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Keywords:

  • Bioaccumulative compounds;
  • Organochlorines;
  • Avian;
  • Migration;
  • Metabolism

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

This study evaluated the interactions of flight, fasting, and 1,1,1-trichloro-bis(4-chlorophenyl)ethane (p,p′-DDT) loading on residue metabolism and distribution in recently exposed white-crowned sparrows (Zonotrichia leucophrys). Female sparrows were dosed with 5 mg p,p′-DDT per kg body weight over 3 d. Following 1 d of recovery, sparrows were flown in a wind tunnel for up to 140 min, in 15-min blocks. Food was withheld from the start of the flight period until birds were euthanized. DDT, 1,1-dichloro-2,2-bis(4 chlorophenyl)ethane (DDD), and 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) were present in all tissues examined. 1-Chloro-2,2-bis(4-chlorophenyl)ethene (DDµ), 1,1-bis(4-chlorophenyl)ethane (p,p′-DDη), and 2,2-bis(4-chlorophenyl)ethanol (p,p′-DDOH) were not found. Fasting did not significantly affect the rate of residue increase over time in any of the tissues examined. When sparrows flew and fasted simultaneously, fasting seldom contributed to an increase in tissue residues. However, the length of time flown was significantly correlated with increasing toxicant concentrations in the brain, kidney, and liver, effectively demonstrating the potential for brief flights to enhance mobilization of DDT and its metabolites. Dose, flight, and fasting also increased residues in brain tissue. These contaminant redistributions may have important ramifications on the stresses experienced by migratory birds. Environ. Toxicol. Chem. 2012;31:336–346. © 2011 SETAC


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

DDT, other chlorinated hydrocarbons, and many of their metabolites are highly lipophilic and accumulate in fatty tissue. Avian species have proven to be particularly susceptible to deleterious effects from DDT use. One dehalohalogenation product, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (p,p′-DDE), has induced eggshell thinning, which caused dramatic declines of many top-level predators such as the bald eagle (Haliaeetus leucocephalus), peregrine falcon (Falco peregrinus), and brown pelican (Pelecanus occidentalis) 1. DDT effectively controls vector-borne carriers of disease, and despite its environmental hazard, its use continues in many tropical regions 2.

Migratory birds can bioaccumulate DDT and metabolites and other lipophilic compounds during time intervals spent in their tropical overwintering grounds, especially prior to migration when their lipid reserves are greatest. Once migration begins, birds experience high energetic demands, which deplete lipid stores and mobilize stored contaminants. Birds that experience cycles of high and low lipid stores due to migration, breeding, or adverse weather conditions may be susceptible to large pulses of contaminants 3–6. We were particularly interested in the effects of staging and migratory flight on redistribution and transformation of DDT in birds. This issue is especially relevant given the use of DDT for vector control in many tropical and neotropical areas, where migratory birds stage for transcontinental migrations. Also, toxicity manifests if threshold concentrations of organochlorine insecticides are exceeded in the brain 7, 8. Therefore, a need exists for understanding the metabolism and disposition of DDT and its transformation products (ΣDDT) before and during a migratory flight.

Laboratory experiments have demonstrated the relationship between lipid mobilization and energetic stress in birds that had been fasted 9–11. However, limited work has evaluated the role of more strenuous exercise, such as flight, and its ability to alter contaminant storage and metabolism. Fasting is a simple stressor to introduce in laboratory studies. However, it may not mimic metabolism and contaminant partitioning that occurs during strenuous physical activity, including migratory flight. Avian flight in wind tunnels provides a more accurate system within which contaminant movement may be measured during strenuous exercise. However, training birds to fly in a wind tunnel is difficult and limits sample size 12.

Birds can respond to environmental stressors in one of two ways: through the activation of the neurogenic system or through the hypothalamic–pituitary–adrenal (HPA) axis 13. Response by the HPA axis causes an increase in the synthesis and release of steroid hormones from the adrenal cortex. The primary corticoid released in birds, corticosterone 14, is mainly responsible for the overt signs of long-term stress. Typical effects include cardiovascular diseases, hypercholesteremia, gastrointestinal lesions, and immunological changes 13. Environmental stressors that elicit such HPA axis responses include ambient temperature extremes 15, 16, food deprivation 17, muscular exhaustion 18, water deprivation 19, handling 20–23, flight 24, and decreased fat deposits during migration 25.

One effect of corticosterone release is an alteration in locomotor activity 26, 27, although it is associated with many physiological and behavioral activities. These include, but are not limited to, vernal and autumnal migration (garden warbler [Sylvia borin] 25, migratory and sedentary passerines 28, willow tit [Parus montanus] 29, gray catbird [Dumetella carolinensis] 30); hyperphagia and fat deposition (white-crowned sparrow [Zonotrichia leucophrys] 31, dark-eyed junco [Junco hyemalis] 32, reviews 33, 34); seasonal and daily cycles (snow bunting [Plectophenax nivalis] 21, white-throated sparrow [Zonotrichia albicollis] 35, 36, white-crowned sparrow 37–39); and breeding behavior (snow bunting 21, white-crowned sparrow 27, song sparrow [Melospiza melodia] 40).

This study was part of a larger research effort to evaluate the effects of physical stress on circulating corticosteroid 41, DDT, and DDT transformation product concentrations. Gender-specific differences in corticosterone responses to identical stimuli were found in breeding, free-living white-crowned sparrows based on breeding status 37 and ambient temperatures 42. In addition, strong social hierarchies and elevated corticosterone levels may manifest in males grouped together. We chose to limit this study to female sparrows to avoid these confounding factors. The portion of the study presented here examined the metabolism and movement of p,p′-DDT in fasting female white-crowned sparrows that were sedentary or undergoing simulated migratory flight. This study was intended to evaluate the extent to which stressors influenced DDT transformation and distribution among tissues.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

Reagents

Analytical standards included p,p′-DDT, p,p′-DDD (1,1-dichloro-2,2-bis(4-chlorophenyl)ethane), p,p′-DDE, p,p′-DDµ (1-chloro-2,2-bis(4-chlorophenyl)ethene), p,p′-DDη (1,1-bis(4- chlorophenyl)ethane), and p,p′-DDOH (2,2-bis-4-chlorophenyl) ethanol) and were obtained from Aldrich Chemical. All solvents used were of pesticide analysis grade (VWR Scientific), including n-hexane, acetone, dichloromethane, methanol, and diethyl ether (99%). Anhydrous sodium sulfate (granular, 60–100 mesh) and florisil (60–100 mesh) (VWR Scientific) were baked overnight at 400°C and 150°C, respectively. Florisil was deactivated with 1.2% deionized water before use.

Animals

All procedures involving living vertebrates were performed in accordance with protocols approved by the Texas Tech University Animal Care and Use Committee. White-crowned sparrows were collected near Lubbock, Texas, USA, during the early winter and remained in outdoor aviaries until mid-May. Birds were captured with mist nets, transported to an outdoor aviary, housed, and maintained using procedures described previously 41. Briefly, 280 females were housed three to four birds per 2′ × 2′ × 2′ cage. Mazuri® small bird maintenance (15.6% protein, 7% fat, 2.9% fiber, 4.5% ash) and water were provided ad libitum. In addition, diet was regularly supplemented with wild birdseed, romaine lettuce, and meal worms (Tenebrio molitor). Temperature control was maintained in the range of 10 to 33°C, and the photoperiod ranged from 9.8 to 14.0 h during the acclimation, training, and artificial migration activities.

To limit the study population to female sparrows, a chromo-helicase-DNA-binding gene on the avian Z and W chromosomes was amplified with polymerase chain reaction (PCR) to identify the sex of sparrows 43. Polymerase chain reaction products were separated on 2% agarose gels containing 5 µl of ethyl bromide (EtBr) per 100 ml gel. The gels were run for 42 min at 90V with TAE (TRIS-citric acid-ethylene diamine tetraacetic acid) as the running buffer 41.

The local Audubon Society lists this species as abundant to the Lubbock area through the first week in May, tapering down to being absent by June. Therefore, the mid-May timing of our study overlapped the latter portion of the natural migratory window of Z. leucophrys.

Flight training

An open circuit suction type wind tunnel was used for flight training. The tunnel included a 2 m long × 1.25 m high × 1 m wide working/observation section, partitioned from the tunnel and fan using a one-half inch nylon netting. Air velocity was maintained at 16 km/h. The chamber floor and netting were lined with 18G bare copper wires, set 5 mm apart and carried 0.05 to 0.1 mA. The current was sufficient to discourage perching without causing undue stress 44–46. During flight training, investigators observed birds approaching the electrified grid and moving back into flight before even perching, as one toenail contacted the grid.

Flight training was conducted in this system beginning in February and included flights twice a week for six weeks. Each female sparrow was tested to improve chances of identifying individuals capable of flying in the wind tunnel. Birds were placed on a suspended perch within the observation section, initially with the tunnel fan off, and then with the fan on. The perch was removed for increasing amounts of time, thereby forcing the sparrows to attempt flight.

During a five-week interval, sparrow cohorts flew at increasing frequency, beginning with three times per week, and ending with five to six flights per week. Training sessions lasted between 15 and 45 min depending on the individual bird's ability to fly. Flight training continued until 45 birds were able to fly continuously for a minimum of 15 min at 16 km/h. During the latter portion of training and during the experimental phase, three birds were flown simultaneously in the wind tunnel.

Experimental design

After completing flight training, sparrows were administered 5 mg p,p′-DDT per kg per day in 70 to 110 µl of corn oil, delivered by gavage over three consecutive days. Half of the birds (n = 108) were given the DDT solution and the other half (n = 108) received an equivalent amount of corn oil (Fig. 1). No regurgitation of ingested doses was noted by investigators during the dosing procedures, which lasted several hours. After dosing, all birds rested for 1 d. On the fifth day, birds were assigned to treatment groups (Fig. 1): 15 dosed and 15 control birds comprised the unstressed (UNS) cohort, 59 dosed and 59 control birds were fasted only (FO), while 34 of both dosed and control sparrows were fasted and flown (FF). Each treatment group contained subgroups that were used to assess temporal effects of stressors over time intervals as long as 9 h, during which FF birds flew for up to 2.5 h.

thumbnail image

Figure 1. Dosing paradigm for white-crowned sparrows (Zonotrichia leucophrys). Fasted and flown groups comprised of birds in respectively labeled levels. Flight and fasting times are nominal. *{n} = sample size.

Download figure to PowerPoint

Sparrows were transported to the testing facility and divided into groups. The first group contained UNS birds, held in their cages with food and water. Unstressed birds were euthanized by decapitation throughout the experiment as near as possible to the same times as FF birds to evaluate hormonal fluctuations. Remaining sparrows were divided so that half remained in their cages and half were flown (FF). Groups were paired so that when FF sparrows began their 20-min flights, food was removed from the cage of a corresponding FO group. After 20 min, the first FF groups and the corresponding FO groups of birds were euthanized by decapitation.

During the second flight interval, flight times were reduced to 15 min, after which birds were returned to their cages, provided water, and allowed to rest for 45 min. Following this schedule and using the space constraints of the wind tunnel, it took 4 h of fasting to attain 1 h of flight and 9 h of fasting for 2.5 h of flight. The FF birds flew for one to nine intervals.

Upon completion of flight or fasting, birds were immediately decapitated. Sufficient personnel were present to allow simultaneous euthanasia of all birds comprising a given cohort. For each FF cohort that completed its designated flight duration, a corresponding FO cohort was similarly euthanized. Dissections were completed within 10 min of decapitation, and all tissues were frozen in liquid nitrogen to prevent postmortem metabolism. Brain, subcutaneous fat, abdominal fat, liver, kidney, and muscle were stored at −20°C until processed for residue analysis.

Residue analysis

Tissues were homogenized with 30 g of anhydrous sodium sulfate before extracting with an accelerated solvent extractor (Dionix ASE 200). Extracts were purified with 1.2% deactivated florisil 47 before quantitation with a Hewlett Packard 6890 gas chromatograph equipped with a 5973 mass selective detector. Separations were achieved with a 30 m × 0.25 mm DB-5 column. Analytes were identified by congruence of retention time and spectral similarity to National Institute of Standards and Technology traceable standards. Selected positive ions were monitored with the mass spectrometer as described in the U.S. Environmental Protection Agency test methods SW846 (www.epa.gov/epawaste/hazard/testmethods/sw846/online/index.htm). One spiked chicken liver and one blank was analyzed with each batch of 20 tissues. Recoveries for DDT (74 ± 4%), DDD (76 ± 5%), DDE (73 ± 4%), DDη (68 ± 3%), DDµ (75 ± 4%), and DDOH (65 ± 5%) were based on 80 spiked chicken livers. The limit of quantification for each analyte was 25 ng/g wet weight.

Statistical analysis

In general, residue data departed from normal distribution according to the Shapiro–Wilk test for normality. Statistical tests were performed on log-transformed residues under the assumption of normally distributed data. Two-way analysis of variance (2-way ANOVA) was used to compare mean analyte concentrations for birds receiving DDT doses and vehicle control. The total time each bird was stressed was not considered for this analysis. An analysis of covariance (ANCOVA) was used to determine effects of dose (DDT or vehicle), time fasted, time flown, and combinations thereof on residues in tissues. Unstressed, FO, and FF sparrow cohorts experienced the same treatment at time 0. Therefore, these three cohorts were combined for the time 0 residue data when evaluating temporal distributions of DDT and metabolites in sparrows. For some assessments, responses were evaluated with respect to DDT dosing. The group of birds receiving corn oil only are hereafter referred to as vehicle birds (VBs), and those receiving DDT are referred to as dosed birds. Statistical analyses were performed using JMP Start Statistics (SAS Institute) using p ≤ 0.05 to indicate significance.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

Mean tissue residues

No quantifiable residues of DDµ, DDη, and DDOH were found in sparrow tissues. While considering summaries of mean contaminant residues in each tissue type (Table 1), it should be noted that mean concentrations encompass samples from sparrows that were stressed for varying amounts of time. For example, the FO group contains birds that fasted from 0 min to 9 h, while the FF group contains sparrows which flew from 26 min to 2.5 h while fasting from 30 min to 10 h. Residue means were significantly higher (p < 0.0001) in all tissues of birds dosed with DDT (Table 2) compared with those that received corn oil vehicle only (VBs). Treatment effects were found in all tissues except muscle (p ≤ 0.05). Concentrations of DDE in tissues of dosed FO and dosed UNS groups were 30 to 90% of concentrations in the dosed FF group, but approximately two to four times greater than concentrations in VBs (Table 1). 1-Trichloro-bis(4-chlorophenyl)-ethane and DDD concentrations followed a similar pattern in the brain, kidney, and subcutaneous adipose (Table 1). Dose × treatment interactions were observed for DDT and DDD in brain and DDD in muscle, because concentrations in VBs were significantly lower than in dosed birds and did not change among treatment groups (Table 2). In DDT-dosed birds, mean DDD concentration in muscle of the FF group (0.198 mg/kg) was twice the concentration observed in the US group (0.099 mg/kg; see Table 1). Abdominal adipose tissue of dosed birds contained large fractions of DDT, DDD, and DDE among analyzed tissues. Approximately 70% of the DDT (Table 3), 55% of the DDE, and 45% of the DDD moved into adipose abdominal tissues. Brain and kidney generally contained less than 10% of total DDT, DDD, and DDE. Liver contained the greatest percentage of DDD, approximately twice the amount of either DDT or DDE, regardless of fasting or flight stress.

Table 1. Mean DDT, DDD, and DDE concentrationsa in selected tissues from stressed white-crowned sparrows (Zonotrichia leucophrys)
Sparrows dosed with corn oil vehicle only
 Abdominal adiposeBrainKidneyLiverMuscleSubcutaneous adipose
DDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDT
Unstressed                  
 Meanb0.0560.1970.0680.0230.0210.0230.0340.0340.0250.0210.0350.0250.0240.0290.0220.0380.1270.062
 95% LCLc0.0390.0870.0360.0190.0190.0180.0230.0240.0210.0150.0220.0150.0190.0210.0180.0320.0570.032
 95% UCL0.0810.4470.1300.0280.0230.0290.0490.0470.0310.0290.0560.0420.0300.0410.0260.0440.2790.118
 nd151515151515101010151515151515151515
Fasted
 Mean0.0410.1380.0480.0210.0220.0210.0250.0330.0290.0220.0300.0260.0210.0330.0210.0370.1200.038
 95% LCL0.0380.0800.0390.0190.0200.0190.0230.0270.0220.0170.0220.0180.0190.0230.0180.0330.0650.034
 95% UCL0.0450.2380.0590.0230.0250.0230.0270.0400.0380.0280.0410.0380.0230.0470.0230.0410.2200.043
 n282828282828282828262626272727282828
Flown and fasted
 Mean0.0550.3400.0660.0210.0250.0210.0290.0490.0310.0200.0580.0210.0210.0370.0230.0430.3510.056
 95% LCL0.0470.1860.0540.0190.0200.0190.0220.0340.0210.0170.0310.0180.0160.0220.0150.0380.1610.041
 95% UCL0.0630.6220.0810.0220.0310.0220.0380.0710.0450.0230.1090.0240.0280.0600.0340.0500.7640.076
 n181818171717181818181818181818181818
Sparrows dosed with DDT
 Abdominal adiposeBrainKidneyLiverMuscleSubcutaneous adipose
DDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDT
  • a

    (mg/kg wet wt).

  • b

    Geometric means within each treatment without considering time stressed for individual sparrows.

  • c

    LCL and UCL represent the lower and upper confidence levels, respectively.

  • d

    n = number of birds.

    DDT = 1,1,1-trichloro-bis(4- chlorophenyl)ethane; DDE = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene; DDD = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane.

Unstressed                  
 Mean0.4020.4553.7140.0240.0320.0620.1320.0880.7090.2420.1110.7650.0990.0590.4140.4130.58511.059
 95% LCL0.1300.1920.7830.0190.0220.0280.0850.0470.2800.0800.0450.3020.0470.0270.1960.2300.2696.460
 95% UCL1.2471.08117.6190.0310.0460.1370.2060.1641.7940.7350.2721.9370.2060.1300.8750.7431.27318.931
 n101010101010444101010101010101010
Fasted
 Mean0.6370.5197.5850.0260.0320.0810.0740.0500.4630.1960.0660.4070.1710.0720.7950.5410.41612.921
 95% LCL0.3940.3374.0500.0220.0260.0540.0540.0370.3310.1090.0450.2320.1260.0470.5880.3000.2335.984
 95% UCL1.0300.79914.2040.0310.0390.1190.1020.0660.6470.3510.0970.7150.2320.1101.0740.9750.74427.900
 n323232323232313131313131313131323232
Flown and fasted
 Mean0.9250.98513.1730.0430.0510.2290.1540.0960.8470.3660.1300.8820.1980.0890.7931.1271.34328.654
 95% LCL0.3850.4285.0170.0310.0360.1540.1030.0760.5680.1670.0730.4440.1630.0510.6430.6340.68614.179
 95% UCL2.2212.26534.5840.0600.0720.3430.2300.1211.2620.8060.2291.7530.2400.1530.9782.0032.63157.906
 n202020202020202020191919202020202020
Table 2. Results from two-way analysis of variance examining the effects of dose (DDT or vehicle only) and treatment (unstressed, fasted, or flown) on mean DDT, DDD, and DDE concentrations in white-crowned sparrows (Zonotrichia leucophrys)
 Whole modelaDoseTreatmentDose × treatmentb
  • DDT = 1,1,1-trichloro-bis(4-chlorophenyl)ethane; DDE = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene; DDD = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane.

  • a

    Mean values do not take into account the time stressed for each individual. Therefore, no time effect occurred.

  • b

    Dose × treatment indicates level of interaction between effects.

  • c

    p values are tabulated.

Abdominal adipose
 DDT<0.001c<0.0010.3640.487
 DDD<0.001<0.0010.4040.520
 DDE0.002<0.0010.0400.822
Brain
 DDT<0.001<0.0010.0070.003
 DDD<0.001<0.0010.0390.010
 DDE<0.001<0.0010.0130.224
Kidney
 DDT<0.001<0.0010.1610.196
 DDD<0.001<0.0010.0030.126
 DDE<0.001<0.0010.0010.460
Liver
 DDT<0.001<0.0010.3950.130
 DDD<0.001<0.0010.6200.407
 DDE0.002<0.0010.0200.858
Muscle
 DDT<0.001<0.0010.1050.088
 DDD<0.001<0.0010.1960.029
 DDE0.003<0.0010.4980.964
Subcutaneous adipose
 DDT<0.001<0.0010.1140.095
 DDD<0.001<0.0010.0480.190
 DDE<0.001<0.0010.0020.989
Table 3. Percentages of DDT, DDD, and DDE in abdominal adipose, brain, kidney and liver in white-crowned sparrows (Zonotrichia leucophrys) dosed with DDT
 Abdominal adiposeBrainKidneyLiver
DDDDDEDDTDDDDDEDDTDDDDDEDDTDDDDDEDDT
  • a

    Geometric means within each treatment without considering time stressed for individual sparrows.

  • b

    Standard error.

  • c

    LCL and UCL represent the lower and upper confidence levels, respectively.

  • d

    n = number of birds.

    DDT = 1,1,1-trichloro-bis(4-chlorophenyl)ethane; DDE = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene; DDD = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane.

Unstressed            
 Meana40.8752.4671.083.346.151.784.605.803.9351.1835.5923.22
 SEb11.357.297.471.240.650.390.921.571.1712.767.397.00
 95% LCLc18.6238.1656.430.904.881.022.812.711.6426.1721.109.50
 95% UCL63.1366.7685.725.777.422.546.408.886.2176.2050.0836.93
 nd777777777777
Fasted
 Mean50.4964.8274.876.6510.974.415.524.793.7137.3419.4217.01
 SE5.554.325.712.011.872.421.450.700.725.423.754.92
 95% LCL39.6056.3663.682.717.31−0.322.683.432.2926.7212.087.37
 95% UCL61.3773.2886.0510.5914.629.158.366.165.1347.9526.7626.65
 n282828282828282828282828
Fasted and flown
 Mean38.1957.0368.146.4512.965.328.866.217.2446.5023.8119.29
 SE7.026.417.021.923.372.273.391.622.866.985.005.02
 95% LCL24.4244.4754.382.696.350.882.213.031.6432.8114.019.46
 95% UCL51.9669.5881.9110.2119.579.7615.529.3812.8460.1833.6029.13
 n181818181818181818181818

Weight loss was observed in fasted birds and weight loss was correlated with fasting time for birds that fasted only (p < 0.001; r2 = 0.29). Weight loss also correlated with flight stresses (p < 0.001; r2 = 0.71) 41. Twelve to 19% weight loss was observed for birds that underwent extended flight (80–141 min) and fasting times (510–600 min). The cohort that fasted only for this period of time experienced −2 to +5% weight loss. Weight gain likely resulted from water consumption. For fasting times shorter than 500 min, weight loss was the same among those fasting and flying cohorts 41.

Neither fasting nor flight influenced the percentage of DDT, DDD, or DDE in the abdominal adipose. However, in other tissues percentages of DDT, DDD, and DDE were significantly lower in the UNS birds and highest in the FF sparrows (p < 0.05). 1,1-Dichloro-bis(4-hlorophenyl)ethene in liver was unique in that the greatest percentage, 36%, was in dosed-unstressed birds and least in the dosed-fasted-only sparrows, 19.4%.

Treatment effects over time

Among the FO sparrows, DDT administration (dose) was the dominant factor in explaining DDT, DDD, and DDE in all tissues (p ≤ 0.0299) with the exception of DDD in brain (p = 0.1294; Table 4). Residues were significantly greater in all tissues of dosed birds (p ≤ 0.0203), with the exception of DDD (p ≤ 0.1183) and DDE (p ≤ 0.0610) in brain. The lack of significance (p > 0.05) during fasting indicates that DDT, DDD, and DDE concentrations did not change relative to the length of time fasted. Lacking that difference, no interactions could occur between dose (DDT or vehicle) and time fasted.

Table 4. Analysis of covariance analysis of DDT, DDD, and DDE residues in fasted white-crowned sparrows (Zonotrichia leucophrys)
 Whole modelDoseTime fastedDose × time fasted
  1. The p valuesa are tabulated. Whole model values indicate significance of dose (DDT or vehicle) and length of time fasted on residue concentrations within each tissue. Dose and time fasted are effect tests. Dose × time fasted indicates level of interaction between effects.

  2. DDT = 1,1,1-trichloro-bis(4-chlorophenyl)ethane; DDE = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene; DDD = 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane.

  3. ap = probability of accepting a false hypothesis.

Abdominal adipose
 DDT<0.0010.00010.9350.710
 DDD<0.0010.00010.4110.719
 DDE0.0020.00680.7640.875
Brain
 DDT<0.001<0.0010.3750.336
 DDD0.1290.1180.1660.723
 DDE0.0080.0610.4020.645
Kidney
 DDT<0.001<0.0010.0500.708
 DDD<0.001<0.0010.8400.254
 DDE0.0300.0200.7900.530
Liver
 DDT<0.001<0.0010.7710.460
 DDD<0.001<0.0010.7080.872
 DDE0.0040.0150.2280.969
Muscle
 DDT<0.001<0.0010.5340.059
 DDD<0.001<0.0010.7440.288
 DDE0.006<0.0010.8820.147
Subcutaneous adipose
 DDT<0.001<0.0010.9500.881
 DDD<0.001<0.0010.7000.401
 DDE0.0040.0030.4870.394

DDT administration (dose, time fasted and flown) affected DDT, DDD, and DDE concentrations in all tissues (p ≤ 0.0366) except DDE in abdominal adipose (p ≤ 0.0626), subcutaneous adipose (p ≤ 0.0613), and liver (p ≤ 0.0652: Table 5). Similar to linear associations observed for FO birds, differences between toxicant concentrations in most tissues of the FF groups were primarily related to dose. Notably, DDT and DDD in brain were linearly associated with flight time and the dose × flight interaction (p ≤ 0.04). DDT concentrations increased in brain (p ≤ 0.0373) and kidney (p ≤ 0.0260), but DDT concentrations in livers were negatively associated with time flown (p ≤ 0.0110). Concentrations of DDD similarly increased with time flown in sparrow brains (p ≤ 0.0094) and kidney (p ≤ 0.0211), and DDE accumulated in the kidney (p ≤ 0.0075). Unlike results for FO sparrows, dosing alone was insufficient to explain DDE concentrations in organs as the duration of stress was increased.

Table 5. Analysis of covariance of DDT, DDD, and DDE in fasted and flown white-crowned sparrows
 WholeDoseTimeTimeDose×Dose×Time flown×Dose × time flown
model flownfastedtime flowntime fastedtime fasted×time fasted
  1. The p valuesa are tabulated. Whole model values indicate significance of dose group, length of time flown, and length of time fasted on residue concentrations within each tissue. Dose, time flown, and time fasted are effect tests. Dose × time flown, dose × time fasted, time flown × time fasted, and dose × time flown × time fasted indicate level of interaction between effects.

  2. DDT = 1,1,1-trichloro- bis-(4chlorophenyl) ethane; DDE = 1,1-dichloro-2,2-bis (4 chlorophenyl) ethylene; DDD = 1,1-dichloro-2,2-bis (4 chlorophenyl) ethane.

  3. ap = probability of accepting a false hypothesis.

Abdominal adipose
 DDT<0.0010.0020.9480.7920.7720.5320.7410.998
 DDD<0.0010.0420.8550.9370.9720.6600.4150.781
 DDE0.0630.4750.4770.4560.6800.9630.6490.414
Brain
 DDT<0.0010.0790.0370.9730.0110.6670.1400.046
 DDD<0.0010.6100.0090.4010.0060.4360.0770.059
 DDE0.0010.9240.1330.7110.0790.7170.4040.323
Kidney
 DDT<0.001<0.0010.0260.8890.6670.8620.0360.932
 DDD<0.0010.0030.0210.8680.7930.5600.0140.586
 DDE0.0050.5100.0070.5280.7330.3340.0080.957
Liver
 DDT<0.001<0.0010.0110.9530.1680.2610.0060.234
 DDD<0.001<0.0010.3140.5240.6520.7970.0680.278
 DDE0.0650.1420.7250.5510.0810.6400.9130.097
Muscle
 DDT<0.001<0.0010.3000.5970.4220.8630.3480.424
 DDD<0.0010.0030.3920.1340.1990.1060.2040.069
 DDE0.0370.6040.65030.2200.2920.1430.5260.646
Subcutaneous adipose
 DDT<0.001<0.0010.1480.6870.1100.4700.3850.148
 DDD<0.001<0.0010.1310.5910.2280.4500.1340.134
 DDE0.0610.3810.0700.9790.6920.9980.2290.820

Redistribution of DDT, DDD, and DDE in sparrows increased toxicant concentrations in organs with increasing time stressed. This relationship is demonstrated in the dose × time flown interaction in DDT and DDD in brain. Similarly, fasting was ineffective in mobilizing metabolites into the kidney and liver. This finding directly contrasts effects of flying. The difference in slopes along the fasting and flight axes of response surfaces (DDT, DDD, and DDE in liver and DDT in kidney) indicated significant interactions in time flown × time fasted (Figs. 2–5). Vehicle birds had background concentrations that infrequently exceeded quantification limits for our methods. Therefore, the metabolite concentrations of the vehicle birds were not dependent on, and did not increase with duration of stress.

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Figure 2. Model of fasting and flight effects on 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) concentration in liver of flown white-crowned sparrows (Zonotrichia leucophrys) dosed with DDT. Whole model estimates significance of model and p values represent the probability of accepting a false hypothesis. Model based on 23 residue data entries. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

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thumbnail image

Figure 3. Model of fasting and flight effects on 1,1-dichloro-2,2-bis(4-chlorophenyl)ethylene (DDE) concentration in liver of flown white-crowned sparrows (Zonotrichia leucophrys) dosed with DDT. Whole model estimates significance of model and p values represent the probability of accepting a false hypothesis. Model based on 23 residue data entries. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

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thumbnail image

Figure 4. Model of fasting and flight effects on DDT concentration in liver of flown white-crowned sparrows (Zonotrichia leucophrys) dosed with DDT. Whole model estimates significance of model and p values represent the probability of accepting a false hypothesis. Model based on 23 residue data entries. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

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Figure 5. Model of fasting and flight effects on DDT concentration in kidney of flown white-crowned sparrows (Zonotrichia leucophrys) dosed with DDT. Whole model estimates significance of model and p values represent the probability of accepting a false hypothesis. Model based on 23 residue data entries. [Color figure can be seen in the online version of this article, available at wileyonlinelibrary.com]

Download figure to PowerPoint

Both DDE and DDD concentrations in liver increased with increasing time fasted and flown (Figs. 2, 3). However, the increase due to time flown caused a much more dramatic effect when fasting times were short. With short fasting times, flight time rapidly decreased DDT concentrations in the liver (Fig. 4). However, when stress involved longer fasting and flight times, DDT concentrations increased in liver, although at a much reduced rate compared to DDE and DDD. The pattern for DDT in the liver (p = 0.0376) and kidney (p = 0.0425) of dosed flown birds is distinctly different. DDT concentrations in the kidney increased with flight time regardless of the amount of time fasted (Fig. 5). Fasting appears to increase DDT concentration initially, but concentrations decreased with prolonged fasting.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

Residue metabolism in the fasted sparrows

The classic model of fasting involves three phases 48. Phase I involves a rapid loss in body mass from carbohydrate metabolism. Body mass loss is slowed in phase II as triglycerides, having a high mass specific energy content, become the major source of fuel. Phase III begins with another rapid loss of body mass as lipids become exhausted and protein catabolism becomes the major source of energy. The length of phase I varies and depends on individual bird species and their ability to tolerate extended fasts. Barn owls (Tyto alba) are in phase I for less than a day 49, quail for 2 to 3 d 50, and penguins (Pyoscelis adeliae) up to a week 51. Based on their body size, white-crowned sparrows are unable to fast for long periods 52. In addition, increasing DDT, DDD, and DDE concentrations in lipid pools indicates utilization of the pools for energy 3. Mobilization of lipid reserves defines phase II of the classic model of fasting. Given the weight loss of up to 19% observed in fasted birds 41, phase II was likely to have begun, but phase III had not been reached. We base the initiation of phase II on the fact that all birds still had fat layers just over the breast muscle. The presence of subcutaneous fat pads over the breast muscle in all birds indicates that phase III had not begun in birds.

Nutritional status in birds, as measured by lipid reserves, is critical in the distribution of lipophilic contaminants such as ΣDDT. In the FO sparrows, analyte concentrations in all tissues were different between the dosed and VB groups, with the exception of DDD in brain. The lack of correlation between residues in tissues and time fasted (Tables 4, 5) suggests that even though fasting depletes fat deposits 3, 6, 53, the depletion is not rapid enough to release DDT and its transformation products from lipid pools. This phenomenon has been observed in cases where other chlorinated pollutants are retained by lipid pools that serve as the sole energy source in developing embryos 54. During periods of rapid lipid metabolism, lipophilic contaminants will be mobilized into other tissues, including the brain 3, 6, 55–58. Therefore, it is more likely that more rapid or thorough depletion of fat reserves would mobilize ΣDDT more widely within dosed sparrows.

Residue mobilization in flown sparrows

The sizeable fractions of DDT, DDE, and DDD found in abdominal adipose suggest that these toxicants had moved into this reasonably stable lipid depot by the time that flight or fasting stress began. Contaminant movement into the brain, which has a more stable lipid pool, is well known to be impaired but not precluded by the blood–brain barrier 59, 60. Thus, retention of DDT in the abdominal adipose is a clear indication of the extent to which the dose had come to equilibrium in tested tissues.

Fasted only and FF groups are similar in that whole model significance is driven by the administered DDT (that is, dose). Flying affected residues in brain, kidney, and liver tissues (Figs. 2–5). Clear indications exist that flying increased the absolute concentration of DDT and its metabolites in tissues of dosed sparrows (Table 1), and a similar, but insignificant, trend appears to be followed for birds that fasted without physical stress. One important aspect of the data interpretation involves the temporal relation of fasting and flight. In general, birds that fasted for short periods flew for short intervals. Similarly, birds that fasted for longer periods flew for longer intervals. Therefore, stressors within in the FF groups are not completely independent.

The ubiquitous nature of p,p′-DDE in the environment and metabolic processes explains the fact that birds from dosed FF groups contained p,p′-DDE concentrations in adipose tissue and liver were not different from those in the corresponding VB groups (Table 1). Sparrows in this study were taken from the wild and can be assumed to carry background DDE concentrations. The ad libitum feeding would allow birds to retain fat stores, as well as lipophilic contaminants, that were present at capture. Furthermore, lipid stores are thought to be used for energy early in flight that could release recently deposited DDE, thereby returning DDE in lipid stores to predosing concentrations. Plasma free fatty acids resulting from lipid hydrolysis increase rapidly within the first hour of flight 61, 62. Therefore, the FF group should more rapidly mobilize lipid pools relative to the FO group. Also, concentrations exhibited high variance for the dosed FF group. The elevated concentration of DDT and DDD in all dosed birds relative to VBs further supports the hypothesis that DDE in adipose tissue represented environmentally derived toxicant concentrations.

This is the first study to demonstrate that flying time enhances the movement of chlorinated pesticides into the brain well beyond the concentrations induced by fasting alone (Table 5). DDT and DDD concentrations in the brain increased with duration of flight, but DDD did not exhibit an interaction between fasting and flight duration. These data effectively demonstrate the potential for brief flights to enhance the mobilization of ΣDDT from brain tissue.

Implications of differential transfer based on fasting or physical activity are significant for chlorinated hydrocarbon transfer to critical organ tissues. This is especially true of the brain, which is highly susceptible to impairment when chlorinated hydrocarbons cross the blood–brain barrier. Our observation that DDT and its metabolites move into brain tissue of sparrows during flight is important because DDT and related compounds are neurotoxic 1. As little as 10 mg/kg DDT or 20 mg/kg DDD in the brain is diagnostic of death. In contrast, adipose tissue may contain hundreds of mg/kg of ΣDDT with no adverse effects 1. The summation of concentrations for DDT and its metabolites in brains of VBs, dosed UNS birds, and FF birds were <0.07 mg/kg, 11.2 mg/kg, and 24.7 mg/kg, respectively. The demonstrated mobilization of these toxicants into brain at concentrations that are within the range of lethality is strong evidence that flight stress should be incorporated into any evaluation of DDT mobilization during migration. Inclusion of such data may significantly alter the outcome of risk assessments for migratory birds.

DDT, DDD, and DDE concentrations increased in the kidney relative to flight duration. Kidneys filter large amounts of blood and may receive 20% or greater of the cardiac output. However, they are not typically fatty organs and do not accumulate large quantities of lipophilic contaminants. Clearance rates of DDT, DDD, and DDE are not significantly different from muscle, liver, brain, heart, gonad, or abdominal adipose 63. Therefore, increased concentrations of DDT and its metabolites in the kidneys of exercised sparrows may result from increased plasma concentration and increased cardiac output.

DDT in the liver represents the only analyte that was negatively correlated with flight duration. Lipid in the liver represents a readily available lipid pool that should release organochlorine compounds upon energetic stress. Also, liver and muscle are more metabolically active and have the greater ability to metabolize DDT 64. The role of increased mobilization is clearly demonstrated by the fact that DDT loss from sparrow liver was greater for birds experiencing longer flight time relative to fasting duration. Increased metabolite:DDT ratios have been observed in liver of grackles (Quiscalus quiscula) dosed with DDT 65. In addition, chlorinated hydrocarbons induce hepatic cytochrome P450 enzymes in other avian species 66, 67 and mammals 68, 69 that might affect tissue burdens.

Comparisons to previous DDT metabolism studies

The absence of DDµ, DDη, and DDOH in sparrow tissue does not readily support DDT metabolic pathways that have been hypothesized by prior research in laboratory rodent species. Based on rat studies, Peterson and Robison 70 suggested that DDT was metabolized into DDE and DDD. 1,1-Dichloro-bis(4-chlorophenyl)ethylene (DDE) and DDD were then metabolized into DDµ, which cascaded through a series of metabolites including DDMS, DDη, DDOH, finally to be excreted as DDA (2,2-bis(4-chlorophenyl)acetic acid). Based on mouse and hamster studies 71, DDT can be primarily converted into DDD that is transformed directly into DDA and excreted. 1-Chloro-bis(4-chlorophenyl)ethylene) (DDµ) and DDE exist as minor pathways at best, the former a derivative of DDD and the latter of DDT. Fawcett and King 72 injected 14C-labeled DDT, DDD, DDE, and DDµ intraperitoneally into rats and Japanese quail (Coturnix japonica) and traced the pharmacokinetics of each compound. Their findings differ from the above studies in that the metabolism of DDT to DDA does not generate DDµ as an intermediate product. Rather, DDA is produced from DDµ, but DDµ is not a direct metabolic product of DDT. They also found that the metabolic pathways are similar between rats and quail. However, the clearance of metabolites is much quicker in rats, indicating that quail have a much poorer ability to metabolize DDT and the other metabolites into hydrophilic DDA. Quail were able to excrete DDµ as rapidly as the rat. In addition, rats produced DDOH as a metabolite of DDT, unlike the quail.

There are several explanations for the lack of DDµ, DDη, and DDOH in the present study. First, metabolites may have been present but below detection limits. Sparrows in our study received 5 mg DDT/kg body weight. Other metabolite studies dosed animals with single doses of 100 ppm DDT or greater. Because our sparrows received 20 times less DDT, metabolite quantities may have been below quantitation limits. Second, species-specific metabolism of DDT has been recorded for mammals 73. It is therefore possible that the white-crowned sparrow transforms DDT to these metabolites sparingly if at all. Third, some metabolites may have been short-lived intermediates that were quickly metabolized and/or excreted.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES

Increasing concentrations of DDD and DDE in tissues (Table 5; Figs. 2–5) clearly demonstrate that physical activity transforms and mobilizes DDT from lipid reserves, allowing movement into highly perfused organs, specifically liver and brain. These data indicate the need to incorporate actual physical activity when evaluating the effect of migratory stress on contaminant mobilization and distribution in birds. Simple caloric stress does not represent the situation experienced by migrating wildlife in the environment. Mobilization of DDT into the brain is significant as a potential route for neurotoxicity, which would be particularly detrimental during migration.

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  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. REFERENCES
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