Aquatic Toxicology 144–145 (2013) 332–340

Contents lists available at ScienceDirect

Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox

Locomotor behavior in zebrafish (Danio rerio) larvae exposed to perfluoroalkyl acids Mazhar Ulhaq, Stefan Örn ∗ , Gunnar Carlsson, David A. Morrison, Leif Norrgren Swedish University of Agricultural Sciences, Department of Biomedicine and Veterinary Public Health, SE-750 07 Uppsala, Sweden

a r t i c l e

i n f o

Article history: Received 20 June 2013 Received in revised form 11 October 2013 Accepted 16 October 2013 Keywords: Zebrafish Larvae Behavior Perfluoralkyl acid Locomotor activity

a b s t r a c t Perfluoroalkyl acids (PFAAs) are persistent organic contaminants that have been detected in wildlife, humans and the environment. Studies have shown that the toxicity of PFAAs is determined by the carbon chain length as well as the attached functional group. The locomotor activity of zebrafish larvae has become widely used for evaluation of chemicals with neurotoxic properties. In the present study the behavioral effects of seven structurally different PFAAs (i.e. TFAA, PFBA, PFOA, PFNA, PFDA, PFBS and PFOS) were evaluated in zebrafish larvae. Exposure to high concentrations of TFAA, PFNA, PFBS and PFOS resulted in distinct changes in behavioral patterns. Based on redundancy analysis, our results demonstrate three main factors affecting zebrafish larval locomotor behavior. The strongest effect on behavior was determined by the carbon chain length and the attached functional group. PFAAs with longer carbon chain length as well as PFAAs with attached sulfonic groups showed larger potential to affect locomotor behavior in zebrafish larvae. Also the concentration of the PFAAs determined the behavior responses. The results of the present study are in agreement with previous studies showing correlations between the chemical structure of PFAAs and the toxicological effects. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Perfluoroalkyl acids (PFAAs) are persistent organic contaminants that have been used in surfactants, lubricants, adhesives, cosmetics, paper coatings, firefighting foams, agrochemicals and medicine (Kissa, 2001; Renner, 2001). These organofluorine chemicals have high energy carbon fluorine (C F) covalent bonds that make them resistant to hydrolysis, microbial degradation and photodegradation. The PFAAs can have a carboxylic or sulfonic functional group, and perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are well-known representatives of these respective groups. The ubiquity and stability of PFAAs make them a matter of high concern both for humans and in ecotoxicology. The PFAAs have been shown to be protein-binding and non-biodegradable, making them persistent and bioaccumulative in both humans and wildlife, including animals in remote locations such as polar bears (Calafat et al., 2006; Giesy and Kannan, 2001; Yamashita et al., 2008). Among the PFAAs, PFOS and PFOA have historically been the most widely detected in humans and environmental samples (Domingo et al., 2012; Kannan et al., 2005; Miege et al., 2012; Rudel et al., 2011). In humans, drinking water, fish and shellfish are considered to be the major sources for dietary intake of PFAAs, and are the main

∗ Corresponding author. Tel.: +46 18671178; fax: +46 18673532. E-mail address: [email protected] (S. Örn). 0166-445X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquatox.2013.10.021

routes of exposure, together with occupational sources and inhalation of indoor dust (EFSA, 2008). PFAAs can cross the placental barrier and be excreted via breast milk, resulting in a high risk for fetuses and neonates (Olsen et al., 2009a; Tao et al., 2008a,b). Epidemiological studies from several countries have shown inverse associations between maternal plasma levels of PFAAs and newborn birth weights (Fei et al., 2007; Maisonet et al., 2012; Washino et al., 2009). The brain is, along with the liver, blood and kidney, one of the targets for accumulation, although individual PFAAs seem to accumulate in distinct distribution patterns. In mice, exposure to PFOS and PFOA during pregnancy resulted in four times higher accumulation of PFOS in the brains of newborn mice compared with PFOA (Onishchenko et al., 2011). The nervous system is a sensitive target for various chemicals, and PFAAs have been linked as causative agents of mental disorders (Barkley, 1998; Brown et al., 2005; Hoffman et al., 2010). In epidemiological studies, correlations between PFAA exposure levels and ADHD (attention deficit hyperactivity disorder) in children have been reported (Fei et al., 2008; Hoffman et al., 2010). PFAAs are considered to be developmental neurotoxicants, and effects on spontaneous behavior, habituation capability, learning and memory later at adulthood have been observed in prenatally or neonatal exposed mice (Johansson et al., 2008; Onishchenko et al., 2011). For aquatic organisms, discharges from municipal sewage treatment plants are assumed to be significant sources of contamination in aquatic environments. Several studies have shown that PFAAs are present in wastewater and released into sludge and natural

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

waters (Olofsson et al., 2013; Pan et al., 2011; Yeung et al., 2009). Measurements of PFOS in the aquatic environment indicate a high bioconcentration factor, and also biomagnification in organisms at higher trophic levels (Kannan et al., 2005). Studies have shown that the toxicity of PFAAs is determined by the carbon-chain length as well as the functional group attached (Hagenaars et al., 2011; Zheng et al., 2012). In experimental fish studies, developmental malformations, physiological disturbances and impaired behavior have been observed after exposure to different PFAAs (Hagenaars et al., 2011; Zheng et al., 2012; Chen et al., 2013; Huang et al., 2010). Long-term studies with zebrafish exposed to PFOS have resulted in adverse effects on offspring development (Wang et al., 2011). The decreased larval survival was correlated to the PFOS body burden in the offspring of exposed female zebrafish, suggesting maternal transfer of the compound (Wang et al., 2011). Maternal transfer has also been indicated in wild fish due to the presence of high concentrations in fish eggs (Kannan et al., 2005). Measurement of zebrafish larval locomotor behavior has become a widely used method for identification of chemicals causing behavioral disturbances. Embryos and larvae of zebrafish are well suited to behavioral testing due to e.g. their small size, ease of handling, and rapid development. Commonly, the locomotor activity is measured both in light and darkness in order to detect changes in the swimming activity in response to physical or chemical stress. The lighting conditions affect the locomotor activity of zebrafish larvae, with less activity recorded during light compared with darkness (Emran et al., 2008; Irons et al., 2010; MacPhail et al., 2009). Sudden darkness normally results in an immediate and robust hyperactivity in locomotor behavior, and deviations from this normal behavior can be recorded after exposure to neurotoxicants. Locomotor activity of zebrafish larvae has been widely used to establish specific behavioral patterns for characterization of drugs and chemicals (Ali et al., 2011; Kokel et al., 2010; Rihel et al., 2010). For evaluation of pollutants, zebrafish behavior is a response indicator of sublethal toxicity, and can provide information on the mode of action of the tested chemicals. Chronic PFOS exposure has been shown to cause behavioral disturbances in both adult zebrafish and their offspring (Chen et al., 2013). In embryonic zebrafish, PFOS exposure affects the expression of specific proteins in the brain leading to motor neuron malformations, as well as affecting cell proliferation causing muscular malformations (Zhang et al., 2011). Disturbances in skeletal muscle fibers and motor neuron development play significant roles in locomotor behavior later in life (Drapeau et al., 1999; Flanagan-Steet et al., 2005). The great stability and high bioaccumulation potential of long carbon chained PFAAs, along with their poor excretion in humans, make them of high concern as global contaminants. Because of the phasing out of some of the longer carbon chain chemicals the manufacturing industry has started to replace these with shorter PFAA homologues, as they have shown a more rapid excretion in animal experiments (Kudo et al., 2001; Ohmori et al., 2003; Olsen et al., 2009b). However, long chained PFAAs are still detected in environmental samples, next to the shorter replacement homologues (Naile et al., 2013; Zhang et al., 2013). There is a lack of toxicological effect data for PFAAs, especially in aquatic organisms. Furthermore, the effects seem to vary depending on factors such as species, gender and age. In a previous study we showed that exposure to seven PFAAs resulted in structure-activity related effects on the embryonic development of zebrafish (Ulhaq et al., 2013). In the present study, the exposed larvae from the earlier study of PFAAs with different numbers of carbons and attached alkyl groups were compared for their potential behavioral effects, in order to further increase the knowledge about potential toxicological effects of PFAAs on fish development.

333

2. Materials and methods 2.1. Test chemicals All of the tested perfluorinated chemicals in the experiments were purchased from Sigma–Aldrich, Germany (Table 1), except PFBA that was purchased from Alfa Aesar® GmbH & Co KG, Germany. A stock solution of each chemical was prepared in reconstituted standardized water at a concentration well below its reported solubility in water at 25 ◦ C. The standardized water was prepared according to ISO (1996). Exposure solutions were prepared freshly prior to testing on zebrafish embryos. 2.2. Zebrafish maintenance and collection of eggs Adult zebrafish (Danio rerio) of the AB strain were maintained in a flow-through system of carbon-filtered tapwater (pH 7.2–7.6; hardness 6.7◦ dH; temperature 26 ± 1 ◦ C; conductivity 468 ␮S/cm; light cycle of 14 h). Stock fish were fed to satiation daily with commercial flakes (SERA Vipan) as a staple food with added freeze-dried chironomids (Nutrafin), frozen chironomids and frozen Artemia nauplii (Akvarieteknik). Breeding groups of male and female (3:2) adult zebrafish were placed in 10-L glass aquaria equipped with spawning nets the evening before the collection of eggs. Half an hour after onset of lights the following morning, eggs were collected from the breeding group tanks and rinsed for removal of debris. Normally developed fertilized eggs were selected under a stereomicroscope for the experimental studies. 2.3. Experimental design This study was preceded by measurements of lethal and sublethal embryo toxicity endpoints recorded up to 144 h post fertilization (hpf), which have been published separately (Ulhaq et al., 2013). In the present study the effects on behavior were evaluated on the same exposed individuals. Briefly, fertilized eggs were randomly distributed individually into flat-bottom, 48-well polystyrene plates (Costar® ) along with 750 ␮L of the exposure medium. The PFAAs were tested at six consecutive concentrations differing by a factor of 3.3 (Table 1). For each PFAA test, four 48-well plates were used, with a total of 24 embryos per PFAA concentration as well as 24 in the water control group. Each treatment group was equally distributed to each of the four well plates, i.e. 6 embryos per concentration per plate, giving a total of 168 embryos for each PFAA test. The plates were covered with parafilm, and the embryos were exposed to the PFAAs until 144 hpf. The data collection involved measurements of sublethal behavioral endpoints at 144 hpf using an automated computerized videotracking system. 2.4. Larval locomotor activity test The larval locomotor activity test was performed using the Viewpoint Zebrabox® behavioral recording system (ViewPoint Life Sciences, Lyon, France). This system monitors movements using automated video recording, with a multi-well plate holder Zebrabox equipped with internal LED lights (light recordings), infrared illumination (darkness recordings) and a mounted camera. Each 48-well plate was transferred to the Zebrabox, where the larvae were allowed to acclimatize to the environment for 10 min in light. Studies were conducted to determine the effect on larvae activity both in light and darkness. After the acclimation phase in light, the locomotive activity was recorded in four consecutive 10-min phases of alternate dark and light. For generation of the data protocol of locomotor activity, a subtraction method was used for detection of objects darker than

334

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

Table 1 Description of the perfluoroalkyl acids (PFAAs) tested in the zebrafish larvae, their groups, acronyms, structures, CAS numbers and nominal exposure concentration ranges. Test compound

Acronym

Formula

Perfluoroalkyl carboxylic acids Trifluoroacetic acid Perfluorobutyric acid Perfluorooctanoic acid Perfluorononanoic acid Perfluorodecanoic acid

PFCAs TFAA PFBA PFOA PFNA PFDA

Cn F2n+1 COOH CF3 COOH C3 F7 COOH C7 F15 COOH C8 F17 COOH C9 F19 COOH

Perfluoroalkane sulfonic acids Perfluorobutane sulfonic acid Perfluorooctane sulfonic acid

PFSAs PFBS PFOS

Cn F2n+1 SO3 H C4 F9 SO3 H C8 F17 SO3 H

a

CAS number (purity %)

Number of carbons

Exposure concentration range (mg/L)

76-05-1 (>98) 375-22-4 (99) 335-67-1 (96) 375-95-1 (97) 335-76-2 (98)

2 4 8 9 10

10–3000 10–3000 3–1000 0.03–10 0.1–30

375-73-5 (>98) 1763-23-1 (98)

4 8

10–3000 0.03–10

EC50 a (mg/L) 700 2200 350 16 5 450 1.5

Values based on combined sublethal and lethal embryotoxicity effect data from Ulhaq et al. (2013).

background with a minimum object size. To remove system noise, a threshold of 0.135 mm (minimum distance moved) was used for filtering all of the data. Locomotor endpoints were designed to express the changes in the general swimming activity in response to the physical or chemical stress of exposure. Analysis of the data protocols for each larva, which were based on the behavioral endpoints quantified through movement analysis described by Alvarez and Fuiman (2005) and Murphy et al. (2008), are presented and defined in Table 2. For each larva the data from the endpoint measurements of each 1-min interval were compiled and used as response variables in the statistical evaluations. At first, the total activity data were calculated based on all active and non-active larvae. Second, the data from only the active larvae were further evaluated. 2.5. Repeatability of the test The repeatability between the zebrafish locomotor tests was assessed by calculating the coefficient of variation (CV = s/x) of the larvae in the water control groups (n = 24 for each compound) of all seven independent experiments of PFAAs during the first dark phase of the test. In the formula ‘s’ is the standard deviation of any behavioral parameter of the larvae in the control group and ‘x’ is the mean value of that parameter in the same group in the specific phase of visual response. These coefficients are presented as percentage values. 2.6. Data analysis As the studies were conducted separately and involved different clutches of eggs for each of the tested PFAAs, each treatment was compared only to its matched corresponding control. For each larva, the number of 1-min intervals where the larvae showed either activity or no activity was recorded. The data were then Table 2 Definition of locomotor activity endpoints. Behavioral endpoints

Definition

Activity counts (n)

Number of times the activity of the larvae exceeded the minimum threshold level of movement during each measurement period of 60 s. Total time the larvae exceeded the minimum threshold level of movement during each measurement period of 60 s. Total swimming distance of the larvae when exceeding the minimum threshold level of movement during each measurement period of 60 s. Swimming time of the larvae relative to the total measurement period. Swimming distance for each measurement period of 60 s. Swimming distance per swimming time.

Swimming time (s)

Swimming distance (mm)

Relative swimming time (%) Average swimming speed (mm/s) Active swimming speed (mm/s)

compiled and expressed as a mean proportion of the activity of larvae in each treatment group during the integration period (1min period of measurement) for each of the 10-min alternate light and dark periods. For the final statistical analyses, dead larvae and larvae with no activity quantified at all were excluded. In the experiments there were multiple response parameters, including the ambulatory, observatory and inferential behavioral endpoints. Ambulatory and observatory responses are the primary parameters describing the motor activity of the larvae. The ambulatory response is related to the displacement of the larva in the field (swimming distance), the observatory response include the time taken to travel this distance (swimming time) and the number of events when the larva was active during the integration period (activity counts). The inferential endpoints are the secondary behavioral parameters (average swimming speed, active swimming speed and relative swimming time) calculated from the ambulatory and observatory values. Furthermore, there were several groups of explanatory variables: chemicals (PFDA, PFNA, TFAA, PFOA, PFOS, PFBA, PFBS), phases (dark period 1, light period 1, dark period 2, light period 2), concentrations (Table 1), and atomic (number of carbons: C2, C4, C8, C9, C10, and attached group: CO3 H, SO4 H). The relationships between these two datasets (response and explanatory) were analyzed using multivariate data analyses. The effects of the different explanatory characteristics on the zebrafish behavior were analyzed by redundancy analysis (RDA; ter Braak, 1995). This is a constrained ordination technique based on principal components analysis that, in a joint analysis of the two datasets (i.e. behavioral and explanatory), assesses the degree to which they show co-variation (ter Braak and Prentice, 1989). That is, it seeks patterns among the samples that occur in both datasets, while ignoring patterns that are unique to either one of the datasets alone. The RDA analysis used the CANOCO version 4.54 program (Biometris, Wageningen, Netherlands). For evaluation of the chemical concentrations, the chemicals were “standardized” for comparison by expressing the concentrations relative to the EC50 values as calculated for lethal and/or sub-lethal endpoints in the embryotox test at 144 hpf (Ulhaq et al., 2013).The concentrations immediately below and above the EC50 were coded as “Medium” for each chemical, while all lower values were coded “Low” and all higher values were coded “High”. The concentrations were not equally distributed around their EC50 values. By this there were variations in number of contributing concentrations in the “Low” and “High” categories between the different PFAAs. Thus, only PFOS and TFAA had concentrations in the “High” category. Various transformations of the data variables were examined, but none of these improved on the analysis of the untransformed data. All of the variables had variance inflation factors EC50 ). Differences in toxicities were thus not evaluated between the chemicals but rather are correlations between chemical structure and type of activity response.

Locomotor behavioral analysis often serves as a sensitive tool for detection and evaluation of sublethal effects of chemicals (Kane et al., 2005). Moreover, behavior endpoints can be used to provide important information about the ecological consequences of environmental pollutants. Zebrafish locomotor activity has previously been used for specific high throughput behavioral profiling, leading to the characterization of psychotropic drugs (Kokel et al., 2010; Rihel et al., 2010). The behavioral analysis in the present study has shown that exposure of zebrafish larvae to PFAAs cause disturbances in locomotor activity. This study was based on the same individuals tested for embryotoxicity of the PFAAs in our previous study (Ulhaq et al., 2013). The toxicity of the different PFAAs recorded in the previous study was generally low concerning lethal

Table 3 Eigenvalues and percentage of variance explained by the redundancy analysis (RDA). RDA

Axis 1

Axis 2

Axis 3

Axis 4

Eigenvalues Species–environment correlations Cumulative percentage variance of species occurrence data Cumulative percentage variance of species occurrence–environment relation

0.628 0.890 62.8 81.8

0.118 0.819 74.6 97.1

0.021 0.922 76.8 99.9

0.001 0.353 76.8 100.0

Table 4 Measurements of the behavioral endpoints in zebrafish larvae exposed to different PFAAs. Data are expressed as mean values per 1-min interval. For explanation of the endpoints see Table 2. Concentrations in bold refer to the category “Medium” used in the multivariate analysis. “N” is the number of larvae subjected to behavior analyses. D1 and D2 represent the first and second dark periods, while L1 and L2 represent the corresponding light periods. PFAA

Conc. (mg/L)

N

Activity counts (n)

D1

L1

D2

Swimming time (s)

L2

Swimming distance (mm)

Relative swimming time (%)

Average swimming speed (mm/s)

Active swimming speed (mm/s)

D1

L1

D2

L2

D1

D1

L1

D2

L2

D1

L1

D2

L2

D1

L1

D2

L2

L1

D2

L2

0 10 30 100 300 1000 3000

23 22 23 22 21 17 8

15.2 16.9 14.5 15.4 15.4 16.7 5.20

6.66 7.89 7.25 6.64 6.92 6.90 2.04

13.2 14.8 13.1 13.9 14.2 16.1 4.40

6.59 5.71 5.71 5.14 6.35 3.83 2.63

12.3 14.3 11.6 12.4 12.9 14.3 4.42

5.60 7.25 6.25 5.98 6.51 6.24 1.60

10.6 12.6 10.7 11.4 12.4 14.7 3.74

5.61 5.20 4.76 4.55 5.89 3.37 2.20

131 138 119 144 126 157 56

43.7 51.9 45.2 44.6 41.3 53.9 14.0

106 115 103 119 109 145 46

41.4 32.9 33.5 29.0 35.5 25.8 19.6

20.5 23.8 19.4 20.7 21.5 23.9 7.31

9.34 12.0 10.4 9.98 10.8 10.4 2.67

17.6 21.1 17.8 19.1 20.7 24.5 6.24

9.35 8.67 7.94 7.58 9.82 5.61 3.67

2.19 2.30 1.98 2.40 2.10 2.61 0.94

0.72 0.86 0.75 0.74 0.68 0.90 0.23

1.77 1.92 1.71 2.00 1.82 2.43 0.78

0.69 0.54 0.56 0.48 0.59 0.43 0.32

11.1 14.3 10.6 13.2 9.96 11.4 12.5

8.61 7.26 7.72 8.19 6.64 9.42 9.34

9.90 12.7 10.2 11.3 8.70 10.1 11.6

7.92 5.20 7.44 6.96 6.50 8.62 11.2

PFBA

0 10 30 100 300 1000 3000

22 23 23 22 20 21 17

16.8 17.6 15.3 15.1 15.5 19.0 17.8

6.66 5.90 4.91 6.30 7.08 8.68 6.06

16.1 17.5 14.3 15.0 15.7 19.7 17.4

5.50 4.90 4.26 5.35 5.17 6.33 5.65

13.9 13.6 11.7 11.8 12.4 16.8 16.0

6.09 4.55 3.84 5.23 6.13 7.88 5.62

13.2 13.6 10.9 11.7 12.6 17.7 15.9

5.03 3.91 3.43 4.38 4.44 5.95 5.26

121 145 133 125 120 162 174

3.5 33.9 27.9 32.3 43.3 59.2 48.5

112 138 117 114 114 165 162

28.5 25.3 23.5 25.5 29.1 37.0 47.0

23.2 22.6 19.5 19.6 20.8 28.0 26.8

10.1 7.58 6.41 8.72 10.2 13.1 9.37

22.1 22.7 18.3 19.5 21.1 29.4 26.6

8.38 6.50 5.72 7.31 7.41 9.91 8.78

2.02 2.42 2.22 2.08 2.01 2.70 2.90

0.63 0.57 0.46 0.56 0.70 1.01 0.79

1.87 2.30 1.95 1.90 1.91 2.76 2.71

0.48 0.40 0.37 0.45 0.45 0.65 0.75

8.98 10.9 12.0 11.9 10.1 9.63 11.2

6.80 8.42 8.98 8.12 7.57 7.55 8.86

8.57 10.3 11.2 10.6 9.29 9.19 10.6

7.19 6.42 7.78 7.32 6.52 6.36 8.48

PFOA

0 3 10 30 100 300

24 24 23 23 23 20

19.4 17.8 18.8 20.6 20.0 16.8

8.16 7.40 9.00 9.33 10.4 7.56

18.4 17.4 18.5 19.4 19.5 16.0

6.11 5.52 6.12 6.26 6.77 5.23

16.8 15.7 16.2 17.6 17.1 13.5

7.80 6.93 7.67 7.56 9.34 6.58

16.3 15.3 16.1 17.1 17.0 13.2

5.81 5.35 5.80 5.06 6.15 4.51

155 153 179 206 235 175

52.3 50.4 75.7 82.8 105 67.2

142 144 174 191 211 166

36.0 31.7 44.1 49.0 52.8 41.6

28.1 26.1 27.0 29.3 28.5 22.5

13.0 11.5 12.8 12.6 15.5 10.9

27.2 25.6 27.0 28.5 28.3 22.0

9.68 8.92 9.68 8.43 10.2 7.52

2.60 2.56 2.99 3.44 3.91 2.92

0.87 0.84 1.26 1.38 1.76 1.12

2.37 2.40 2.90 3.19 3.52 2.77

0.60 0.53 0.73 0.82 0.88 0.69

9.42 10.1 11.2 12.4 14.4 13.4

6.77 7.38 9.27 9.76 10.3 10.6

8.78 9.62 10.9 11.5 12.5 13.1

6.20 6.22 7.02 8.11 7.82 9.05

8.39 7.53 7.12 6.61 7.18 9.76 8.69

19.6 16.5 16.7 18.0 17.0 17.4 20.2

6.81 6.58 6.26 4.11 4.62 5.62 5.36

17.1 15.1 15.1 15.6 15.5 14.9 18.1

7.89 7.46 6.73 6.00 6.78 8.07 7.86

16.8 14.3 14.7 15.8 15.2 14.6 18.2

6.48 6.17 5.98 3.82 4.65 5.01 5.36

169 144 141 184 154 186 221

55.2 52.1 49.9 52.8 51.6 81.0 73.2

165 133 132 172 143 175 216

41.8 40.3 39.4 22.9 28.5 40.4 37.1

28.5 25.1 25.2 26.0 25.8 24.8 30.3

13.1 12.4 11.2 10.0 11.3 13.4 13.0

28.0 23.9 24.5 26.3 25.3 24.4 30.3

10.8 10.2 9.98 6.36 7.76 8.36 8.94

2.82 2.40 2.36 3.06 2.57 3.11 3.70

0.92 0.86 0.83 1.88 0.86 1.35 1.22

2.74 2.22 2.21 2.86 2.39 2.92 3.60

0.69 0.67 0.66 0.38 0.48 0.67 0.61

10.0 9.60 9.47 11.7 9.91 12.9 12.3

6.86 6.77 7.35 8.29 7.21 9.25 7.90

9.87 9.20 8.95 10.4 9.06 12.3 11.6

6.16 6.21 6.37 6.08 6.36 7.85 7.43

PFNA

0 0.03 0.1 0.3 1 3 10

24 23 23 23 23 20 20

19.9 17.4 17.4 18.3 17.6 18.1 20.6

PFDA

0 0.1 0.3 1 3 10

24 21 23 24 23 4

21.8 21.9 21.7 23.6 22.0 10.9

21 22 22 22 23 10

11.2 9.42 10.9 10.2 11.0 8.25

20 21 19 14 19 12 10

19.5 14.9 17.5 14.4 14.5 5.28 2.14

PFBS

PFOS

0 10 30 100 300 1000 0 0.03 0.1 0.3 1 3 10

14.9 13.6 16.0 15.9 14.6 12.0

22.4 23.5 22.8 23.8 23.4 7.02

13.1 10.5 13.2 14.5 11.9 6.24

10.7 11.7 10.9 12.8 12.2 4.02

4.98 4.49 5.94 5.91 6.05 1.78

11.1 13.0 11.6 12.8 12.9 2.67

3.80 2.73 4.36 5.12 4.08 1.47

131 161 133 154 206 65.0

41.3 40.4 56.0 49.8 79.7 16.3

130 168 134 152 198 57.4

26.4 20.9 33.2 40.8 38.1 16.3

18.0 19.6 18.2 21.3 20.3 6.71

8.30 7.49 9.90 9.85 10.0 2.96

18.4 21.7 19.3 21.3 21.6 4.45

6.34 4.56 7.28 8.54 6.81 2.46

2.19 2.70 2.21 2.57 3.44 1.08

0.68 0.67 0.93 0.83 1.32 0.27

2.16 2.80 2.23 2.54 3.30 0.95

0.44 0.34 0.55 0.68 0.63 0.27

12.7 14.8 12.6 13.2 17.8 20.5

8.81 10.0 9.57 9.71 14.1 15.2

12.2 13.8 12.2 12.6 15.6 29.0

7.85 10.2 8.62 8.25 10.3 21.3

3.95 3.67 4.00 4.32 5.40 7.25

10.6 8.82 11.2 8.94 10.8 6.58

3.94 3.08 3.58 3.50 4.48 6.61

8.34 6.58 7.87 7.34 8.16 5.66

2.92 2.72 3.12 3.20 4.14 5.07

8.24 6.28 8.15 6.40 8.08 4.42

3.15 2.37 2.71 2.59 3.47 4.53

87.9 105 107 112 126 110

24.0 28.9 25.8 36.5 49.1 98.8

82.7 91.6 102 93.9 121 91.6

22.1 20.4 22.0 25.4 33.7 82.6

13.9 10.9 13.1 12.2 13.6 9.42

4.85 4.54 5.21 5.34 6.90 8.46

13.7 10.4 13.6 10.6 13.4 7.38

5.25 3.95 4.52 4.32 5.79 7.56

1.46 1.75 1.79 1.88 2.11 1.84

0.40 0.48 0.43 0.60 0.81 1.64

1.38 1.52 1.70 1.56 2.03 1.52

0.37 0.34 0.36 0.42 0.56 1.38

10.5 17.0 13.8 15.6 16.0 20.8

8.04 11.9 9.48 12.5 13.0 22.0

10.0 15.9 12.0 15.0 16.2 23.0

7.62 10.3 8.78 10.6 10.9 22.0

8.04 7.30 8.3 8.31 6.62 3.01 2.09

18.8 14.4 17.5 14.4 15.1 3.77 1.68

5.48 5.50 6.80 6.28 3.81 3.68 2.17

16.5 12.3 14.6 11.6 11.8 4.16 1.40

6.98 6.67 7.26 6.93 5.43 2.09 1.47

16.2 12.2 14.7 11.6 12.2 2.73 1.09

4.63 4.88 6.52 5.54 3.18 2.58 1.54

165 120 149 140 166 59.9 17.8

60.3 45.1 75.2 73.4 71.2 35.7 12.3

152 112 147 136 170 39.0 10.6

34.8 33.5 46.2 43.6 37.3 36.4 12.9

27.5 20.5 24.4 19.4 19.6 6.93 2.34

11.6 11.1 12.0 11.6 9.05 3.49 2.45

27.1 20.4 24.5 19.3 20.4 4.56 1.82

7.72 8.13 10.8 9.24 5.29 4.29 2.56

2.75 2.01 2.49 2.34 2.77 1.00 0.30

1.00 0.75 1.25 1.22 1.18 0.60 0.20

2.54 1.88 2.45 2.27 2.84 0.65 0.17

0.58 0.55 0.77 0.72 0.65 0.60 0.21

10.1 9.91 10.6 13.4 15.9 15.0 13.2

8.03 6.63 10.1 10.4 13.6 16.2 13.5

9.55 9.27 10.3 13.0 15.1 15.2 14.8

6.99 7.10 8.4 9.38 13.4 14.3 8.98

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

TFAA

337

338

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

Table 5 Results of the multivariate analysis of variance. Factor

DF

F

P

Chemical Phase Concentration Chemical * phase Concentration * phase Residual

6 3 3 18 9 144

21.20 139.23 25.37 1.54 1.49

0.0001 0.0001 0.0001 0.0505 0.1151

and sublethal effects (Table 1). The results from both of our studies suggest that PFAAs affect embryo development as well as behavior, although at concentrations higher than commonly measured in natural surface waters. Generally, we have observed effects on both the embryo development as well as the behavior at mg/L levels, while environmental water concentrations usually are below ng/L levels. When evaluating the effects on behavior each PFAA was tested individually, resulting in a total of seven different tests. To determine the robustness of the test, the inter-assay coefficient of variation (CV) was calculated for all endpoints of the controls using the data from the first dark phase. For the controls the CV was 46, 46, 44, 32, 38 and 44% for the endpoints swimming distance, average swimming speed, swimming time, active swimming speed, activity counts and relative swimming time, respectively. Similar CV values were reported by Winter et al. (2008) in a comparative test using zebrafish larvae. These results indicate that the test is repeatable and does not hinder the detection of the exposure effects. Also, water quality parameters were measured in the tests and found to be within an acceptable range (7.4 ± 0.6, 295–430 ␮S/cm and 92–98%, for pH, electric conductivity and oxygen saturation, respectively). The sum total of affected larvae (mortalities and malformations) was less than 10% in water controls throughout the exposure period. The time of activity for all larvae, including both active and inactive, subjected to behavioral analyses during the light and dark phases is presented for each of the seven PFAAs tested in Fig. 1 Generally, the locomotor activity in the dark phase (D1) increased after transition from the acclimation (A) light phase. The increase in activity was reduced in the following light phase (L1). A similar pattern was observed in the next alternate phases (D2 and L2). This is in agreement with normal behavior of zebrafish larvae. Zebrafish are normally active during day and become inactive at night (Cahill et al., 1998; Hurd et al., 1998). The larvae have preference for light and aversion for darkness (Champagne et al., 2010; MacPhail et al., 2009). After sudden transition to darkness the larvae become more active and then slow down (Hurd and Cahill, 2002; Prober et al., 2006). In the present study elevated larval activity in darkness was not observed in the highest tested concentrations of PFOS, PFNA and PFBS (Fig. 1). For TFAA, PFBS, PFOS and PFNA there was a reduction in the overall activity at the highest concentrations as compared to that of the matching controls (Fig. 1). Disturbances in behavioral response patterns have been observed in zebrafish larvae after exposure to known neuroactive drugs, such as ethanol, amphetamine, and cocaine (Irons et al., 2010). For ethanol, high concentrations caused total inactivity, while exposure to lower concentrations resulted in hyperactivity (Irons et al., 2010). Similarly as observed for PFOS, PFBS and PFNA in the present study, a reduced activity in the dark phases was observed after exposure to both cocaine and amphetamine (Irons et al., 2010). The disturbances in larval activities observed after exposure to PFAAs in the present study might be due to similar mechanisms as for the neuroactive drugs, or caused by other mechanisms, such as developmental delay or disturbed energy metabolism. The mean values for all behavior endpoints at the four consecutive measurement phases are presented in Table 4. The species

(behavioral responses) and environmental variables (qualitative factors for PFAAs, their exposure concentrations, types, nature and chemistry) were subjected to redundancy analysis (RDA). The RDA shows that the behavioral characteristics form three simple patterns of variation (Fig. 2a). In the figure, the behavioral similarities among the samples are indicated by the spatial relationship of the points; points near each other show more similarity among themselves (based on the zebrafish behavior) than they do to points further away. The variation of each of the behavioral characteristics among the samples is indicated by the vector arrows, with longer arrows showing more variation. A correlation among the variables is positive when the behavioral vectors are long and pointing roughly into the same direction, whereas perpendicular arrows indicate no correlation. The behavioral characteristics formed three groups of correlated behavior: swimming distance, average swimming speed, and relative swimming time were highly correlated, as were activity counts and swimming time, with active swimming speed being separate (Fig. 2a). The RDA shows a relatively weak relationship between the behavioral characteristics and the explanatory characteristics (Fig. 2b–d), with the two axes shown accounting for 97% of the total sum of squares of the first two axes of the equivalent unconstrained ordination (Table 3). In Fig. 2, the influence of each of the explanatory characteristics is indicated by the star symbols, which indicate the centroids (multivariate medians) of those samples having each characteristic. In spite of the weak relationship, all three of the types of explanatory variables are statistically significant at P = 0.0001 (Table 5). The “High” concentration resulted in higher active swimming speed (Fig. 2c). However, it should be noted that only two of the tested PFAAs contributed to this category, i.e. TFAA and PFOS. The lack of other PFAAs in this category was mainly due to high mortalities in concentrations above the EC50 values (Ulhaq et al., 2013). The higher active swimming speed for the “High” concentrations might be due to changes in swimming patterns because of developmental malformations, such as the oedemas observed in the individuals (Ulhaq et al., 2013). However, these effects might also be due to neurotoxic mechanisms causing rapid bursts of activity. For example, the neuroactive drug ethanol has been shown to cause rapid and short movements resulting in high active swimming speed in zebrafish larvae (De Esch et al., 2012). The carboxylic PFAAs as a group was positively correlated to activity counts and swimming time, while the sulfonic PFAAs were negatively correlated to these endpoints. The strongest effect on behavior was determined by the differences between the chemicals (Fig. 2b), notably their differences in number of carbons and attached functional group (Fig. 2d). Thus, PFAAs with long carbon chains and/or attached sulfonic groups have stronger impacts on the behavior responses. In particular the active swimming speed was higher after exposure to PFDA, PFBS and PFOS compared with the other tested PFAAs.The sulphonic PFAAs are considered to be more toxic than the equivalent carboxylic PFAAs based on structure activity relationship evaluations in zebrafish embryos (Hagenaars et al., 2011; Shi et al., 2008; Ulhaq et al., 2013; Zheng et al., 2012). Notably, the replacement chemical for PFOS, PFBS, was the compound that excerted the highest impact on the tested behavioral endpoints. PFBS was positively correlated to active swimming speed and negatively correlated to all other endpoints measured. There is currently a lack of studies on the ecotoxicological impact of the short-chained PFAAs. The results from the present study call for further evaluation of PFBS and other short-chained PFAAs when considering environmental impact. The present study indicates that the environmental pollutants PFAAs can cause disturbances in zebrafish behavior that might imply ecological consequences for wild fish. Exposure of wild fish populations to high levels of PFAAs may lead to changed behavior patterns, such as foraging, reproduction, predator avoidance and social interactions, which are essential for fitness and survival.

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

4. Conclusions The intention of the present study was to compare and evaluate the toxicity of different PFAAs on locomotor behavior in zebrafish larvae. The behavioral analysis showed that early embryonic exposure to PFAAs can cause disturbances in locomotor behavior in zebrafish larvae. Exposure to high concentrations of TFAA, PFNA, PFBS and PFOS resulted in distinct changes in behavioral patterns. Our results demonstrate three main factors affecting zebrafish larval locomotor behavior. The strongest effect on behavior was determined by the carbon chain length and the attached functional group. PFAAs with longer carbon chain length, as well as PFAAs with attached sulfonic groups, showed larger potential to affect locomotor behavior in zebrafish larvae. This might be due to a higher bioconcentration potential for PFAAs with these caracteristics. The concentration of the PFAAs also determined the behavior responses but less than did the carbon chain length and the attached functional group. The results of the present study are in agreement with previous studies showing correlations between the chemical structure of PFAAs and their toxicological effects. Conflicts of interest The authors have nothing to disclose. Acknowledgements This work was supported by The Swedish Research Council, grant 2011-27237-89648-39. Mazhar Ulhaq was funded PhD scholarship by PMAS, Arid Agriculture University, Rawalpindi, Pakistan. References Ali, S., Champagne, D.L., Alia, A., Richardson, M.K., 2011. Large-scale analysis of acute ethanol exposure in zebrafish development: a critical time window and resilience. PLoS ONE 6, e20037. Alvarez, M.D., Fuiman, L.A., 2005. Environmental levels of atrazine and its degradation products impair survival skills and growth of red drum larvae. Aquat. Toxicol. 74, 229–241. Anderson, M.J., 2001. Permutation tests for univariate or multivariate analysis of variance and regression. Can. J. Fish Aquat. Sci. 58, 626–639. Barkley, R., 1998. Attention-Deficit/Hyperactivity Disorder. A Handbook for Diagnosis and Treament, 2nd ed. Guilford Press, New York, NY, USA. Besse, P., 2001. Pratique de la Modélisation Statistique. Publications du Laboratoire de Statistique et Probabilité, Université Paul Sabatier, Toulouse. Brown, C., Gardner, C., Braithwaite, V.A., 2005. Differential stress responses in fish from areas of high- and low-predation pressure. J. Comp. Physiol. B 175, 305–312. Cahill, G.M., Hurd, M.W., Batchelor, M.M., 1998. Circadian rhythmicity in the locomotor activity of larval zebrafish. Neuroreport 9, 3445–3449. Calafat, A.M., Needham, L.L., Kuklenyik, Z., Reidy, J.A., Tully, J.S., Aguilar-Villalobos, M., Naeher, L.P., 2006. Perfluorinated chemicals in selected residents of the American continent. Chemosphere 63, 490–496. Champagne, D.L., Hoefnagels, C.C.M., de Kloet, R.E., Richardson, M.K., 2010. Translating rodent behavioral repertoire to zebrafish (Danio rerio): relevance for stress research. Behav. Brain Res. 214, 332–342. Chen, J., Das, S.R., La Du, J., Corvi, M.M., Bai, C., Chen, Y., Liu, X., Zhu, G., Tanguay, R.L., Dong, Q., Huang, C., 2013. Chronic PFOS exposures induce life stage-specific behavioral deficits in adult zebrafish and produce malformation and behavioral deficits in F1 offspring. Environ. Toxicol. Chem. 32, 201–206. De Esch, C., Van, D.L.H., Slieker, R., Willemsen, R., Wolterbeek, A., Woutersen, R., 2012. Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol. Neurotoxicol. Teratol. 34, 425–433. Domingo, J.L., Ericson-Jogsten, I., Perello, G., Nadal, M., Van Bavel, B., Karrman, A., 2012. Human exposure to perfluorinated compounds in Catalonia, Spain: contribution of drinking water and fish and shellfish. J. Agric. Food Chem. 60, 4408–4415. Drapeau, P., Ali, D.W., Buss, R.R., Saint-Amant, L., 1999. In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish. J. Neurosci. Methods 88, 1–13. EFSA, 2008. Opinion of the scientific panel on contaminants in the food chain on perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) and their salts. EFSA J. 653, 1–131.

339

Emran, F., Rihel, J., Dowling, J.E., 2008. A behavioral assay to measure responsiveness of zebrafish to changes in light intensities. J. Vis. Exp. 20, 1–6. Fei, C., McLaughlin, J.K., Lipworth, L., Olsen, J., 2008. Prenatal exposure to perfluorooctanoate (PFOA) and perfluorooctanesulfonate (PFOS) and maternally reported developmental milestones in infancy. Environ. Health Perspect. 116, 1391–1395. Fei, C.Y., McLaughlin, J.K., Tarone, R.E., Olsen, J., 2007. Perfluorinated chemicals and fetal growth: a study within the Danish National Birth Cohort. Environ. Health Perspect. 115, 1677–1682. Flanagan-Steet, H., Fox, M.A., Meyer, D., Sanes, J.R., 2005. Neuromuscular synapses can form in vivo by incorporation of initially aneural postsynaptic specializations. Development 132, 4471–4481. Giesy, J.P., Kannan, K., 2001. Global distribution of perfluorooctane sulfonate in wildlife. Environ. Sci. Technol. 35, 1339–1342. Hagenaars, A., Vergauwen, L., De Coen, W., Knapen, D., 2011. Structure–activity relationship assessment of four perfluorinated chemicals using a prolonged zebrafish early life stage test. Chemosphere 82, 764–772. Hoffman, A.F., Laaris, N., Kawamura, M., Masino, S.A., Lupica, C.R., 2010. Control of cannabinoid CB1 receptor function on glutamate axon terminals by endogenous adenosine acting at A1 receptors. J. Neurosci. 30, 545–555. Huang, H., Huang, C., Wang, L., Ye, X., Bai, C., Simonich, M.T., Tanguay, R.L., Dong, Q., 2010. Toxicity, uptake kinetics and behavior assessment in zebrafish embryos following exposure to perfluorooctanesulphonicacid (PFOS). Aquat. Toxicol. 98, 139–147. Hurd, M.W., Cahill, G.M., 2002. Entraining signals initiate behavioral circadian rhythmicity in larval zebrafish. J. Biol. Rhythms 17, 307–314. Hurd, M.W., Debruyne, J., Straume, M., Cahill, G.M., 1998. Circadian rhythms of locomotor activity in zebrafish. Physiol. Behav. 65, 465–472. Irons, T.D., MacPhail, R.C., Hunter, D.L., Padilla, S., 2010. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish. Neurotoxicol. Teratol. 32, 84–90. ISO, 1996. Water Quality – Determination of the Acute Lethal Toxicity of Substances to a Freshwater Fish [Brachydanio rerio Hamilton–Buchanan (Teleostei, Cyprinidae)] Part 1. Static Method ISO. Johansson, N., Fredriksson, A., Eriksson, P., 2008. Neonatal exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) causes neurobehavioural defects in adult mice. Neurotoxicology 29, 160–169. Kane, A.S., Salierno, J.D., Brewer, S.K., 2005. Fish models in behavioral toxicology: automated techniques, updates and perspectives. In: Ostrander, G.K. (Ed.), Methods in Aquatic Toxicology, vol. 2. Lewis Publishers, Boca Raton, FL, pp. 559–590 (chapter 32). Kannan, K., Tao, L., Sinclair, E., Pastva, S.D., Jude, D.J., Giesy, J.P., 2005. Perfluorinated compounds in aquatic organisms at various trophic levels in a Great Lakes food chain. Arch. Environ. Contam. Toxicol. 48, 559–566. Kissa, E., 2001. Fluorinated Surfactants and Repellants, 2nd ed. Marcel Dekker, New York. Kokel, D., Bryan, J., Laggner, C., White, R., Cheung, C.Y., Mateus, R., Healey, D., Kim, S., Werdich, A.A., Haggarty, S.J., Macrae, C.A., Shoichet, B., Peterson, R.T., 2010. Rapid behavior-based identification of neuroactive small molecules in the zebrafish. Nat. Chem. Biol. 6, 231–237. Kudo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R., Kawashima, Y., 2001. Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats. Chem. Biol. Interact. 134, 203–216. MacPhail, R.C., Brooks, J., Hunter, D.L., Padnos, B., Irons, T.D., Padilla, S., 2009. Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol. Neurotoxicology 30, 52–58. Maisonet, M., Terrell, M.L., McGeehin, M.A., Christensen, K.Y., Holmes, A., Calafat, A.M., Marcus, M., 2012. Maternal concentrations of polyfluoroalkyl compounds during pregnancy and fetal and postnatal growth in British girls. Environ. Health Perspect. 120, 1432–1437. Miege, C., Peretti, A., Labadie, P., Budzinski, H., Le Bizec, B., Vorkamp, K., Tronczynski, J., Persat, H., Coquery, M., Babut, M., 2012. Occurrence of priority and emerging organic compounds in fishes from the Rhone River (France). Anal. Bioanal. Chem. 404, 2721–2735. Murphy, C.A., Rose, K.A., Alvarez, M.D., Fuiman, L.A., 2008. Modeling larval fish behavior: scaling the sublethal effects of methylmercury to population-relevant endpoints. Aquat. Toxicol. 86, 470–484. Naile, J.E., Khim, J.S., Hong, S., Park, J., Kwon, B.O., Ryu, J.S., Hwang, J.H., Jones, P.D., Giesy, J.P., 2013. Distributions and bioconcentration characteristics of perfluorinated compounds in environmental samples collected from the west coast of Korea. Chemosphere 90, 387–394. O’brien, R.M., 2007. A caution regarding rules of thumb for variance inflation factors. Qual. Quant. 41, 673–690. Ohmori, K., Kudo, N., Katayama, K., Kawashima, Y., 2003. Comparison of the toxicokinetics between perfluorocarboxylic acids with different carbon chain length. Toxicology 184, 135–140. Olofsson, U., Brorstrom-Lunden, E., Kylin, H., Haglund, P., 2013. Comprehensive mass flow analysis of Swedish sludge contaminants. Chemosphere 90, 28–35. Olsen, G.W., Butenhoff, J.L., Zobel, L.R., 2009a. Perfluoroalkyl chemicals and human fetal development: An epidemiologic review with clinical and toxicological perspectives. Reprod. Toxicol. 27, 212–230. Olsen, G.W., Chang, S.C., Noker, P.E., Gorman, G.S., Ehresman, D.J., Lieder, P.H., Butenhoff, J.L., 2009b. A comparison of the pharmacokinetics of perfluorobutanesulfonate (PFBS) in rats, monkeys, and humans. Toxicology 256, 65–74.

340

M. Ulhaq et al. / Aquatic Toxicology 144–145 (2013) 332–340

Onishchenko, N., Fischer, C., Ibrahim, W.N.W., Negri, S., Spulber, S., Cottica, D., Ceccatelli, S., 2011. Prenatal exposure to PFOS or PFOA alters motor function in mice in a sex-related manner. Neurotox. Res. 19, 452–461. Pan, Y.Y., Shi, Y.L., Wang, J.M., Cai, Y.Q., 2011. Evaluation of perfluorinated compounds in seven wastewater treatment plants in Beijing urban areas. Sci. China Chem. 54, 552–558. Prober, D.A., Rihel, J., Onah, A.A., Sung, R.J., Schier, A.F., 2006. Hypocretin/orexin overexpression induces an insomnia-like phenotype in zebrafish. J. Neurosci. 26, 13400–13410. Renner, R., 2001. Growing concern over perfluorinated chemicals. Environ. Sci. Technol. 35, 154A–160A. Rihel, J., Prober, D.A., Arvanites, A., Lam, K., Zimmerman, S., Jang, S., Haggarty, S.J., Kokel, D., Rubin, L.L., Peterson, R.T., Schier, A.F., 2010. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science 327, 348–351. Rudel, H., Muller, J., Jurling, H., Bartel-Steinbach, M., Koschorreck, J., 2011. Survey of patterns, levels, and trends of perfluorinated compounds in aquatic organisms and bird eggs from representative German ecosystems. Environ. Sci. Pollut. Res. 18, 1457–1470. Shi, X., Du, Y., Lam, P.K., Wu, R.S., Zhou, B., 2008. Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS. Toxicol. Appl. Pharmacol. 230, 23–32. Tao, L., Kannan, K., Wong, C.M., Arcaro, K.F., Butenhoff, J.L., 2008a. Perfluorinated compounds in human milk from Massachusetts, USA. Environ. Sci. Technol. 42, 3096–3101. Tao, L., Ma, J., Kunisue, T., Libelo, E.L., Tanabe, S., Kannan, K., 2008b. Perfluorinated compounds in human breast milk from several Asian countries, and in infant formula and dairy milk from the United States. Environ. Sci. Technol. 42, 8597–8602. ter Braak, C.J.F., 1995. Ordination. In: Jongman, R.H.G., ter Braak, C.J.F., van Tongeren, O.F.R. (Eds.), Data Analysis in Community and Landscape Ecology. , 2nd ed. Cambridge University Press, Cambridge. ter Braak, C.J.F., Prentice, I.C., 1989. A theory of gradient analysis. Adv. Ecol. Res. 18, 272–317.

Ulhaq, M., Carlsson, G., Örn, S., Norrgren, L., 2013. Comparison of developmental toxicity of seven perfluoroalkyl acids to zebrafish embryos. Environ. Toxicol. Pharmacol. 36, 423–426. Wang, M.Y., Chen, J.F., Lin, K.F., Chen, Y.H., Hu, W., Tanguay, R.L., Huang, C.J., Dong, Q.X., 2011. Chronic zebrafish Pfos exposure alters sex ratio and maternal related effects in F1 offspring. Environ. Toxicol. Chem. 30, 2073–2080. Washino, N., Saijo, Y., Sasaki, S., Kato, S., Ban, S., Konishi, K., Ito, R., Nakata, A., Iwasaki, Y., Saito, K., Nakazawa, H., Kishi, R., 2009. Correlations between prenatal exposure to perfluorinated chemicals and reduced fetal growth. Environ. Health Perspect. 117, 660–667. Winter, M.J., Redfern, W.S., Hayfield, A.J., Owen, S.F., Valentin, J.P., Hutchinson, T.H., 2008. Validation of a larval zebrafish locomotor assay for assessing the seizure liability of early-stage development drugs. J. Pharmacol. Toxicol. Methods 57, 176–187. Yamashita, N., Taniyasu, S., Petrick, G., Wei, S., Gamo, T., Lam, P.K.S., Kannan, K., 2008. Perfluorinated acids as novel chemical tracers of global circulation of ocean waters. Chemosphere 70, 1247–1255. Yeung, L.W.Y., Yamashita, N., Taniyasu, S., Lam, P.K.S., Sinha, R.K., Borole, D.V., Kannan, K., 2009. A survey of perfluorinated compounds in surface water and biota including dolphins from the Ganges River and in other waterbodies in India. Chemosphere 76, 55–62. Zhang, L., Li, Y., Chen, T., Xia, W., Zhou, Y., Wan, Y., Lv, Z., Li, G., Xu, S., 2011. Abnormal development of motor neurons in perfluorooctane sulphonate exposed zebrafish embryos. Ecotoxicology 20, 643–652. Zhang, W., Zhang, Y., Taniyasu, S., Yeung, L.W., Lam, P.K., Wang, J., Li, X., Yamashita, N., Dai, J., 2013. Distribution and fate of perfluoroalkyl substances in municipal wastewater treatment plants in economically developed areas of China. Environ. Pollut. 176, 10–17. Zheng, X.-M., Liu, H.-L., Shi, W., Wei, S., Giesy, J., Yu, H.-X., 2012. Effects of perfluorinated compounds on development of zebrafish embryos. Environ. Sci. Pollut. Res. 19, 2498–2505.