1,6,7,8,9,14,15,16,17,17,18,18-dodecachloropentacyclo[12.2.1.16,9.02,13.05,10]octadeca-7,15-diene

Toxicity to birds

Link to relevant study record(s)

Reference

Endpoint:

toxicity to birds

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2013

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Not a classical study design and for a subchronic toxicity study only very few parameters were recorded (mortality, liver weight). No information on behaviour changes or body weight, nor information on other organs than liver. Rather high levels of DP in the control groups tissue are questioning the study performance.

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the present study, male common quails (Coturnix coturmix) were consecutively exposed to commercial DP-25 by gavage for 90 days at 1, 10, and 100 mg/kg/d dosages. Concentrations of DP isomers in liver, muscle, and serum were determined after exposure. Liver enzyme activities involved in xenobiotic biotransformation processes and oxidative stress were measured, as well as the contents of glutathione and maleic dialdehyde.

GLP compliance:

not specified

Dose method:

gavage

Vehicle:

yes

Details on preparation and analysis of diet:

oral administration of 0, 1, 10 and 100 mg/kg bw/d of DP dissolved in cornoil.

Test organisms (species):

Coturnix coturnix japonica

Details on test organisms:

A total of 60 male quails (6-8 weeks of age, with an average weight of 125 g) were obtained from a farm in Beijing, China. The animals were raised individually in suspended, unattached cages in a mass air-displacement room at 20-26 °C. After 14 days acclimatisation, they were randomly separated into four groups.

Limit test:

no

Total exposure duration (if not single dose):

90 d

Post exposure observation period:

none

No. of animals per sex per dose and/or stage:

15 males/group (only males were used)

Control animals:

yes, concurrent no treatment

Nominal and measured doses / concentrations:

0, 1, 10 and 100 mg/kg bw/d dose groups were applied

Details on test conditions:

A total of 60 male quails (6-8 weeks of age, with an average weight of 125 g) were obtained from a farm in Beijing, China. The animals were raised individually in suspended, unattached cages in a mass air-displacement room at 20-26 °C. After 14 days acclimatisation, they were randomly separated into four groups and orally administered com oil with commercial DP-25 dosages of 0, 1, 10, and 100 mg/kg/d for 90 days, respectively. All quails were euthanized by decapitation after the experiment. Livers were quickly perfused in situ with ice-cold 0.9% w/v sodium chloride until the entire organ became pale. Livers were removed from the bodies and rinsed with ice-cold 0.9% w/v sodium chloride, and were then weighed, cut into pieces, frozen in liquid nitrogen, and stored at -80 °C until chemical and enzyme activity analyses. Pectoral muscle and serum were also immediately collected, frozen in liquid nitrogen, and stored at -80 °C for chemical analyses. All experiments and procedures were approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences.

Details on examinations and observations:

GC/MS analysis: The DP isomers and their dechlorinated analogs in the samples were identified by an Agilent 6890 gas chromatograph (GC) system coupled with a 5975C mass spectrometer (MS) using electron capture negative ionization (ECNI) in the selective ion-monitoring (SIM) mode. Target chemicals, along with surrogate and internal standards, were separated using a 30 m DB-XLB capillary column (0.25 mm i.d., 0.25 µm thickness; Agilent Technologies). Ions were scanned as foliows: m/z 653.8 and 651.8 for DP isomers (syn-DP and anti-DP), m/z: 618 and 620 for anti-Cl11-DP and syn-
Cl11-DP, m/z 584 and 586 for anti-Cl10-DP, and m/z 79 and 81 for the PBDE congeners. All GC/MS peaks were calculated by their peak area. Semi-quantitative determination was achieved for syn-Cl11-DP by reference to the response of anti-Cl11-DP on GC/MS.
Sample Preparation: For liver and muscle, 1 g of tissue was homogenized with ashed anhydrous sodium sulfate, spiked with recovery standard BDE-181, and Soxhlet extracted with 50% acetone in hexane for 48 h. A portion of the extract was used for gravimetric lipid determination. The remainder was subjected to gel permeation chromatography (GPC) and eluted for lipid removal with dichloromethane/hexane (l:l, v/v). Fractions from 90 to 280 mL containing target compounds were collected and concentrated to 1 mL under a gentle stream of nitrogen. The extract was further purified on a silica column (i.d. = 1 cm) packed with 8 cm neutral silica and 8 cm acidified silica, and eluted with 40 ml of hexane/dichloromethane (v/v = 1:1). The elute was concentrated and redissolved in 270 µL of isooctane, and then spiked with an internal standard (BDE-128) before instrumental analysis.
Serum extraction and purification were performed as per previous research [20], with some modification. In brief, 2 mL of serum was spiked with surrogate (BDE-181). The protein was denatured with 1 mL of hydrochloric acid (6M) and 6 mL of 2-propanol, and the mixtures were shaken vigorously. Both DP and its metabolites were twice extracted with 6 mL of hexane/methyl tert-butyl ether (MTBE) (v/v = 1:1) mixture. The combined organic extracts were washed with potassium chloride solution (1%; 3 mL) and concentrated to dryness under N; for gravimetric lipid weight determination. Extracts were redissolved with 6 mL of hexane. The aqueous phase compounds were separated from neutrals by partitioning with potassium hydroxide (0.5 M in 50% ethanol). The aqueous phase was re-extracted with hexane (6 mL) for complete extraction. Neutral phase was treated with 2 mL of concentrated sulfuric acid to remove lipids, and subsequently purified by silica column as described above. The elutes were concentrated to near dryness under
N2, redissolved in 270 µL of isooctane, and spiked with a known amount of intemal standard (BDE-128) before instrumental analysis.
QA/QC: Quality assurance was conducted by analyses of procedural blanks, blank spikes, and sample duplicates. Procedural blanks were used in parallel with the treated samples for every extraction, with a procedural blank performed for each batch of eleven samples. Recoveries of surrogates for
all tissue and serum samples ranged from 70.4% to 90.6%, with an average of 80.9%. The relative standard deviation (RSD) of duplicates among triplicate samples was less than 20%. The method detection limit (MDL) was set as the average value plus 3-fold standard deviation for analytes, which were detected in the procedural blanks (n = 6). For analytes not detected in the blanks, a signal-to-noise ratio of ten was set as MDL. The MDLs of syn-DP, anti-DP, and anti-Cl11-DP were 27.85, 46.12, and 0.68 ng/g lw in muscle and liver, and 0.20, 0.054, and 0.042 ng/ml in serum, respectively.
Enzyme Activity Analyses: Liver microsomal alkoxyresorufin O-dealkylase assays (resorufin assays) were performed, where alkoxyresorufins are metabolized by O-dealkylases to a resorufin product. The alkoxyresorufins used (and name of their assays) were 7-ethoxyresorufin (EROD), 7-methoxyresorufin (MROD), and 7-pentoxyresorufin (PROD). Hepatic microsomal erythromycin N-demethylase (ERND) and 7-benzyloxy-4-trifluoromethylcoumarin debenzylase (BFCD) assays were also performed. Detailed information on microsomal preparation and enzyme activity assays are shown in Supplementary Methods. Enzyme activities of CAT and SOD, and the content of GSH and MDA were detected using commercial kits, with detailed information on sample preparation and methods shown in Supplementary Methods.

Details on reproductive parameters:

not assessed

Reference substance (positive control):

no

Duration (if not single dose):

90 d

Dose descriptor:

LD50

Effect level:

> 100 mg/kg bw/day (nominal)

Conc. / dose based on:

test mat.

Basis for effect:

mortality

Duration (if not single dose):

90 d

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Conc. / dose based on:

test mat.

Basis for effect:

organ weights

Mortality and sub-lethal effects:

Quail mortality during the 90 day exposure experiment was zero. At the end of the trial, body and liver weight were not significantly altered between the control and exposure groups.

Effects on reproduction:

not assessed

Results with reference substance (positive control):

not applied

Further details on results:

Concentrations of syn-DP, anti-DP, syn-Cl11-DP, and anti-Cl11-DP were detected in all examined samples, as well as in the control group. For example, average syn-DP and anti-DP in muscle of the control group were 5800 ±2000 and 1000 ±330 ng/g lipid weight (lw), respectively, which may partly relate to the widespread distribution of DP in the environment, including the exposure room and feedstuff.
Accumulation of DP isomers was observed in the exposed quails. The highest concentrations of syn-DP occurred in liver of the 10 mg/kg/d group, being nearly ten times greater than observed in the 1 mg/kg/d group and three times greater than observed in the 100 mg/kg/d group. For anti-DP, however, the highest concentrations occurred in the 1 mg/kg/d group. Additionally, anti-DP concentrations decreased with increasing DP exposure dosage. These findings indicate that isomer-specific and concentration-related pharmacokinetics of DP existed in the exposed quails. However, the mechanism for the observed dose-dependent accumulation of DP isomers is not understood based on the present data.
Hepatic Enzyme Activities: Research has shown that DP isomers are widespread in birds, such as herring gull eggs, peregrine falcon eggs, and bald eagle plasma. However, little information exists on the adverse effects of DP isomers on bird species. Cytochrome P450s have critical roles in the metabolism of endogenous chemicals and process the burden of drugs and other xenobiotics. In the present study, the enzyme activities of EROD, MROD, PROD, and BFCD were lower in the 1, 10, and 100 mg/kg/d groups compared to that in the control group. The activities of PROD significantly decreased in the exposed groups compared to the control. This may have adverse effects on quails since PROD activity is an indicator of CYP2B in mammals, which is, in turn, involved in the metabolism or inactivation of several endogenous chemicals such as steroid and gonadal hormones. For ERND, the 10 and 100 mg/kg/d dosed groups showed higher enzyme activities than the control group. ERND activity is mainly CYP3A-dependent in mammals. Forms of CYP3A are among the most abundant and important xenobiotic-metabolizing CYP enzymes, mediating the metabolism of numerous xenobiotics, including pollutants, pesticides, and food contaminants, as well as endogenous compounds such as steroid hormones and bile acids. In our study, the increased ERND activity in the 10 and 100 mg/kg/d dosed groups might alter the detoxification and metabolism activities of CYP3A to endogenous and exogenous compounds, and partly contribute to the biological effects of DP.
Defense system antioxidants are composed of enzymatic or nonenzymatic molecules that exist in extra- and intra-cellular compartments. These molecules act upon reactive oxygen species through reduction reactions in order to render them harmless. In the present study, the activities or contents of GSH and antioxidants CAT, SOD, and MDA were determined. An increasing trend was observed among the exposed groups compared to the control group. A significant increase occurred in the 10 and 100 mg/kg/d groups for CAT (p = 0.009 and 0.004, respectively) and in the 1 mg/kg/d group for SOD (p = 0.006), suggesting that DP exposure may induce oxidative stress. With H2O2; as its substrate, CAT is involved in protection against oxidative reaction and the level of SOD reflects the body’s ability to scavenge oxygen free radicals. The increase in SOD levels in the exposed groups may be a manifestation of the body’s stress reaction.

Reported statistics and error estimates:

All statistical analyses were conducted using SPSS 16.0 for Windows (SPSS Inc., Chicago, IL). Statistical significance in concentrations and other experimental statistics was determined using one-way analysis of variance (ANOVA) followed by Fisher’s least significant difference (LSD)test. Normal distribution and homogeneity of variance were confirmed before ANOVA. Significant difierences were set at p < 0.05.

Validity criteria fulfilled:

not specified

Conclusions:

No toxicity or growth effects on organs were seen in the study with 90 days exposure up to 100 mg/kg bw DP/d. No NOEL was derived by the authors but based on reported information, the highest dose applied in this study can be seen as No-Observed-Adverse-Effect-Level, as no sign of toxicity were seen. Only accumulation was seen but was not dose-dependent (increase from 1 mg/kg bw/d dose group to 10 mg/kg bw/d dose group but decrease to 100 mg/kg bw/d dose group again). As no recovery period was applied, it is unclear whether DP was really accumulated or whether tissue concentrations in muscle liver and serum was only an effect of exposure. Enzyme activity was altered but not dose dependent and the meaning of this remained unclear.

Description of key information

No signs of toxicity were seen up to a dose of 100 mg/kg bw/d, delivered to quails for 90 days of exposure. No NOEL/NOAEL was derived by the authors.

Additional information

In a subchronic toxicity studies with birds (quails) no toxicity or growth effects on organs were seen following 90 days exposure up to 100 mg/kg bw DP/d via gavage. No NOEL was derived by the authors but based on reported information, the highest dose applied in this study can be seen as No-Observed-Adverse-Effect-Level, as no sign of toxicity were seen. Only accumulation was seen but was not dose-dependent (increase from 1 mg/kg bw/d dose group to 10 mg/kg bw/d dose group but decrease to 100 mg/kg bw/d dose group again). As no recovery period was applied, it is unclear whether DP was really accumulated or whether tissue concentrations in muscle liver and serum was only an effect of exposure. Enzyme activity was altered but not dose dependent and the meaning of this remained unclear. In summary, no toxic effects to quails were seen despite the high exposure (100 mg/kg bw/d). However, as the study was not following guidelines and only mortality, body weight and liver weight were assessed, besides liver enzyme activity and some other parameters, the study is considered to be of limited validity. Although mainly accumulation of DP was in focus of the study, the results were not dose dependent and thus of no use for bioaccumulation assessment.