food items.
The presence of bromide enhanced the formation of THMs
by 25e124%, on a mass basis in all samples (Fig. 3). In addition
to the increase in THM production, bromide changed the
speciation of THMs. Before bromide addition, chloroform was
the only THM formed in all samples. With 1 ppm of bromide,
other forms of THMs, including bromodichloromethane,
dibromochloromethane, and bromoform, were produced in
all samples. Utilizing ‘drain scat’ as an example, the primary
species was bromodichloromethane, accounting for 42%, followed by chloroform (31%), dibromochloromethane (23%), and
bromoform (4%).
The cancer risks from exposure to THMs in marine mammal
pool water through oral ingestion, dermal absorption, and
inhalation for both male and female trainers are shown in
Table 1. Among the three exposure routes, inhalation of THMs
poses the highest cancer risk (5.16 104 to 1.30 103) for
marine mammal trainers who regularly swim in aquaria,
followed by dermal absorption (4.63 108 to 2.86 107) and
oral ingestion (5.40 109 to 8.64 108). This risk from
inhalation is much higher than the negligible cancer risk level
(106) defined by the US EPA, while those from dermal absorption and oral ingestion were within the negligible range.
Comparing different genders, female trainers have slightly
higher cancer risks than male trainers through all different
exposure routes. For noncarcinogenic risks, the hazard index
of THMs for the oral ingestion (maximum of 4.52 104) and
dermal absorption (maximum of 1.20 103) were below the
EPA acceptable level of 1 by a factor of about 1000 (Table 1).
4.
Discussion
3.5.
4.1.
DOC and THM loads in marine mammal facilities
Correlations among DOC, THMs, and UVA254
Positive linear correlations (R2 ¼ 0.64e0.74; both P < 0.001) between WEOC concentration and UVA254 in the food items or
The variation of DOC and THM concentrations along different
compartments of the marine mammal water treatment
224
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
pools in 5 different countries reviewed by Lee et al. (2009)). Of
70 pool water samples taken at TMMC, 11 had a higher THM
concentration than 80 mg/L, the MCL of THM in drinking water
implemented by USEPA.
The positive correlations between monthly THM concentration and both DOC concentration and the number of marine mammals admitted to TMMC (Fig. 1) indicate that the
increase in food inputs and waste generation yielded higher
risk of exposure to unsafe levels when high numbers of marine mammals were present.
4.2.
UV254 reflected DOC sources but failed to predict
THM formation
Fig. 3 e Water extractable organic carbon (a) and specific
trihalomethane formation potential (b) from potential
carbon sources. Error bar shows the standard deviation. In
(b), different letters on top of the bars indicate significant
differences (P < 0.05) among different groups (uppercase
for bromide addition and lowercase for no bromide
addition), and the number at the bottom of bars indicates
the percentage increase in STHMFP of samples after
bromide addition. aThe values of urine in (a) represent the
volume concentration of DOC (in g/L) instead of WEOC.
systems (Fig. 2) clearly revealed THM formation during the
water disinfection process and its contribution to the THM
load in pool water. Due to the dilution effect and evaporation,
the concentration of THMs in pool water was less than half of
that generated in storage tank. In the pool water, the monthly
DOC load ranged from 4.91 mg/L to 34.7 mg/L on average
(Fig. 1), which is in agreement with observations in other
marine mammal aquaria (Spotte and Adams, 1979), but much
higher than those commonly found in municipal water
(commonly <5 mg/L) or swimming pool water (commonly
<10 mg/L) (Lee et al., 2009; Zwiener et al., 2007). However,
evaluating the THMs, the concentration in pool water at
TMMC (1.1e144.2 mg/L with average of 44.1 mg/L) was comparable to that observed in seawater/saltwater aquaria (up to
140 mg/L) by Shi and Adams (2012), and those reported in
swimming pools (average of 17.5e132.4 mg/L for swimming
Ultraviolet absorption spectrophotometry provides an inexpensive and convenient tool to characterize the aromaticity of
DOC. The regression between DOC and UV254 (Fig. 4c) in pool
water at TMMC is consistent with the regression between
WEOC and UV254 of food and wastes (Fig. 4a), with slopes
greater than those for soil extract (DOC ¼ 17.85 UV254 - 0.071;
R2 ¼ 0.98) or natural water (DOC ¼ 30.3 UV254 - 0.212;
R2 ¼ 0.93) (Chow et al., 2008), suggesting that the food input
and waste production are the major DOC sources in marine
mammal aquaria. Humic substances from plant or soil
leachates or natural water that commonly have high aromaticity have been well identified as THM precursor in tap water
(Croue et al., 2000). In contrast to the humic substance, DOC
from food and waste primarily contained readily biodegradable nitrogen-enriched organic matter, such as fatty acids and
protein, which are relatively smaller molecules with less aromatic characteristics (Her et al., 2002; Leenheer and Croue,
2003; Stevenson, 1994), contributing to the STHMFP of WEOC
of marine mammal pool water (36 mg-THM/mg-C on average)
being significantly lower than those of natural waters (typically 60e100 mg-THM/mg-C) (Chow et al., 2005).
Because of its low cost and convenience, UV254 has been
widely applied to predict DOC levels and THM formation in the
drinking water industry (commonly R2 > 0.8 (Diaz et al., 2009;
Dobbs et al., 1972; Edzwald et al., 1985)). However, our documentation of weak correlations with monitored THM levels in
marine mammal aquaria (R2 ¼ 0.44, Fig. 4d) and THMFP from
food and waste materials (R2 ¼ 0.40, Fig. 4b) suggests it can
hardly be a good surrogate to predict THM levels in marine
mammal aquaria.
4.3.
Contribution of different DOC sources to THM
formation
Combining the WEOC and STHMFP results, we can calculate
the mass-normalized THMFP from different sources. The
average THM formation potential per mass sample followed
the order of: herring (1066 mg-THM/g-wet-sample) squid
(1054 mg-THM/g-wet-sample) > sardine (836 mg-THM/g-wetsample) > pool scat (768 mg-THM/g-wet-sample) > urine
(168 mg-THM/mL(g)-wet-sample) > drain scat (116 mg-THM/gwet-sample).
Consider the example of a fully grown male sea lion with a
body weight of 200 kg in a pool being fed 6 kg (3% of its body
weight) of squid daily with all food consumed: 3.0e7.4 g DOC
and 0.15e0.35 g THM would likely be dissolved in the water
225
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
Fig. 4 e Comparisons of linear correlations of organic carbon and trihalomethane concentrations with the corresponding
ultraviolet absorbance at 254 nm (UVA254). Organic carbons were dissolved organic carbon in marine mammal pool water
(n [ 50, a & b), and water extractable organic carbon from solid carbon sources in an isolated tank system (n [ 25, c & d) at
The Marine Mammal Center, Sausalito, California, USA.
from his fecal production, according to the relatively stable
metabolic capability (range of the mass ratio of feces to food in
Table A.1: 3.2e7.7%). Although few data on urine volume of
the adult sea lion are available, based on a urine volume of
800e950 mL per day for a 50 kg sub-adult male California sea
lion (Pilson, 1970), the DOC and THM generated per day by
urine would equal 9.6e11.4 g and 0.13e0.16 g, respectively.
However, all offered food is not always completely
consumed, and thus the percentage of uneaten food should
influence the total DOC and THM productions. Taking the
same example above, if we assumed 0%, 1%, 5%, and 10% of
the 6 kg of squid input went uneaten, then the total DOC yield
from food and waste would be 15.7 g, 17.6 g, 25.1 g, and 34.5 g,
respectively, while the corresponding THM potential production would amount to 0.39 0.10 g, 0.45 0.10 g, 0.69 0.10 g,
and 0.99 0.09 g, respectively (Fig. 5). The food-derived THM
contribution would increase rapidly with the percentage of
food remaining, contributing almost half (46%) of the THM
when even just 5% of food remained. In an extreme case, if all
6 kg of squid were left uneaten and not removed, the potential
generation would be 204 g of DOC and 6.32 g THM. Under this
condition, a minimum of 79,000 L of water would be required
to house only one adult sea lion in captivity to guarantee a safe
level of THMs below the 80 mg/L threshold.
4.4.
Risk assessment
We calculated the cancer and noncarcinogenic health risks of
THMs to marine mammal trainers to help workers understand
their risks and add to the body of literature for different
exposure pathways. The average risk from inhalation exposure to THMs in marine mammal pool water ranged from
Table 1 e Lifetime cancer risks and hazard indices (for noncarcinogenic risks) from THM exposure for male and female
trainers working in marine mammal aquaria.
Male Trainer
Arithmetic mean
Cancer risks:
Oral
2.80E-08
Dermal
1.51E-07
Inhalation
5.88E-04
Noncarcinogenic hazard indices:
Oral
1.16E-04
Dermal
4.57E-04
Female trainer
Minimum
Maximum
Arithmetic mean
Minimum
Maximum
5.40E-09
4.63E-08
5.16E-04
7.20E-08
2.75E-07
1.09E-03
3.36E-08
1.57E-07
6.99E-04
6.48E-09
4.82E-08
6.15E-04
8.64E-08
2.86E-07
1.30E-03
1.80E-05
2.49E-04
3.76E-04
1.16E-03
1.39E-04
4.75E-04
2.16E-05
2.59E-04
4.52E-04
1.20E-03
226
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
water and duration of captivity), as well as lack of information
on cancer development in sea lions.
Notably, our study has not accounted for the health risk
from other, more toxic, DBPs such as haloacetic acids, haloacetonitriles and halonitromethanes (Shi et al., 2013). The
water in marine mammal aquaria have low organic C/N ratio
(here <3.5) and large amounts of protein, which suggests the
haloacetonitriles yield could account for a large fraction of
DBPs (Wang et al., 2013, 2012). Much is still unknown about the
risks of DBPs to marine mammals in captivity and marine
mammal workers, which should not be simply considered as
“negligible” but should be further explored.
Fig. 5 e The trihalomethane (THM) formation potential
when different percentages of food are left uneaten and not
removed from a marine mammal pool. Estimates based
upon 6 kg of squid offered to a 200 kg adult sea lion in an
aquaria, and the feces and urine production are in
proportion to food intake.
5.16 104 to 1.30 103, two orders of magnitude higher than
the negligible risk level (106) defined by the US EPA. This estimate highlights the significant risk from the inhalation of
THMs around marine mammal pools. Inhalation exposure
was the primary risk for THM exposure, similar to what has
been found for recreational swimming pools in other studies
(Erdinger et al., 2004; Lee et al., 2009). The calculated hazard
index for noncarcinogenic risk ranging from 105 to 103 was
much lower than 1, which is the EPA-defined acceptable level.
Notably, the health risk we calculated here is only from
occupational exposure to the THMs in marine mammal pools.
The marine mammal trainers are also exposed to THMs when
they drink tap water daily and swim in recreational swimming
pools; therefore, their total THM exposure and the cancer or
noncarcinogenic risks should be higher depending on other
activities.
Compared to humans, marine mammals have a greater fat
and lipid content, predisposing marine mammals to accumulate THMs. By implication, the marine mammals in
aquaria may also face potential health risks from THMs. To
our knowledge, the only available document discussing the
possible impact of THM on marine mammals is Spotte’s risk
assessment (1991). His assessment considered THM intake by
ingestion only and concluded that the risk THM posed to
marine mammals is negligible. However, dermal contact and
inhalation are also important routes for THM exposure
(Erdinger et al., 2004; Miles et al., 2002). Considering an adult
California sea lion (200 kg) with a lung volume of 55 mL/kg and
an average resting respiration rate of 438 breaths/h (Wise
et al., 2009), its inhalation rate would be 4.82 m3/h. Also,
because of the increased sea lion exposure time to water in
aquaria (assuming 14 h/day), we calculated the potential dose
rate via inhalation exposure to be at least one order of
magnitude higher than that of a trainer. However, the lifetime
cancer health risks are difficult to estimate because of likely
varying exposure frequency and duration (how much time in
4.5.
Implications for marine mammal aquaria
management
We have shown that food and waste materials are important
DOC sources and THM precursors in marine mammal aquaria.
If not collected by the pool-operator, the uneaten foods and
feces (16 1 mg-WEOC/g-wet sample) compose the drain scat
(2 0 mg-WEOC/g-wet sample) with the water flow, therefore
most of the DOC (14 mg-WEOC/g-wet sample) is released into
pool water. It is noteworthy that drain scat still releases DOC
with the highest STHMFP, even after long retention time in
water. Thus, it is necessary to inspect the pool regularly and
remove all solid waste materials from the water systems as
soon as possible to minimize the THM generation.
According to the monitoring results along the water
treatment system (Fig. 2), approximately 30% of the DOC on
average was retained and re-entered the pool water after
treatment through the settling tank, protein skimmer, sand
filter, and chlorination. This finding is consistent with a previous study, which showed carbon fed to the animals could
remain in the water systems after water treatment, along with
the organic carbon which accumulated linearly with time in
the closed water system (Spotte and Adams, 1979). By implication, the THM generation likely increases with the accumulating DOC level due to water recirculation. The pool
operator should therefore add and exchange water on a regular basis to avoid deteriorating water quality. Also, as bromide has long been identified as one of the major precursors
in forming more toxic brominated THM (Richardson et al.,
2007) during water sanitization (Bull et al., 2001; Croue et al.,
2000; Hua et al., 2006; Xie, 2004), marine mammal holding
facilities should avoid using Br-containing disinfectants to
minimize the production of more toxic THMs.
5.
Conclusions
(1) Trihalomethanes were detected in a model marine
mammal rehabilitation facility at levels ranging from
1.1 to 144.2 mg/L with an average of 44.1 mg/L. Monthly
average THM levels increased with the number of animals admitted.
(2) Uneaten food in the tank and animal wastes were
identified as major sources of DOC and THM precursors.
Each gram of food or waste material can potentially
generate 22e34 mg or 2e16 g of DOC in water, respectively. The THMFP of food and waste ranged from 14 to
w a t e r r e s e a r c h 5 9 ( 2 0 1 4 ) 2 1 9 e2 2 8
58 mg-THM/mg-C. The presence of bromide in water
increased the THM formation.
(3) Because uneaten food left in pools can dramatically
increase the DOC concentration and THM formation,
careful pool management to remove uneaten food, as
well as wastes, is highly recommended. In fact if even
only 5% of uneaten food remains in the pool, the foodderived DOC was estimated to contribute 46% to the
THM budget, followed by feces (34%) and urine (20%).
(4) Unlike natural water with humic substances (source of
DOC in tap water), ultraviolet absorption at 254 is not be
a reliable indicator for THM formation for marine
mammal pool water, as evidenced by the poor correlation between THM level and UV254 (R2: 0.40e0.44).
(5) Health risk analysis suggests that trainers have a high
risk from inhalation exposure of THM (5.16 104 to
1.30 103) from marine mammal pool water, which is
higher than the negligible risk level (106) defined by the
USEPA. The risks from oral ingestion or dermal absorption are both below 106. The marine mammals in
the pool may, also, be at risk.
Acknowledgments
The authors gratefully acknowledge financial support for this
project from the National Swimming Pool Foundation Postdoctoral Research Fellowship. Alex Chow was also supported
in part by an NIGMS MORE Institutional Research and Academic Career Development Award to UC Davis and San
Francisco State University, http://prof.ucdavis.edu, grant
number K12GM00679. Joelle Sweeney was supported by the
National Marine Fisheries Service no. 40ABNF001144, the Earl
and Ethyl Myers Oceanographic and Marine Biology Trust
scholarship, and the David and Lucile Packard Foundation
Grant, under the supervision of Larry Young and the SJSU
IACUC protocol no. 841, TMMC IACUC protocol no. SJ 1/03, and
SFSU IACUC protocol no. A3-048. The California sea lion
feeding study was supervised by Dr. James T. Harvey of Moss
Landing Marine Laboratories, Moss Landing, CA. Jun-Jian
Wang appreciated the financial support from the China
Scholarship Council (CSC[2011]3010). Special thanks to The
Marine Mammal Center and all volunteers for their help.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.04.007.
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