Our research program involving the biological effects of ALAN on birds has received funding from the FWO Flanders (to M.E. and R.P., project ID: G.0A36.15N), the University of Antwerp and the European Commission (to M.L.G, Marie Sk&#322;odowska-Curie fellowship ID: 799667). We acknowledge the intellectual and technical support of members of the Behavioral Ecology and Ecophysiology Research group at the University of Antwerp, especially Peter Scheys and Thomas Raap.

All applications of this system to animal experiments were approved by the University of Antwerp&#39;s ethical committee and conducted in accordance with Belgian and Flemish laws. Methodology adhered to the ASAB/ABS guidelines for the use of animals in behavioral research. The Belgian Royal Institute for Natural Sciences (Koninklijk Belgisch Instituut voor Natuurwetenschappen; KBIN) provided licenses for all researchers and personnel.
1. Creating the experimental system
<ol>...

The authors declare that they have no conflicts of interest.

This nest box-based system of LED lights and a paired IR camera has allowed researchers to assess a range of intriguing questions regarding the biological effects of ALAN. Moreover, there are many more research directions that can be pursued with the system. In addition, expanding the use of the system to other species could help foster an understanding of interspecific differences in sensitivity to ALAN. Below some non-exhaustive possibilities for future research are presented in the hope that this paper will help motiv...

Animals have evolved with the natural patterns of light and darkness that define day and night. Thus, circadian rhythms in hormonal systems orchestrate rest and activity patterns and allow animals to maximize fitness1,2,3. For instance, the circadian rhythm in glucocorticoid hormones, with a peak at the onset of daily activity, primes vertebrates to behave appropriately across the 24-h period via effects on glucose metabolism and responsiveness to environmental stressors4. Similarly, the pineal hormone melatonin, which is released in response to darkness, is integrally involved in governing patterns of circadian rhythmicity and also has antioxidant properties5,6. Entrainment of many aspects of circadian rhythmicity, such as melatonin release, is affected by the photoreception of levels of light in the environment. Thus, the introduction of artificial light into the environment to support human activity, recreation, and infrastructure has the potential to have wide-reaching effects on the behavior, physiology and fitness of free-ranging animals7,8. Indeed, diverse effects of exposure to artificial light at night (ALAN) have been documented9,10, and ALAN has been highlighted as a priority for global change research in the 21st century10.
Measuring the effects of ALAN on free-ranging animals poses non-trivial challenges for a number of reasons. First, mobile animals moving through the environment constantly experience different levels of light. Thus, how does one quantify the level of light that individual animals are exposed to? Even if levels of light on the territory of the animal can be quantified, the animal may employ avoidance strategies that affect patterns of exposure, thus demanding simultaneous tracking of animal location and light levels. Indeed, in most field studies, the mean and variation in light exposure levels are unknown11. Second, exposure to ALAN is often correlated with exposure to other anthropogenic disturbance factors, such as noise pollution, chemical exposure, and habitat degradation. For instance, animals occupying habitats along the margins of roadways will be exposed to light from street lamps, noise from vehicular traffic, and air pollution from vehicular emissions. How then does one effectively isolate the effects of ALAN from the effects of confounding variables? Rigorous field experiments that enable good measurements of both light exposure levels and response variables are essential to evaluating the severity of the biological effects of ALAN, and to developing effective mitigation strategies11.
This article describes an experimental approach that, although not without its limitations (see discussion section), helps assuage, if not eliminate the difficulties identified above. The approach entails experimentally manipulating ALAN levels inside the nest boxes of a free-living, diurnal bird species, the great tit (Parus major), using a system of light-emitting diode (LED) lights and an infrared (IR) camera installed within nest boxes. The setup enables simultaneous acquisition of video recordings, including audio, which allows researchers to assess effects on behaviors and vocalizations. Great tits utilize nest boxes for breeding, and sleep in the nest boxes between November and March. Females also sleep inside the nest boxes during the breeding season12. The system has also been used to a lesser extent to study effects of ALAN on blue tits (Cyanistes caeruleus). The first difficulty, involving knowing light levels encountered by the animal, is mitigated in that, given that an individual is willing to enter the nest box (or is already in the nest box in the case of immobile nestlings), light levels can be precisely determined by the researcher. The second difficulty, involving correlations to confounding variables, can be controlled by using nest boxes in similar environments, and/or measuring the levels of confounding variables near nest boxes. In addition, in cavity-nesting birds, adopting an experimental approach is powerful because nest boxes or natural cavities can shield nestlings and adults from ALAN13, which may explain why some correlative studies find little effect of ALAN (or anthropogenic noise)14, whereas experimental studies more often find clear effects (see below). Moreover, a repeated measures experimental design can be adopted in which individuals serve as their own control, which further increases statistical power, and the probability of detecting meaningful biological effects. The sections below: (1) explain the details of the design and implementation of the system, (2) summarize the important results that have been thus far derived using the system, and (3) propose future research directions that could be pursued, both in tits and other animals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Broad spectrum; 15 mm x 5 mm; LED headlight</td>
			<td>RANEX; Gilze; Nederlands</td>
			<td>6000.217</td>
			<td>A similar model could also be used</td>
		</tr>
		<tr>
			<td>Battery</td>
			<td>BYD</td>
			<td>R1210A-C</td>
			<td>Fe-battery 12 V 120 Wh ( lithium iron phosphate battery)</td>
		</tr>
		<tr>
			<td>Dark green paint</td>
			<td></td>
			<td></td>
			<td>Optional. To color nest boxes/electronic enclosures</td>
		</tr>
		<tr>
			<td>Electrical tape</td>
			<td></td>
			<td></td>
			<td>For electronics</td>
		</tr>
		<tr>
			<td>Homemade timer system</td>
			<td>Amazon</td>
			<td>YP109A 12V</td>
			<td>A similar model could also be used</td>
		</tr>
		<tr>
			<td>Infrared camera</td>
			<td>Koberts-Goods, Melsungen, DE</td>
			<td>205-IR-L</td>
			<td>Mini camera; a similar model could also be used</td>
		</tr>
		<tr>
			<td>Light level meter</td>
			<td>ISO-Tech ILM; Corby; UK</td>
			<td>1335</td>
			<td>To calibrate light intensity</td>
		</tr>
		<tr>
			<td>Mini DVR video recorder</td>
			<td>Pakatak, Essex, UK</td>
			<td>MD-101</td>
			<td>Surveillance DVR Recorder Mini SD Car DVR with 32 GB</td>
		</tr>
		<tr>
			<td>Passive integrated transponder (PIT) tags</td>
			<td>Eccel Technology Ltd, Aylesbury, UK</td>
			<td>EM4102</td>
			<td>125 Kh; Provides unique electronic ID</td>
		</tr>
		<tr>
			<td>Radio frequency identification (RFID) Reader</td>
			<td>Trovan, Aalten, Netherlands</td>
			<td>GR-250</td>
			<td>To scan PIT tags and determine bird identity</td>
		</tr>
		<tr>
			<td>Resistor</td>
			<td>RS Components</td>
			<td></td>
			<td>Value depending on voltage battery and illumination</td>
		</tr>
		<tr>
			<td>SD card</td>
			<td>SanDisk</td>
			<td></td>
			<td>64 GB or larger</td>
		</tr>
		<tr>
			<td>SongMeter</td>
			<td>Wildlife Acoustics; Maynard, MA</td>
			<td></td>
			<td>Optional. Provides a means of monitoring vocalizations outside of nest boxes</td>
		</tr>
		<tr>
			<td>TFT Color LED Portable Test Monitor</td>
			<td>Walmart</td>
			<td></td>
			<td>Allows verification that the camera is on and recording the image correctly</td>
		</tr>
		<tr>
			<td>Wood</td>
			<td></td>
			<td></td>
			<td>To construct nest boxes/electronic encolsures</td>
		</tr>
	</tbody>
</table>

an experimental approach to investigating effects of artificial light at night on free-ranging animals: implementation, results, and directions for future research

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from human infrastructure and recreational activity. Artificial light at night (ALAN) has the potential to have widespread effects on animal behavior, physiology, and fitness, which can translate into broader-scale effects on populations and communities. Understanding the effects of ALAN on free-ranging animals is non-trivial due to challenges such as measuring levels of light encountered by mobile organisms and separating the effects of ALAN from those of other anthropogenic disturbance factors. Here we describe an approach that allows us to isolate the effects of artificial light exposure on individual animals by experimentally manipulating light levels inside nest boxes. To this end, a system can be used consisting of light-emitting diode (LED) light(s) adhered to a plate and connected to a battery and timer system. The setup allows exposure of individuals inside nest boxes to varying intensities and durations of ALAN while simultaneously obtaining video recordings, which also include audio. The system has been used in studies on free-ranging great tits (Parus major) and blue tits (Cyanistes caeruleus) to gain insight into how ALAN affects sleep and activity patterns in adults and physiology and telomere dynamics in developing nestlings. The system, or an adaptation thereof, could be used to answer many other intriguing research questions, such as how ALAN interacts with other disturbance factors and affects bioenergetic balance. Furthermore, similar systems could be installed in or near the nest boxes, nests or burrows of a variety of species to manipulate levels of ALAN, evaluate biological responses, and work towards building an interspecific perspective. Especially when combined with other advanced approaches for monitoring the behavior and movement of free-living animals, this approach promises to yield ongoing contributions to our understanding of the biological implications of ALAN.

The peer-reviewed research articles published using this system are summarized in Table 2. Several other manuscripts are in progress. These studies address three major suites of research questions. First, the system has been used to study the effects of light exposure on sleep behavior and activity levels in adults. To this end, a repeated measures experimental design was employed, in which the same individual was first recorded sleeping under natural conditions and subsequently recorded sleeping in a li...

Watch this Scientific Journal Video about An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Res

An Experimental Approach to Investigating Effects of Artificial Light at Night on Free-Ranging Animals: Implementation, Results, and Directions for Future Research

Artificial light at night (ALAN) has wide-reaching biological effects. This article describes a system for manipulating ALAN inside nest boxes while monitoring behavior, consisting of LED lights coupled to a battery, timer, and audio-capable infrared video camera. Researchers could employ this system to explore many outstanding questions regarding the effects of ALAN on organisms.

Animals have evolved with natural patterns of light and darkness. However, artificial light is being increasingly introduced into the environment from ...

Our method for manipulating artificial light at night, or ALAN, inside nest boxes, is powerful for determining the effects of ALAN on the behavior, physiology and fitness of animals. This technique helps overcome the challenges inherent to studying how ALAN affects free-ranging animals, because researchers can expose animals to light at night in a controlled fashion. This system can be used in any avian or mammalian species that uses cavities for breeding or resting, and also has the potential to be modified for use in other contexts.Start by habituating the animals to the experimental setup by placing a dummy box on the top of the nest box at least one day prior to the experiment, to minimize the effects of novelty aversion. To survey the focal individuals, fit the animals in the study population with a passive integrative transponder or PIT tag, to allow for identification within the nest box, without disturbing the birds. In experiments involving the effect of artificial light at night or ALAN on sleep behavior, visit the nest boxes on the night before the experiment, and scan the boxes with a radio frequency indication or RFID reader, to determine which birds are roosting inside.To record undisturbed sleep in the absence of ALAN, implement a repeated measures design by recording sleeping under conditions of darkness for one night, and then in the presence of ALAN, for one night. For both nights, before taking the infrared, or IR camera into the field, synchronize the time with the local time. Next, insert an MDSD card into the SD slot into the mini DVR recorder adjacent to the battery.At least two hours prior to the onset of darkness, remove the dummy box from the top of the nest box and open the nest box lid to place the plate containing the IR camera inside, with the camera objective oriented downward. Then extend the electronic connectors out of the groove in the nest box. After closing the nest box lid, place the enclosure containing the battery, recorder and timer, on top of the nest box.Then connect the red connector from the recorder to the white connector from the camera, the yellow connector from the recorder to the yellow connector from the camera, and the black connector from the battery to the red connector from the camera. And for the ALAN night, the LEDs to the timer, and the timer to the power source. Once connected, put the wires inside the enclosures that houses the system.As the recording starts, check with a small TFT screen to ensure the correct image, and then press record. One hour after dark, return to the nest box to check the identity of the birds sleeping inside, by moving an RFID transponder reader around the bottom and sides of the nest box, and recording the unique identification number communicated from the PIT tag. On the morning following the control recording, two hours after sunrise, collect the battery system from the nest box before returning the dummy box on top of the nest box.In the laboratory, charge the battery and remove and download the SD card from the recorder to collect the behavioral data. After successfully downloading the data, erase the data from the SD card, followed by inserting the card into the mini DVR recorder. On a subsequent night, implement the light exposure treatment by setting up the timer system for the desired time of light exposure, and control recording as described before, with the timer connected to the power and the LEDs connected to the timer.For experiments involving multiple nights of light exposure and video recording, collect the systems each morning to recharge the batteries during the day, and then replace the system in the evening. Later, collect data on the response variables of interest. The study documented the dramatic effects of ALAN exposure on the sleep behavior.The birds exposed to ALAN at a relatively low intensity of 1.6 lux, woke up half an hour earlier, left the nest box 20 minutes earlier, and slept 40 minutes less than the control birds. Here we focus on using the system to assess effects on sleep, but the system can also be used to test effects on developing nestlings during the breeding season. To assess the physiological effects of ALAN exposure, animals can be captured, blood samples obtained, and morphometric measurements can also be taken at this time.This nest box based system has allowed researchers to assess a range of intriguing questions regarding biological effects of ALAN, that could not previously be assessed, such as effects on sleep.

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2.3K Views.  University of Antwerp. Il nostro metodo per manipolare la luce artificiale di notte, o ALAN, all'interno di cassette nido, è potente per determinare gli effetti di ALAN sul comportamento, la fisiologia e la forma fisica degli animali. Questa tecnica aiuta a superare le sfide inerenti allo studio di come ALAN colpisce gli animali ruspanti, perché i ricercatori possono esporre gli animali alla luce di notte in modo controllato. Questo sistema può essere utilizzato in qualsiasi specie aviaria o mammifera che utiliz...

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