The Effect of Polychlorinated Biphenyls on the Song of Two Passerine Species

1. The Effect of Polychlorinated Biphenyls on the Song of Two Passerine Species By Sara DeLeon, Rayko Halitschke, Ralph S. Hames, Andre Kessler, Timothy J. DeVoogd, and Andre A. Dhondt PLoS ONE 8(9):e73471. doi:10.1371/journal.pone.0073471, 2013 Presented by ErumHadi, MPH, MsEd

2. Useful Terms and Abbreviation • PCB – Poly Chlorinated Biphenyls • Higher Vs Lower Chlorinated PCBs – sites where chlorine is attached to the benzene ring • Specific PCB congener – Highly chlorinated or low chlorinated • Trill – repeating sounds • Glissando – series of notes

3. Introduction • In 1991, Environmental Protection Agency (EPA) reported the General Electric (GE) plants at Ford Edward and Hudson Falls illegally discharged about 1.3 million pounds of PCBs in to Hudson River between 1947 and 1977. • There are 209 congeners of PCB each with different toxicity, environmental persistence, and biological effect.

4. Introduction • Unchlorinated PCB toxic effects are like dioxin, some PCBs are endocrine disruptors, and low chlorinated have estrogenic actions. • PCB congener profiling is difficult to do in smaller organism without destroying the organism. This study sampled small volumes of blood to quantify congener-specific PCB without killing the organism.

5. Introduction • Since PCB interfere with endocrine, which is related to neurological functioning, they could have behavioral effects. • This study aims to determine the magnitude and extent of PCB effects on behavior to control sub lethal PCB exposure. • Song is key behavior for bird species’ mate identification, attraction, and territory defense.

6. Introduction • Bird song is effected by DDT, heavy metals, natural and synthetic estrogen mimics, and BDE-99. • The effect of PCBs on birdsong is not known. PCBs may influence song quality by interrupting androgenic and estrogenic metabolism of testosterone, which regulates song control nuclei n the passerine brain. • In lab experiments, PCB has decreased the volume of robust nucleus of arcopallium, a motor song region in bird brains.

7. Introduction • First objective: quantify congener-specific PCB levels using small volumes of blood from individual birds. • Second objective: whether song characteristics co-vary with PCB levels along a contamination axis.

8. Literature Review • Polychlorinated biphenyls (PCBs) are chemicals made in US that have polluted the world. In 1979, PCBs were banned due to their negative effects on the environment, but 108 Kilograms still remain. • Waid JS (1986) PCBs and the environment. Florida: CRC Press. • Boyle A, Silvin C, Hassett J, Nakas J, Tanenbaum SW (1992) Bacterial PCBbiodegradation. Biodegradation 3: 285–298. • PCBs are complex, hydrophobic and lipophilic chemicals that decompose slowly and accumulate in living things. • Harris ML, Elliot JE (2011) Effects of polychlorinated biphenyls, dibenzo-pdioxins and dibenzofurans, and polybrominateddiphenyl ethers in wild birds.In: Beyer WN, Meador JP, editors. Environmental contaminants in biota: interpreting tissue concentrations. 2nd ed. Boca Raton, FL: CRC Press. pp. 477–528.

9. Literature Review • PCB congeners and their effects are determined by the number of chlorines on di-benzene ring. PCB congener data is collected on large animals, which give large enough samples due to biomagnification. • Waid JS (1986) PCBs and the environment. Florida: CRC Press. • Destructive sampling is used to get large samples for congener PCB data or carcasses are tested in case of endangered species. • Dell’Omo G (2002) Behaviouralecotoxicology. In: Weeks JM, O’Hare S,Rattner BA, editors. Ecological and environmental toxicology series. Chichester:Wiley. • Senthil Kumar K, Kannan K, Giesy JP, Masunaga S (2002) Distribution and elimination of polychlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls, and p,p9-DDE in tissues of bald eagles from the upper peninsula of Michigan.Environ SciTechnol 36: 2789–2796.

10. Literature Review • PCBs are similar to Dioxin and endocrine disruptors, while PCBs with fewer chlorines have estrogenic effects, and others have androgenic and anti-androgenic effects. • Van den Berg M, Birnbaum LS, Denison M, De Vito M, Farland W, et al. (2006) The 2005 world health organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. ToxicolSci 93: 223–241. • Crisp TM, Clegg ED, Cooper RL, Wood WP, Anderson DG, et al. (1998) Environmental endocrine disruption: an effects assessment and analysis. Environ Health Perspect 106: 11–56. • Plı´sˇkova´ M, Vondra´cˇek J, Fernandez Canton R, Nera J, Kocˇan A, et al. (2005) Impact of polychlorinated biphenyls contamination on estrogenic activity in human male serum. Environ Health Perspect 113: 1277–1284.

11. Literature Review • Behavior is controlled by hormones. PCB interferes with endocrine regulation and thus effects behavior. Song is essential behavior that helps birds with mate identification, attraction and territory defense. Song is affected by other chemicals, but PCB effect is not known. • Dell’Omo G (2002) Behaviouralecotoxicology. In: Weeks JM, O’Hare S,Rattner BA, editors. Ecological and environmental toxicology series. Chichester:Wiley. • Catchpole CK, Slater PJ (2003) Bird song: biological themes and variations. Cambridge University Press. • Iwaniuk AN, Koperski DT, Cheng KM, Elliott JE, Smith LK, et al. (2006) The effects of environmental exposure to DDT on the brain of a songbird: changes in structures associated with mating and song. Behav Brain Res 173: 1–10. • 18. Gorissen L, Snoeijs T, Duyse EV, Eens M (2005) Heavy metal pollution affects dawn singing behaviour in a small passerine bird. Oecologia 145: 504–509. • Markman S, Leitner S, Catchpole C, Barnsley S, Mu¨ ller CT, et al. (2008) Pollutants increase song complexity and the volume of the brain area HVC in a songbird. PLoS ONE 3: e1674. • 20. Eng ML, Elliott JE, MacDougall-Shackleton SA, Letcher RJ, Williams TD (2012) Early exposure to 2,29,4,49,5-pentabromodiphenyl ether (BDE-99) affects mating behavior of zebra finches. ToxicolSci 127: 269–276. • 21. Ball GF, Riters LV, Balthazart J (2002) Neuroendocrinology of song behavior and avian brain plasticity: multiple sites of action of sex steroid hormones. Front Neuroendocrinol 23: 137–178.

12. Literature Review • In lab experiments, PCB effects motor song region of bird brains and volume of this region is related to song structure. PCBs can be influencing song characterisitics. • Hoogesteijn AL, Kollias GV, Quimby FW, De Caprio AP, Winkler DW, et al. (2008) Development of a brain nucleus involved in song production in zebra finches (Taeniopygiaguttata) is disrupted by Aroclor 1248. Environ Toxicol Chem27: 2071–2075. • Moore JM, Sze´kely T, Bu¨ ki J, DeVoogd TJ (2011) Motor pathway convergence.

13. Literature Review • PCB loads in small amounts from blood, feathers or hair need more testing in the field. • Rivera-Rodriguez LB, Rodriguez-Estrella R, Ellington JJ, Evans JJ (2007) Quantification of low levels of organochlorine pesticides using small volumes (#100 mL) of plasma of wild birds through gas chromatography negative chemical ionization mass spectrometry. Environ Pollut 148: 654–662. • Van den Steen E, Covaci A, Jaspers VLB, Dauwe T, Voorspoels S, et al. (2007) Experimental evaluation of the usefulness of feathers as a non-destructive biomonitor for polychlorinated biphenyls (PCBs) using silastic implants as a novel method of exposure. Environ Int 33: 257–264.

14. Hypothesis • If birds are exposed to different levels of PCB exposure then their bird song will differ between birds exposed to higher levels of PCB pollution and birds with lower levels of PCB exposure.

15. Materials and Methods • Study sites: From 2006 to 2009, 48 recording sites & 7 mist-netting sites were established in five New York State region with documented variability in levels of PCBs in Hudson River basin. • Two regions with historic PCB inputs were labeled as +Hudson (downstream) and ++ Hudson (at point). • Three regions with no local point source PCB input were control and labeled –Ithaca & -Adirondacks (within 40 m of a river/lake), -Hudson. 2. EPA USA (1997) Phase 2 report, further site characterization and analysis: volume 2C, data evaluation and interpretation report: Hudson River PCB reassessment RI/FS. New York.

16. Figure 1. Map of recording and mist-netting sites. The five study regions are indicated by grey rectangles. 2 Ithaca is in Ithaca, NY. 2 Adirondacks is in the Adirondack Mountains. 2 Hudson is directly above the point source pollution at General Electric (GE, indicated by a star), +Hudson and ++ Hudson are downstream of GE. Recording sites are indicated with dark grey points, and mist-netting sites are indicated with white points.

17. Materials and Methods • Study species: Black Capped Chickadees and Song Sparrows. • Adults eat insects and feed young insects too. • Both forage near the river. • Both have well-studied songs and researchers have identified specific song characteristics important in communication.

18. Materials and Methods • Subjects: were captured using birdfeeders with sunflower seeds to attract them to mist nets. • Playbacks from previous seasons were played to attract territorial males. • All birds measured and tested within 20 minutes of capture and released. • Species identified and sexed according to Pyle [2], and banded with metal USFWS bands. • Wing chord, tarsus length, weight, muscle score, fat score and mites were recorded.

19. Materials and Methods • Blood samples for PCB were collected and stored in centrifuge tubes, and placed on ice. • PCB Blood Analysis: PCB levels were determined by gas chromatography mass spectrometry. • Mercury Blood Analysis: A large enough blood sample was obtained from only song sparrows to conduct mercury blood test. • Song Recording: All recordings made with Fostex FR-2 Field Memory Recorder or a Tascam HD-P2 Recorder. Males of both species were recorded. • Subset of n=29 song sparrows were tested blood samples, banded and recorded.

20. Materials and Methods • Song Analysis: • Glissando ratio for identity of species varies <2% • Interval ratio male quality (high quality males produce both ratios consistently and at all frequencies) • Song sparrows sing 4-12 types of songs. Trill rate (# of syllables/unit time) were recorded. Trill rate of <4Hz=high quality.

21. Materials and Methods • Statistical Analysis: • Total PCB data were log-transformed and analyzed with a two way ANOVA in which species identity and regions were fixed effects, followed by a Tukey post-hoc test. • Chi-square test was used to see whether proportions of stereotypical and variable individual black-capped chickadee singers differed between regions. • Chi-square test was used to see individual song sparrows singing high performance and low performance trills differed between regions.

22. Results • Black-capped chickadee total PCB concentration in ++ Hudson region was higher than + Hudson region, but not significantly so. • Control sites had lower levels. • +Hudson region had large proportion of higher chlorinated PCBs than all other regions. • Song Sparrows: significantly higher total PCB concentrations in ++ Hudson. • Song sparrows in the ++Hudson region have largest proportion of lower Chlorinated PCBs. • +Hudson song sparrows had higher proportions of lower chlorinated PCbs.

23. Figure 3. Black-capped chickadees had more variable glissando ratios in regions of PCB contamination. Average glissando ratio (A) and average interval ratio (B) of black-capped chickadees by region. Bars represent mean6SE and non-overlapping letters indicate statistical difference between regions. Proportion of stereotyped/variable glissando ratios (C) and interval ratios (D) of individual black-capped chickadees. Individuals singing stereotyped ratios (grey, CV,0.03) and variable ratios (black, CV.0.03, numbers in bars) are shown by region.

24. Figure 4. Song sparrow trill performance varied between study regions. A) Trill rate-frequency bandwidth trade-off of song sparrows. The regression line is calculated from all recorded trills, and each point represents a randomly chosen trill type of one individual song sparrow. The dotted lines represent the trill rate-bandwidth cut-off of 4 kHz distance from the trill performance regression line (solid). Low performance trills are between the origin and the lower dotted line. B) The proportion of song sparrows singing high performance (grey, distance from regression line,4 kHz) or low performance (black, distance from regression line.4 kHz) trills by region. The numbers in bars are the actual percentages of individual sparrows singing low performance trills.

25. Discussion • Both black-capped chickadees and song sparrows have elevated total PCB concentrations in ++Hudson, which is the region with the highest historical PCB contamination. • Song sparrows niche is near the river. • Black capped chickadee live in higher canopy in the woodlands by the river. • PCB profile of song sparrow was similar to the PCB profile of Hudson river. • Both species have higher chlorinated PCBs away from source due to bioaccumulation.

26. Discussion • Black capped chickadee song glissando ratio varied highest in areas of highest PCB concentration. • This indicates a biologically significant deviation of species identity signal (glissando ratio) in both + and ++ Hudson river. • Black capped chickadee in the +Hudson region had the strongest song differences not ++Hudson. (Due to U-shaped)

27. Discussion • Song sparrow trill performance did vary by region indicating that song sparrow trill performance may be affected by PCB pollution. • Song sparrows in ++ Hudson region have highest PCB levels and high trill performers, scientist conclude that this is due to PCBs acting directly as hormones to increase trill performance.

28. Future Research • Studies that record song and measure PCB congener composition in the same individual rather than averages at a site is needed. • Additional field studies are needed to understand how suites of different PCB congeners effect behavior. • Laboratory research is needed to determine a causal relationship between sub lethal dietary PCB levels and behavioral changes, including songs.