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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
Deviations:
yes
Remarks:
besides effects on mortality and body weight also various biomarkers were observed.
GLP compliance:
not specified
Remarks:
not reported in publication
Analytical monitoring:
not required
Details on sampling:
not required
Vehicle:
no
Details on preparation and application of test substrate:
no details on preparation of test soil were provided despite of the nominal concentrations (0, 0.1, 1, 10 and 50 mg DP/kg dw) and composition of soil (artificial soil of pH 6.0 ±0.5, containing 10% dried cow manure, 20% kaolin clay, and 70% industrial sand).
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
Animal treatment: Earthworms Eisenia fetida were purchased from a farming factory in Jurong, Jiangsu Province, China. Healthy adult earthworms (60-days old, 300 - 400 mg, and well-developed clitellum) were used for all exposure experiments. Artificial soil of pH 6.0 ±0.5, containing 10% dried cow manure, 20% kaolin clay, and 70% industrial sand was applied to raise the earthworms. A water content of 35% (w/w) in the artificial soil was used in exposure period according to OECD 207. Before exposure experiment, the earthworms were acclimated in the artificial soil, without any DP for one week. Then E. fetida were added to contaminated soil of different DP concentrations (0, 0.1, 1, 10, 50 mg/kg) in wide-mouth bottles (1 L) supplied with continuous light source at 20 ±1 °C for 14 days. Cow manure which was dried at 60 °C and ground to pass through a 2 mm sieve was added as the food. Each treatment contained 3 replicates and each bottle kept 12 earthworms. Water was sprayed into the room of the bottle regularly to keep air humidity at 80%, and the bottles were covered with plastic film that had been punched with small holes using needles. Prior to all further tests, the earthworms were extracted from the culture media and placed in enclosed petri dishes for 24 h on moist filter paper at 20 ±1 °C to force them to purge their gut contents.
Study type:
extended laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
14 d
Test temperature:
20 ±1 °C
pH:
soil pH: 6.0 ±0.5
Moisture:
A water content of 35% (w/w) in the artificial soil was used during the exposure period according to OECD 207; air humidity was kept at 80%.
Details on test conditions:
Cow manure(dried at 60 °C and ground to pass through a 2 mm sieve) was added as food. Each treatment contained 3 replicates and each bottle kept 12 earthworms. Water was sprayed into the room of the bottle regularly to keep air humidity at 80%, and the bottles were covered with plastic film that had been punched with small holes using needles.
Nominal and measured concentrations:
0, 0.1, 1, 10, 50 mg/kg
Reference substance (positive control):
no
Key result
Duration:
14 d
Dose descriptor:
LC50
Effect conc.:
> 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
and body weight
Duration:
14 d
Dose descriptor:
LC10
Effect conc.:
> 50 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
and body weight
Details on results:
Body weight and death rate: During the exposure period, no significant treatment-related changes in death rate and body weight were found (p > 0.05). Acute toxicity of DP was therefore very low. Even for highest exposure concentration (50 mg/kg), which is much higher than the detected concentration in environmental matrices, the mortality rate was still lower than 10%.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
none, as effects on mortality and body weight were higher than the maximum tested concentration (50 mg/kg soil dw)

Antioxidant defenses induced by DP: SOD activity and GSH level in E. fetida were measured after a 3-, 7-, and 14-day exposure to characterize the changes of antioxidant defenses induced by DP. For SOD activity, no significant change was found in four treatment groups following a 3-day exposure. After a 7-day exposure, significant increases (p < 0.05) were found in all treatment groups compared to control. However, after a 14-day exposure, the SOD activity in the highest concentration DP-treated group was inhibited. For GSH, a 3-day exposure of DP significantly increased the GSH levels in E. fetida. After a 7-day exposure, the GSH levels were further elevated. Similar to SOD activity, on day 14, the GSH levels were inhibited as compared to controls, which showed no change throughout the experimental period. Biochemical responses of organisms to environmental stress are often used as early warning indices of pollution in the environment and enzymatic activities have been regarded as biomarkers of environmental pollution. These enzymes include antioxidant activities and they protect cells against adverse effects of ROS. Generally, cells will reduce oxygen to water through their electron transport chains and protect themselves from ROS damage through the use of enzymes such as SOD. SOD and GSH are the common antioxidant defenses which represent two general classes of antioxidant defenses: enzymatic and water-soluble reductants, respectively. Along with the increase of exposure period, both parameters were first increased, and then inhibited. The initial increase indicated that the exposure of DP resulted in the formation of reactive oxygen species (ROS), which then stimulated the biosynthesis of SOD to protect the cells against oxidant damage. However, upon exposure to DP for longer times, SOD activity and GSH level in the high concentration group decreased compared with those of the control on day 14. This phenomenon was assumed to be due to the excessive generation of ROS, which has been shown previously to inhibit SOD activity and GSH level. These results showed that DP could induce antioxidant defenses in the earthworm, and allows to hypothesize that oxidative damage was occurring at greater concentrations as well as longer exposures to DP.

Oxidative damage induced by DP: Two types of oxidative damage for lipids and nucleic acids were characterized by the measurement of MDA and 8-OHdG, respectively. In the high exposure group, significant increases of both MDA and 8-OHdG were found. MDA is an important indicator of lipid peroxidation, which is regarded as one of the major peroxidation products under ROS stress conditions. It is known that antioxidant defenses can inactivate ROS. When ROS generation overloads antioxidant defenses, free radicals can act on lipids and nucleic acids and alter their structure and function. The highest MDA levels were found on day 3, and the level decreased on day 7, but increased again on day 14. The results could be explained by scenario that when earthworms were exposed to DP, DP increased the ROS levels causing an increase in lipid oxidation and thus MDA. Then the antioxidative systems were likely activated which reduce ROS and thus there was a decrease in oxidized lipid and thus MDA levels. Along with the increase of exposure time was the increased generation of ROS; at this point the anti-oxidative system could not effectively eliminate ROS. Thus, MDA levels increased again. This hypothesis is validated by the results of SOD and GSH activities. However, in the case of DNA damage, there was a closer relationship between ROS and 8-OHdG. Accumulation of highly reactive ROS induces subsequent damaging events including formation of 8-OHdG. Moreover, increased 8-OHdG indicates that its ability to pair with adenine, instead of cytosine, leading to G:C into T:A transversions if the damage is not repaired prior to DNA replication. In this study, the responses of 8-OHdG at different exposure levels showed that the highest 8-OHdG levels were found on day 7. Based on our data, we suggest that the repair of DNA damage is delayed and thus the observed increase in 8-OHdG levels.

AChE activity induced by DP: As shown by data, a 7-day exposure of DP significantly increased AChE levels. The highest AChE activities were found in the 1 mg/kg DP group. On day 14, the AChE activities were inhibited in two high-concentration groups. It is known that AChE is a key enzyme in the biological neural conduction and performs important functions in neurotransmission. The changes of AChE activities induced by DP indicated that DP exposure could cause neurotoxicity.

DNA damage induced by DP: The comet assay has been proven to be a useful tool to identify genotoxic effects of chemicals on invertebrates. In this study, we chose both tail DNA and olive tail moment (OTM) from the comet assay to evaluate DNA damage in E. fetida. DP exposure increased the levels of tail DNA and OTM, however, only long-term exposure (14-day) or high concentrations (50 mg/kg DP) significantly increased the levels of DNA strand breaks (p < 0.05). The results showed that DP poses a potential genotoxic threat to earthworms. In our previous studies, we found that DP could not induce DNA strand breaks in mouse liver cells. These differences sug gest that DP may exhibit species specificity in its toxicity. The potential value of the comet assay in environmental monitoring has been reported by Mitchelmore et al. They suggested that DNA strand breaks, which were measured by the comet assay, can act as a biomarker of genotoxicity. However they also emphasized that this approach should be combined with the use of other biomarkers. Combined with the results of enzyme activity and oxidative damage, the data from the comet assay suggests that earthworms experience more stress from the DP at higher concentrations during long time exposure, as would be predicted.

Alteration of transcriptomic profiles induced by DP: A total of 5824 genes were compared and analyzed between control and treatment groups. The DEGs for treatment groups were chosen based on the fold change and statistical p-value. On day 7, a total of 1464, 1362, 1750, and 1512 DEGs were found in 0.1, 1, 10, and 50 mg/kg DP-treatment groups, respectively. After a 14-day exposure, the number of DEGs in 0.1, 1, 10, and 50 mg/kg DP-treatment groups were 1243, 1368, 1134, and 1094, respectively. The DEGs found in all treatment groups were then extracted and compared. The results showed that 425 and 395 DEGs were all altered on day 7 and day 14, respectively. No obvious changes in the number of DEGs were found in the different concentration groups and exposure period. Similar results were found in the transcriptomic profiles of mouse exposed to DP. For the range of fold changes, most DEGs in DP-treated groups changed by ±1.50 - 1.99 fold (41.6 - 53.0%) and ±2.00 - 4.00 fold (31.8 - 39.7%). qRT- PCR was applied to verify the results from RNA-Seq. For most genes, the fold changes from qRT- PCR analysis were very similar to those determined by RNA-Seq analysis, but overall tended to show less pronounced changes. In order to identify the biological significance of DEGs, they were mapped into the KEGG pathways and GO terms. The significantly changed KEGG pathways and GO process were identified on the basis of the criterion that has 4 or more DEGs. These pathways mainly involved the large subunit ribosomal protein, myosin heavy chain, chitinase and cytochrome c oxidase subunit. 109 and 201 DEGs in 24 KEGG pathways were found on day 7 and day 14, respectively. On day 14, a total of 63, 95, 130, and 201 DEGs in 24 KEGG pathways were identified in 0.1, 1, 10, and 50 mg/kg DP-treatment groups, respectively. These results demonstrate that the increase of exposure concentration and time period can significantly enhance the changes in transcriptomic profiles. These GO terms mainly refer to gene expression (GO: 0010467), metabolic process (GO: 0008152), cellular process (GO: 0009987) and so on. Like the KEGG pathway analysis, similar dose-dependent and time-dependent effects on the DEGs were found, indicating that DP caused the changes in transcriptomic profiles of earthworm. To further identify the effects on transcriptomic profiles of DEGs, the DEGs found in all treatment groups and exposure period were chosen. A total of 98 DEGs were identified. However, only 31 DEGs could be functionally annotated, which were used for further analyses. We found that there were significant differences in gene expression profiles between the two exposure periods (day 7 and day 14). But no significant dose-dependent changes were found. After a 14-day exposure, most DEGs were up-regulated, especially from EW1 R1P07 G05 to EW2 R1P09 C06. These DEGs all involve chitinase, which has been shown to relate to cuticle formation. In addition, some other genes such as BB998284, EW1 F1P06 F02 and EW1 F1P01 G01, were also up-regulated. These genes relate to signaling transduction and stress response. As such, these results indicated that DP exposure caused changes in transcriptomic profiles related to neurotoxicity.

Systemic analysis of DP toxicity: Based on the results of biochemical and RNA-Seq analyses, the possible toxicological effects and mechanism of DP actions on earthworm E. fetida were systematically characterized by combining the genotype and phenotype. The toxicity of DP could be divided into two fields, which were oxidative damage and neurotoxicity. In the transcriptomic analysis, we found genes coding flap endonuclease (Gene ID:Lr CHECF 23H01 M13R), NADH dehydrogenase(ubiquinone) Fe-S protein (Gene ID:Lr PAHCF 01E12 M13R), peroxiredoxin (GeneID: EW1 F1P09 B09), and catalase (Gene ID: Lr JV2CF 59C05 SKplus) were all significantly altered. The pathway analysis indicated that the biological processes related to cellular stress response, responses to oxidative stress, and antioxidant enzymes expression were significantly altered. Besides these pathways, we found the NADP–NADPH redox cycling and some signaling pathways were significantly altered by DP exposure. It has been shown that the changes of NADP–NADPH redox cycling could promote ROS production. These results indicate that DP might cause stress responses as well as oxidative stress. It has been demonstrated that some traditional toxic contaminants can enhance oxidative stress and several cellular and metabolic alterations, including lipid peroxidation of membranes or DNA damage. In the biochemical analysis for this study, DP exposure significantly altered SOD activity as well as GSH levels in earthworms, and increased MDA and 8-OHdG levels. The increases in oxidative stress and damage may be attributed to multiple modes of action identified by genomic analysis. Based on these results, we deduce that oxidative stress might be one mechanism of DP actions. The conclusion is similar with our previous study, in which we found oxidative damage might be the mechanism of action of DP toxicity in mouse livers. Oxidative stress is closely related to function of the nervous system. In this study, we found changes in AChE activity, which is an indicator of neurotoxicity from DP. In our transcriptomic analysis we also found that changes in genes and pathways related to neuronal function. For example, acyl carrier protein (Gene ID: EW1 F1P04 C04) is involved in modulation of GABAergic transmission; polypyrimidine tract-binding protein (Gene ID: BP524431), and semaphoring (Gene ID: EW1 F1P01 G01) have been shown to be responsible for neuronal differentiation; hexosaminidase (Gene ID: EW1 F2P14 D06) plays important roles in the degradation of ganglioside GM2 in neurons. The pathway analysis further indicates that DP exposure could change biological processes related to neurological dysfunction, calcium binding, and signal transduction. Besides the direct changes in neurological dys-function, the alterations of calcium binding and signal transduction are involved in the neuronal damages. Calcium binding proteins and calcium signals, as intracellular messengers, play important roles in all aspects of the cell’s function. It has been shown that oxidative stress can affect calcium binding and its homeostasis. Furthermore, disruption of calcium homeostasis can result in neuronal diseases. We predict that calcium binding and homeostasis alterations might be important reasons for the DP-induced neuronal damage, however, this hypothesis needs to be addressed with additional analyses.

Conclusions: This study is the first to demonstrate DP toxicity on a soil animal. Based on the results from biochemical and transcriptomic analyses, our data indicate that the acute toxicity of DP is very low. However, DP exposure can induce oxidative damage to earthworms. In addition, we found that DP exposure can change the AChE activity of earthworms as well as affecting neuronal damage-related genes and pathways. These results provide an insight into toxicological effects and mechanism of actions of DP on earthworms, and should be very useful for the risk assessment of DP in soil ecosystems. However, almost all biological indicators did not show dose-response relationship.

Validity criteria fulfilled:
yes
Conclusions:
Body weight and death rate: During the exposure period, no significant treatment-related changes in death rate and body weight were found (p > 0.05). Acute toxicity of DP was therefore very low. Even for highest exposure concentration (50 mg/kg), which is much higher than the detected concentration in environmental matrices, the mortality rate was still lower than 10%.
Executive summary:

Based on the results from biochemical and transcriptomic analyses, our data indicate that the acute toxicity of DP is very low. No mortality and no changes in body weight were observed in any dose group up to 50 mg/kg soil dw. However, DP exposure may induce oxidative damage to earthworms. In addition, it was found that DP exposure can change the AChE activity of earthworms as well as affecting neuronal damage-related genes and pathways. These results provide an insight into toxicological effects and mechanism of actions of DP on earthworms. However, almost all biological indicators did not show dose-response relationship.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:

Description of key information

Dechlorane Plus did not induce mortality at concentrations tested (0, 0.1, 1, 10 and 50 mg/kg soil dw) in an acute study to earthworms (eisenia fetida). Thus, the LC10 and LC50 were > 50 mg/kg soil dw.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:
50 mg/kg soil dw

Additional information

Zhang et al. published in 2014 their findings on a study, they have performed similar to OECD207 guideline on acute toxicity to earthworms (eisenia fetida), in which nominal DP concentrations of 0, 0.1, 1, 10 and 50 mg/kg soil dw were applied.

During the exposure period, no significant treatment-related changes in death rate and body weight were found (p > 0.05). Acute toxicity of DP was therefore very low. Even for the highest exposure concentration (50 mg/kg), the mortality rate was still lower than 10%. Thus, the LC10 and LC50 both were > 50 mg/kg soil dw.

Based on the results from biochemical and transcriptomic analyses, the data reported indicate that DP exposure may induce oxidative damage to earthworms. In addition, it was found that DP exposure can change the AChE activity of earthworms as well as affecting neuronal damage-related genes and pathways. These results provide an insight into toxicological effects and mechanism of actions of DP on earthworms. However, almost all biological indicators did not show dose-response relationship, and thus these findings have to be seen with caution.