Registration Dossier

Ecotoxicological information

Toxicity to other aquatic organisms

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although not a standard OECD method the suitability of the method is well documented in peer-reviewed scientific literature (Olive, P.L., Banáth, J.P., Durand, R.E., 1990. Heterogenity in radiationinduced DNA damage and repair in tumor and normal cells measured using the ‘‘comet’’ assay. Radiat. Res. 122, 86–94). Thus, the results are considered reliable with restrictions.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Comet assay with T. thermophile
Principles of method if other than guideline:
The comet assay is a well-established and sensitive approach to measure DNA damage in single cells. The assay is widely accepted as a standard method applied to genotoxicology, human biomonitoring, and ecological monitoring. In the comet assay test, treatment of T. thermophile cells with a toxicant results in a concentration-dependent increase in DNA damage.
GLP compliance:
not specified
Remarks:
not indicated in the publication
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
The test was performed with two controls (T. thermophile in culture with and without 0.5% DMSO) and different concentrations of DP solutions (2.4, 12, 60, 300, 1500 µg/L) for 30 min. The DP solutions were diluted by distilled water from DP stock solution containing DMSO, with the highest final concentration of DMSO 0.5% in 1500 µg/L.
Test organisms (species):
other: Tetrahymena thermophila
Details on test organisms:
The ciliated protozoan T. thermophile was from Key Laboratory of School of Environment, Nanjing University. It was grown axenically at 28 °C for 48 h in a complex medium (pH 6.8 – 7.0) containing 15% proteose–peptone, 5% yeast extracts, and 1% glucose.
Before treated with the test substances, the cells were centrifuged for 20 min at 3500g. The pellets were resuspended at a ratio of 10:1 in 10 mM Tris–HCl buffer (pH 7.4).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Post exposure observation period:
none
Hardness:
not reported
Test temperature:
not reported
pH:
not reported
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
nominal 2.4, 12, 60, 300, 1500 µg/L
Reference substance (positive control):
no
Duration:
30 min
Dose descriptor:
other: DAN damage
Effect conc.:
>= 300 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: DNA damage
Details on results:
In the comet assay test, treatment of T. thermophile cells with DP resulted in a concentration-dependent increase in DNA damage (Fig. 5). The results showed that concentrations of DP from 300 to 1500 µg/L elicited high levels of DNA damage, while the lower concentrations failed to show any treatment-related differences (Fig. 6). Thus, the comet assay is a relatively sensitive tool to estimate the genotoxicity of DP. However it should be noted that previous studies have shown that 5000 mg/kg of DP by gavage for 10 d could not induce DNA strand breaks in mouse livers (Wu et al., 2012), which suggests that the genotoxicity of DP is species specific (Ma et al., 1999). Additionally, it is noteworthy that the tested concentrations showing effects (300 and 1500 µg/L) are more than three orders of magnitude higher than the water solubility of DP and could only be achieved by using DMSO. Thus, the relevance for environmental organisms of the effects at 300 and 1500 µg/L is negligable.
Results with reference substance (positive control):
none applied
Reported statistics and error estimates:
Results were analyzed by SPSS 18.0 software (SPSS Inc.). Statistical differences of biological parameters between DP-treated groups and control group were evaluated using one-way ANOVA test. A probability value < 0.05 (p < 0.05) was accepted as significance.

The comet assay is a well-established and sensitive approach to measure DNA damage in single cells. The assay is widely accepted as a standard method applied to genotoxicology, human biomonitoring, and ecological monitoring. Tail DNA%, Tail Moment (TM) and Olive Tail Moment (OTM) were chosen as the relevant measure of genotoxicity. Among these, OTM is recognized as the most relevant measures, which is computed to be OTM = (tail mean - head mean) * Tail DNA% in arbitrary units (Olive et al., 1990).

Validity criteria fulfilled:
yes
Conclusions:
Although DNA damage was observed at 300 and 1500 µg/L, no effects were seen at lower concentrations of Dechlorane plus. Considering the water solubility of DP being < 300 ng/L and thus more than three orders of magnitude lower than concentration at which effects were seen, these finding are not considered being relevant to aquatic organisms in real life environment.
Executive summary:

In this study, tetrahymena thermophila was chosen as testing organisms to investigate the mutagenicity of DP in the aqueous environment. The concentration gradient of DP used in this study was chosen based on its environmental levels (comet assay: 2.4, 12, 60, 300, 1500 µg/L).

The comet assay conducted with t. thermophila was relatively sensitive as there was a significant increase in DNA damage when the concentrations of DP increased from 300 to 1500 µg/L, while the lower concentrations failed to show any treatment-related differences. Therefore, DP may pose a potential risk at concentration ≥ 300 µg/L, which is more than three orders of magnitude above the limit of water solubility for DP.

Endpoint:
toxicity to other aquatic vertebrates
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although not a standard OECD method the suitability of the method is well documented in peer-reviewed scientific literature (Monarca, S., Zani, C., Richardson, S.D., Thrustonm, A.D., Jr, M., Feretti, M.D., Villarini, M.A., 2004. New approach to evaluating the toxicity and genotoxicity of disinfected drinking water. Water Res. 38, 3809–3819). Thus, the results are considered reliable with restrictions.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Vicia faba micronucleus test
Principles of method if other than guideline:
The V. faba micronucleus test is a short-term bioassay that can predict the genotoxic potential of the pollutant from water (Monarca et al., 2004).
GLP compliance:
not specified
Remarks:
not indicated in the publication
Analytical monitoring:
not specified
Vehicle:
yes
Details on test solutions:
The effects of cosolvent (DMSO) on MCN frequency in vicia faba root tip cells were assessed first. It is clear that the first two groups of DMSO concentrations have no observable effect on cells, while the 1% DMSO shows significant effects. According to the results, 0.5% as the cosolvent concentration in the study was chosen.
Test organisms (species):
other: vicia faba
Details on test organisms:
Root tips of V. faba were chosen for this experiment. Dry broad bean seeds, which were stored in kraft paper bags at 4 °C, were soaked in distilled water for 24 h in the incubator at 25 °C, and then wrapped in wet gauze for another 24 h, before germinating on moist cotton in the dark. When primary roots reached 2 – 3 mm in length, they were selected for germination of lateral roots for 48 h. Lateral roots, when reaching 1.0 – 1.5 cm in length were used for the tests.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
6 h
Post exposure observation period:
exposure was followed by a 24h recovery period
Hardness:
not reported
Test temperature:
not reported during exposure period
pH:
not reported
Dissolved oxygen:
not reported
Salinity:
not reported
Nominal and measured concentrations:
nominal 2.4, 12, 60, 300, 1500 µg/L of DP
Details on test conditions:
The roots chosen were treated for 6 h with distilled water and DMSO solution as two controls, or with DP solutions at various concentrations (2.4, 12, 60, 300, 1500 µg/L). These DP solutions were diluted by distilled water from DP stock solution, with the highest final concentration of DMSO 0.5% in 1500 µg/L. After a 24 h recovery period, roots were cut and fixed in a methanol-acetic acid solution (3:1, v/v) for 24 h. Root tips were rinsed with distilled water twice (5 min duration each time) hydrolyzed in 5 M HCl at 28 °C for 10 min until they were softened. The roots were then strained by Feulgen technique and washed with sulfur dioxide solution for slide preparation.
The mitotic zone (1 mm) was cut off from the root tips with a dissecting needle, immersed in a drop of 45% acetic acid on a slide, mashed with the dissecting needle and squashed under a cover glass. Randomly selected views on the slides were monitored to determine the number of micronucleated cells and the total number of scored cells taken from 6 separate seedlings for each group.
MCN‰ = the number of cells with micronuclei/the total number of scored cells * 1000‰ .
Reference substance (positive control):
no
Duration:
6 h
Dose descriptor:
other: genotoxicity to plants
Effect conc.:
>= 300 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: genotoxicity
Details on results:
Fig. 4(b) indicates the effects of DP on MCN‰ in root tip cells. After exposure to DP solutions for 6 h, MCN‰ neither increases with DP concentration nor shows significant differences between DP treatments and the negative control, with exception for 300 µg/L DP group. This suggests that DP of environmental levels (200 – 2000 pg/L in China) (Jia et al., 2011; Ma et al., 2011) is unlikely to cause genotoxicity following acute exposure.
Results with reference substance (positive control):
none applied
Reported statistics and error estimates:
Results were analyzed by SPSS 18.0 software (SPSS Inc.). Statistical differences of biological parameters between DP-treated groups and control group were evaluated using one-way ANOVA test. A probability value < 0.05 (p < 0.05) was accepted as significance.

The V. faba micronucleus test is a short-term bioassay that can predict the genotoxic potential of the pollutant from water ( Monarca, S., Zani, C., Richardson, S.D., Thrustonm, A.D., Jr, M., Feretti, M.D., Villarini, M.A., 2004. New approach to evaluating the toxicity and genotoxicity of disinfected drinking water. Water Res. 38, 3809–3819). The assay is easy to conduct, inexpensive, rapid, and sensitive in detecting both clastogenic and aneugenic effects in order to assess the genotoxicity of the contaminants (Duan et al., 2000; Hajjouji et al., 2007; Marcato-Romain et al., 2009).

Validity criteria fulfilled:
yes
Conclusions:
The micronucleus test showed no significant difference between treatment and control groups, indicating no genotoxicity of DP.
Executive summary:

In this study, vicia faba was chosen as testing organisms to investigate the mutagenicity of DP to environmental organisms. The concentration gradient of DP used in this study was chosen based on its environmental levels (micronucleus tests: 2.4, 12, 60, 300, 1500 µg/L).

The micronucleus test showed no significant difference between treatment and control groups, indicating no genotoxicity of DP.

Description of key information

In a very recent publication by Dou et al, 2015, genotoxicity to aquatic organisms was investigated showing no genotoxic effects in a vicia faba micronucleus test and no effects in a comet assay with tetrahymena thermophile up to 60 µg/L. The comet assay showed DNA damage at 300 and 1500 µg/L, but such concentrations are more than 5 orders of magnitude above the water solubility of Dechlorane Plus and thus not environmentally relevant.

Additional information

In one study, vicia faba was chosen as testing organisms to investigate the mutagenicity of DP to environmental organisms. The concentration gradient of DP used in this study was chosen based on its environmental levels (micronucleus tests: 2.4, 12, 60, 300, 1500 µg/L). The micronucleus test showed no significant difference between treatment and control groups, indicating no genotoxicity of DP.

In another experiment by the same authors, tetrahymena thermophila was chosen as testing organisms to investigate the mutagenicity of DP in the aqueous environment. The concentration gradient of DP used in this study was identical (comet assay: 2.4, 12, 60, 300, 1500 µg/L). The comet assay conducted with t. thermophila was relatively sensitive as there was a significant increase in DNA damage when the concentrations of DP increased from 300 to 1500 µg/L, while the lower concentrations failed to show any treatment-related differences. Therefore, DP may pose a potential risk at concentration ≥ 300 µg/L, which is more than five orders of magnitude above the limit of water solubility for DP (< 1.67 ng/L), and thus of no relevance.