The effect of NPS on the development of the blowfly, compared to the control population. the control population

Day 1- Results

Initial observations regarding larval length at this time point did not appear to vary substantially. No larvae stood out as particularly larger or smaller than those of other sample populations. Larval lengths ranged from 3.05mm to 6.92mm (Table 9), showing a natural and expected variation.

When tested using a mixed effects model comparing lengths across the different diets, statistically significant differences were present (F=2.4397, P=0.0018). Data set was normally distributed and uniform in variance.

Larval Length

Further analysis (Figure 85) showed the majority of the data at day 1 to not be significantly different, with one exception. Larvae feeding on 5EAPB were significantly shorter (mean=4.4806mm, SD=0.7727mm) than those feeding on Paracetamol (mean=5.2395mm, SD=0.9988mm), (p<0.01), Pink Panther (mean=5.2125mm, SD=0.9335), (p=0.0119) and Benzofury Blue (mean=5.1528mm, SD=0.9764), (p=0.0354).

However this is not significantly different from the control population (mean=4.8825mm, SD=0.5371) so it is not as important within this investigation as the focus remains on comparison with non-drugged larvae. A number of outliers can be seen within the population feeding on 5EAPB so it can be speculated that there are similar range of uneven data resulting in a lower mean.

165 | P a g e Larval Weight

As observed with larval length, no obvious visual differences, other than natural variance, were observed across the test populations at day 1 (Figure 86). Larval weights ranged from 0.0005g to 0.0032g (Table 9). Testing with a mixed effects model returned no statistically different results (F=1.2597 , P=0.2227). A few outliers are observed within the population but it is hypothesized that this is due to an uneven spread of data as they are not beyond what would be expected. The observed range of data is approximately equal across all drug diets.

A scatter graph of all data from individual larval samples displays both weight and length and shows positive correlation between the two variables. This is expected because as larval length increases, as does larval weight, comparison was drawn at a later experimental time point after further exposure to the research drugs.

Principle component analysis was carried out using PC 1 and PC 2, together explaining 100% of sample variance, shown in Figure 88. Hoteling’s 𝑇² statistic at a 5% significance level is included to provide information on potential outliers or those statistically very different. Data points are spread across the plot showing no pattern, no clustering is observed and numerous overlapping values are shown.

166 | P a g e Figure 85: Box plot showing larval length for day 1 across all samples (n=40).

167 | P a g e Figure 86: Box plot showing larval weight for day 1 across all samples (n=40).

Larval We igh t ( g)

168 | P a g e Table 9: The minimum, maximum and mean lengths and weights (including standard deviation) at 24 hours (n=40).

Drug Min Max Mean Standard Deviation % Standard Deviation

5EAPB Length (mm) 3.05 6.51 4.4806 0.773 17.25

Weight (mg) 0.5 2.8 1.4 0.600 42.86

6APB Length (mm) 3.75 6.91 4.6403 0.921 19.85

Weight (mg) 0.8 2.9 1.5 0.500 33.33

6+5APB Length (mm) 3.75 6.91 4.8158 0.903 18.76

Weight (mg) 0.6 3.2 1.7 0.700 41.18

AMT Length (mm) 3.64 6.91 4.823 0.937 19.44

Weight (mg) 0.8 2.6 1.5 0.500 33.33

Benzocaine Length (mm) 3.64 6.11 4.8565 0.621 12.79

Weight (mg) 0.7 3.2 1.7 0.600 35.29

Benzofury-Blue Length (mm) 3.75 6.91 5.1528 0.976 18.95

Weight (mg) 0.7 3.1 1.7 0.600 35.29

Benzofury-Green Length (mm) 3.75 6.91 4.7463 0.919 19.35

Weight (mg) 0.7 2.6 1.7 0.500 29.41

Benzofury-Beige Length (mm) 3.7 6.43 4.7653 0.855 17.94

Weight (mg) 0.8 3 1.6 0.500 31.25

Blow Length (mm) 3.7 6.92 4.6983 0.942 20.04

Weight (mg) 0.6 2.9 1.5 0.600 40.00

Caffeine Length (mm) 3.7 6.92 4.688 0.893 19.04

Weight (mg) 0.6 2.8 1.5 0.500 33.33

Control Length (mm) 4.01 5.92 4.8825 0.537 11.00

Weight (mg) 0.6 2.3 1.5 0.500 33.33

Ivory Wave Length (mm) 3.7 6.91 4.837 0.887 18.33

Weight (mg) 0.5 3.1 1.7 0.600 35.29

Paracetamol Length (mm) 3.75 6.91 5.2395 0.999 19.06

Weight (mg) 0.7 3.1 1.6 0.600 37.50

Pink Panther Length (mm) 3.75 6.91 5.2125 0.934 17.91

Weight (mg) 0.5 2.9 1.6 0.500 31.25

Synthacaine Length (mm) 3.75 5.93 4.892 0.622 12.71

Weight (mg) 0.5 3.1 1.5 0.600 40.00

MDA Length (mm) 3.746 6.914 4.6403 0.921 19.84

Weight (mg) 0.7 2.8 1.5 0.500 33.33

169 | P a g e Figure 87: Scatter graph showing relationship between length and weight data observed at 24 hours.

170 | P a g e Figure 88: PCA plot showing all drug additions at 24 hours.

171 | P a g e

Day 1: Discussion

At 24 hours, most sample populations showed no significant differences for both larval length and weight. One exception to this was length of larvae from the experimental population given 5EAPB, which was shown to be significantly different to Paracetamol, Benzofury Blue, and Pink Panther diets but not to the control. It is hypothesized that this is due to an uneven spread of data across the sample population as the length range is in line with the range observed across other diets and a number of outliers are identified in the higher larval length region, for this drug only. Principle component analysis agreed with this analysis, showing no population clustering and no patterns emerging. This was expected at such an early time point, only 24 hours after introduction to the diet; any expected effects on development would become obvious after further exposure to the drugs of interest. Based on the results shown, at 24 hours after exposure to one of several Novel Psychoactive Substances or common adulterants, no significant differences were observed. This means that larval age at this point would not be incorrectly estimated due to drug presence and PMI estimations would not be affected.

For ease, on all following sampling days, significant differences (p < 0.001) are shown with a star on box plots.

172 | P a g e using a mixed effects model (Figure 85), comparing the lengths across the different diet, significant differences were shown (F=22.28, P<0.0001). Further analysis to define where the differences lay showed a larger number of differences than seen previously at 24 hours. Data analysis will focus mainly on the differences found between the control and other diet populations as this is of most forensic importance. No larvae feeding on any of the researched diets were shown to be significantly shorter than the control population (mean= 6.811mm, SD=0.3325mm) at 48 hours. Larvae feeding on a number of diets were, however, shown to be significantly longer than those within the control population, 5EAPB, Benzofury Beige, Benzofury Blue, Caffeine, Pink Panther (P<0.01) and Blow (p=0.0117). Significance values are shown, the reader is directed to Table 10 for data values. All other treatments were shown not to cause significant changes within the larval length. As expected, a large number of populations were shown to be significantly different from one and other. Notably 5EAB, Benzofury Beige and Benzofury Green have a larger range of data points.

Larval Weight

Preliminary observations when sampling larval weight showed an obvious development increase in comparison with 24 hours prior, especially when comparing to the control

173 | P a g e population. Four populations immediately showed to be larvae of a larger weight, 5EAPB, Benzofury Beige, Benzofury Blue and Pink Panther. Across all populations, the larval weight ranged from 0.0024g (AMT) to 0.0141g (Pink Panther, Benzofury Blue and Benzofury Beige). A mixed effects model was initially used to determine if results were significantly different. (F=43.666, P<0.0001). This was investigated further using Tukey pairwise tests. Four drug diets were found to be significantly different to the control population. 5EAPB was the only diet found to increase the larval weight significantly (P=<0.01), which had also previously been found to significantly increase larval length.

Three diets were found to have significantly reduced weights; AMT (P=<0.01), Paracetamol (P=<0.01) and Blow (P=<0.01). Blow had previously been found to have significantly increased in length at this time point. All other treatments were shown not to cause a difference deemed significant. The range of weight measurements seems larger for most excluding the control population. Data set was normally distributed and uniform in variance.

Principle component analysis was carried out using PC 1 and PC 2, which accounted for 100% of the explained variance (Figure 91).

As explained earlier in this chapter, Hoteling’s 𝑇² statistic at a 5% significance level is shown, the outliers defined by this agree with those selected during the pairwise testing.

Development data is beginning to show clustering of individual variables, although with overlap. The control samples can be seen in lower centre of the plot and arguably, the data does appear to already be spread across the plot, suggesting that after 48 hours exposure, the drug additions are having an effect on development.

174 | P a g e Figure 89: Box plot showing larval length for day 2 across all samples (n=40).