Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes

Marine Environmental Research xxx (2015) 1e9

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Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes Tiffany L. Hedrick-Hopper a, *, Lauren P. Koster a, Sandra L. Diamond a, b a b

Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409, USA Western Sydney University, School of Science and Health, Hawkesbury Campus, Penrith, NSW 2751, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 May 2015 Received in revised form 30 August 2015 Accepted 13 September 2015 Available online xxx

Rising water temperatures due to climate change may increase the uptake and effects of triclosan in aquatic organisms. Our objectives were to investigate the accumulation of dietary triclosan and its neuroendocrine effects in Atlantic croaker, an estuarine fish, under two temperatures and during depuration. A pilot study was used to select a dietary exposure of 50 mg/kg. For 10 days, fish were exposed to one of four diet/temperature treatments (n ¼ 16/treatment): normal diet at 26  C and 29  C and triclosan-treated diet at 26  C and 29  C. Fish exposed to triclosan at 26  C accumulated 2.6 mg/kg wet weight on average versus 5.6 mg/kg wet weight at 29  C. Triclosan exposure significantly impacted reflexes, resulting in the loss of the dorsal fin reflex (DS) in 53% of fish, while temperature and triclosan etemperature interactions were not significant. Triclosan body burden did not significantly predict DS loss. There were no significant differences in thyroid hormone levels among groups. Triclosan-treated fish at 26  C were fed untreated pellets for 5 additional weeks. Two fish lost the DS during the first depuration week, and no affected fish recovered the reflex. These results have important implications for fish and their predators, as the DS may be important for swimming performance and social patterning. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Body burden Estuarine contaminants Reflex impairment Climate change Uptake Depuration Thyroid hormones

1. Introduction Triclosan (2,4,40-trichloro-20-hydroxydiphenyl ether) is an anti-microbial agent used in a growing number of products such as deodorants, toothpastes, and soaps. Although wastewater treatment removes the majority of triclosan found in residential wastewater, residual triclosan enters watersheds (Reiss et al., 2002), and is subsequently transported to coastal estuaries. Despite having a half-life in the environment of only 12 days (Gatidou et al., 2010), triclosan is one of the most frequently detected chemicals associated with wastewater worldwide. It was detected in 58% of US waters surveyed during a 1999e2000 USGS study of 139 streams across 30 states with a median concentration of 140 ng/L and a maximum measured concentration of 2300 ng/L (Kolpin et al., 2002). Similarly, a 2011 review of all published studies available at the time found triclosan was detected in 56.8% of surface water samples at a mean concentration of 50 ng/L (Brausch and

* Corresponding author. E-mail address: [email protected] (T.L. Hedrick-Hopper).

Rand, 2011). Sampling of German estuaries found triclosan in the surface waters of the Ems (0.012e0.11 ng/L), Weser (0.018e0.620 ng/L), and Elbe (1.20e6.87 ng/L) estuaries (Xie et al., 2008). Triclosan was detected in rivers and lakes in Switzerland at concentrations of up to 74 ng/L (Lindstrom et al., 2002). Triclosan was detected ubiquitously during a two year study in the Jiulong River in China and its estuary with concentrations of up to 64 ng/L detected (Lv et al., 2014). It has been detected at levels up to 134 ng/ L in the surface waters of the Ton Canal in Japan (Nishi et al., 2008). Some of the highest recorded surface water levels come from rivers in India where concentrations up to 5160 ng/L were recorded (Ramaswamy et al., 2011). Increased use of triclosan in personal care products has also led to drastically increased environmental levels over short periods of time in some systems. For example, in 2006, the mean triclosan concentration in Charleston, South Carolina estuaries was 0.63 ng/L (DeLorenzo et al., 2008). By 2008, the mean triclosan concentration had risen to 7.5 ng/L in the same estuary (Fair et al., 2009). Aquatic organisms are highly sensitive to triclosan exposure. The 48-h LC50 (concentration at which 50% of the subjects died) for Japanese medaka (Oryzias latipes) fry is 0.352 mg/L (Foran et al.,

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2000). Tatarazako et al. (2004) found that a bacterium (Vibrio fisheri), a crustacean (water flea Ceriodaphnia dubia), and two species of fish (Japanese medaka, O. latipes, and zebrafish, Danio rerio) all had an IC25 less than 0.30 mg/L and an IC50 less than or equal to 0.40 mg/L (IC is the concentration that causes a reduction in growth or reproduction of the population by the specified percentage). However, a microalga (Selenastrum capricornutum) was 30e80 times more sensitive than the bacterium, crustacean, and fishes with an IC25 of 0.0034 mg/L and an IC50 of 0.0047 mg/L (Tatarazako et al., 2004). While current levels of triclosan in estuaries do not appear to pose an acute mortality risk to aquatic organisms (Lyndall et al., 2010), continually increasing environmental triclosan levels may lead to a variety of sublethal effects. Triclosan can act as an androgen antagonist (Ahn et al., 2008; Rostkowski et al., 2011) and can also impact estrogenic pathways (Ishibashi et al., 2004). Triclosan disrupts postembryonic development in anurans (Veldhoen et al., 2006), and acts as an endocrine disrupter in male mosquitofish (Raut and Angus, 2010). Triclosan has been shown to negatively affect thyroid hormone homeostasis by decreasing thyroxine (T4) concentrations in rats (Crofton et al., 2007). Triclosan inhibits muscle contraction in rats, mice, and freshwater fishes (Cherednichenko et al., 2012; Kjærheim et al., 1995). It can also alter swimming performance in freshwater fish at levels as low as 0.075 mg/L (Fritsch et al., 2013). In addition, because triclosan is stored in the fatty tissues of animals, it can bioaccumulate at higher trophic levels. For example, triclosan has been found in the bile of fish (Adolfsson-Erici et al., 2002) and in the plasma of wild bottlenose dolphins (Tursiops truncatus) (Fair et al., 2009). Triclosan has also been found in human breast milk (Adolfsson-Erici et al., 2002). Environmental factors such as water temperature can amplify the lethal and sublethal impacts on aquatic organisms from chemicals such as triclosan, and water temperatures are predicted to increase in estuaries and oceans due to climate change. Higher temperatures may lead to increased deposition of organic chemicals in aquatic ecosystems (Noyes et al., 2009), although warming temperatures may also lead to faster degradation of these chemicals. Higher water temperatures are also associated with an increased uptake rate for pollutants in fish because at higher temperatures, ventilation rates increase due to increases in metabolic rates and decreases in oxygen solubility (Kennedy and Walsh, 1997). Climate changes affecting contaminant bioavailability and uptake for fish species may also have large impacts on the contaminant levels found in top predators such as larger fish, sharks, seabirds, and marine mammals. The International Panel on Climate Change (IPCC) currently projects a global air temperature increase of 1.8e4.0  C by the year 2100 (IPCC, 2007). Ocean temperatures are expected to rise by a similar amount, as sea temperatures closely track air temperatures (IPCC, 2007). For the midAtlantic Coastal region (central New Jersey to central North Carolina), an increase in seawater temperature of 1.0e1.5  C above the average temperatures in 1990 is predicted by 2030 with an increase of 2.7e5.3  C by 2095 (Najjar et al., 2000). Sublethal effects of environmental stressors on aquatic organisms can take the form of small changes in behavior or physiology that result in higher mortality rates over the long term (Rose et al., 2003). In fish, overall condition and vitality can be measured by testing a variety of reflexes that are present in healthy fish, called Reflex Action Mortality Predictors (RAMPs). Impairment of these reflexes is associated with increased stress and delayed mortality in a variety of species (Davis, 2007). RAMP tests have primarily been used to investigate delayed mortality from barotrauma in discarded or released fish caught during commercial or recreational fishing operations (Campbell et al., 2010). However, because RAMP tests measure overall fish performance, they are likely a good measure of

the sublethal effects on fish from any environmental stressor, including contaminant exposure. The major objective of this study was to explore the effects of triclosan exposure on estuarine fish body burden, reflex behaviors, and physiology (thyroid hormone levels). We also wanted to explore whether the triclosan effects on these response variables would change with the higher water temperatures predicted under future climate scenarios. Finally, due to the short half-life of triclosan in the environment, we wanted to study the duration of fish responses to triclosan after exposure had ended. Our hypotheses were: 1) that elevated water temperatures would result in higher accumulations (body burdens) in fish, 2) that triclosan exposure would impair fish reflexes and would result in reduced levels of thyroid hormones, 3) that both higher triclosan body burdens and higher temperatures would impair fish reflexes to a greater extent, and 4) that any adverse effects of exposure would disappear shortly after exposure stopped. Atlantic croaker (Micropogonias undulatus) was chosen as the test species since juveniles are commonly found throughout estuaries in the southern and eastern US, where they are potentially exposed to increasing triclosan concentrations (Lassuy, 1983). These fish are a major prey species in estuaries along the US Atlantic coast such as Charleston Harbor, South Carolina, where triclosan bioaccumulation in bottlenose dolphins has been previously shown (Fair et al., 2009). This study is unique because it represents, to our knowledge, the first exploration of the effects of triclosan on an estuarine fish, the first to look for effects of triclosan on fish reflexes, and the first investigation of the effects of water temperature on the accumulation of triclosan. 2. Methods As an overview, three experiments were conducted to test the effects of temperature and triclosan on Atlantic croaker. The first experiment was a pilot test to determine the concentration needed in food pellets for fish to accumulate measurable levels of triclosan using the planned chemical analysis method. The second experiment tested control and triclosan-exposed fish at two different water temperatures (26  C and 29  C) for effects on triclosan body burdens, fish reflexes, and thyroid hormones. The third experiment determined the duration of reflex impairment after triclosan exposure had ceased. 2.1. Fish collection Fish were acquired in June 2011 from a live bait facility in Corpus Christi, Texas and transported to the aquaculture facility on the Texas Tech University campus at Lubbock, Texas. To kill any external parasites, fish were kept in a freshwater bath for 1 min. Fish treatment was compliant with all requirements of The Code of Ethics of the World Medical Association for Animal Experiments (http:// europa.eu.int/scadplus/leg/en/s23000.htm), The Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Texas Tech University Institutional Animal Care and Use Committee (Approval No. 10071-01). Fish used in this study were 140e200 mm in size and considered similar to the average size of Atlantic croaker consumed by wild bottlenose dolphins in South Carolina and similar estuaries (Pate, 2008). Fish were acclimated to the lab environment in a 2000 L tank at 27.5  C for approximately 10 days until they began to feed and behave normally. During acclimation, fish were given a diet of 45% protein with 8% lipid in 3.0 mm pellets (Silver Cup Fish Feed, N02670000301) ad libitum since this was the recommended diet for Atlantic croaker aquaculture (Davis and Arnold, 1997).

Please cite this article in press as: Hedrick-Hopper, T.L., et al., Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes, Marine Environmental Research (2015), http://dx.doi.org/10.1016/ j.marenvres.2015.09.006

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2.2. Experiment 1 e pilot test Because fish needed to accumulate easily measurable body burdens (at least 0.22 mg/kg wet weight for the planned chemical analysis method) over a relatively short period of time, a pilot test was conducted using food pellets at 1 mg/kg, 10 mg/kg, and 50 mg/ kg of triclosan. Triclosan impregnated food pellets for fish in triclosan exposure groups were created by mixing triclosan (97% HPLC, SigmaeAldrich, 72779-5G-F) with ultra-pure water to create a 10 mg/L solution. Pellets were soaked in the dark in this solution at a rate of 10 ml, 100 ml, and 500 ml of triclosan solution for 100 g of pellets at approximately 20  C. Pellets were then allowed to dry until all excess water had evaporated. Pellets were tested using the methods described below to ensure they had an average concentration of triclosan of 1 mg/kg, 10 mg/kg, and 50 mg/kg respectively. Five fish were fed each of the dosages for 10 days at 26  C, euthanized using decapitation with pithing, and analyzed as described in Section 2.3.1 below to see if triclosan body burdens could be easily measured. 2.3. Experiment 2 e temperature and triclosan exposure For this experiment, two temperature levels were investigated: a representative ambient temperature and a projected future temperature. The mean summer temperature from stations sampled in 2005 as part of the South Carolina Estuarine and Coastal Assessment Program (SCECAP) was used to select the ambient summer temperature of 26  C. Future climate projections for the mid-Atlantic coast were used to select the projected future temperature of 29  C (Najjar et al., 2000). Fish were randomly assigned to one of four treatment groups (n ¼ 16 per group): 1) low temperature and triclosan, 2) low temperature and no triclosan (low temperature control), 3) high temperature and triclosan, and 4) high temperature and no triclosan (high temperature control). For the experiment, individual fish were housed in 75 L tanks. After being placed into the tanks, fish were acclimated to their experimental temperature over a period of 3 days. Temperatures were slowly shifted from the 27.5  C holding tank temperature to either 26  C or 29  C at a rate of 0.5  C per day. Croaker are tolerant of a wide range of temperatures and tolerate small changes in temperature well (Lassuy, 1983). This small increase or decrease in temperature over a 24 h period is much less than the amount required to induce temperature shock (Barton and Peter, 1982; Coutant et al., 1974; Donaldson et al., 2008). Additionally, this temperature shift is within the range of daily temperature variations seen at single stations in the SCECAP study. Fish assigned to control groups were fed unadulterated food pellets. For treated pellets, three 5 g samples of food pellets were tested with the same extraction and liquid chromatography methods described below to ensure pellets contained 50 mg/kg triclosan on average (range: 48.78e50.25 mg/kg). Once the experimental period had started, fish were fed ad libitum either pellets impregnated with 50 mg/kg triclosan or untreated food pellets for 10 days. Food consumption was recorded daily; any pellets not consumed after several minutes were removed. After 10 days of dietary exposure, fish were tested for reflexes and blood was sampled for thyroid hormones as discussed below, after which fish were euthanized using decapitation with pithing. Carcasses were stored for later body burden analysis at 20  C in the dark to reduce any triclosan degradation. 2.3.1. Body burden analyses For analysis of body burdens, fish were thawed and any tissue that would be digestible by a predator was dissected and saved. Tissue was homogenized in a laboratory blender at 20,000 rpm so that subsequent analysis would represent the whole-body triclosan

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concentration that would be available to a predator consuming the fish. A 5 g tissue sample was analyzed for each individual using a QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) extraction method with LC-MS analysis (Anastassiades et al., 2003). QuEChERS is a widely used method for examining the concentrations of organic chemicals in organic samples (e.g. fruits, vegetables, fish, etc.). Generally, samples are homogenized, and chemical residues are extracted using a combination of salts and a solvent. The extracted chemicals are then cleaned up for analysis. For this analysis, each homogenized sample was first spiked with b-Estradiol 17-acetate (>99%, SigmaeAldrich Corp, E-7879) to act as a surrogate. The sample was then added to a tube containing QuEChERS extraction salts (Restek Corp, 23991) along with 5 ml of H2O and 10 ml of acetonitrile (HPLC grade, Fisher Scientific, A998). The mixture was vortexed and allowed to sit for 2 h before being centrifuged at 3200 rpm for 20 min. The clear organic layer was then removed from the top and added to a tube for dispersive cleanup (United Chemical Technologies, ECMPSC1815CT). This tube was vortexed and allowed to sit another 2 h. It was then centrifuged for 20 min at 3200 rpm. A syringe with a 0.45 mm filter was used to filter the extract into labeled amber vials. Samples were analyzed using liquid chromatography with UV detection on a Grace Prevail C18 column (4.6  250 mm; W.R. Grace & Co., 99210) at a wavelength of 200 nm. An acetonitrile:water (70:30) mobile phase and a 0.85 ml min1 flow rate were used. A 5 point external standard calibration curve was used to calculate concentrations of both triclosan and b-Estradiol 17-acetate from each sample chromatogram. 2.3.2. Reflex tests Because most studies of triclosan have focused on specific effects on endocrine function, development, or qualitative activity level rather than on overall negative impacts on fish condition and reflexes, we explored the effects of triclosan on fish reflexes using RAMP responses. Although RAMP responses have not been previously used as a toxicological endpoint, they provide insight into overall fish stress and condition. We conducted RAMP tests on day 0 (after temperature acclimation but prior to the start of the experimental period) and on day 10. The four reflexes tested were gag, vestibulo-ocular, dorsal fin, and tail flex. For the gag test, a small probe was inserted into the fish's throat to see if the fish gagged. In the vestibulo-ocular test (VO), the fish was rotated to see if the fish continued to focus its eye on the handler. The dorsal fin test (DS) consisted of seeing whether the fish erected its dorsal fin in response to a brushing stimulus. Similarly, to test the tail flex response a brushing stimulus was applied to the tail to see if the fish flexed it. Presence of a given reflex was scored as a 1 while absence was scored as a 0. 2.3.3. Thyroid hormone analyses After the RAMP tests on day 10, blood samples for analysis of thyroid hormones were drawn from the caudal vasculature of each fish using 24 gauge needles. Blood was allowed to clot for 30 min in individually labeled centrifuge tubes, then was centrifuged for 5 min at 5500 rpm. Plasma was removed from the top of the tube and placed into a second tube where it was stored at 20  C. Thyroid hormones in thawed plasma samples were analyzed using enzyme-linked immunosorbent assay (ELISA) kits. For each fish, a 50 ml sample of plasma was analyzed for total T3 (Abnova Corp., KA0198) and a 25 ml sample of plasma was analyzed for total T4 (Abnova Corp., KA0200). Single samples were analyzed for each fish due to the small amount of plasma available from each individual fish. However, all standards were done in triplicate. Plates for both assays were read on a spectrophotometer at 450 nm wavelength. For the T3 analysis, the limit of detection was 0.2 ng/ml of plasma.

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For the T4 analysis, the limit of detection was 0.4 mg/dL of plasma.

3.2. Experiment 2

2.4. Experiment 3 e reflex recovery testing

3.2.1. Food consumption There was no significant difference between the experimental groups in fish growth or food consumption during the 10-day treatment period. Although most fish grew in length, the percent increases in size were very small and were similar between treatment groups (Table 2). All fish continued to eat throughout the experiment, with total food consumption during the experimental period ranging from 0.2 to 2.4 g. Interestingly, there were some patterns in food consumption by treatment group (Table 2). The total amount of food consumed by each fish during the treatment period was higher on average in the two high temperature groups than in the two low temperature groups. Additionally, within each temperature, food consumption was higher on average for triclosan-exposed fish than for control fish. However, for all groups there was a large amount of individual variation in food consumption and the 95% confidence intervals for all 4 groups overlapped, indicating no significant differences between food consumption among groups.

To see if lost reflexes might be recovered after triclosan exposure had ceased, 15 fish were randomly assigned to either a triclosanexposure or a control group (n ¼ 10 for triclosan-exposed group and n ¼ 5 for control group). For this experiment, all fish were kept at the previously described representative ambient water temperature of 26  C. Fish were acquired, housed, and exposed to triclosan or control food pellets as in the previously described experiment. The same RAMP tests were conducted both prior to and immediately following the exposure period. Unlike the first experiment, fish were not sacrificed after testing on day 10. Starting on day 11, all fish were fed untreated food pellets and RAMP tests were conducted weekly for an additional 5 weeks (a total of 6 tests after the exposure period). After this period, fish were sacrificed as previously described. 2.5. Statistical analyses All statistical analyses were conducted using R 2.15.1 (R Core Team, 2012). Because reflex data were binary and longitudinal in nature, they were analyzed using Generalized Estimating Equations (GEE) methods for testing the main effects of temperature and triclosan treatments and their interaction. Reflex data were then analyzed for a doseeresponse relationship with fish triclosan concentrations as well as thyroid hormone levels. Thyroid hormone data were analyzed for treatment group differences using a randomized MANOVA. Because T3 and T4 levels are related, relationships between accumulated triclosan and thyroid hormones were explored using a combination of a principal components analysis (PCA) and regression analyses. The PCA was used to convert the T3 and T4 measurements to linearly uncorrelated variables (principle components). A regression was then used to explore the relationship between triclosan body burdens and each of the principle components. Because different fish consumed different quantities of food and food consumption would affect the amount of triclosan consumed, food consumption was used as a covariate in the statistical analysis of fish triclosan concentrations. Fish triclosan body burden concentrations were analyzed using a randomized ANCOVA. 3. Results 3.1. Experiment 1 No fish in the 1 mg/kg dosage group showed measurable triclosan body burdens (Table 1). For the 10 mg/kg group, the mean accumulation was 0.271 mg/kg wet weight, and 2 fish showed measurable triclosan body burdens. In the 50 mg/kg group, the average triclosan body burden was 2.059 mg/kg wet weight with all fish showing measurable body burdens. Based on these tests, a dietary dosage of 50 mg/kg was selected for triclosan-exposed fish since this was the only group where triclosan was measurable in all tested fish.

3.2.2. Body burden analyses Chromatograms for each fish tissue sample as well as the 5 standards were generated. Retention times for triclosan and bEstradiol 17-acetate were approximately 9.3 min and 10.8 min respectively. The quantitation limit for triclosan was estimated to be 0.22 mg/kg wet weight. Mean recovery for the b-Estradiol 17acetate surrogate was 87.8% (95% CI ¼ 77.8e97.8). Fish body burdens ranged from 1.8 to 16.9 mg/kg wet weight. Fish in the high temperature group accumulated significantly more triclosan than fish in the low temperature group (randomized ANCOVA, F ¼ 5.22, p ¼ 0.02). For high temperature fish, mean triclosan concentration was 5.6 mg/kg wet weight (95% CI ¼ 4.4e6.9, Table 2). Mean triclosan concentration for low temperature fish was 2.6 mg/kg wet weight (95% CI ¼ 2.4e2.8, Table 2). 3.2.3. Reflex tests Exposure to triclosan significantly affected the dorsal fin reflex of the fish, while higher water temperature had no significant effect on any reflex (Fig. 1). All fish displayed all four reflexes during preexperimental testing. During post-experiment tests, all fish continued to display the vestibulo-ocular and tail flex responses. One fish did not display the gag reflex during the post-experiment tests, and that fish was a member of the low temperature triclosan group. The gag, VO, and tail flex reflexes were not significantly different based on triclosan exposure (GEE, Gag-Wald ¼ 0.01, p ¼ 0.99; VO-Wald ¼ 0.01, p ¼ 0.99; Tail Flex-Wald ¼ 0.01, p ¼ 0.99), or temperature (GEE, Gag-Wald ¼ 0.01, p ¼ 0.99; VO-Wald ¼ 0.01, p ¼ 0.99; Tail Flex-Wald ¼ 0.01, p ¼ 0.99). For the dorsal fin response, all fish displayed the response in the low temperaturecontrol group (95% CI ¼ 82.9e100%), and 93.8% (95% CI ¼ 69.7e99.9%) showed the response in the high temperaturecontrol group (indicative of one fish not displaying the DS response). In the low temperature-triclosan group, 62.5% (95% CI ¼ 38.5e81.2%) of fish lifted their dorsal fins in the postexperiment test, while 43.8% (95% CI ¼ 23.1e66.9%) of fish in the

Table 1 Triclosan accumulation in pilot test fish. 95% confidence intervals are shown in parentheses. n ¼ 5 per treatment group. Dosage

Total triclosan body burden (mg/kg wet wt)

% Fish with measurable triclosan accumulated

1 mg/kg 10 mg/kg 50 mg/kg

Not measurable 0.271 (0.261e0.281) 2.059 (1.974e2.144)

0 40 100

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Table 2 Mean values for measured parameters by group. 95% confidence intervals are shown in parentheses. Values with different letters are significantly different from one another. n ¼ 16 per treatment group.

Percent growth of fish Total food consumption (g) Total triclosan body burden (mg/kg wet wt) Plasma total T3 (ng/ml) Plasma total T4 (mg/dL)

Low temperature-control

Low temperature-triclosan

High temperature-control

High temperature-triclosan

n ¼ 16

n ¼ 16

n ¼ 16

n ¼ 16

A

A

A

0.005 (0.002e0.008) 0.34A (0.27e0.41) 0A (NA) 2.49A (1.99e2.99) 1.21A (0.71e1.71)

0.006 (0.001e0.01) 0.48A (0.36e0.64) 2.6B (2.4e2.8) 2.45A (1.93e2.97) 1.67A (1.15e2.19)

0.002 (0e0.004) 0.52A (0.38e0.67) 0A (NA) 2.19A (1.7e2.69) 1.67A (1.17e2.17)

0.004A (0.001e0.007) 0.65A (0.37e0.93) 5.7C (4.3e7.0) 2.48A (2.04e2.88) 1.60A (1.16e2.04)

Fig. 1. Percent of fish displaying each reflex response after 10 day experimental period (error bars represent 1 s.e.). Bars with different letters are significantly different from one another.

high temperature-triclosan group showed a positive DS response (Fig. 1). There was a significant main effect of triclosan exposure on the DS reflex (GEE, Wald ¼ 2.42, p ¼ 0.02), while the main effect of temperature was not significant (GEE, Wald ¼ 0.01, p ¼ 0.99). The interaction of temperature and triclosan on the dorsal fin reflex was also not significant (GEE, Wald ¼ 0.01 p ¼ 0.99). Fish body concentration of triclosan was not a significant predictor of DS reflex loss (Fig. 2, logistic regression, z ¼ 1.175, df ¼ 55, p ¼ 0.24). In addition, neither total plasma T3 (logistic regression, z ¼ 0.29,

df ¼ 55, p ¼ 0.78) nor total plasma T4 (logistic regression, z ¼ 0.10, df ¼ 55, p ¼ 0.94) had significant relationships with reflex loss.

3.2.4. Thyroid hormone analyses Total T3 and T4 levels were similar between treatment groups (Table 1), and there were no statistically significant differences in T3 and T4 levels between the 4 treatment groups (randomized MANOVA, F ¼ 1.54, p ¼ 0.16). There were also no significant effects of fish triclosan body burdens on the two principle components of the thyroid hormone measurements (PC1: multiple regression, t ¼ 0.56, p ¼ 0.58; PC2: multiple regression, t ¼ 0.365, p ¼ 0.72).

3.3. Experiment 3

Fig. 2. Dose-response relationship between triclosan body burden (mg/kg wet weight) and post-test dorsal fin reflex (DS: 1 ¼ reflex present, 0 ¼ reflex absent).

During the reflex recovery experiment, all fish showed all reflex responses prior to the exposure period. None of the control group fish ever lost any reflex responses. In the triclosan-exposed group, no fish lost the gag, vestibulo-ocular, or tail flex responses. However, only 60% of triclosan exposed fish displayed the dorsal fin reflex at the end of the 10-day triclosan exposure period. Two additional fish that displayed the dorsal fin reflex at the end of the exposure period did not do so during the test for reflex recovery a week later. None of the fish that lost the dorsal fin reflex recovered the response at any point during the rest of the trial, which lasted an additional 5 weeks, despite the lack of further exposure to triclosan (Fig. 3).

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T.L. Hedrick-Hopper et al. / Marine Environmental Research xxx (2015) 1e9

Fig. 3. Dorsal fin reflex loss after triclosan exposure (error bars represent 1 s.e.). Triclosan exposure started on day 0 after testing and ended on day 10. Graph includes only fish given triclosan-infused pellets (n ¼ 10).

4. Discussion In keeping with our hypotheses, increased water temperature was associated with increased accumulation of triclosan, with fish at the higher water temperature accumulating nearly twice as much triclosan on average as fish at the lower water temperature. In addition, triclosan exposure did have negative impacts on fish reflexes; specifically on the ability of croaker to erect their dorsal fins. Almost half of the fish lost this dorsal fin reflex, but gag, vestibulo-occular, and tail flex responses were unaffected. Contrary to our hypotheses, we found no impacts of either triclosan exposure or temperature on plasma T3 or T4 levels. In addition, although triclosan exposure was related to reflex impairment, triclosan accumulation as measured by body burden levels was not related to either reflex impairment or to the levels of thyroid hormones. Increased water temperature did not lead to reflex impairment, and there was no significant interaction between triclosan and water temperature. Surprisingly, fish continued to lose the dorsal fin reflex up to one week after exposure ceased, and the lost dorsal fin reflex was not recovered by any fish even 5 weeks after exposure to triclosan had ceased. This impairment, along with the increased accumulation of triclosan in fish tissues at higher water temperatures has important implications for both Atlantic croaker and for higher trophic level organisms such as dolphins that eat Atlantic croaker, particularly under predicted climate conditions. 4.1. Triclosan accumulation The results of this experiment show that temperature does affect accumulation of triclosan in Atlantic croaker. Fish exposed to triclosan via their diet in 29  C water had significantly higher concentrations of triclosan than fish exposed in 26  C water. This pattern is consistent with other studies of temperature effects on organic contaminants. Increased temperature was correlated with increased accumulation of an organic pesticide in three different aquatic insects (Buchwalter et al., 2003). Similarly, exposure to organophosphate pesticides at higher temperatures increased uptake rates and elimination rates in midge larvae (Lydy et al., 1999). A study of PAHs in sunfish found that while elimination rates did increase at higher temperatures, uptake rates increased to an even greater degree resulting in a net increase in contaminant concentrations at higher temperatures (Jimenez et al., 1987). Because croaker in this study were exposed to the triclosan via their diet, differences in food consumption could play a role in triclosan accumulation. Fish in the higher water temperature group

consumed more food on average than fish in the lower temperature treatment, so their dose of triclosan would also have been higher. However, it is important to note that the differences in food consumption between these groups were not significant and there is a large amount of variability in food consumption within each group. Thus, increased triclosan dosage due to increased food consumption alone does not appear to explain the higher accumulation of triclosan in the higher water temperature group. Additionally, these differences in food consumption were taken into account in the ANCOVA, which found significantly different triclosan accumulation between the high and low temperature treatment groups. Since increased water temperatures do result in increased metabolic rates in fish, it is likely that in the high temperature group both the uptake rate for triclosan and its elimination rate are higher since these processes are both dependent on fish metabolism (Jimenez et al., 1987). However, it would appear that the uptake rate is increased to a greater degree than the elimination rate resulting in the increased concentrations seen in the higher temperature treatment group. Although the exposure to 50 mg/kg food pellets experienced by the fish in this study was considerably higher than ambient environmental levels, this dosage resulted in body burden levels that are similar to recorded values of triclosan found in the wild. Specifically, body burden levels in the current study ranged from 1.8 to 16.9 mg/kg wet weight, while bile levels in wild fish living near wastewater treatment plants in Sweden ranged from 0.24 to 4.4 mg/kg wet weight depending on species, wastewater treatment plant, and distance from the outfall (Adolfsson-Erici et al., 2002). Fish caged near outfalls had up to an order of magnitude higher levels of triclosan in bile than free-ranging fish. In estuaries, Atlantic croaker are likely exposed to triclosan through both diet and exposure to sediments. Atlantic croaker diets include benthic crustaceans, polychaetes, molluscs, and fishes (Overstreet and Heard, 1978). While estuarine invertebrates have not been studied specifically, the highest reported body burdens for freshwater invertebrates have been around 0.3e0.4 mg/kg (Coogan and Point, 2008). While foraging, croaker spend large amounts of time in close proximity to sediments (Overstreet and Heard, 1978; Parker, 1971). Although estuarine systems have not been heavily surveyed, the highest reported levels in estuarine sediments were 0.8 mg/kg (Miller et al., 2008). Therefore, although experimental conditions of a short exposure to a high level of dietary triclosan do not exactly replicate the exposures that would be found in nature, the pattern of higher accumulation with higher water temperature displayed by the experimental fish are applicable to fish exposed to environmental concentrations. If current trends continue, environmental concentrations of triclosan will continue to increase as triclosan is increasingly incorporated into additional products. For example, the amount of triclosan found in one estuary in South Carolina increased more than 10 fold between 2006 and 2008 (DeLorenzo et al., 2008; Fair et al., 2009). In addition, as coastal water temperatures continue to rise due to climate change, triclosan concentrations in exposed organisms will likely increase. Since triclosan is known to bioaccumulate, the combination of increased environmental concentration and increased temperature will lead to even further increases in body concentrations, particularly at higher trophic levels (e.g. predator species such as sharks and dolphins). Interestingly, the 29  C temperature used as the high temperature treatment in this study is within the range of temperatures recently recorded at some shallow stations along the South Carolina coast during summer months (Van Dolah et al., 2006; Van Dolah et al., 2013). These areas could be currently experiencing this increased triclosan accumulation during periods when waters warm. Our results therefore suggest that there might be a seasonal

Please cite this article in press as: Hedrick-Hopper, T.L., et al., Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes, Marine Environmental Research (2015), http://dx.doi.org/10.1016/ j.marenvres.2015.09.006

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component to triclosan accumulation in coastal ecosystems that should be considered in future environmental studies. 4.2. Reflexes Generally, the loss of any reflex is indicative of increased stress and an increased risk of mortality (Davis, 2007). The loss of the dorsal fin reflex as opposed to the gag, vestibulo-ocular, or tail flex reflexes may provide further insight into triclosan's sublethal effects. One possibility is that the lack of dorsal fin reflex reflects problems with muscular movements due to triclosan exposure. Triclosan has been shown to lead to a general depression of metabolism in freshwater mussels (Binelli et al., 2011; Falfushynska et al., 2014) and to impair mitochondrial function in rats (Newton et al., 2005; Weatherly et al., 2015) and human cells (Vera et al., 2015; Weatherly et al., 2015). Triclosan has been shown to specifically inhibit muscle contraction in rats and mice (Cherednichenko et al., 2012; Kjærheim et al., 1995). Triclosan exposure negatively impacted swimming performance in fathead minnows, including a decrease in swimming activity, endurance, and predator avoidance ability (Cherednichenko et al., 2012). Another study in fathead minnows also found exposure to triclosan decreased distance traveled while swimming as well as negatively impacting the expression of proteins associated with muscle excitation and contraction (Fritsch et al., 2013). Zebrafish exposed to triclosan similarly showed poor swimming performance, abnormal operculum movement, and a loss of equilibrium (Oliveira et al., 2009). Yet, if muscle function inhibition in these fish is responsible for the lost dorsal fin reflex, impacts on other reflexes like the gag and tail flex might also be expected. Since we saw no impacts on any other reflex responses, the loss of the dorsal fin reflex may not be the result of an inability on the part of the fish to raise the dorsal fin, but some more indirect mechanism. Although it might be expected that higher triclosan body burdens lead to increased impairment or that body burden levels over a given triclosan threshold lead to an impaired dorsal fin response, our results do not show any such patterns. We did not find any linear relationship between body burdens and the lost dorsal fin response, and there was no obvious threshold of body burden concentrations that separated fish that did and did not lift their dorsal fin. Thus, there may be an unmeasured variable or pathway for this negative effect. The sex of fish used in this study was not recorded, and the only hormones measured were total plasma T3 and T4. It is possible that fish sex or one of these unmeasured hormones may play a role in the loss of this reflex response. In many fish species, erection of the dorsal fin is associated with aggressive or dominance behaviors (Cole and Noakes, 1980; Iersel, 1953; Laming and Ebbesson, 1984; Rosenau and McPhail, 1987). Compression of this fin can also be associated with fear or fright responses (Laming and Ebbesson, 1984). The missing dorsal fin reflex may indicate changes in aggression or fright responses in the fish. Similar shifts in aggressive behavior have been seen when fish are exposed to either triclocarbon (a similar chemical to triclosan) or a mixture of triclocarbon and triclosan. Fish exposed to these compounds show significantly decreased levels of aggression as compared to non-exposed fish (Schultz et al., 2012). Atlantic croaker live in schools and form dominance hierarchies within these groups (Gibbard et al., 1979). Thus, if triclosan reduces aggressive behaviors in fish, it may have impacts on dominance hierarchies and other social patterning. Additionally, the vulnerability of Atlantic croaker to predation may be increased by triclosan exposure, particularly if exposure causes decreased muscle function, reduction in swimming performance, or shifts in anti-predator behavioral strategies used by the fish. Reduction in aggression or social status could also push

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Atlantic croaker into less desirable habitats with higher predation risk. If Atlantic croaker are more vulnerable to predation due to triclosan exposure, this would potentially impact both the Atlantic croaker population as well as the populations of Atlantic croaker predators such as bottlenose dolphins. If fish with higher contaminant loads are also easier for predators to capture, accumulation in predators could be increased. The difference of only 3  C between the two temperature treatments as well as the ability of croaker to tolerate a wide range of temperatures likely account for the lack of any effects of water temperature on reflexes. The loss of reflexes is indicative of stress. Juvenile croaker are particularly resilient and are captured in water temperatures as high as 35.5  C (Lassuy, 1983). They have been suggested to grow well with minimal stress at temperatures up to 28.4  C with some indications they may be able to live with minimal stress at temperatures as high as 32  C (Parker, 1971). 4.3. Thyroid hormones Contrary to our hypotheses, there were no significant effects of either water temperature or triclosan exposure on plasma total T3 or T4. A correlation between thyroid hormones and water temperature has been seen in catfish, but the temperatures sampled varied by up to 15  C (Suchiang and Gupta, 2011). Similarly, impacts of water temperature on thyroid hormones were seen in white grouper, but temperatures in this study varied by 22  C (Abbas et al., 2012). A study where Atlantic cod were held for 9 months at two temperatures that varied 4e6  C found a only a weak correlation between T4 and water temperature (r2 ¼ 0.28) and no correlation between water temperature and T3 (Comeau et al., 2000). The 3  C difference between treatment groups for only 10 days in this experiment may be too small to see any potential impacts on thyroid hormones. Impacts on thyroid hormones after triclosan exposure have been reported previously in other aquatic species (Crofton et al., 2007; Raut and Angus, 2010; Veldhoen et al., 2006). Exposure periods in these studies ranged from a 24 h acute exposure to a 35 day subchronic exposure. The 10 day subchronic exposure used in this study would fall within the range of other studies that saw thyroid effects, but none were seen in the current study. One possible reason for the lack of thyroid effects in this study may be the high level of individual variation on plasma total T3 and T4 levels (Table 1). Effects of triclosan or water temperature on these two hormones would have been more easily examined if each fish had been tested for plasma total T3 and T4 prior to the experimental period to calculate a baseline hormone level as well as at the end of the period. Unfortunately, blood sampling prior to the experimental period was not possible for this study due to the large amount of blood required for the tests relative to the total blood volume of the fish. This problem may be mitigated in future studies by employing methods for examining fish hormones that do not require blood sampling. Methods for extracting cortisol and similar hormones from water in which fish are held have since been developed and used to examine impacts of stressors on these hormones (Archard et al., 2012; Ellis et al., 2004; Wong et al., 2008). Although these methods have not yet been specifically developed for thyroid hormones, it is likely that thyroid hormones could be explored in this fashion. This would allow multiple replicate samples to be analyzed for each individual as well as samples before and after treatment without injuring or stressing the fish. Additionally, it is possible that in Atlantic croaker, thyroid effects might be more easily observed via thyroid histology instead of circulating plasma thyroid levels. Future studies of triclosan effects in fish should examine this alternative method as well to more clearly understand whether thyroid function is actually altered due to

Please cite this article in press as: Hedrick-Hopper, T.L., et al., Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes, Marine Environmental Research (2015), http://dx.doi.org/10.1016/ j.marenvres.2015.09.006

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triclosan exposure. 4.4. Reflex recovery It is interesting to note that in the reflex recovery experiment, no fish ever recovered the lost dorsal fin response. The biological halflife of triclosan is estimated to be fairly fast at approximately 12 days (Gatidou et al., 2010). The 5 week depuration period would result in concentrations approximately 1/8 of those in fish tested at the end of the exposure period. Specific body burdens were not measured for these fish. However, based on the average accumulated triclosan for the 26  C group during the accumulation experiment, these fish would have concentrations of approximately 0.3 mg/kg wet weight. This body burden is comparable to the environmental values of 0.8 mg/kg reported in marine sediments (Miller et al., 2008) and 0.3e0.4 mg/kg seen in freshwater invertebrates (Coogan and Point, 2008). This indicates that either small, environmentally available concentrations of triclosan are required to elicit this response, that recovery takes longer than 5 weeks of non-exposure, or that negative effects on these reflexes from higher doses are not reversible. In summary, increased water temperatures led to increased dietary accumulation of triclosan in exposed fish. As coastal water temperatures continue to increase, triclosan concentrations in exposed organisms will likely increase as well. Because triclosan bioaccumulates, increasing water temperatures will likely lead to even higher triclosan concentrations at higher trophic levels. At the same time, triclosan exposed fish demonstrate a loss of the dorsal fin reflex. This reflex does not return even 5 weeks after triclosan exposure has ended. The loss of this reflex may be tied to decreased muscle performance or dominance behavior in these fish, meaning that triclosan exposure could potentially negatively impact swimming performance or shift dominance hierarchies and social patterning in these fish. It is also possible that anti-predator behavior may be impacted in these fish. Any impacts on antipredator behavior could impact both croaker and croaker predators. If highly contaminated fish are more vulnerable to predation, patterns of accumulation in croaker predators may be impacted. Acknowledgments We thank Dr. Todd Anderson for assistance with development of extraction and LC-MS methods, Dr. Richard Strauss for statistical ~ o and Tim Grabowski for technical expertise, Drs. Reynaldo Patin assistance, and our team of undergraduate researchers: Jason Barker, Jason Baca, Tiara Smith, Marylyn Matthew, and Apollo Castillo. We thank Dr. Lou Densmore and the Texas Tech University Department of Biological Sciences for the funding that made this project possible. References Abbas, H.H., Authman, M.M., Zaki, M.S., Mohamed, G.F., 2012. Effect of seasonal temperature changes on thyroid structure and hormones secretion of White Grouper (Epinephelus Aeneus) in Suez Gulf, Egypt. Life Sci. J. 9, 700e705. Adolfsson-Erici, M., Pettersson, M., Parkkonen, J., Sturve, J., 2002. Triclosan, a commonly used bactericide found in human milk and in the aquatic environment in Sweden. Chemosphere 46, 1485e1489. Ahn, K.C., Zhao, B., Chen, J., Cherednichenko, G., Sanmarti, E., Denison, M.S., Lasley, B., Pessah, I.N., Kültz, D., Chang, D.P.Y., 2008. In vitro biologic activities of the antimicrobials triclocarban, its analogs, and triclosan in bioassay screens: receptor-based bioassay screens. Environ. Health Perspect. 116, 1203. Anastassiades, M., Lehotay, S.J., Stajnbaher, D., Schenck, F.J., 2003. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J. AOAC Int. 86, 412e431. Archard, G.A., Earley, R.L., Hanninen, A.F., Braithwaite, V.A., 2012. Correlated behaviour and stress physiology in fish exposed to different levels of predation pressure. Funct. Ecol. 26, 637e645.

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Please cite this article in press as: Hedrick-Hopper, T.L., et al., Accumulation of triclosan from diet and its neuroendocrine effects in Atlantic croaker (Micropogonias undulatus) under two temperature Regimes, Marine Environmental Research (2015), http://dx.doi.org/10.1016/ j.marenvres.2015.09.006