Survival of T. molitor larvae exposed to tested chemicals.
Eugenol caused time and concentration-dependent lethality of T. molitor larvae (Fig. 1). In the case of three methods: injection, filter paper tests, and immersion mortality exceeded 50% within 7 days, for the majority of concentrations. During the inhalation and oral toxicity tests, mortality did not exceed 50%. Therefore, no LT50 values were calculated, or they were very high. For the majority of obtained LT50 values, they were lower than 3 days, with the lowest ones being observed in the case of filter paper tests, for 200 µL/mL and immersion tests for 100 and 300 µL/mL. On the other hand, the longest periods were calculated for immersion tests, 50 µL/mL, injection test for 50 µL/mL and oral toxicity test for 100 µL/mL. trans-Cinnamaldehyde caused time-dependent lethality of T. molitor larvae, too (Fig. 2). In the majority of cases the effects depended on concentration, too. There were only insignificant differences between the adjacent concentrations. In the case of oral toxicity tests, only the highest concentrations resulted in mortality exceeding 50% within 7 days. Interestingly, for filter paper tests toxicity above 50% was obtained very quickly, for all concentrations higher than 15 µL/mL. That led to a very steep increase in toxicity and a rapid increase in mortality approaching 100% within the first two days of the experiment. Cinnamaldehyde showed the lowest toxicity of the tested compounds. In many cases, the lethality did not reach 50% after 7 days of exposure (Fig. 3). Curiously enough, the middle concentration – 100 µL/mL caused the highest toxicity in filter paper tests, higher than the lower and the higher concentrations. Oral and inhalation toxicity of cinnamaldehyde was very low for all tested concentrations.
Table 1 shows the calculation of the statistical significance of LC50 values obtained in all types of tests, for the three tested substances. For eugenol, injection tests were significantly different from all other methods, except the filter paper test. Also, there was no statistically significant difference between both contact toxicity tests: immersion and filter paper. No statistically significant differences were found between any of the measured LC50 values for trans-cinnamaldehyde. Within cinammaldehyde tests, statistically significant differences were observed between injection vs. immersion and immersion vs. filter paper tests.
Table 1. Comparison of LC50/7d values for T. molitor larvae, calculated for eugenol, trans-cinnamaldehyde and cinnamaldehyde in five toxicity tests.
|
Eugenol
|
trans-cinnamaldehyde
|
Cinammaldehyde
|
Comparison
|
Significance
|
p-value
|
Significance
|
p-value
|
Significance
|
p-value
|
Injection vs. Inhalation
|
****
|
<0,0001
|
ns
|
>0,9999
|
-
|
-
|
Injection vs. Immersion
|
*
|
0,0111
|
ns
|
>0,9999
|
****
|
<0,0001
|
Injection vs. Filter paper
|
ns
|
0,6803
|
ns
|
>0,9999
|
ns
|
0,6803
|
Injection vs. Oral toxicity
|
****
|
<0,0001
|
ns
|
>0,9999
|
-
|
-
|
Inhalation vs. Immersion
|
**
|
0,0065
|
ns
|
>0,9999
|
-
|
-
|
Inhalation vs. Filter paper
|
****
|
<0,0001
|
ns
|
>0,9999
|
-
|
-
|
Inhalation vs. Oral toxicity
|
ns
|
>0,9999
|
ns
|
>0,9999
|
-
|
-
|
Immersion vs. Filter paper
|
ns
|
0,2737
|
ns
|
>0,9999
|
****
|
<0,0001
|
Immersion vs. Oral toxicity
|
**
|
0,0061
|
ns
|
>0,9999
|
-
|
-
|
Filter paper vs. Oral toxicity
|
****
|
<0,0001
|
ns
|
>0,9999
|
-
|
-
|
Statistical significance between administration rout of each compound was determined using One-way ANOVA with Tukey's multiple comparisons test and is indicated by p ≤ 0.05 (*), p ≤ 0.01 (**) p ≤ 0.001 (***) or p ≤ 0.0001 (****), ns – not significant, “-“ – could not be calculated
Of the three tested substances, usually eugenol showed the higher toxicity, expressed as the lowest LC50/7d values, when the same way of application was used (Table 2). On the other hand, cinnamaldehyde never had higher toxicity than eugenol nor trans-cinnamaldehyde.
Table 2. Comparison of toxicity of tested substances to T. molitor larvae in particular tests
Compared substances
|
Statistical significance
|
p-value
|
Injection (a)
|
eugenol vs. trans-cinnamaldehyde
|
*
|
0,0228
|
eugenol vs. cinnamaldehyde
|
****
|
<0,0001
|
trans-cinnamaldehyde vs. cinnamaldehyde
|
***
|
0,0009
|
Inhalation (b)
|
eugenol vs. trans-cinnamaldehyde
|
****
|
<0,0001
|
Immersion (a)
|
eugenol vs. trans-cinnamaldehyde
|
****
|
<0,0001
|
eugenol vs. cinnamaldehyde
|
****
|
<0,0001
|
trans-cinnamaldehyde vs. cinnamaldehyde
|
****
|
<0,0001
|
Filter paper (a)
|
eugenol vs. trans-cinnamaldehyde
|
ns
|
>0,9999
|
eugenol vs. cinnamaldehyde
|
ns
|
>0,9999
|
trans-cinnamaldehyde vs. cinnamaldehyde
|
ns
|
>0,9999
|
Oral toxicity (b)
|
eugenol vs. trans-cinnamaldehyde
|
****
|
<0,0001
|
Statistical significance was determined using either (a) One-way ANOVA with Tukey's multiple comparisons test or (b) Student's t-test and is indicated by p ≤ 0.05 (*), p ≤ 0.001 (***) or p ≤ 0.0001 (****), ns – not significant. Substances in bold showed lower LC50/7d values in the compared pairs
Probit analysis
In all performed tests - except trans-cinnamaldehyde in contact toxicity test on filter paper and cinnamaldehyde in inhalation toxicity test- we observed mortality of tested T. molitor larvae (Table 3). The 7-day LC20 and LC30 values were rather low for the majority of methods of administration (10 and 8 of the obtained values were lower than 30 µL/mL, respectively). The correlation coefficient in the majority of tests was higher than 0.7, which indicates a high or very high positive correlation between the concentration of substances and mortality. Only in one case (cinnamaldehyde, oral toxicity test) we did not find the correlation. However, the B coefficient was higher than 1 only in some cases. which indicates a slow increase of toxicity, with increasing concentrations of the tested substances. In consequence, the values of LC95 were very high. When considering LC50 values, the highest toxicity of eugenol and cinnamaldehyde was observed in tests on filter paper, in the case of trans-cinnamaldehyde – in the injection test. For eugenol, the toxic effects, estimated on the basis of LC50 values were as follows: filter paper > injection > immersion > oral toxicity > inhalation. For trans-cinnamaldehyde, the order of toxicity was as follows: injection > > filter paper > inhalation > immersion > oral toxicity. For cinnamaldehyde, we observed the highest toxicity for filter paper > injection > immersion > oral toxicity. To sum up, the highest toxicity seems to be obtained in filter paper and injection. No high toxicity was obtained during inhalation tests and when the substances were added to food.
Table 3. Probit analysis of five types of toxicity tests of eugenol, trans-cinnamaldehyde and cinnamaldehyde, performed for T. molitor
eugenol
|
Type of toxicity test
|
Equation
|
R2
|
LC20/7d
|
LC30/7d
|
LC50/7d
|
LC95
|
|
LC [µL/mL]
|
95% CI Lower –Upper [µL/mL]
|
LC [µL/mL]
|
95% CI Lower –Upper [µL/mL]
|
LC [µL/mL]
|
95% CI Lower –Upper [µL/mL]
|
LC [µL/mL]
|
95% CI Lower –Upper [µL/mL]
|
injection
|
y = 0.2253x + 4.8023
|
0.65
|
1.32
x 10-3
|
0.06 x 10-3- 2.724
x 10-2
|
3.464
x 10-2
|
1.68 x 10-3 - 0.71624
|
7.70539
|
0.37271 - 159.30106
|
-
|
-
|
inhalation
|
y = 0.5366x + 3.2633
|
0.90
|
45.009
|
10.310 - 196.484
|
178.133
|
40.805 -
777.633
|
1731.374
|
396.608 -
7558.233
|
-
|
-
|
filter paper
|
y = 0.4694x + 4.9729
|
0.76
|
3.620
x 10-2
|
7.26 x 10-3 - 0.181
|
0.165
|
3.299 x 10-2-
0.821
|
2.010
|
0.403 - 10.025
|
5159.491
|
1034.5531 -
25731.247
|
immersion
|
y = 1.2346x + 2.6445
|
0.89
|
15.365
|
8.224 -
28.709
|
28.732
|
15.377 - 53.684
|
80.855
|
43.274 - 151.072
|
2075.545
|
1110.841 -
3878.040
|
oral toxicity
|
y = 1.1327x + 2.8199
|
0.90
|
15.194
|
7.767 -
29.72278
|
28.956
|
14.802 -
56.64291
|
84.081
|
42.982 - 164.478
|
2381.477
|
1217.407 -
4658.615
|
trans-cinnamaldehyde
|
injection
|
y = 1.1129x + 4.1047
|
0.92
|
1.064
|
0.502 -
2.257
|
2.078
|
0.980 -
4.407
|
6.281
|
2.962 -
13.321
|
201.768
|
95.138 -
427.904
|
inhalation
|
y = 1.4886x + 2.7336
|
0.91
|
7.824
|
4.549 -
13.457
|
13.122
|
7.629 -
22.570
|
30.851
|
17.937 -
53.063
|
450.558
|
261.953 -
774.957
|
filter paper
|
|
|
|
|
|
|
14.7*
|
|
|
|
immersion
|
y = 1.3216x + 2.4544
|
0.94
|
18.633
|
10.296 - 33.721
|
32.928
|
18.195 - 59.592
|
84.406
|
46.640 - 152.755
|
1616.759
|
893.359 - 2925.933
|
oral toxicity
|
y = -4.987x + 17.976
|
0.54
|
589.925
|
431.336 -
806.821
|
509.550
|
372.568 - 696.895
|
399.975
|
292.450-
547.032
|
187.155
|
136.842 -
255.966
|
cinnamaldehyde
|
injection
|
y = 1.0455x + 3.6736
|
0.83
|
2.50305
|
1.213 -
5.163
|
5.316
|
2.577 -
10.967
|
18.468
|
8.953 -
38.096
|
917.743
|
444.999 -
1893.126
|
inhalation
|
No mortality stated
|
filter paper
|
y = 0.7549x + 4.0979
|
0.93
|
1.198
|
0.460 - 3.117
|
3.156
|
1.213 - 8.215
|
15.661
|
6.017 - 40.760
|
2381.399
|
914.956 -
6198.181
|
immersion
|
y = 0.9463x + 2.587
|
0.93
|
44.058
|
18.704 -
103.782
|
96.157
|
40.821 -
226.507
|
349.395
|
148.326 -
823.031
|
19993.240
|
8487.585 -
47095.806
|
oral toxicity
|
y = 0.1888x + 3.9299
|
0.01
|
16.063
|
0.293 -
880.581
|
740.158
|
13.501 -
40576.843
|
4161. 85 x 102
|
7591.601 -
2281.6 x 104
|
1757.6
x 1011
|
3206.01 x 109 -
9635.5 x 1012
|
R2 – correlation coefficient, LCn – value of lethal concentration for n=20, 30, 50, and 95% of tested population. * - the value is only an estimation due to the very small difference between 0 and 100% mortality
Morphological changes
The representative T. molitor control larva is shown in Figure 4A. The coloration of larvae is uniform, and no morphological malformations were noticed. T. molitor larvae treated with selected compounds from cinnamon bark at appropriate concentrations are shown in Figures 4B-I. Significant differences were noted among larvae treated with eugenol, trans-cinnamaldehyde or cinnamaldehyde at concentrations of 50, 100, 200 and 300 µL/mL compared to the control samples. At lower concentrations, no changes in the morphology of the tested organisms were noted.
Injection
When the compounds were administered by injection, changes were seen as early as 24 hours after the beginning of the experiment. After injecting insects with compounds at a concentration of 50 µL/mL, changes in the pigmentation of their bodies were observed, especially above the injection site (Fig. 4B), while after administration of compounds at a concentration of 200 µL/mL, a complete change of pigmentation to black compared to the control proving necrosis of the larval tissues was observed (Fig. 4C).
Filter paper tests and contact toxicity
In T. molitor larvae, morphological changes have been observed 24 hours since the start of the experiment and after administration of compounds from cinnamon bark only at the two highest tested concentrations - 200 and 300 µL/mL. Tissue necrosis was also noted (Fig. 4E, F). At lower tested concentrations, no changes in the morphology of the tested insects were observed (Fig. 4D).
Oral toxicity tests
After ingestion by T. molitor larvae of food with the addition of eugenol, trans-cinnamaldehyde or cinnamaldehyde, changes in body pigmentation were mainly observed 48 hours after the start of the experiment. Attempts to molt and body deformations were also noted (Fig. 4G, H).
Inhalation
After inhalation of eugenol or trans-cinnamaldehyde, changes in pigmentation and morphology of larvae were observed only at concentrations of 200 and 300 µL/mL. Changes were observable 72 hours after the start of the experiment. In addition to the darker coloration of the body of the examined invertebrates, attempts to molt were also observed (Fig. 4I).