Influences of combined traffic noise on anxiety in mice

STOTEN-21446; No of Pages 7 Science of the Total Environment xxx (2016) xxx–xxx

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Influences of combined traffic noise on anxiety in mice Guoqing Di ⁎, Yaqian Xu Institute of Environmental Pollution & Control Technology, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Combined traffic noise (CTN) impacts on anxiety were studied by an animal experiment. • Results in this study were compared to prior studies on single traffic noise effects. • No obvious impacts of 70 dB(A) CTN on anxiety in mice was found in this study. • CTN had less impact on anxiety than single high-speed railway and aircraft noises. • Noise events parameters and auditory sensitivity may affect anxiety levels.

a r t i c l e

i n f o

Article history: Received 16 August 2016 Received in revised form 3 November 2016 Accepted 20 November 2016 Available online xxxx Editor: D. Barcelo Keywords: Combined traffic noise Anxiety High-speed railway noise Aircraft noise Acoustical parameters Auditory sensitivity

a b s t r a c t With the rapid development of traffic facilities in China, traffic noise pollution is increasingly prominent. This research aims to explore the influences of combined traffic noise on receptors' anxiety. Institute of cancer research mice were exposed to combined traffic noise (CTN) from highway and high-speed railway for 52 days, whose day-night equivalent continuous A-weighted sound pressure level (Ldn) was 70 dB(A). The impacts of CTN on anxiety were explored by behavior tests and monoamine neurotransmitter assays. The results were in depth discussed in comparison to two previous studies on the impacts of single high-speed railway noise (HSRN) and aircraft noise (AN), but data from the three studies were not merged and statistically compared. No significant differences were shown in the behavioral indicators and the monoamine levels between the experimental and control groups after CTN exposure, indicating no obvious impacts of 70 dB(A) CTN on anxiety in mice were found in this study. When Ldn was approximately 70 dB(A), CTN had less obvious impacts on anxiety than HSRN and AN, which is mainly related to that both the acoustical parameters of noise events [maximum noise level (LAmax), noise events duration, slope of rise, difference of LAmax from 1-min background equivalent continuous A-weighted sound pressure level] and modified day-night equivalent continuous R-weighted sound pressure level (considering animal auditory sensitivity to different sound frequencies and circadian rhythms) of CTN are smaller than those of HSRN and AN. © 2016 Elsevier B.V. All rights reserved.

Abbreviations: AN, aircraft noise; CTN, combined traffic noise; DA, dopamine; HSRN, high-speed railway noise; ICR, Institute of Cancer Research; LAeq, equivalent continuous Aweighted sound pressure level; LAmax, maximum noise level; Ldn, day-night equivalent continuous A-weighted sound pressure level; LDBT, light-dark box test; NE, norepinephrine; LR, ' dn, day-night equivalent continuous R-weighted sound pressure level; L R,dn, modified day-night equivalent continuous R-weighted sound pressure level; OFT, open-field test; SD, Sprague-Dawley; 5-HT, serotonin. ⁎ Corresponding author. E-mail addresses: [email protected] (G. Di), [email protected] (Y. Xu).

http://dx.doi.org/10.1016/j.scitotenv.2016.11.144 0048-9697/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144

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G. Di, Y. Xu / Science of the Total Environment xxx (2016) xxx–xxx

1. Introduction Recently, with the rapid development of highway and high-speed railway in China, the density of traffic networks has increased continuously. To save land resources, transport lines, such as highways and high-speed railways, are always constructed in parallel, which results in the increasingly prominent pollution of combined traffic noise (CTN). There are significant differences in both time and frequency domain properties between single and combined traffic noises from highway and high-speed railway. Highways with large traffic flow produce continuous noise; while high-speed railways produce intermittent noise with a shorter duration, a higher peak sound level and more low-frequency components (Di and Zheng, 2013). Noises with different acoustic characteristics could induce different influences on the receptors (Di et al., 2014; Elmenhorst et al., 2012). In order to confirm whether noise emission standards for combined and single traffic noises need to be established separately, it is necessary to conduct a comparative study on the effects due to combined and single traffic noises. Most studies regarding the health effects of traffic noises focus on annoyance (e.g., Gidlof-Gunnarsson et al., 2012; Bartels et al., 2015; Xie et al., 2016), sleep disturbance (e.g., Marks et al., 2008; Lercher et al., 2010) and cardio-cerebrovascular disease (e.g., Aydin and Kaltenbach, 2007; Belojevic et al., 2008). Only a few studies have investigated the impacts of traffic noises on receptors' anxiety. It has been found that positive and negative results coexist in those studies. Hardoy et al. (2005) discovered that the airport noise exposure of high levels was associated with an increased risk of syndromal anxiety states. In addition, Bocquier et al. (2014) reported that the exposure to nighttime road traffic noise might increase the individual use of anxiolytics-hypnotics. However, a study conducted by Halonen et al. (2014) received a negative result, namely, road traffic noise exposure was irrelevant to the usage of anxiolytics. All the research mentioned above adopted the method of field survey to explore the impacts of traffic noises on anxiety. Nevertheless, field surveys may be influenced by bias and confounding, and their observational nature makes it difficult to draw firm causal relationships. That may be an important reason of the inconsistent findings from the above studies. On the contrary, the research carried out through animal models in laboratories with controlled conditions may efficiently avoid the impacts of bias and confounding. As an environmental stimulus, noise can cause stress responses on the receptors which are composed of alterations in behavior, autonomic function and the secretion of multiple neurotransmitters (Carrasco and de Kar, 2003). Stress responses are considered to be one of the major causes of mental disorders such as anxiety (McEwen, 2000). Due to the fact that the behaviors and related neurotransmitter concentrations of the animals are subject to changes in the situation of anxiety, the anxiety level can be assessed by particular behavior models (Ramos and Mormede, 1997; Gould et al., 2009) and measurements of related neurotransmitter levels (Lechin et al., 1998; Habr et al., 2014). Behavioral models to evaluate stress and anxiety are well established in murine, among which the open-field and the light-dark box tests are two kinds of classic models (Archer, 1981; Belzung and Griebel, 2001; Crawley, 2007). Specifically, the open-field test (OFT) is potentially sensitive to both activity and exploration, and to emotionality, which tends to suppress the expression of the former (Deacon et al., 2002). The commonly used indices to assess emotionality, like anxiety, are center time, locomotion and defecation (Ramos and Mormede, 1997; Prut and Belzung, 2003; Gould et al., 2009). The light-dark box test (LDBT) rests on the conflict between the aversion to light of murine and the tendency to explore a novel environment, hence, the measures of exploration in the lit compartment (time and entrances) are often regarded as experimental indices of anxiety (Crawley, 1989; Bourin and Hascoet, 2003). Many researchers characterize the degree of anxiety by monoamine neurotransmitters levels, among which norepinephrine (NE),

dopamine (DA) and serotonin (5-HT) concentrations are common indicators (Pitchot et al., 1992; Inoue et al., 1994; Lechin et al., 1998; Silva and Brandao, 2000). Noise stress has been documented to activate the autonomic nervous system and neuroendocrine system, and induce variations of plasma NE, DA, and 5-HT concentrations (Babisch, 2003; Di et al., 2011b; Di and He, 2013). To investigate anxiety of receptors caused by combined traffic noise from highway and high-speed railway, both behavioral responses (the OFT and the LDBT) and levels of plasma monoamine neurotransmitters (NE, DA, 5-HT) were evaluated in this study. Meanwhile, in 2011 and 2013, two similar animal experiments were conducted (Di et al., 2011a; Di and He, 2013). In these two previous experiments, SpragueDawley (SD) rats and Institute of Cancer Research (ICR) mice were respectively exposed to single aircraft noise (AN) and single high-speed railway noise (HSRN), whose intensities measured by day-night equivalent continuous A-weighted sound pressure level (Ldn) were both (70 ± 1.5) dB(A). According to the present experiment and the two previous experiments, the differences of impacts on anxiety between combined and single traffic noises were discussed, and the reasons for the differences were analyzed in terms of acoustical parameters of traffic noise events and subject auditory sensitivity. 2. Materials and methods 2.1. Animals Healthy male ICR mice (n = 60, 4 weeks of age, weighing 15– 20 g) obtained from Experimental Animal Center of Zhejiang Province (Hangzhou, China) were used for the experiments. The mice were randomly assigned into two groups: the control group (n = 30) and the experimental group (n = 30). They were housed five per cage and kept under controlled ambient temperature (22 ± 2 °C), humidity (50%– 60%) and a 12 h/12 h light/dark cycle (light on from 08:00 to 20:00). They had free access to water and food. All procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals established by the National Institutes of Health (1996). Every possible effort was made to minimize animal suffering and to reduce the number of animals used. 2.2. Combined traffic noise sampling and exposure A four-channel dynamic signal analyzer (Photon II, Royston, England) was used to record highway and high-speed railway noises at different time of the day, respectively. The two single traffic noises collected were reasonably arranged and superimposed to get CTN according to the 24 h traffic flux of highway (2092 passenger car units per hour) and high-speed railway (24 vehicles per hour in the daytime and 12 vehicles per hour at night). The CTN was played through a dodecahedron non-directional sound source (Nor270, Norsonic, Lierskogen, Norway) in a sound insulation lab. The Ldn of the experimental group was (70 ± 1.5) dB(A) (the intensity of noise exposure varied slightly in different cages, and 70 dB was the average exposure intensity in all cages, while ± 1.5 dB was the range of exposure intensity difference). The equivalent continuous Aweighted sound pressure level (LAeq) of the background noise was no N35 dB(A), which was the intensity presented to the control group. After 7-day adaptation in the laboratory, the experimental group was exposed to the CTN 24 h per day for 52 days, while the control group was not exposed. 2.3. Behavioral tests and monoamine neurotransmitter assays Behavioral tests, as well as monoamine neurotransmitter assays, were carried out at three different points in time during the period of noise exposure. Ten mice from each group were tested at each point in time. Detailed experiment schedule is shown in Table 1. The OFT

Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144

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was carried out on days 4, 24, and 44, and the LDBT on days 5, 25, and 45. For the purpose of reducing the possible impacts of behavioral tests on monoamine levers, blood sampling from the same mice examined in the behavioral tests was carried out 7 days later of the LDBT (on days 12, 32, and 52). In addition, all experiments were conducted at the same time of the day (between 17:00 and 19:00) in order to avoid circadian rhythm induced variations. The OFT was performed in a well illuminated wooden square arena (72 cm × 72 cm) with 30 cm high walls. The floor and walls were painted black and the floor was divided into 64 grids (9 cm × 9 cm) by white lines. The thirty-six girds located in the center of arena were regarded as “center area”. The test was conducted in a sound insulation lab. The individual mouse was placed in the center of arena and its behaviors were recorded by a camera for 5 min. Through playing back videos, the center time, number of line crossing, rearing and defecation, was calculated by three individuals independently. The mean values of the results from the three individuals were considered as the final results. The light-dark box was one third for the dark and two thirds for the lit compartment with an exterior size of 18 cm × 10.5 cm × 10.5 cm (l × w × h). The lit and dark compartments were separated by a partition. An opening in the center of the partition at floor level allows the animal to shuttle freely from one part to another. The lit compartment was painted white and illuminated by a 20-W light source at the height of 30 cm above the floor, while the dark compartment was painted black and covered by a light-proof lid. The animal was placed in the center of the lit compartment with its back to the opening at the beginning of the test, and its behaviors were also recorded by a camera for 5 min. The time spent in the lit compartment and the number of transitions between the two compartments were analyzed. 1.0 mL blood was collected in a 1.5 mL centrifuge tube by eyeball removal. 40 μL disodium ethylene diamine tetraacetic acid solution of 50 g/L was added to the sample and oscillated. After 15 min of quiescence, the mixture was centrifuged at 3000 r/min for 20 min at 4 °C. Then, 150 μL supernatants were extracted, to which 150 μL perchloric acid of 5% was added. The mixture was thoroughly shaken and stood for 20 min to fully precipitate the plasma proteins, followed by centrifugation at 10000 r/min for 15 min at 4 °C. 150 μL supernatants were extracted for the determination of plasma NE, DA, 5-HT levels by high performance liquid chromatography-fluorimetric detection (Di et al., 2011b; Di and He, 2013).

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2.5. Exposure intensity calculations considering subject auditory sensitivity and circadian rhythms Other than acoustic properties of noise events, differences in the hearing sensitivity and circadian rhythms of different receptors can also bring variances in anxiety degree under the same noise exposure intensity (Ldn). According to rat auditory sensitivity, Bjork et al. (2000) introduced a sound pressure level weighting called R-weighting. Based on R-weighting, the spectrums of CTN, HSRN and AN, as well as the audiograms of mice (Ehret, 1974) and rats (Kelly and Masterton, 1977), the actual noise exposure intensities in the three animal experiments on CTN, HSRN (Di and He, 2013) and AN (Di et al., 2011a) were calculated (Table 3) as Eqs. (1)–(4). The actual noise exposure intensity was termed as the day-night equivalent continuous R-weighted sound pressure level (LR, dn) in this study. F ij ¼ Lij −T i LR j ¼ 10 log

ð1Þ n  X

100:1 F ij



ð2Þ

i¼1

0 1 ¼ 10 log@ m

m X

1 0:1LR j A

10

ð3Þ

  16 0:1LReq;d 8 þ 10 100:1ðLReq;n þ10Þ LR;dn ¼ 10 log 24 24

ð4Þ

LReq;T

j¼1

where Fij is the R-weighted sound pressure level of i-th 1/3 octave band within the j-th time interval of Δt; Lij is the sound pressure level of i-th 1/3 octave band within the j-th time interval of Δt; Ti is the hearing threshold of mice or rats at the center frequency of i-th 1/3 octave band; LRj is the R-weighted sound pressure level within the j-th time interval of Δt; n is the number of 1/3 octave bands; LReq,T is the equivalent continuous R-weighted sound pressure level within the time interval of T; m is the number of Δt within the time interval of T, m = T / Δt; LR,dn is the day-night equivalent continuous R-weighted sound pressure level; LReq,d is the equivalent continuous R-weighted sound pressure level of 16 h in the daytime from 06:00 to 22:00; LReq,n is the equivalent continuous R-weighted sound pressure level of 8 h at night from 22:00 to 06:00 the next day.

2.4. Traffic noise events acoustic properties analysis In order to explore the reasons for the differences of impacts on anxiety between combined and single traffic noises, the acoustic properties of noise events of CTN in this study, and HSRN (Di and He, 2013) or AN (Di et al., 2011a) in previous studies were analyzed (Table 2). According to Elmenhorst et al. (2012), a noise event was defined as an increase in sound pressure level that exceeded statistic sound level L90 by 3 dB(A); slope of rise for a noise event was represented by the steepest slope of the noise event curve (Elmenhorst et al., 2012); the 1-min background LAeq was defined as LAeq during 1-min time span directly preceding the start of a noise event (Elmenhorst et al., 2012).

Table 1 Experiment schedule. Experiment

First test (n = 10)

Second test (n = 10)

Third test (n = 10)

OFT LDBT Monoamine neurotransmitter assays

Day 4 Day 5 Day 12

Day 24 Day 25 Day 32

Day 44 Day 45 Day 52

Table 2 Acoustic parameters of noise events of combined traffic noise (CTN) from highway and high-speed railway, single high-speed railway noise (HSRN) and single aircraft noise (AN). Parameters

The number of noise events

Exposure Range sound sources

CTN HSRN AN Noise events duration CTN (s) HSRN AN Slope of rise CTN [dB(A)/s] HSRN AN CTN LAmax [dB(A)] HSRN AN CTN 1-min background HSRN LAeq [dB(A)] AN CTN Difference of LAmax from 1-min HSRN background LAeq AN [dB(A)]

10,800/day 524/day 205/day 0.1– 56.6 7.8– 12.4 25.1– 77.9 9.6– 279.6 100.4– 648.7 84.4– 1335.4 61.2– 82.6 84.5– 90.9 81.3– 90.2 62.0– 69.2 38.5– 42.8 29.2 −7.6– 16.2 41.7– 52.4 52.1– 61.0

Median 25th 75th percentile percentile / / / 0.2 11.0 52.8 20.5 315.4 272.1 61.3 87.7 82.8 64.1 42.8 / −2.8 47.0 53.6

/ / / 0.1 7.8 43.4 19.0 100.4 132.3 61.2 84.5 86.6 63.6 38.5 / −2.9 41.7 57.4

/ / / 9.2 12.3 63.4 64.7 648.7 430.8 65.6 90.8 89.2 68.1 42.8 / 1.2 48.0 60.0

Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144

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Table 3 Exposure levels of combined traffic noise (CTN) from highway and high-speed railway, single high-speed railway noise (HSRN) and single aircraft noise (AN) considering animal hearing sensitivity and circadian rhythms. Exposure sound sources

LR,dn [dB(R)]

L′R,dn [dB(R)]

CTN HSRN AN

42.7 45.6 45.4

45.8 53.4 50.4

differences in plasma NE (Fig. 1a), DA (Fig. 1b), and 5-HT (Fig. 1c) levels between the two groups over the period of CTN exposure (P N 0.05). Post-hoc power analyses displayed that the statistical power of the tests of the NE level and 5-HT level on day 52 was respectively 0.88 and 0.80.

4. Discussion The calculation of LR,dn above is based on the circadian rhythm of humans. In other words, LR,dn is computed by adding 10 dB to LReq,n considering that noise has a greater influence at night (22:00– 06:00 the next day) on humans when they have a rest than in the daytime. However, the circadian rhythm of mice and rats is different from that of human. In general, mice and rats take a rest at 08:00– 20:00 and stay active at 20:00– 08:00 the next day (Stinus et al., 1998; Li et al., 2015). So LR,dn needs to be modified to meet the circadian rhythm of mice and rats. The modified LR,dn is represented as L′R,dn, which is calculated by adding 10 dB to the equivalent continuous R-weighted sound pressure level of 08:00– 20:00 (L′Req,d) (Table 3) as Eq. (5).    0 12 0:1L0Req;n 12 0:1 L0Req;d þ10 þ 10 10 LR;dn ¼ 10 log 24 24

ð5Þ

where L′R,dn is the modified day-night equivalent continuous R-weighted sound pressure level; L′Req,n is the equivalent continuous R-weighted sound pressure level of 12 h from 20:00 to 08:00 the next day; L′Req,d is the equivalent continuous R-weighted sound pressure level of 12 h from 08:00 to 20:00. 2.6. Statistical analysis All data from the behavioral tests and monoamine assays are expressed as mean ± SEM. Differences in the experimental results between the experimental and control groups were evaluated by unpaired Student's t-tests using SPSS 20.0. Significance was accepted at the level of P b 0.05. Furthermore, power analyses were conducted using the G*Power 3.1. 3. Results 3.1. The behavioral tests After CTN of 70 dB(A) exposure for different time, the results of the OFT and LDBT are shown in Tables 4 and 5. There were no significant differences in the center time, the number of line crossing, rearing, and defecation from the OFT between the experimental and control groups under different noise exposure durations, as well as the time spent in the lit compartment and the number of transitions from the LDBT (P N 0.05). Post-hoc power analyses showed that the statistical power of the test of line crossing number on day 24 was 0.83. 3.2. Concentrations of plasma monoamines Fig. 1 shows the average levels of plasma monoamines (NE, DA, 5HT) in the experimental and control groups. There were no significant

Generally, in the OFT, the animal anxiety level is positively correlated with the center area duration and the number of defecation, whereas it is negatively correlated with the center time, the number of line crossing, grooming and rearing (Lister, 1990; Prut and Belzung, 2003); in the LDBT, the animal anxiety level is negatively correlated with the time spent in the lit compartment and the number of transitions (Bourin and Hascoet, 2003; Ohl, 2005); in the experiment of plasma monoamines, the animal anxiety level is positively correlated with NE, DA, and 5-HT concentrations (Pitchot et al., 1992; Inoue et al., 1994; Silva and Brandao, 2000). In the present study, over the duration of CTN exposure, all the behavioral indicators from the OFT and LDBT, as well as the levels of the three kinds of plasma monoamines showed no significant differences between the experimental and control groups. When considering the statistical power, that reached the specification of 0.80 (Cohen, 1992) in the tests of three indices (the number of line crossing on day 24, the NE level and 5-HT level on day 52). Therefore, for the three indices, it was certain in statistics that there were no significant differences between the experimental and control groups. Taking the results above into account, no obvious evidences demonstrated that 70 dB(A) CTN could cause significant impacts on anxiety in mice in the present study. However, it should be pointed out that lack of statistical power in the tests of other indicators (center time, time spent in the lit compartment, DA level, etc.) except the three indices above probably led to that non-significant differences between the experimental and control groups in these indicators were false negative results, which should be further confirmed through additional animal experiments with a larger sample size. To obtain results with statistical certainty, planning the sample size based on a priori power analysis is vital in future studies. The previous study on impacts of HSRN with a exposure duration of 53 days found that behavioral indicators (center time, the number of line crossing, grooming and rearing, the time spent in the lit compartment and the number of transitions) in the experimental group were significantly less than those in the control group, while the plasma DA level was obverse (Di and He, 2013). And the research on impacts of AN with a exposure duration of 36 days discovered that the number of line crossing was reduced significantly in the experimental group, whereas the center area duration and the plasma NE level were significantly increased (Di et al., 2011a). The results in those two previous studies indicated that (70 ± 1.5) dB(A) HSRN exposure for 53 days and AN exposure for 36 days would cause anxiety in mice and rats, respectively. Thus, when Ldn was 70 dB(A) approximately, CTN was less influential to anxiety compared with HSRN (Di and He, 2013) and AN (Di et al., 2011a). The discrepancy above could be caused by the differences in noise acoustic characteristics and experimental subjects, which will be concretely discussed below.

Table 4 The results of OFT in experimental group (EG, n = 10) and control group (CG, n = 10). Noise exposure duration

4 days 24 days 44 days

Center time (s)

Number of line crossing

Number of rearing

Number of defecation

CG

EG

CG

EG

CG

EG

CG

EG

112.9 ± 5.9 89.9 ± 10.1 78.0 ± 3.9

122.5 ± 4.2 89.3 ± 6.2 78.4 ± 6.8

392.0 ± 16.6 313.0 ± 11.8 361.2 ± 11.0

403.1 ± 24.9 355.2 ± 17.6 356.6 ± 17.9

5.8 ± 2.2 9.2 ± 2.8 2.5 ± 0.9

2.6 ± 0.7 5.5 ± 0.9 4.2 ± 1.7

3.2 ± 0.6 3.1 ± 0.6 4.6 ± 0.9

2.8 ± 0.6 1.8 ± 0.4 3.3 ± 0.7

Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144

G. Di, Y. Xu / Science of the Total Environment xxx (2016) xxx–xxx Table 5 The results of the LDBT in experimental group (EG, n = 10) and control group (CG, n = 10). Noise exposure duration

Time spent in the lit compartment (s)

Number of transitions

CG

EG

CG

EG

5 days 25 days 45 days

135.0 ± 10.0 120.5 ± 10.5 133.5 ± 8.0

141.9 ± 11.0 139.6 ± 8.6 118.4 ± 9.6

35.0 ± 1.0 29.4 ± 1.5 30.9 ± 2.2

39.9 ± 4.2 31.5 ± 3.4 25.6 ± 1.6

4.1. The impact of noise events acoustic properties on anxiety levels of the receptors Concerning the relationship between acoustic characteristics of traffic noise events and noise effects, studies mainly focus on annoyance and sleep disturbance (Lambert et al., 1996; De Coensel et al., 2007; Marks et al., 2008; Lercher et al., 2010; Elmenhorst et al., 2012). Scarce number of studies about the impact of noise events acoustic properties on receptors' anxiety has been found. Fyhri and Aasvang (2010) discovered links between noise annoyance, sleep disturbances and anxiety. Beutel et al. (2016) recently demonstrated in a cohort study that depression and anxiety increased with the degree of noise annoyance. In consequence, acoustic characteristics of noise events which are related to the degree of annoyance and sleep disturbance, would also affect anxiety levels. As the analysis results of noise events acoustic properties shown in Table 2, except the number of noise events and 1-min background LAeq, all the acoustic parameters of noise events analyzed [noise events duration, slope of rise, maximum noise level (LAmax), difference of

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LAmax from 1-min background LAeq] of CTN are much smaller than those of HSRN and AN. Studies have shown that traffic noise annoyance significantly increases with the slope of rise (Lambert et al., 1996; De Coensel et al., 2007; Elmenhorst et al., 2012). Sleep disturbance due to the traffic noise significantly increases with the noise events duration, the slope of rise, LAmax and the difference of LAmax from 1-min background LAeq (Marks et al., 2008; Lercher et al., 2010; Elmenhorst et al., 2012), among which LAmax plays a decisive role (Basner et al., 2006; Marks et al., 2008; Brink et al., 2010). Thus, the fact that LAmax, the noise events duration, the slope of rise and the difference of LAmax from 1-min background LAeq of CTN are smaller than those of HSRN and AN, is one of the important reasons for CTN being less influential on anxiety than HSRN (Di and He, 2013) and AN (Di et al., 2011a) when Ldn = (70 ± 1.5) dB(A). With regard to the correlation between the number of noise events and annoyance, as well as between the number of noise events and sleep disturbance, Sato et al. (1999) and Janssen et al. (2014) indicated that the number of traffic noise events was irrelevant to annoyance and sleep quality, respectively. However, some other studies suggested that the number of traffic noise events was positively correlated with noise annoyance (Gidlof-Gunnarsson et al., 2012; Lambert et al., 2015) and sleep disturbance (Smith et al., 2013). In the present study, anxiety was less affected by CTN than by HSRN and AN, whose number of noise events is far less than that of CTN. That is, the number of traffic noise events is not related to anxiety. Therefore, this article supports the hypothesis of Sato et al. (1999) and Janssen et al. (2014). The above analysis implies that when evaluating noise effects and developing noise limits, researchers should not only concern the parameters representing macroscopic exposure intensity of noise (e.g., Ldn), but also the microstructure of noise (e.g., the acoustic characteristics of the single noise event).

Fig. 1. Concentrations of plasma monoamines in experimental group (EG, n = 10) and control group (CG, n = 10) after noise exposure for different time, (a) norepinephrine (NE), (b) dopamine (DA), (c) serotonin (5-HT).

Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144

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4.2. The impact of subject auditory sensitivity and circadian rhythms on anxiety levels

participated in and devoted their efforts to this program during animal feeding, data collection and analysis.

In the animal experiments on CTN and HSRN (Di and He, 2013) effects, ICR mice were used as subjects, while SD rats were used in the animal experiments on AN effect (Di et al., 2011a). Nevertheless, humans, rats and mice vary in hearing sensitivity to different sound frequencies according to their audiograms (Sivian and White, 1933; Kelly and Masterton, 1977; Ehret, 1974). At frequencies of 20 Hz to 8000 Hz, hearing sensitivity decreases in the order of humans, rats and mice. At the same time, frequency spectrums vary with different traffic noises. Thus, Ldn based on A-weighting according to auditory sensitivity of humans cannot reflect the noise exposure intensity the receptors actually received, which depends both on the hearing ability of the receptors at each frequency and on the spectrum of the noise. As the calculations results of actual exposure intensity shown in Table 3, when the Ldn of CTN, HSRN and AN was approximately equal, the LR,dn of CTN is 2.9 dB(R) smaller than that of HSRN and 2.7 dB(R) smaller than that of AN. On the basis of L′R,dn which was achieved according to the circadian rhythm of mice and rats, the gap in noise exposure intensity between CTN and the two single traffic noises is further increased. The L′R,dn of CTN is 7.6 dB(R) and 4.6 dB(R) smaller than that of HSRN and AN, respectively. Thus, influenced by noise frequency spectrums, animal auditory sensitivity and circadian rhythms, the noise exposure intensity experimental animals actually received (L′R,dn) of CTN is smaller than that of HSRN and AN, which may be another reason why anxiety from CTN was less than that from HSRN and AN when the Ldn of noise was approximately equal. It gives us an important enlightenment that there are great differences in effects of noise between different experimental animals even when they are exposed to the same noise. To make the results among each study comparable, the strain of experimental animals should keep consistent as far as possible. Likewise, in order to better analogize the noise effects of animal to those of human, it is recommended to use animals whose auditory sensitivity is similar to humans, such as the gerbil (Turner et al., 2005), when using the rodent as models to conduct noise effects experiments.

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5. Conclusions The study has found no significant impacts of CTN on behaviors and plasma NE, DA, 5-HT levels, namely, no obvious evidences indicated that 70 dB(A) CTN could cause impacts on anxiety in ICR mice. The results in this study were further discussed in contrast to previous studies on impacts of single traffic noise, but data were not merged and statistically compared. It was found that CTN had less obvious impacts on anxiety compared with HSRN and AN, which is mainly owing to two reasons. Firstly, acoustic parameters of noise events (LAmax, noise events duration, slope of rise, difference of LAmax from 1-min background LAeq) of CTN are much smaller than those of HSRN and AN. Secondly, L′R,dn (i.e. exposure intensity that animals actually received by taking noise frequency spectrums, animal auditory sensitivity and circadian rhythms into consideration) of CTN is smaller than that of HSRN and AN. Those suggest that when evaluating noise effects and establishing noise limits, not only should the parameters representing noise macroscopic exposure intensity (e.g., Ldn) be concerned, but also the noise microstructure (e.g., the acoustic characteristics of the single noise event) should be taken into account. In future similar animal studies, animals whose auditory sensitivity is similar to humans, such as gerbil, should be employed consistently for the comparison between different studies and applying the results into humans. Acknowledgments The authors show sincere thanks to the National Nature Science Foundation of China (Grant No. 11174251) for funding this research, and show special gratitude to the teachers and students who have

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Please cite this article as: Di, G., Xu, Y., Influences of combined traffic noise on anxiety in mice, Sci Total Environ (2016), http://dx.doi.org/10.1016/ j.scitotenv.2016.11.144