Effects of Clonazepam on developmental stages, larval weight, and length measurements of Calliphora vicina
Description
Benzodiazepines such as clonazepam can cause overdose-related deaths when used either therapeutically or illicitly. In cases where routine forensic methods are unable to determine the time and cause of death due to advanced decomposition, these drugs may affect the development of necrophagous insects and lead to deviations in PMI estimations. Investigating these potential effects on necrophagous insects provides valuable comparative data for minPMI estimates. The present study investigate the effects of different clonazepam concentrations on the larval and pupal developmental stages, larval weight, and length measurements of C. vicina. Experimental studies were conducted under controlled laboratory conditions. Laboratory studies were initiated with newly hatched first-instar larvae and continued with checks at six-hour intervals until adult emergence. For the larval weight measurements presented in the study, 10 larvae were randomly selected from each experimental group and weighed (in milligrams) on a precision balance. The subjects were then subjected to a boiling water bath, and their body lengths (mm) were meticulously recorded. The posterior spiracles were examined under an Olympus BX51 stereo microscope in order to ascertain the developmental stages. The present dataset supplies reference data for the determination of PMI in cases of suspected death due to clonazepam by means of C. vicina.
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The data was obtained from experiments under controlled laboratory conditions. Environmental conditions were standardized at 23 ± 1°C, 65–70% relative humidity, and a photoperiod of 10:14 h (L:D). Clonazepam (Rivotril®, 2 mg tablets) approval was granted by the Turkish Medicines and Medical Devices Agency. A single tablet was weighed and the active pharmaceutical ingredient concentration calculated for preparing stock solution. Subsequently, 14.5833 g of the tablet was adequately ground and dissolved in 200 ml of pure water using a magnetic stirrer until complete dissolution was achieved. From this 200-ml stock solution, 10, 15, 25, 50, and 100-ml aliquots were incorporated into 300 g of minced fresh bovine lung and homogenized to ensure uniform drug distribution. For comparison, a control group was prepared using 300 g of bovine lung without any pharmaceutical agent. 250 newly hatched first-instar larvae were transferred to each experimental group (n = 250 per treatment). The complete experimental design included six treatment groups (five drug concentrations plus control). To determine the effects of clonazepam on the developmental stages of C. vicina, each stage was monitored continuously and recorded through adult emergence. The length and weight of 10 randomly selected larvae from each treatment group were measured and recorded every six hours until pupation. For each sampling point, larvae were removed and weighed, measured for body length then photographed for morphological documentation. Larvae were killed in boiling water for 30 seconds and preserved in 80% ethanol for subsequent analysis. Larval instar determination was performed by examinations of posterior spiracles using an Olympus BX51 stereo microscope. IBM SPSS Statistics version 25.0 was used to analyze the effects of clonazepam on larval weight, length, and developmental stages. Developmental stages were analyzed using one-way MANOVA. Levene’s and Kolmogorov–Smirnov tests were applied to test the assumptions of homogeneity of variances and normality, respectively. Post hoc Tukey HSD tests were performed to identify differences between groups. The significance level was set at 0.01 with a 99% confidence interval. Two-way MANOVA was conducted to analyze larval weight and length. Levene’s and Kolmogorov–Smirnov tests were again applied to assess variance equality and normal distribution. Because this assumption was not met, attempts were made to adjust the variances, but these were unsuccessful, necessitating the use of non-parametric analyses. Accordingly, Kruskal–Wallis tests were conducted. Paired post hoc Mann–Whitney U tests were performed against the control group, with Holm correction applied for multiple comparisons. To control the family-wise error rate (FWER) at the 5% level, Holm correction was applied to the five simultaneously generated p-values using SPSS.
Institutions
- Jandarma ve Sahil Guenlik Akademisi
- Eskisehir Teknik Universitesi