Effects of Pine Needle Accumulation on Tadpole Development, Oxidative Stress and Genetic Damage in Odontophrynus reigi (Anura: Odontophrynidae)

Abstract

Large extensions of the Atlantic Forest in Brazil and Argentina have been replaced by monoculture tree plantations (mainly Pinus spp.). Small changes in the chemistry of pools where leaf litter tend to accumulate can have major effects on amphibian physiology and ecology. This work studied effects of pine needles on Odontophrynus reigi tadpoles. We exposed tadpoles to different concentrations of pine needles (0, 0.5, 1.5 and 2.5 g/L). In tadpoles, we determined: 1) morphological endpoints, 2) growth rate and development stage, 3) oxidative stress parameters (catalase, glutathione-S-transferase, lipid peroxidation (LPO) and protein oxidation (PO)) and 4) genetic damage (micronucleus, erythrocytes nuclear abnormalities and mitotic index). Dissolved oxygen (DO), pH, ammonia and total phenols (TP) were determined in water. As pine needle concentration increased, DO, pH and ammonium decreased in water, and TP increased. All morphological endpoints increased as pine needle increased, except body width that decreased. Oxidative damage had opposite effects: LPO decreased 50%, and PO had a 3-fold increase at 2.5 g/L. Catalase and glutathione-S-transferase enzymatic activities did not change significantly. Mitotic index increased with pine needle concentration. These physiological responses, likely driven by changes in water chemistry, suggest a potential long-term cost for individuals inhabiting pine monocultures.

Key words
Natural Habitat; Odontophrynus Reigi; Agrosystem; Oxidative Stress; Total Phenols

INTRODUCTION

Exotic monoculture tree plantations have replaced vast areas of the world’s native forest (Iglesias-Carrasco et al. 2022), which reduces landscape heterogeneity and the number and diversity of habitats available for species (Lindenmayer & Franklin 2002, Magura et al. 2000, Gangenova et al. 2018). Monoculture tree plantations have been associated with reductions in the number of species of several animal groups: arthropods, reptiles, amphibians, and birds (Iglesias-Carrasco et al. 2022, Gangenova et al. 2018, Zurita & Bellocq 2012, Fork et al. 2015, Glor et al. 2001).

Native wildlife remaining in monoculture tree plantations can be affected by the changes caused by plant litter, which can in turn vary the phenotypic responses (e.g., development of morphological traits and immune responses) of resident animals, particularly those in pools and other water bodies where plant litter tends to accumulate (e.g., Burraco et al. 2018, Gangenova et al. 2020). Lentic environments can retain allochthonous subsidies for long periods, enabling litter to have a more sustained effect on environmental attributes and provide a less transitory source of energy and nutrients (Brinson et al. 2003). As stated by Stoler & Relyea (2020), leaf litter is a major driver of ecological dynamics in forested wetlands, and these inputs can generate unique ecological conditions.

Forest ephemeral ponds support a high diversity and abundance of amphibians (Iglesias-Carrasco et al. 2022), and small changes in the chemistry of these aquatic habitats, as well as surrounding terrestrial areas (depending on the life cycle phase) can have major effects on amphibian ecology (Hickman & Watling 2014, Watling et al. 2011a, b). It has been observed that amphibians growing in resource-limited environments can alter behavior, morphology, development, and life history traits (Stoler & Relyea 2013, 2020, Walls & Gabor 2019). These phenotypic changes are likely adaptive responses that improve individual performance, affect ecological interactions, and may lead to species diversification (Miner et al. 2005, Agrawal 2001).

In the last decades, large extensions of the Atlantic Forest in Brazil and Argentina have been replaced by monoculture tree plantations (mainly Pinus spp.) (Zurita 2019, Gangenova et al. 2020). Non-native tree plantations of Pinus spp. have replaced more than 332,255 ha of natural forests in the NE Argentina (Izquierdo et al. 2008). One-third of these pine plantations is for production of cellulose-paper, the remaining for wood and resin (Zaninovich & Gatti 2020). The Atlantic Forest of South America is one of the most biodiverse areas with the highest number of endemic species worldwide; simultaneously, it is one of the most threatened ecosystems due to anthropogenic activities (Mittermeier et al. 2005, 2011), such as agriculture and livestock (Myers et al. 2000) with only 11–16% of its original cover remaining (Ribeiro et al. 2009). Studies show that anurans use monoculture pine ponds for reproduction, but number of species and abundance are low (Gangenova et al. 2018, Machado et al. 2012). The reduction in richness and change in the species composition suggests that some species that inhabited these ecosystems are excluded, leaving only the most resistant to environmental changes induced by monoculture pine plantations (Guerry & Hunter 2002, Hazell et al. 2001).

Odontophrynusreigi is an ecologically relevant species, given its abundant populations; like other species of the genus, adults have a generalist carnivorous diet based mainly on arthropods (unpublished data); have a relatively prolonged larval development, and show significant resilience to environmental disturbances. In this sense, O. reigi maintains its abundance in monoculture pine plantations in Misiones, Argentina (referred as Odontophrynus sp., Gangenova et al. 2018), and tolerates soils from monoculture pine plantations (Schvezov et al. 2023). Odontophrynus reigi occurs in the grassland of the Southern Cone Mesopotamian savanna, Alto Paraná Atlantic forest, Araucaria moist forests, Cerrado, and Humid Chaco Ecoregions in Argentina, Brazil, and Paraguay (Frost 2025). The species shows explosive reproduction mainly during autumn and winter (between March and late November). It is noteworthy that O. reigi seems tolerant to other anthropogenic disturbances, with several records from urban environments (e.g., along roadways, inside sewers, gardens, and backyards) (Rosset et al. 2021).

The objective of this work is to study effects on O. reigi tadpoles by pine needles. We studied the effect of quantity of pine needles in O. reigi tadpoles, exposing them to different concentrations of pine needles. We proposed the following hypothesis: increasing pine needle quantity affects negatively on the development of tadpoles, and antioxidant enzyme activity increases as well as oxidative damage parameters and genetic damage, due to the chemical changes in water. For this, we studied in O. reigi tadpoles: 1) morphological endpoints (total length, body length, body width, body depth, tail fin length, tail muscle depth, tail fin depth and interorbital width); 2) growth rate and development stage; 3) biochemical endpoints (catalase (CAT), glutathione-S-transferase (GST), lipid peroxidation (LPO) and protein oxidation (PO)); and 4) genetic damage (micronucleus, erythrocytes nuclear abnormalities and mitotic index). We considered the following physical-chemical parameters in water: pH, dissolved oxygen (DO), ammonia and total phenols (TP), as parameters that affect development of O. reigi tadpoles.

MATERIALS AND METHODS

Sampling area

We carried out this study in the semi-deciduous Atlantic Forest in northeast Argentina, province of Misiones. Climate is subtropical humid with no dry season (Cabrera & Willink 1980); the mean annual rainfall is about 1500–2000 mm, and the mean annual temperature is 20 °C, with a minimum of 15 °C in July (Tortorelli 2009) and a maximum of 35 °C in January (INTA 2022). Soils are mostly Ultisols (Moretti & Navarro de Rau 2019). Non-native tree plantations of Pinus spp. have replaced more than 332,255 ha of natural forests in the South Atlantic Forest of North-East Argentina, mostly in the last 60 years (Izquierdo et al. 2008, Zaninovich & Gatti 2020), and only a few understory native species (Ctenitis submarginalis (Langsd. & Fisch.) Ching and Thelypteris scabra (C. Presl) Lellinger) grow below the pine plantation canopy cover (Trentini et al. 2017).

Samplings of pine needles were performed in Corpus, Misiones, Argentina, in a monoculture pine plantation (27° 09’ 22.4”S, 55° 28’ 55.5”W) 25 years old, used only for resin collection. Site preparation methods of the monoculture pine plantation if any, are unknown. By interview with the landowner, the stand had not been thinned, had no known history of fire, and used no insecticides nor herbicides, nor had streams or rivers nearby. We collected in April 2021 ≈ 1 kg of pine needle that recently had fallen to the ground and hence corresponded to the top- most layer of litter in small 20 m2 patches. The sampling site was 500 m within the limits of the pine plantation, where the forest edge has no effect (Gatti et al. 2019). Only the pine trees planted, with no understory native species nearby gave shadow. The pine needles were separated from organisms, sticks, and pebbles. All samples were transported to the laboratory in black plastic bags and were dried at air temperature, and once they were dried, they were kept in a dry plastic bag protected from light at low temperature.

Tadpole sampling

Odontophrynus reigi tadpoles were collected in June 2021 (27° 26’ 46.7” S, 55° 57’ 31.6” W) from a temporary pond, with no pine plantations nearby, affected mainly by anthropogenic noise given by the street near it. The collection method was done by passing nets in the ponds, and tadpoles that seemed at plain sight in Gosner stages 20-22 were selected. They were taken to the laboratory and kept in dechlorinated tap water at 20 ± 3 °C (seasonal temperature) and 11:13 L/D photoperiod (seasonal photoperiod) until reaching Gosner stage 25. They were fed with commercial rabbit chow (Schvezov et al. 2023) and naturally present algae (Janes et al. 2023) during the acclimation period and the experiments.

Experimental design

In the experiment, we assessed how tadpoles are affected at different concentrations of pine needles. For this, we assessed four different concentrations of pine needles: 0, 0.5, 1.5 and 2.5 g/L. There is no known literature as the amount of litter produced in the pine forest, nor the possible pine needle concentrations a tadpole is exposed in the temporary pond during development in the studied area. Therefore, we took 0.5 g/L pine needle concentration as guide, since it has been used before in previous experiments (Burraco et al. 2018, Maerz et al. 2005).

The experiment was performed in semi-natural conditions (Manenti et al. 2023). Rearing containers were placed at room temperature and exposed to natural daily photoperiod (11:13 L/D). Aquariums consisted of plastic containers (35 cm long x 25 cm wide x 17 cm deep). Before the experiment, we put in the aquariums the pine needle (concentration of pine needle g/L specified above) and 10 L water on top and left for seven days so the water: leaf system reached equilibrium. Three replicates for each treatment were done. To determine Gosner stage, we weighed and observed ten tadpoles in stereomicroscope previously. Considering that all tadpoles were from the same pond and were reared in the same conditions, we considered that tadpoles with similar weight were in Gosner stage 23. This was done to avoid stress during tadpole manipulation. On day zero of the experiment 10 tadpoles on Gosner stage 23 (mean body mass: 0.15 ± 0.03 g) were randomly distributed in each aquarium. We changed the water in each aquarium every 3 to 5 days without removing the animals or leaf litter. Furthermore, to minimize disturbances, we did not remove 70% of the water in each aquarium (Savage et al. 2002). We fed tadpoles every four days ad-libitum (0.007 g/day per individual). We observed that tadpoles fed on algae found in the aquariums, which subsequently provided a resource of food during the experiment. At the end of the experiment, we dried the remaining pine needles at room temperature, and weighed it. Remaining pine needle in aquariums was (82 ± 2) %.

We checked aquariums daily throughout the experimental period for the presence of dead tadpoles, and if we found one, they were removed. When more than three tadpoles showed Gosner stage 38, the experiments ended to avoid morphological changes due to metamorphosis near to metamorphic climax. The experiment was done from tadpole development Gosner stage 23 to 38, which lasted 61 days. Water pH, DO and temperature were taken every two days, and ammonium was determined at the end of the experiment by nessler method (SM 4500.NH₃-B and C) in PEIU-FCEQyN-UNaM. Total phenols in water samples were determined by the Folin-Ciocalteu’s method (Kupina et al. 2019) on day zero and at the end of the experiment. Gallic acid was used as standard, therefore concentrations are determined as galic acid equivalents.

Biological endpoints: survival, development, and growth

At the end of the experiments, growth was immediately measured by weighing each larva (electronic balance 0.001 g precision (N= 30)). Before larvae were weighed, the water excess was gently removed. Then, fifteen larvae from each concentration were euthanized (TMS-MS222 buffered at pH 7.8) and preserved in 10 % formalin to measure development and growth endpoints. These specimens were deposited at the LGE (Laboratorio de Genética Evolutiva- Instituto de Biología Subtropical) herpetological collection.

Tadpoles were staged (Gosner 1960) and morphological variations were determined on a Nikon SMZ1270 stereoscopic microscope and with a digital caliper (to the nearest 0.1 mm). We recorded eight morphological variables: total length, body length, body width, body depth, tail fin length, tail muscle depth, tail fin depth and interorbital width according to Teplitsky et al. (2003). The growth rate (GR) was calculated as stated by Teplitsky et al. (2003) as follows: GR: exp (ln (final weight – initial weight)/number of days of the experiment).

All procedures followed the Ethics and Integrity Committee of the National University of Misiones (16Q1314-PI).

Biochemical endpoints: antioxidants, oxidative damage and genetic damage

Catalase (CAT) and Glutathione-S-transferase (GST) were determined as antioxidants biomarkers and protein oxidation (PO) and lipid peroxidation (LPO) as oxidative damage. Ten tadpoles of each treatment were rinsed with distilled water, dabbed dry on tissue paper, weighed, placed in Eppendorf tubes, and refrigerated on ice. Immediately they were taken to -80 ºC freezer until analysis of CAT, GST, LPO, and PO. The homogenates were prepared using a relation wet tissue:buffer of 1:4, using cold Tris–HCl buffer (20 mM TRIS, 25 mM sucrose, 1 mM EDTA. pH = 7.4. (Lajmanovich et al. 2019)). Samples were homogenized in ice for 5 min and then centrifuged at 10,000 ×g for 15 min. The supernatants collected were employed as antioxidant and oxidative damage source. The activity of CAT was determined according to Aebi (1984). GST activity was measured by the method described by Habig et al. (1974). Enzymatic activities of GST and CAT were expressed as U mg protein-1, where 1 U is equivalent to 1 µmol of substrate consumed or product formed per minute at 25 °C. Lipid peroxidation (LPO) was determined by the method proposed by Ohkawa et al. (1979). Data are expressed as µmol TBARS g wet tissue-1. Protein oxidation (PO) was analyzed by Reznick & Packer (1994) and expressed as nmol carbonyl mg protein-1. Total protein concentration was estimated following Lowry method (Lowry et al. 1951), using bovine albumin as standard. All samples were analyzed by triplicate.

Micronucleus (MN) test was performed as a biomarker of DNA damage. Five tadpoles from each treatment were collected and anesthetized with benzocaine (De Lima Barros et al. 2020, Leary et al. 2020). Peripheral blood was obtained from the caudal vein and smeared onto clean slides. Criteria for distinguishing and counting a micronucleus were: circular or ovoid chromatin bodies with a smaller diameter than the principal nucleus, intensity of stain and focus similar to the principal nucleus, non-refractive, located within the cytoplasm and not connected or overlapped with the principal nucleus (Al-Sabti & Metcalfe 1995, Ferreira et al. 2003). Additionally, other erythrocyte nuclear abnormalities (ENA) like lobed nuclei, binucleate or segmented nuclei, kidney-shaped nuclei, notched nuclei, and pyknotic nuclei were assessed (Guilherme et al. 2008, Lajmanovich et al. 2015). The results were expressed as ENA, the mean value of the sum of all the lesions observed.

Data analyses

Data are expressed as the mean and standard deviation (mean ± SD). Briefly, all data had their distribution tested using the Shapiro–Wilk normality test and the Bartlett test of homogeneity. A Spearman correlation analyses was done for morphometric data, and since Body depth and Total length presented a high correlation with at least two morphometric variables (Supplementary Material – Table SI), these were removed from further analyses on morphometric parameters.

To test our hypothesis we did linear models. Physical chemical variables presented normality but not homogeneity, and since we had a single data point per tank, a Generalized least square model (GLS) was done with gls from the nlme package. We fitted linear mixed models with Gaussian error distributions using lmer from lme4 package Growth rate, Gosner, CAT, GST, PO, and morphometric data, except Body width; and we fitted generalized mixed models using gmler form lmer4 package for LPO and Body width using a Gamma error distributions (Burraco et al. 2017) and MN, ENA and MI with Poisson error distribution (counts). We included tank as a random factor in the mixed models.

For all statistical tests, the significance level was set at p < 0.05 (Sokal & Rohlf 1995). All data were statistically analyzed with software R (version 4.4.0.; R Development Core Team (2021)).

RESULTS

The experiment lasted 61 days, and survival was 100% in all aquariums. Growth rate presented a significant increase as pine needles concentration increased (F = 25.94, p < 0.001. Table I), and tadpoles in higher pine needle concentration presented higher Gosner stage (F = 7.43, p < 0.01. Table I).

Table I
Growth rate and Gosner stage of tadpoles (N=15) exposed to different concentrations of pine needles in laboratory.

Pine needle concentration	Growth rate	Gosner
0 g/L	0.959 ± 0.003	29.9 ± 2.7
0.5 g/L	0.970 ± 0.007	32.8 ± 3.9
1.5 g/L	0.972 ± 0.003	33.3 ± 3.8
2.5 g/L	0.979 ± 0.001	33.4 ± 1.7

Physical-chemical data presented significant changes as pine needle concentration increased (Tables SI-SV). pH, DO and ammonium decrease as pine needle concentration increase, and only Total phenols presented a 30% increase with pine needle concentration (Table II).

Table II
Physical-chemical parameters of water from aquariums at different concentrations of pine needles (N= 3). Ammonium (NH₄+): mg/L. Total phenols: g/mL. DO: mg/L.

Pine needle concentration	pH	DO mg/L	Total phenols g/mL	NH4+ mg/L
0 g/L	7.03 ± 0.02	5.6 ± 0.3	0.43 ± 0.08	1.40 ± 0.01
0.5 g/L	6.76 ± 0.01	4.1 ± 0.3	0.49 ± 0.01	1.35 ± 0.02
1.5 g/L	6.44 ± 0.01	2.1 ± 0.2	1.52 ± 0.01	0.51 ± 0.03
2.5 g/L	6.23 ± 0.01	2.3 ± 0.2	1.39 ± 0.08	0.72 ± 0.04

All morphological parameters presented a significant increase (Supplementary Information), except Body width (F = 21.61, p < 0.0001) that decreased at higher pine needle concentration (Fig. 1), and Interorbital width that showed no significant changes (F = 3.01, p = 0.09).

Figure 1
Tables SI-SV.

The antioxidant enzyme activity was 74 ± 24 UCAT / mg protein and 1.14 ± 1.95 µUGST / mg protein, with no significant differences between treatments (CAT: F = 0.545, p= 0.465; GST: F = 1.54, p = 0.223). On the other hand, LPO (F = 42.66, p < 0.001) and PO (F= 23.47, p < 0.001) presented opposite results, since as pine needle increased, LPO decreased 50% but PO had a 3-fold increase (Fig. 2a, b). Micronucleus and ENA were observed in O. reigi tadpoles (Fig. 3), but their frequencies (MN: 0.0006 ± 0.0009. ENA: 0.007 ± 0.003) had no differences at different concentrations (MN: F = 0.067, p = 0.799. ENA: F = 0.354, p = 0.559), but the MI presented a 20-fold increase (Fig. 2c).

Figure 2
Morphological endpoints (mm) of Odontophrynus reigi (N= 15) exposed to Pine needles (g/L) in laboratory that presented significant changes. BW: Body weight. BL: Body length. TMD: Tail muscle depth. TFD: Tail fin depth. Data expressed as mean ± standard deviation.

Figure 3
Biochemical endpoints of Odontophrynus reigi tadpoles (N= 10) exposed to Pine needles (g/L) in laboratory that presented significant changes. a) Protein oxidation (nmol carbonyl mg protein-1). b) Lipid peroxidation (µmol TBARS g wet tissue-1) c) Mitotic Index (MI) frequency (‰).

All model’s results are presented in Table SIII.

DISCUSSION

Odontophrynus reigi larvae does tolerate pine needles, its survival has been observed as pine needle quantity increased, although, it presented some changes in the morphological traits, and in the oxidative stress response, which could bring consequences in later stages. Overall, the physical-chemical changes in water at different pine needle concentrations have accelerated O. reigi growth rate and development, with morphological differences. Response towards plant litter is very species specific, as observed by Burrow & Maerz (2021) work, in which larval development of gopher frog (Lithobates capito, referred as Rana capito) and ornate chorus frog (Pseudacris ornata) tadpoles was extended in certain plant litters, but in others not.

The accelerated growth rate was largely caused by the chemical variations in water, which produces the physiological changes observed in O. reigi tadpoles in our work. Iglesias-Carrasco et al. (2022) observed that growth was affected in three anuran species exposed to an exotic plant. The authors explain that growth did not have an equal response due to their variation in their respiratory capacity and ability to cope with potential reduction in oxygen levels. Odontophrynus reigi individuals are usually buried when they are not breeding or feeding (Rosset et al. 2021), where they might have lower levels of oxygen, and could explain the tolerance towards the lower levels of DO given by pine needles in our work.

The morphological and development differences observed at different concentrations in our work, along with the accelerated growth rate, may cause long-term fitness implications. Gangenova et al. (2020) has observed in adults of O. reigi (referred as O. americanus in their work) in pine plantations from Misiones an increment in their overall body size and body mass, compared to those one found in the Atlantic Forest, its natural habitat. The authors suggest that this difference may be to improve their walking and burrowing performances, enhancing their ability to search for dispersed preys and penetrate into concentrated food sources. These phenotypic changes may be adaptive responses that improve the individual performance, affect ecological interactions, and may lead to species diversification (Miner et al. 2005, Stoler & Relyea 2020).

Additionally, the accelerated growth rate could have affected the concentration of protein carbonyls. There is evidence that protein synthesis is crucial during formation of muscle and new adult structures, in which ROS interaction with SH groups during metamorphic changes can produce conformational changes that allow for better protein-protein or protein-DNA interactions (Magder 2006). Sohal et al. (1993) associated carbonyl content with the life expectancy or the physiological age, and not with the chronological age, in houseflies. These authors observed that a decreased level of flies’ physical activity (determined as metabolic rate) in conditions of hyperoxia, contributed to a lower rate of carbonyl accretion. Although, they could not get to a conclusion, they emphasize on the fact that the deleterious effects of sublethal exposure to hyperoxia seem irreversible, as indicated by both the overall carbonyl content and the depression of metabolic rate.

Furthermore, the antioxidant system of O. reigi responded towards increase of pine needle concentration. To our knowledge, there are few studies observing protein oxidation and lipid peroxidation at the same time as oxidative stress parameters. In our work, we did not determine metabolic rate, but a lower DO concentration in the water of higher pine needle concentrations may have contributed to the increase of PO in O. reigi tadpoles, but this effect was not observed in the lipid level. There is evidence that in hypoxic environments, organisms diminish their antioxidant enzyme activities and there is low or no effect in lipid peroxidation, e.g., king crab Lithodes santolla (Schvezov et al. 2017) and the common carp Cyprinus carpio (Lushchak et al. 2005). We observed that the two antioxidant enzymes determined did not respond to the chemical changes in water (CAT and GST), but the oxidative stress parameters did show differences, indicating that the antioxidant metabolism is affected somehow during this exposure, which could be due to the lowering of DO. This is also reflected in the MI found in the tadpoles exposed to higher pine needles concentrations, since the mitotic cell proliferation could represent a short-term means to increase the oxygen carrying capacity of the blood in amphibian species (Barni et al. 2007, Pollo et al. 2017). The presence of mitotic erythrocytes suggests that erythropoiesis can be directly stimulated in the peripheral circulation under certain conditions, such as the presence of contaminants, hypoxia or reduced oxygen levels (Barni et al. 2007, González et al. 2021). Seasonal changes, environmental stressors and parasites can also promote a regenerative response that increases the mitosis count in erythrocytes (González et al. 2021). The higher MI obtained in our study was similar to that obtained in frog tadpoles sampled in rice field areas from Italy, but did not appear to affect the overall health of the animals (Barni et al. 2007). However, the effect of experimental stressors on mitotic activity rates in tadpoles is still not clearly understood (Lajmanovich et al. 2014), and it is known that chronic exposure to adverse environmental conditions can lead to sublethal effects in populations, making them more susceptible to infection and diseases (Kiesecker et al. 2001).

A hypoxic environment could affect the organism during development or when it enters an environment with normal oxygen levels (Hermes-Lima & Zenteno-Savín 2002). To our knowledge, studies focused on tadpoles in hypoxic environments are mainly in lung development and behavior (Janes et al. 2023, Feder 1983a, McIntyre & McCollum 2000). Physiological responses that have been observed in larval anurans to low DO include altering blood flow (Feder 1983b), modifying gill ventilation rates (Simard et al. 2003), and changing lung, skin, and gill morphology (McIntyre & McCollum 2000). During acute hypoxia, pond-tadpoles mainly respond by air breathing (Phillips et al. 2024), and under sustained hypoxia atmospheric oxygen is necessary for long term survival, hence some species start developing their lung more rapidly, affecting its development, since metamorphosis requires the possession of well developed lungs (McIntyre & McCollum 2000). In this work, we did not analyze behavior of tadpoles, and no studies of tadpoles oxidative stress response towards hypoxic environments have been done before, to our knowledge, thus making it difficult to compare our results. However, Earl et al. (2012) have observed a lower survival of Pseudacris maculata and P. crucifer tadpoles exposed to red oak litter, probably caused by the lowered DO. These authors also found that DO were lower in treatments with higher tannin concentrations, maybe caused by the increased chemical oxygen demand from leached plant compounds. We observed in our work an increase of total phenols and a lowering of DO in treatments at higher pine needle concentration, which could have caused the response in the oxidative metabolism of O. reigi tadpoles. Phenolic compounds have been linked to decreased survival, increased developmental time, and decreased mass in some amphibian species but not others (Burrow & Maerz 2021, Maerz et al. 2005, Cohen et al. 2012, Stephens et al. 2013). In Rana japonica tadpoles acute toxicity tests, the main mode of action for most substituted phenols proved to be polar narcosis, and a few were bio-reactive (Wang et al. 2001). Phenolic compounds may cause damage to tissues such as gills, as well (Burrow & Maerz 2021, Ventura et al. 2021, Temmink et al. 1989). Ventura et al. (2021) associated the damage in gills due to phenolic compound in Piaractus mesopotamicus with difficulty capturing oxygen leading to hypoxia and mortality.

Tadpoles can use pine needles as food source and stratus. Although, in the tanks (82 ± 2) % of pine needle remained at the end of the experiment. Experiments done in same conditions as described here with native forest leaf litter only (36 ± 2) % of native forest leaf litter remained (unpublished data). Zaninovich et al. (2016) has observed in forest from the same study region that pine needle have a slow decomposition rate. The litter produced in exotic pine plantations is mechanically harder to break down, offers poorer nutrient content, and contains a higher concentration of toxic compounds (Iglesias-Carrasco et al. 2022). Martinez et al. (2016) observed that for detritivore animals the quality of the detritus in exotic pine plantations as food source was lower than in the natural forest. Although more studies are needed, we consider that tadpoles did not use pine needles as food source, but did use it as stratus since there was a lower movement when pine needle was present (personal observation). Still, our results indicate that there is an effect in the oxidative damage due to the presence of pine needle concentration, since the response of O. reigi tadpoles are mainly observed above 1.5 g/L of pine needle concentration.

Overall, pine needles did affect O. reigi tadpoles, mainly observed in the protein damage, lipid peroxidation, mitotic index and accelerated growth rate. Odontophrynus reigi, in our work, shows a tolerance towards the new habitat given by monoculture pine plantations, but we still have to consider the stress effect caused by the increment on pine needles, considering that these tadpoles develop in temporary ponds that could have different concentrations of pine needle. This work is a first showing changes in a native species that inhabits monoculture pine plantations, and has been observed to tolerate the changes given by pine plantations (Schvezov et al. 2023, Gangenova et al. 2018), but at what cost? Little is known as what is the real pine needle concentration that encounters this species, but there is a break, as observed in Fig. 2, above 1.5 g/mL in which tadpoles respond towards the chemical changes in water due to pine needles. The response of O. reigi towards pine needles may be indicative of the adaption of the species to this new habitat given by the monoculture pine plantations. Still, more works are needed in field, and we should consider the possibility that tadpoles are exposed to higher concentrations of pine needles in the ponds where larval development is achieved in the monoculture pine plantations, and combined with effects given by climate change e.g. increase of temperature and drastic drought seasons, making stress conditions much higher for tadpole development.

CONCLUSIONS

Our study demonstrates that pine needle accumulation alters the development of O. reigi tadpoles by accelerating growth and developmental rates and inducing oxidative protein damage, despite no effect on survival. These responses, likely driven by changes in water chemistry, suggest a potential long-term cost for individuals inhabiting pine monocultures. Further research is needed to understand the ecological consequences of these sublethal effects on native amphibian populations in modified landscapes.

SUPPLEMENTARY MATERIAL

Tables SI-SV.

Acknowledgements

We are thankful to Bismarck Sommerfelt for his assistance in field, Carla Silva from PEIU-FCEQyN-UNaM for the ammonium analysis in water samples and Mauricio Benitez for his assistance with the micronucleus test. This research was funded by Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT- PICT 2019-00495 and PICT 2019-03895) and Universidad Nacional de Misiones (UNaM- 16/Q1295-TI).

Data availability

Data is available in Mendeley Data: https://data.mendeley.com/datasets/55y8tcs8ys/1.

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Correspondence

Correspondence to: Natasha Schvezov [e-mail]

Handling editor

Mirco Solé

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  3 July 2025
• Accepted
  1 Nov 2025