Metal pollution indirectly increases oxidative stress in great tit (Parus major) nestlings $, $$ Miia J. Koivula a,n , Mirella Kanerva b , Juha-Pekka Salminen c , Mikko Nikinmaa b , Tapio Eeva a a Department of Biology, Section of Ecology, University of Turku, FI-20014 Turku, Finland b Department of Biology, Division of Genetics and Physiology, University of Turku, FI-20014 Turku, Finland c Laboratory of Organic Chemistry and Chemical Biology, 20014 University of Turku, FI-20014 Turku, Finland article info Article history: Received 6 August 2010 Received in revised form 21 December 2010 Accepted 6 January 2011 Available online 4 February 2011 Keywords: Enzyme activities Great tit Metals Oxidative stress Plumage colour abstract Metals can cause oxidative stress by increasing the formation of reactive oxygen species (ROS), when there are insufficient amount of antioxidants to defend against the growing amount of free radicals. We aimed to find out the most reliable biomarkers to detect pollution-related oxidative stress in wild birds by comparing oxidative stress status in great tit (Parus major) nestlings at populations in polluted and unpolluted areas. We also studied with experimental manipulations whether dietary carotenoid levels have any role in great tits’ antioxidant defence and whether their carotenoid-based plumage colour was connected to an oxidative stress status. We used antioxidants (GSH, carotenoids) and several antioxidant enzymes (GP, GR, GST, SOD, and CAT) as indicators of the oxidative stress. We found no direct connections between dietary metal exposure and antioxidant or antioxidant enzyme levels. The activity of GP was, however, slightly higher in the polluted environment. This was due to poorer condition and subsequently higher level of oxidative stress in the nestlings in the polluted area. We also found a positive association between GP and an ambient temperature during the nestling period, which may be due to higher metabolic activity of partly poikilothermic nestlings in warm weather. The activity of GST was positively related to the number of nestlings at the sampling time. Fledging success was better in an unpolluted area, where also the nestling body mass was higher. Carotenoid treatment increased the plasma carotenoid concentrations 2.1 fold in carotenoid-supplemented birds, but was not associated with the oxidative stress biomarkers or metal levels. The yellow plumage colour was associated with dietary carotenoid levels in both study areas, but not with the metal exposure or the oxidative stress status. Our results suggest that at the exposure levels found in our study area, the enzyme activities do not indicate metal-related oxidative stress. Instead, GP can be used as an indicator of growth related oxidative stress, which is greater in the polluted area. The activity of this enzyme was, however, not directly related to metal exposure, but more likely to some secondary pollution-related change in the nestling condition. & 2011 Elsevier Inc. All rights reserved. 1. Introduction Metals are very reactive elements and thus toxic to many orga- nisms when interfering with metabolism and important biochem- ical reactions (Scheuhammer, 1987; Stohs and Bagchi, 1995; Pinto et al., 2003). Metals can have direct and indirect effects on the wildlife, affecting directly to the egg quality and number, growth rate, nestling condition and morphology of the animals (Hoffman et al., 1985; Nyholm, 1994; Janssens et al., 2003; Bel’skii et al., 2005) as well as an alteration in biochemical processes, such as changes in enzyme activities and free radical levels (Pinto et al., 2003). On the other hand, metal pollution can indirectly change the habitat, community structure and ecological relationship between species (reviewed by Heli ¨ ovaara and V¨ ais¨ anen, 1993; Kiikkil¨ a, 2003). For example, by changing food chains metal pollution can affect the food quality and quantity and thus negatively affect the survival of animals (Eeva et al., 1997; Eeva and Lehikoinen, 2004). Recent find- ings suggest that the metal pollution may also increase oxidative stress levels in wild birds (Berglund et al., 2007; Geens et al., 2009; Kamin ´ ski et al., 2009). However, it is still unclear whether this is caused by the direct pro-oxidant effect of metals or by some secon- dary change, e.g. in diet quality. Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/envres Environmental Research 0013-9351/$ - see front matter & 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.envres.2011.01.005 $ Financial support: the study was financed by the Academy of Finland (T.E. project 8119367) and metal analyses by Boliden Company, Harjavalta, Finland. $$ Ethic approval: the study was conducted in full compliance with Finnish laws and regulation, including the licences of the Animal Care & Use Committee of Turku University and Southwest Finland Regional Environment Centre for our studies. n Corresponding author. Fax: + 358 2 333 6550. E-mail addresses: miikoi@utu.fi (M.J. Koivula), mmkane@utu.fi (M. Kanerva), j-p.salminen@utu.fi (J.-P. Salminen), miknik@utu.fi (M. Nikinmaa), teeva@utu.fi (T. Eeva). Environmental Research 111 (2011) 362–370